Scientific Bibliography

Nanobiology and Cancer Nanotechnology
Nanotechnology Devices and Nanomaterials
Nanotechnology-Enabled Therapeutics Development and Delivery
Cancer Diagnostics and Biosensors
Nanotechnologies in Advanced Imaging
Environment, Health and Safety

Cancer Diagnostics and Biosensors

2007  2006   2005   2004   2003   2002

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2007

Multi-reservoir device for detecting a soluble cancer biomarker.
Daniel KD, Kim GY, Vassiliou CC, Jalali-Yazdi F, Langer R, Cima MJ.
Lab Chip. 2007 Oct;7(10):1288-93.
[ expand abstract ]

By combining the sensing capabilities of nanoscale magnetic relaxation switches (MRS) within multi-reservoir structures, a potentially powerful implantable multiplexed sensor has been developed. MRS are magnetic nanoparticles that decrease the transverse relaxation time (T(2)) of water in the presence of an analyte. The switches encased in polydimethylsiloxane (PDMS) devices with polycarbonate membranes (10 nm pores) have demonstrated in vitro sensing of the beta subunit of human chorionic gonadotrophin (hCG-beta), which is elevated in testicular and ovarian cancer. Devices showed transverse relaxation time (T(2)) shortening by magnetic resonance imaging (MRI) when incubated in analyte solutions of 0.5 to 5 microg hCG-beta mL(-1). The decrease in T(2) was between 9% and 27% (compared to control devices) after approximately 28 h. This prototype device is an important first step in developing an implantable sensor for detecting soluble cancer biomarkers in vivo.

In Situ mocelcular profiling of breast cancer biomarkers with multicolor quantum dots.
Yezhelyev MW, Al-Hajj, Morris C, Marcus AI, Liu T, Lewis M, Cohen C, Zrazhevskiy P, Simons JW, Rogatko A, Nie S, Gao X, O'Regan RM.
Advanced Materials. 2007;19(20);3146-51.
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No abstract available. (Citation link)

Detecting and quantifying biomarkers of risk for colorectal cancer using quantum dots and novel image analysis algorithms.
Bostick RM, Kong KY, Ahearn TU, Chaudry Q, Cohen V, Wang MD.
Conf Proc IEEE Eng Med Biol Soc. 2006;1:3313-6.
[ expand abstract ]

Colorectal cancer, the second leading cause of cancer deaths in the United States, is a molecular disease that is largely lifestyle determined and preventable. While heart disease has been sharply declining, in large part from widespread use of biological measurements that indicate risk ("biomarkers of risk"), such as blood cholesterol, to motivate and guide preventive treatment, colorectal cancer is a disease for which mortality rates have changed little and for which there have been no biomarkers of risk. Based on new knowledge about the molecular basis of colorectal cancer we developed and validated a panel of treatable biomarkers of risk that can be measured in rectal biopsies using automated immunohistochemistry and semi-automated image analysis. The methodology is now being made practical for clinical application through the use of 1) quantum dots, so that all of the biomarkers can be detected simultaneously on the same histologic sections (i.e., multiplexed), and 2) novel, automated image analysis algorithms to measure the quantities and tissue distributions of the biomarkers. Herein we summarize our methods, results, current directions, and progress.

In vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles.
Lee D, Khaja S, Velasquez-Castano JC, Dasari M, Sun C, Petros J, Taylor WR, Murthy N.
Nat Mater. 2007 Oct;6(10):765-9.
[ expand abstract ]

The overproduction of hydrogen peroxide is implicated in the development of numerous diseases and there is currently great interest in developing contrast agents that can image hydrogen peroxide in vivo. In this report, we demonstrate that nanoparticles formulated from peroxalate esters and fluorescent dyes can image hydrogen peroxide in vivo with high specificity and sensitivity. The peroxalate nanoparticles image hydrogen peroxide by undergoing a three-component chemiluminescent reaction between hydrogen peroxide, peroxalate esters and fluorescent dyes. The peroxalate nanoparticles have several attractive properties for in vivo imaging, such as tunable wavelength emission (460-630 nm), nanomolar sensitivity for hydrogen peroxide and excellent specificity for hydrogen peroxide over other reactive oxygen species. The peroxalate nanoparticles were capable of imaging hydrogen peroxide in the peritoneal cavity of mice during a lipopolysaccharide-induced inflammatory response. We anticipate numerous applications of peroxalate nanoparticles for in vivo imaging of hydrogen peroxide, given their high specificity and sensitivity and deep-tissue-imaging capability.

Microfluidic platform for single nucleotide polymorphism genotyping of the thiopurine S-methyltransferase gene to evaluate risk for adverse drug events.
Chowdhury J, Kagiala GV, Pushpakom S, Lauzon J, Makin A, Atrazhev A, Stickel A, Newman WG, Backhouse CJ, Pilarski LM.
J Mol Diagn. 2007 Sep;9(4):521-9.
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Prospective clinical pharmacogenetic testing of the thiopurine S-methyltransferase gene remains to be realized despite the large body of evidence demonstrating clinical benefit for the patient and cost effectiveness for health care systems. We describe an entirely microchip-based method to genotype for common single nucleotide polymorphisms in the thiopurine S-methyltransferase gene that lead to serious adverse drug reactions for patients undergoing thiopurine therapy. Restriction fragment length polymorphism and allele-specific polymerase chain reaction have been adapted to a microfluidic chip-based polymerase chain reaction and capillary electrophoresis platform to genotype the common *2, *3A, and *3C functional alleles. In total, 80 patients being treated with thiopurines were genotyped, with 100% concordance between microchip and conventional methods. This is the first report of single nucleotide polymorphism detection using portable instrumentation and represents a significant step toward miniaturized for personalized treatment and automated point-of-care testing.

Ultra-sensitive surface plasmon resonance based immunosensor for prostate-specific antigen using gold nanoparticle–antibody complex.
Jeong-Woo Choia, Da-Yeon Kanga, Yong-Hark Janga, Hyun-Hee Kima, Junhong Mind, Byung-Keun Oha.
Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2008 Feb 1; 313-314:655-659.
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Prostate-specific antigen (PSA) is a marker for prostate cancer and it has been identified as a potential marker for breast cancer in women. In order to early diagnose the prostate cancer in men and the breast cancer in women, an ultra-sensitive diagnostic tool be required. In this study, to improve the sensitivity of the immunosensor for PSA, signal enhancement of surface plasmon resonance (SPR) was done by means of the conjugation of gold (Au) nanoparticle–antibody complex. Biomolecular film on gold surface and the Au nanoparticle-conjugated antibody were fabricated. The fabrication of Au nanoparticle–protein complex was investigated through UV/vis spectroscopy. The surface structure of fabricated biosensing element was investigated by using SPR and scanning tunneling microscopy (STM). The plot of SPR angle difference versus PSA concentration shows the achievement of their linear correlation, and a limit of detection is 300 fM.

Autonomous microfluidic multi-channel chip for real-time PCR with integrated liquid handling.
Frey O, Bonneick S, Hierlemann A, Lichtenberg J.
Biomed Microdevices. 2007 Oct;9(5):711-8.
[ expand abstract ]

We report on a novel, polymer-based, multi-channel device for polymerase chain reaction that combines, for the first time, rapid sample processing in less than 5 min with high throughput at low costs. This is achieved by sample shuttling, during which submicroliter sample plugs (approximately 100 nl) are oscillated rapidly over three constant-temperature zones by pneumatic actuation with integrated system. The accuracy and the speed of the liquid handling have been significantly increased, while the design of the device can be kept very simple and allows for mass production using conventional low-cost polymer fabrication processes. Massive parallelization can lead to a throughput up to 100 samples in 10 min including the preparation time. The amplification can be optically monitored by means of online fluorescence detection. Successful real-time PCR and the determination of the threshold cycle, Ct, using the developed device were demonstrated with plasmid DNA in a fluorescent real-time format.

Cell-based sensor for analysis of EGFR biomarker expression in oral cancer.
Weigum SE, Floriano PN, Christodoulides N, McDevitt JT.
Lab Chip. 2007 Aug;7(8):995-1003.
[ expand abstract ]

Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. To address this clinical need, we have developed a cell-based sensor to detect oral cancer biomarkers, such as the epidermal growth factor receptor (EGFR) whose over-expression is associated with early oral tumorigenesis and aggressive cancer phenotypes. The lab-on-a-chip (LOC) sensor utilizes an embedded track-etched membrane, which functions as a micro-sieve, to capture and enrich cells from complex biological fluids or biopsy suspensions. Once captured, "on-membrane" immunofluorescent assays reveal the presence and isotype of interrogated cells via automated microscopy and fluorescent image analysis. Using the LOC sensor system, with integrated capture and staining technique, EGFR assays were completed in less than 10 minutes with staining intensity, homogeneity, and cellular localization patterns comparable to conventional labeling methods. Further examination of EGFR expression in three oral cancer cell lines revealed a significant increase (p < 0.05) above control cells with EGFR expression similar to normal squamous epithelium. Results obtained in the microfluidic sensor system correlated well with flow cytometry (r(2) = 0.98), the "gold standard" in quantitative protein expression analysis. In addition, the LOC sensor detected significant differences between two of the oral cancer cell lines (p < 0.01), accounting for disparity of approximately 34 000 EGFR per cell according to quantitative flow cytometry. Taken together, these results support the LOC sensor system as a suitable platform for rapid detection of oral cancer biomarkers and characterization of EGFR over-expression in oral malignancies. Application of this technique may be clinically useful in cancer diagnostics for early detection, prognostic evaluation, and therapeutic selection. Having demonstrated the functionality of this integrated microfluidic sensor system, further studies using clinical samples from oral cancer patients are now warranted.

Quantitative evaluation of sensitivity and selectivity of multiplex nanoSPR biosensor assays.
Yu C, Irudayaraj J.
Biophys J. 2007 Nov 15;93(10):3684-92.
[ expand abstract ]

A new functionalization procedure was developed to replace cyltrimethylammoniumbromide coating on gold nanorods (GNRs) fabricated through seed-mediated growth with chemically active alkanethiols; antibodies were then attached to the GNRs to yield gold nanorod molecular probes (GNrMPs). The functionalization procedure was shown to minimize nonspecific binding. Multiplex sensing was demonstrated for three targets (goat anti-human IgG, goat anti-rabbit IgG, and goat anti-mouse IgG) through the distinct response of the plasmon spectra of GNrMPs to binding events. Quantification of the plasmonic binding events and estimation of ligand binding kinetics tethered to these nanoscale structures was also demonstrated through a mathematical approach. Evaluation of the experimental and theoretical data yields an affinity constant K(a) = 1.34 x 10(7) M(-1), which was in agreement with the IgG-antiIgG binding affinity reported in the literature. The GNrMP sensors were found to be highly specific and sensitive with the dynamic response in the range between 10(-9) M and 10(-6) M. The limit of detection of GNrMPs was found to be in the low nanomolar range, and is a function of the binding affinity: for a higher probe-target affinity pair, the limit of detection can be expected to reach femto molar levels. This technique can play a key role in developing tunable sensors for sensitive and precise monitoring of biological interactions.

Identity profiling of cell surface markers by multiplex gold nanorod probes.
Yu C, Nakshatri H, Irudayaraj J.
Nano Lett. 2007 Aug;7(8):2300-6.
[ expand abstract ]

Gold nanorod molecular probes (GNrMPs) were designed and fabricated for multiplex identification of cell surface markers in HBECs. Cells were probed directly using dark field microscopy integrated with a spectral imager for simultaneous detection of up to three surface markers. The immunophenotype composition of these cell lines indicative of their metastasis potential was assessed using the GNrMPs. The technique has the potential to become an important tool for diagnosis and prognosis of breast and other cancers.

Aptamer-conjugated nanoparticles for the collection and detection of multiple cancer cells.
Smith JE, Medley CD, Tang Z, Shangguan D, Lofton C, Tan W.
Anal Chem. 2007 Apr 15;79(8):3075-82.
[ expand abstract ]

We have extended the use the aptamer-conjugated nanoparticles for the collection and detection of multiple cancer cells. The aptamers were selected using a cell-based SELEX strategy in our laboratory for cancer cells that, when utilized in this method, allow for the selective recognition of the cells from complex mixtures including fetal bovine serum samples. Aptamer-conjugated magnetic nanoparticles were used for the selective targeting cell extraction, and aptamer-conjugated fluorescent nanoparticles were employed for sensitive cellular detection. Employing both types of nanoparticles allows for selective and sensitive detection not possible by using the particles separately. Fluorescent nanoparticles amplify the signal intensity versus a single fluorophore label resulting in improved sensitivity. In addition, aptamer-conjugated magnetic nanoparticles allow for extraction and enrichment of target cells not possible with other separation methods. Fluorescent imaging and a microplate reader were used for cellular detection to demonstrate the wide applicability of this methodology for medical diagnostics and cell enrichment and separation.

Detecting SNPs using a synthetic nanopore.
Zhao Q, Sigalov G, Dimitrov V, Dorvel B, Mirsaidov U, Sligar S, Aksimentiev A, Timp G.
Nano Lett. 2007 Jun;7(6):1680-5.
[ expand abstract ]

We have discovered a voltage threshold for permeation through a synthetic nanopore of dsDNA bound to a restriction enzyme that depends on the sequence. Molecular dynamic simulations reveal that the threshold is associated with a nanonewton force required to rupture the DNA-protein complex. A single mutation in the recognition site for the restriction enzyme, i.e., a single nucleotide polymorphism (SNP), can easily be detected as a change in the threshold voltage. Consequently, by measuring the threshold voltage in a synthetic nanopore, it may be possible to discriminate between two variants of the same gene (alleles) that differ in one base.

Aptamer-functionalized gold nanoparticles for turn-on light switch detection of platelet-derived growth factor.
Huang CC, Chiu SH, Huang YF, Chang HT.
Anal Chem. 2007 Jul 1;79(13):4798-804.
[ expand abstract ]

An aptamer modified gold nanoparticles (Apt-AuNPs) based molecular light switching sensor has been demonstrated for the analysis of breast cancer markers (platelet-derived growth factors (PDGFs) and their receptors) in homogeneous solutions. The PDGF binding aptamer has a unique structure with triple-helix conformation that allows N,N-dimethyl-2,7-diazapyrenium dication (DMDAP) and PDGF bindings. The fluorescence of DMDAP is almost completely quenched by Apt-AuNPs when it intercalates with the aptamers. Owing to high magnitudes of increases (up to 40-fold) in the turn-on fluorescence signals of DMDAP/Apt-AuNP upon PDGFs binding, the approach is highly sensitive for the detection of PDGFs. The DMDAP/Apt-AuNP probe specifically and sensitively detected PDGFs under optimal concentrations of salts and DMDAP. We also demonstrated that the Apt-AuNPs are effective selectors for enrichment of PDGF-AA from large-volume samples. The approach allows detection of PDGF-AA at a concentration down to 8 pM, showing better sensitivity than other signal aptamers. By conducting a competitive assay, we demonstrated the determination of PDGF receptor-alpha with LOD of 0.25 nM when using the DMDAP/Apt-AuNP as a probe.

Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry.
Xing Y, Chaudry, Q, Shen C, Kong KY, Zhau HE, chung LW, Petros JA, O’regan RM, Yezhelyev MW, Simons JW, Wang MD, Nie S.
Nat. Protocols. 2007; 2(5): 1152-65.
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Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD–antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1–3 days depending on the number of biomarkers and QD probes used.

Nanoparticle Self-Assembly Gated by Logical Proteolytic Triggers.
von Maltzahn G, Harris TJ, park JH, Min DH, Schmidt AJ, Sailor MJ, Bhatia SN.
J. Am. Chem. Soc. 2007 May 16;129(19):6064 -65.
[ expand abstract ]

The emergent electromagnetic properties of nanoparticle self-assemblies are being harnessed to build new medical and biochemical assays with unprecedented sensitivity. While current self-assembly assays have displayed superior sensitivity for single molecular targets, the development of systems with the capacity to process multiple inputs may more effectively decipher complex disease signatures such as cancer. Herein, we present the design and synthesis of nanoparticles that perform Boolean logic operations using two proteolytic inputs associated with unique aspects of tumorigenesis (MMP2 and MMP7). Using dynamic light scatting, fluorescence, and MRI, we show that logical AND and OR functions can control the self-assembly of disperse superparamagnetic nanoparticles and enable remote, NMR detection of nanoparticle computation. In the future, by increasing the complexity of assembly triggers, nanoparticles may be tailored to sense a diversity of disease inputs in vitro and potentially in vivo.

EPCA-2: a highly specific serum marker for prostate cancer.
Leman ES, Cannon GW, Trock BJ, Sokoll LJ, Chan DW, Mangold L, Partin AW, Getzenberg RH.
Urology. 2007 Apr;69(4):714-20.
[ expand abstract ]

OBJECTIVES: To describe the initial assessment of early prostate cancer antigen (EPCA)-2 as a serum marker for the detection of prostate cancer and to examine its sensitivity and specificity. METHODS: Serum samples were obtained from 385 men: those with prostate-specific antigen (PSA) levels less than 2.5 ng/mL, PSA levels of 2.5 ng/mL or greater with negative biopsy findings, benign prostatic hyperplasia, organ-confined prostate cancer, non-organ-confined disease, and prostate cancer with PSA levels less than 2.5 ng/mL. In addition, a diverse group of controls was assessed with an enzyme-linked immunosorbent assay to detect an epitope of the EPCA-2 protein, EPCA-2.22. RESULTS: Using a cutoff of 30 ng/mL, the EPCA-2.22 assay had a 92% specificity (95% confidence interval 85% to 96%) for healthy men and men with benign prostatic hyperplasia and 94% sensitivity (95% confidence interval [CI] 93% to 99%) for overall prostate cancer. The specificity for PSA in these selected groups of patients was 65% (95% CI 55% to 75%). Additionally, EPCA-2.22 was highly accurate in differentiating between localized and extracapsular disease (area under the curve 0.89, 95% CI 0.82 to 0.97, P <0.0001) in contrast to PSA (area under the curve 0.62, 95% CI 0.50 to 0.75, P = 0.05). CONCLUSIONS: The results of our study have shown that EPCA-2 is a novel biomarker associated with prostate cancer that has high sensitivity and specificity and accurately differentiates between men with organ-confined and non-organ-confined disease.

Using luminescent nanoparticles as staining probes for Affymetrix GeneChips.
Wang L, Lofton C, Popp M, Tan W.
Bioconjug Chem. 2007 May-Jun;18(3):610-3.
[ expand abstract ]

Microarray technology provides efficient access to genetic information using miniaturized, high-density arrays of DNA probes. We investigated the application of luminescent nanoparticles as probes for Affymetrix GeneChips detection without the need for signal amplification. Our goal is to investigate the feasibility of using luminescent nanoparticles as probes in a commercial microarray system without changing its configurations. With the present imaging modality and existing optical excitation and detection systems of the Affymetrix GeneChips, our early results indicate that nanoparticles not only can be used for GeneChip labeling but also are superior to the traditional fluorescent protein streptavidin-phycoerythrin (SAPE). The advantage of the particles lies in a simplified staining procedure, higher photobleaching threshold, and enhanced luminescence signal. The nanoparticles can be used for detection of low-abundance targets without any amplification step. A concentration detection limit of 50 fM has been achieved. This work demonstrates the feasibility of using luminescent nanoparticles as probes for commercial microarray systems, making them less costly, more reproducible, and potentially quantitative.

A piezoelectric immunosensor for the detection of alpha-fetoprotein using an interface of gold/hydroxyapatite hybrid nanomaterial.
Ding Y, Liu J, Wang H, Shen G, Yu R.
Biomaterials. 2007 Apr;28(12):2147-54.
[ expand abstract ]

The ideal immobilization methods that are suitable for binding immuno-active materials with high efficiency onto the sensing surface are the key target to pursue in the current biosensor design. In this paper, a new hybrid material formed by assembling gold nanoparticles (GNP) onto nano-sized hydroxyapatite (HA) has been employed for the interface design of piezoelectric immunosensor, on which the antibodies were bound. The detection performances of the resulting immunosensor were investigated by use of the antibody-antigen model system of alpha-Fetoprotein (AFP), an important indicator in the diagnosis of clinical cancers. The hybrid material was characterized by the UV-vis spectroscopy, the SEM and TEM measurements. The frequency and electrochemical impedance responses characteristics for the processes of immobilization and immunoreaction of anchored anti-AFP antibodies were studied in detail. The immunoresponse of the proposed immunosensor was compared with those antibodies immobilized by using HA or GNP alone. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of AFP in the concentration range of 15.3-600.0ngml(-1). Such an interface design with the nano-sized hybrid materials should be tailored as a new alternative used for biosensor design.

G-rich oligonucleotide-functionalized gold nanoparticle aggregation.
Wu ZS, Guo MM, Shen GL, Yu RQ.
Anal Bioanal Chem. 2007 Feb 9; [Epub ahead of print].
[ expand abstract ]

Guanine-rich DNA sequences commonly form helical quadruplex structures via Hoogsteen hydrogen bonds. The aggregation behavior of the nanoparticles, which are functionalized with four-guanine-terminated 27-base sequences at a nanoparticle-to-DNA ratio of 1:60, is investigated. To some extent, the guanine-quadruplex structures between the gold nanoparticles (GNPs) promote nanoparticle aggregation. However, the coordination site of the metal ion on the nanoparticle surface is partially passivated: the stability of guanine-rich DNA-GNPs is slightly lower than that of the usual DNA-GNPs, and the metal-ion specificity of nanoparticle assembly is substantially decreased. Thus, a mechanism for the aggregation of guanine-rich sequence-modified GNPs is proposed. It is possible to obtain a stable guanine-rich sequence-functionalized nanoparticle solution at high ionic strength by regulating guanine-rich DNA sequences. The controllability of guanine-rich sequence-modified nanoparticles makes the secondary structure of DNA a potentially useful candidate for DNA analysis and disease diagnostics. Figure Proposed mechanism for the aggregation of G-rich sequence-functionalized GNP.

PCR amplification on magnetic nanoparticles: Application for high-throughput single nucleotide polymorphism genotyping.
Liu H, Li S, Wang Z, Hou P, He Q, He N.
Biotechnol J. 2007 Feb 7; [Epub ahead of print].
[ expand abstract ]

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.

A novel approach to ultrasensitive diagnosis using supramolecular protein nanoparticles.
Lee SH, Lee H, Park JS, Choi H, Han KY, Seo HS, Ahn KY, Han SS, Cho Y, Lee KH, Lee J.
FASEB J. 2007 Feb 5; [Epub ahead of print].
[ expand abstract ]

We report on the ultrasensitive protein nanoprobe system that specifically captures disease marker (autoantibodies of Type I diabetes in this case) with attomolar sensitivity. The system relies on supramolecular protein nanoparticles that bind a specific antibody [65 kDa glutamate decarboxylase (GAD65)-specific autoantibody, i.e., the early marker of Type I diabetes]. The ultrasensitive detection of early marker of Type I diabetes during the early phase of pancreatic beta-cell destruction is important because individuals at high risk of developing Type I diabetes can be identified several years before the clinical onset of the ailment. The bacterial expression of chimera genes encoding N-[human ferritin heavy chain (hFTN-H)]::[specific antigenic epitope]-C produces supramolecular nanoparticles with uniform diameters (10-15 nm), owing to self-assembly activity of hFTN-H. Each nanoparticle, formed by intermolecular self-assembly between the chimera protein molecules, is subjected to carrying a large number (presumably, 24) of epitopes with a homogeneous and stable conformation per autoantibody binding, thereby allowing substantial enhancement of sensitivity. The sensitivity was finally boosted to 3 attomolar concentration of the autoantibodies, 4-9 orders of magnitude more sensitive than conventional immunoassays. Also, this ultrasensitive protein nanoprobe successfully detected natural autoantibodies in the sera from Type I diabetic patients. The attomolar sensitivity was successfully reproduced on the detection of other antibodies, i.e., monoclonal antibodies against hepatitis B surface antigen. With the two antibody markers above, the feasibility of simultaneous and multiplexing-mode detection was also demonstrated.

Sensitized luminescent terbium nanoparticles: preparation and time-resolved fluorescence assay for DNA.
Chen Y, Chi Y, Wen H, Lu Z.
Anal Chem. 2007 Feb 1;79(3):960-5.
[ expand abstract ]

A highly luminescent terbium nanoparticle as the biolabel based on the sensitization of a dye molecule was prepared. The luminescent complexes included in the particles were composed of a quinolone-based dye molecule as the light-energy transfer donor and a polyaminocarboxylate-based chelator with excellent water-solubility and a high binding constant for lanthanides. The structure of two functional entities in the single molecule made the complex highly luminescent in aqueous solution. Silica nanoparticles containing terbium complexes were prepared by the reverse microemulsion method. Such a terbium nanoparticle is as bright as about 340 free terbium complexes, and it has a 1.5-ms fluorescence lifetime that enables it to be used in the time-resolved fluorescence assays. The conjugate of the nanoparticle with oligonucleotide was prepared and used to carry out a DNA sandwich hybridization assay based on magnetic microbeads as solid-phase carrier. The experimental results showed that the detection sensitivity with the nanoparticles is more than 100-fold as high as that with dye Fluorescein isothiocyanate (FITC) molecules.

Recognition and capture of breast cancer cells using an antibody-based platform in a microelectromechanical systems device.
Du Z, Cheng KH, Vaughn MW, Collie NL, Gollahon LS.
Biomed Microdevices. 2007 Feb;9(1):35-42.
[ expand abstract ]

Cancer is one of the most common diseases afflicting humans. The use of biomarkers specific for tumor cells has facilitated their identification. However, technology has not kept pace with the field of molecular biomarkers, leaving their potential unrealized. Here, we demonstrate the efficacy of recognizing and capturing cancer cells using an antibody-based, on-chip, microfluidic device. A cancer cell capture biochip consisting of microchannels of size 2.0 cm long and 500 mum wide and deep, was etched onto Polydimethylsiloxane. Epithelial membrane antigen (EMA) and Epithelial growth factor receptor (EGFR) were coated on the inner surface of the microchannels. The overall chip measured 2.0 cm x 1.5 cm x 0.5 cm. Normal and tumor breast cells in a phosphate buffered saline (PBS) suspension were flowed through the biochip channels at a rate of 15 muL/min. Breast cancer cells were preferentially captured and identified while most of normal cells passed through. The capture rates for tumor and normal cells were found to be >30% and <5%, respectively. This preliminary cancer cell capture biochip design supports our initial effort of moving a BioMEMS device, from the bench top to the clinic.

Nanofluidic structures for single biomolecule fluorescent detection.
Mannion JT, Craighead HG.
Biopolymers. 2007 Feb;85(2):131-43.
[ expand abstract ]

Fluid-filled nanofabricated cavities can be used to increase the spatial resolution of single molecule confocal microscopy based techniques by creating smaller and more uniformly illuminated probe volumes. Such structures may also be used to temporarily stretch single macromolecules, permitting the resolution of molecular details that would otherwise be beyond the capabilities of a diffraction limited system.

Quantum dots for molecular pathology: their time has arrived.
True LD, Gao X.
J Mol Diagn. 2007 Feb;9(1):7-11.
[ expand abstract ]

Assessing malignant tumors for expression of multiple biomarkers provides data that are critical for patient management. Quantum dot-conjugated probes to specific biomarkers are powerful tools that can be applied in a multiplex manner to single tissue sections of biopsies to measure expression levels of multiple biomarkers.

Label-free protein biosensor based on aptamer-modified carbon nanotube field-effect transistors.
Maehashi K, Katsura T, Kerman K, Takamura Y, Matsumoto K, Tamiya E.
Anal Chem. 2007 Jan 15;79(2):782-7.
[ expand abstract ]

We have fabricated label-free protein biosensors based on aptamer-modified carbon nanotube field-effect transistors (CNT-FETs) for the detection of immunoglobulin E (IgE). After the covalent immobilization of 5'-amino-modified 45-mer aptamers on the CNT channels, the electrical properties of the CNT-FETs were monitored in real time. The introduction of target IgE at various concentrations caused a sharp decrease in the source-drain current, and a gradual saturation was observed at lower concentrations. The amount of the net source-drain current before and after IgE introduction on the aptamer-modified CNT-FETs increased as a function of IgE concentration. The detection limit for IgE was determined as 250 pM. We have also prepared CNT-FET biosensors using a monoclonal antibody against IgE (IgE-mAb). The electrical properties of the aptamer- and antibody-modified CNT-FETs were compared. The performance of aptamer-modified CNT-FETs provided better results than the ones obtained using IgE-mAb-modified CNT-FETs under similar conditions. Thus, we suggest that the aptamer-modified CNT-FETs are promising candidates for the development of label-free protein biosensors.

A label-free electrochemical immunoassay for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and nonconductive polymer film.
Tang H, Chen J, Nie L, Kuang Y, Yao S.
Biosens Bioelectron. 2007 Jan 15;22(6):1061-7.
[ expand abstract ]

A simple and sensitive label-free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) has been developed. CEA antibody (CEAAb) was covalently attached on glutathione (GSH) monolayer-modified gold nanoparticle (AuNP) and the resulting CEAAb-AuNP bioconjugates were immobilized on Au electrode by electro-copolymerization with o-aminophenol (OAP). Electrochemical impedance spectroscopy and cyclic voltammetry studies demonstrate that the formation of CEA antibody-antigen complexes increases the electron transfer resistance of [Fe(CN)(6)](3-/4-) redox pair at the poly-OAP/CEAAb-AuNP/Au electrode. The use of CEA antibody-AuNP bioconjugates and poly-OAP film could enhance the sensitivity and anti-nonspecific binding of the resulting immunoassay electrode. The preliminary application of poly-OAP/CEAAb-AuNP/Au electrode for detection of CEA was also evaluated.

Multiplex biosensor using gold nanorods.
Yu C, Irudayaraj J.
Anal Chem. 2007 Jan 15;79(2):572-9.
[ expand abstract ]

Gold nanorods (GNRs) with different aspect ratios were fabricated through seed-mediated growth and surface activation by alkanethiols for the attachment of antibodies to yield gold nanorod molecular probes (GNrMPs). Multiplex sensing was demonstrated by the distinct response of the plasmon spectra of the GNrMPs to binding events of three targets (goat anti-human IgG1 Fab, rabbit anti-mouse IgG1 Fab, rabbit anti-sheep IgG (H+L)). Plasmonic sensors are highly specific and sensitive and can be used to monitor refractive index changes caused by molecular interactions in their immediate vicinity with potential to achieve single-particle biosensing. This technique can play a key role in developing novel optical biosensors for both in vivo and in vitro detection and single-receptor kinetics.

DNA Nucleoside Interaction and Identification with Carbon Nanotubes.
Meng S, Maragakis P, Papaloukas C, Kaxiras E.
Nano Lett. 2007 Jan 10;7(1):45-50.
[ expand abstract ]

We investigate the interaction of individual DNA nucleosides with a carbon nanotube (CNT) in vacuum and in the presence of external gate voltage. We propose a scheme to discriminate between nucleosides on CNTs based on measurement of electronic features through a local probe such as scanning tunneling spectroscopy. We demonstrate through quantum mechanical calculations that these measurements can achieve 100% efficiency in identifying DNA bases. Our results support the practicality of ultrafast DNA sequencing using electrical measurements.

An integrated fluorescence detection system for lab-on-a-chip applications.
Novak L, Neuzil P, Pipper J, Zhang Y, Lee S.
Lab Chip. 2007 Jan;7(1):27-9.
[ expand abstract ]

We present a low-cost miniaturized fluorescence detection system for lab-on-a-chip applications with a sensitivity in the low nanomolar range; a built-in lock-in amplifier enables measurements under ambient light.

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2006

Immunoasssay based on the antibody-conjugated PAMAM-dendrimer-gold quantum dot complex.
Triulzi RC, Micic M, Giordani S, Serry M, Chiou WA, Leblanc RM.
Chem Commun (Camb). 2006 Dec 28;(48):5068-70.
[ expand abstract ]

An immunoassay based upon photoluminescent gold quantum dots aimed at detecting human IgG in aqueous solution from micromolar to nanomolar concentrations is described.

Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays.
Stoermer RL, Cederquist KB, McFarland SK, Sha MY, Penn SG, Keating CD.
J Am Chem Soc. 2006 Dec 27;128(51):16892-903.
[ expand abstract ]

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

Bioelectrocatalytic application of titania nanotube array for molecule detection.
Xie Y, Zhou L, Huang H.
Biosens Bioelectron. 2006 Dec 22; [Epub ahead of print].
[ expand abstract ]

A bioelectrocatalysis system based on titania nanotube electrode has been developed for the quantitative detection application. Highly ordered titania nanotube array with inner diameter of 60nm and total length of 540nm was formed by anodizing titanium foils. The functionalization modification was achieved by embedding glucose oxidases inside tubule channels and electropolymerizing pyrrole for interfacial immobilization. Morphology and microstructure characterization, electrochemical properties and bioelectrocatalytic reactivities of this composite were fully investigated. The direct detection of hydrogen peroxide by electrocatalytic reduction reaction was fulfilled on pure titania nanotube array with a detection limit up to 2.0x10(-4)mM. A biosensor based on the glucose oxidase-titania/titanium electrode was constructed for amperometric detection and quantitative determination of glucose in a phosphate buffer solution (pH 6.8) under a potentiostatic condition (-0.4V versus SCE). The resulting glucose biosensor showed an excellent performance with a response time below 5.6s and a detection limit of 2.0x10(-3)mM. The corresponding detection sensitivity was 45.5muAmM(-1)cm(-2). A good operational reliability was also achieved with relative standard deviations below 3.0%. This novel biosensor exhibited quite high response sensitivity and low detection limit for potential applications.

Quantitative real-time measurements of DNA hybridization with alkylated nonoxidized silicon nanowires in electrolyte solution.
Bunimovich YL, Shin YS, Yeo WS, Amori M, Kwong G, Heath JR.
J Am Chem Soc. 2006 Dec 20;128(50):16323-31.
[ expand abstract ]

The quantitative, real-time detection of single-stranded oligonucleotides with silicon nanowires (SiNWs) in physiologically relevant electrolyte solution is demonstrated. Debye screening of the hybridization event is circumvented by utilizing electrostatically adsorbed primary DNA on an amine-terminated NW surface. Two surface functionalization chemistries are compared: an amine-terminated siloxane monolayer on the native SiO2 surface of the SiNW, and an amine-terminated alkyl monolayer grown directly on a hydrogen-terminated SiNW surface. The SiNWs without the native oxide exhibit improved solution-gated field-effect transistor characteristics and a significantly enhanced sensitivity to single-stranded DNA detection, with an accompanying 2 orders of magnitude improvement in the dynamic range of sensing. A model for the detection of analyte by SiNW sensors is developed and utilized to extract DNA-binding kinetic parameters. Those values are directly compared with values obtained by the standard method of surface plasmon resonance (SPR) and demonstrated to be similar. The nanowires, however, are characterized by higher detection sensitivity. The implication is that SiNWs can be utilized to quantitate the solution-phase concentration of biomolecules at low concentrations. This work also demonstrates the importance of surface chemistry for optimizing biomolecular sensing with silicon nanowires.

A nanocatalyst-based assay for proteins: DNA-free ultrasensitive electrochemical detection using catalytic reduction of p-nitrophenol by gold-nanoparticle labels.
Das J, Aziz MA, Yang H.
J Am Chem Soc. 2006 Dec 20;128(50):16022-3.
[ expand abstract ]

No abstract available

A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability.
Easley CJ, Karlinsey JM, Bienvenue JM, Legendre LA, Roper MG, Feldman SH, Hughes MA, Hewlett EL, Merkel TJ, Ferrance JP, Landers JP.
Proc Natl Acad Sci USA. 2006 Dec 19;103(51):19272-7.
[ expand abstract ]

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

Aptamer-Capped Nanocrystal Quantum Dots: A New Method for Label-Free Protein Detection.
Choi JH, Chen KH, Strano MS.
J Am Chem Soc. 2006 Dec 13;128(49):15584-15585.
[ expand abstract ]

We demonstrate that aptamer-capped near-infrared PbS quantum dots (QDs) can detect a target protein based on selective charge transfer. The water-soluble QDs are synthesized with the thrombin-binding aptamer, which retains the secondary quadruplex structure necessary for binding to thrombin. These QDs have diameters of 3-6 nm and fluoresce around 1050 nm. When the aptamer-functionalized QD binds to its target, a fluorescence quenching occurs due to charge transfer from amine groups on the protein to the QD. Thrombin is detected within 1 min with a detection limit of approximately 1 nM. This selective detection is observed even in the presence of high background concentrations of interfering negatively or positively charged proteins, suggesting that aptamer-capped QDs could be useful for label-free protein assays.

A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability.
Easley CJ, Karlinsey JM, Bienvenue JM, Legendre LA, Roper MG, Feldman SH, Hughes MA, Hewlett EL, Merkel TJ, Ferrance JP, Landers JP.
Proc Natl Acad Sci USA. 2006 Dec 11; [Epub ahead of print].
[ expand abstract ]

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

Resistive-pulse DNA detection with a conical nanopore sensor.
Harrell CC, Choi Y, Horne LP, Baker LA, Siwy ZS, Martin CR.
Langmuir. 2006 Dec 5;22(25):10837-43.
[ expand abstract ]

In this paper, we describe resistive-pulse sensing of two large DNAs, a single-stranded phage DNA (7250 bases) and a double-stranded plasmid DNA (6600 base pairs), using a conically shaped nanopore in a track-etched polycarbonate membrane as the sensing element. The conically shaped nanopore had a small-diameter (tip) opening of 40 nm and a large-diameter (base) opening of 1.5 microm. The DNAs were detected using the resistive-pulse, sometimes called stochastic sensing, method. This entails applying a transmembrane potential difference and monitoring the resulting ion current flowing through the nanopore. The phage DNA was driven electrophoretically through the nanopore (from tip to base), and these translocation events were observed as transient blocks in the ion current. We found that the frequency of these current-block events scales linearly with the concentration of the DNA and with the magnitude of the applied transmembrane potential. Increasing the applied transmembrane potential also led to a decrease in the duration of the current-block events. We also analyzed current-block events for the double-stranded plasmid DNA. However, because this DNA is too large to enter the tip opening of the nanopore, it could not translocate the pore. As a result, much shorter duration current-block events were observed, which we postulate are associated with bumping of the double-stranded DNA against the tip opening.

One-Step Homogeneous Detection of DNA Hybridization with Gold Nanoparticle Probes by Using a Linear Light-Scattering Technique.
Du BA, Li ZP, Liu CH.
Angew Chem Int Ed Engl. 2006 Dec 4;45(47):8022-8025.
[ expand abstract ]

No abstract available

Brain cancer diagnosis and therapy with nanoplatforms.
Koo YE, Reddy GR, Bhojani M, Schneider R, Philbert MA, Rehemtulla A, Ross BD, Kopelman R.
Adv Drug Deliv Rev. 2006 Dec 1;58(14):1556-77.
[ expand abstract ]

Treatment of brain cancer remains a challenge despite recent improvements in surgery and multimodal adjuvant therapy. Drug therapies of brain cancer have been particularly inefficient, due to the blood-brain barrier and the non-specificity of the potentially toxic drugs. The nanoparticle has emerged as a potential vector for brain delivery, able to overcome the problems of current strategies. Moreover, multi-functionality can be engineered into a single nanoplatform so that it can provide tumor-specific detection, treatment, and follow-up monitoring. Such multitasking is not possible with conventional technologies. This review describes recent advances in nanoparticle-based detection and therapy of brain cancer. The advantages of nanoparticle-based delivery and the types of nanoparticle systems under investigation are described, as well as their applications.

Nanopore sequencing technology: research trends and applications.
Rhee M, Burns MA.
Trends Biotechnol. 2006 Dec;24(12):580-6.
[ expand abstract ]

Nanopore sequencing is one of the most promising technologies being developed as a cheap and fast alternative to the conventional Sanger sequencing method. Protein or synthetic nanopores have been used to detect DNA or RNA molecules. Although none of the technologies to date has shown single-base resolution for de novo DNA sequencing, there have been several reports of alpha-hemolysin protein nanopores being used for basic DNA analyses, and various synthetic nanopores have been fabricated. This review will examine current nanopore sequencing technologies, including recent developments of new applications.

Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis.
Becker KF, Schott C, Hipp S, Metzger V, Porschewski P, Beck R, Nahrig J, Becker I, Hofler H.
J Pathol. 2006 Nov 28; [Epub ahead of print].
[ expand abstract ]

Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2 + ) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.

Homogeneous silver-coated nanoparticle substrates for enhanced fluorescence detection.
Xie F, Baker MS, Goldys EM.
J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2006 Nov 23;110(46):23085-91.
[ expand abstract ]

A simple method has been developed for the deposition of uniform silver-coated nanoparticles on glass substrates, with a homogeneous distribution shown by scanning electron microscopy (SEM). UV-visible spectroscopy and energy-dispersive X-ray analysis (EDX) have been used to characterize both the optical density and elemental content of the deposited nanoparticles. The fluorescence enhancement was investigated using a monolayer of FITC-conjugated human serum albumin (FITC-HSA) and tested using laser scanning microscopy at 488 nm excitation wavelength. The enhancement factor was calculated from individual spectra recorded with a Fluorolog-Tau-3 spectrofluorometer. We identified the nanoparticle growth regime which led to fluorescence enhancement. Such enhancement is detectable when Au core-Ag shell nanoparticles increased their size to 47 nm, in agreement with theoretical estimates. The origin of this enhancement for appropriate size nanoparticles is attributed to the effect of an increased excitation rate from the local field enhanced by the interaction of incident light with the nanoparticles and/or higher quantum yield from an increase of the intrinsic decay rate of the fluorophore. We thus demonstrated that the Au core-Ag shell nanostructures on glass surfaces are promising substrates for fluorescence enhancement with outstanding macroscopic homogeneity. This important feature will pave the way for the application of our substrates in biotechnology and life sciences such as imaging and sensing of biomolecules in proteomics.

Label-free immunosensor for prostate-specific antigen based on single-walled carbon nanotube array-modified microelectrodes.
Okuno J, Maehashi K, Kerman K, Takamura Y, Matsumoto K, Tamiya E.
Biosens Bioelectron. 2006 Nov 14; [Epub ahead of print].
[ expand abstract ]

We have fabricated a label-free electrochemical immunosensor using microelectrode arrays modified with single-walled carbon nanotubes (SWNTs). Label-free detection of a cancer marker, total prostate-specific antigen (T-PSA), was carried out using differential pulse voltammetry (DPV). The current signals, derived from the oxidation of tyrosine (Tyr), and tryptophan (Trp) residues, increased with the interaction between T-PSA on T-PSA-mAb covalently immobilized on SWNTs. The selectivity of our biosensor was challenged using bovine serum albumin (BSA) as the target protein. The detection limit for T-PSA was determined as 0.25ng/mL. Since the cut-off limit of T-PSA between prostate hyperplasia and cancer is 4ng/mL, the performance of our label-free electrochemical immunosensor seems promising for further clinical applications.

One-Step Homogeneous Detection of DNA Hybridization with Gold Nanoparticle Probes by Using a Linear Light-Scattering Technique.
Du BA, Li ZP, Liu CH.
Angew Chem Int Ed Engl. 2006 Nov 8; [Epub ahead of print].
[ expand abstract ]

No abstract available

Recognition on the Nanoscale of a DNA Sequence by an Inorganic Crystal Surface.
Scipioni A, Pisano S, Bergia A, Savino M, Samori B, De Santis P.
Chembiochem. 2006 Nov 6;7(11):1645-1648.
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No abstract available

An integrated and sensitive detection platform for magneto-resistive biosensors.
de Boer BM, Kahlman JA, Jansen TP, Duric H, Veen J.
Biosens Bioelectron. 2006 Nov 1; [Epub ahead of print].
[ expand abstract ]

A compact biosensor platform with giant magneto-resistive (GMR) sensors suited for the detection of superparamagnetic nanoparticle labels is presented. The platform consist of disposable biosensor cartridges and an electronic reader, which enables quantitative detection with high analytical performance, combined with robustness, ease of use and at low cost. In order to optimise the signal-to-noise ratio (SNR), magnetic labels are excited at high frequency. Wires, integrated in the silicon of the sensor chip are used to generate a well-defined magnetic field on the sensor surface, thus removing the need for mechanical alignment with external apparatus. A signal modulation scheme is applied to obtain optimal detection accuracy. The platform is scalable and can be adapted according to application-specific requirements. Experimental results indicate that three beads of 300nm diameter can be detected on a sensor surface of 1500mum(2) for a measurement time of 1s.

Attomole microarray detection of microRNAs by nanoparticle-amplified SPR imaging measurements of surface polyadenylation reactions.
Fang S, Lee HJ, Wark AW, Corn RM.
J Am Chem Soc. 2006 Nov 1;128(43):14044-6.
[ expand abstract ]

No abstract available

Versatile apoferritin nanoparticle labels for assay of protein.
Liu G, Wang J, Wu H, Lin Y.
Anal Chem. 2006 Nov 1;78(21):7417-23.
[ expand abstract ]

A versatile bioassay label based on marker-loaded apoferritin nanoparticles (MLANs) has been developed for sensitive protein detection. Dissociation and reconstitution characteristics at different pH as well as the special cavity structure of apoferritin provides a facile route to prepare nanoparticle labels and avoid the complicated and tedious synthesis process of conventional nanoparticle labels. The optical and electrochemical characteristics of the prepared nanoparticle labels are easily controlled by loading different optical or electrochemical markers. A fluorescence marker (fluorescein anion) and a redox marker [hexacyanoferrate(III)] were used as model markers to load into the cavity of apoferritin nanoparticles for microscopic fluorescence immunoassay and electrochemical immunoassay, respectively. Detection limits of 0.06 (0.39 pM) and 0.08 ng mL(-)(1) (0.52 pM) IgG were obtained with fluorescein MLAN and hexacyanoferrate MLANs, respectively. The new nanoparticle labels hold great promise for multiplex protein detection (in connection with nanoparticles loaded with different markers) and for enhancing the sensitivity of other bioassays.

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach.
Pang L, Li J, Jiang J, Shen G, Yu R.
Anal Biochem. 2006 Nov 1;358(1):99-103.
[ expand abstract ]

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.

Microfluidic technologies as platforms for performing quantitative cellular analyses in an in vitro environment.
Martin RS, Root PD, Spence DM.
Analyst. 2006 Nov;131(11):1197-206.
[ expand abstract ]

Quite often, important cellular events occur in environments that are either not amenable to implanted sensors or other types of molecular probes. In such cases, a viable alternative to taking the sensor or probe to the biological sample of interest is to bring the sample of interest out of its natural environment to one that is more conducive to the measurement scheme. The disadvantage of the latter approach is that the sample may not behave in the same manner in vitro as it does in vivo, or that the agonists and other stimuli to which the sample is subjected to in vivo are no longer present. In this Tutorial Review, the authors attempt to provide some guidance, based on their own experiences and those of other scientists, to performing cellular measurements in a quantitative manner under in vitro conditions. Due to the expansive literature on analyses involving cells, the authors have limited this Tutorial Review to those methods involving microfluidic technologies, both in microbore tubing and in microfabricated channels. Initial reports of analyses involving cells in microbore tubing were first reported nearly two decades ago, while those in microfabricated fluidic devices appeared over a decade ago. However, more recently, the complexity of cell analyses using fabricated microfluidic devices (as opposed to microbore tubing) has increased due in part to the improvements in fabrication technologies, fluid handling and delivery capabilities, advances in coatings of the channels within the microfluidic device, and integrated detection schemes. Examples of cellular analyses in microbore tubing and in fabricated microfluidic devices will be given, as well as associated advantages and challenges. Finally, the authors' thoughts on cellular analyses are presented here using the classical steps in an analysis as a guide.

Integrated nanoparticle-biomolecule systems for biosensing and bioelectronics.
Willner I, Baron R, Willner B.
Biosens Bioelectron. 2006 Oct 27; [Epub ahead of print].
[ expand abstract ]

The similar dimensions of biomolecules such as enzymes, antibodies or DNA, and metallic or semiconductor nanoparticles (NPs) enable the synthesis of biomolecule-NP hybrid systems where the unique electronic, photonic and catalytic properties of NPs are combined with the specific recognition and biocatalytic properties of biomolecules. The unique functions of biomolecule-NP hybrid systems are discussed with several examples: (i) the electrical contacting of redox enzymes with electrodes is the basis for the development of enzymatic electrodes for amperometric biosensors or biofuel cell elements. The reconstitution of the apo-glucose oxidase or apo-glucose dehydrogenase on flavin adenine dinucleotide (FAD)-functionalized Au NPs (1.4nm) associated with electrodes, or on pyrroloquinoline quinone (PQQ)-functionalized Au NPs (1.4nm) associated with electrodes, respectively, yields electrically contacted enzyme electrodes. The aligned, reconstituted enzymes on the electrode surfaces reveal effective electrical contacting, and the glucose oxidase and glucose dehydrogenase reveal turnover rates of 5000 and 11,800s(-1), respectively. (ii) The photoexcitation of semiconductor nanoparticles yields fluorescence with a wavelength controlled by the size of the NPs. The fluorescence functions of semiconductor NPs are used to develop a fluorescence resonance energy transfer (FRET) assay for nucleic acids, and specifically, for analyzing telomerase activity in cancer cells. CdSe-ZnS NPs are functionalized by a primer recognized by telomerase, and this is elongated by telomerase extracted from HeLa cancer cells in the presence of dNTPs and Texas-red-functionalized dUTP. The dye integrated into the telomers allows the FRET process that is intensified as telomerization proceeds. Also, the photoexcited electron-hole pair generated in semiconductor NPs is used to generate photocurrents in a CdS-DNA hybrid system associated with an electrode. A redox-active intercalator, methylene blue, was incorporated into a CdS-duplex DNA monolayer associated with a Au electrode, and this facilitated the electron transfer between the electrode and the CdS NPs. The direction of the photocurrent was controlled by the oxidation state of the intercalator. (iii) Biocatalysts grow metallic NPs, and the absorbance of the NPs provides a means to assay the biocatalytic transformations. This is exemplified with the glucose oxidase-induced growth of Au NPs and with the tyrosinase-stimulated growth of Au NPs, in the presence of glucose or tyrosine, respectively. The biocatalytic growth of the metallic NPs is used to grow nanowires on surfaces. Glucose oxidase or alkaline phosphatase functionalized with Au NPs (1.4nm) acted as 'biocatalytic inks' for the synthesis of metallic nanowires. The deposition of the Au NP-modified glucose oxidase, or the Au NP-modified alkaline phosphatase on Si surfaces by dip-pen nanolithography led to biocatalytic templates, that after interaction with glucose/AuCl(4)(-) or p-aminophenolphosphate/Ag(+), allowed the synthesis of Au nanowires or Ag nanowires, respectively.

Detection enhancement in nano-channels using micro-machined silicon groove.
Fekete V, Clicq D, De Malsche W, Gardeniers H, Desmet G.
J Chromatogr A. 2006 Oct 13;1130(1):151-7.
[ expand abstract ]

The present paper reports on an experimental study of the possibility to use a micro-machined detection groove to enhance the detection sensitivity in flat-rectangular nano-channels for ultra-rapid liquid chromatography separations. Transversally running detection grooves with three different axial widths (respectively, 2, 4 and 6mum) and one depth (4.75mum) were tested in glass and silicon channels for the whole range of detectable fluorescein isothiocyanate isomer I, FITC, concentrations. The groove with the most square-like cross-section (i.e., 4mum wide and 4.75mum deep) yielded the best combination of detection gain and minimal additional band broadening. In a 1cm long channel, the effective plate loss caused by the 4mum wide groove would only be of the order of 20%, while the gain in S/N-ratio was of the order of a factor of 5. The detection groove concept yields larger gains in silicon channel substrates than in glass channel substrates, due to the larger stray light losses occurring in the latter.

Cell Detachment Model for an Antibody-Based Microfluidic Cancer Screening System.
Wankhede SP, Du Z, Berg JM, Vaughn MW, Dallas T, Cheng KH, Gollahon L.
Biotechnol Prog. 2006 Oct 6;22(5):1426-1433.
[ expand abstract ]

We consider cells bound to the floor of a microfluidic channel and present a model of their flow-induced detachment. We approximate hydrodynamic force and cell elastic response using static finite-element simulation of a single cell. Detachment is assumed to occur when hydrodynamic and adhesive forces are roughly equal. The result is extended to multiple cells at the device level using a sigmoidal curve fit. The model is applied to a microfluidic cancer-screening device that discriminates between normal epithelial cells and cells infected with human papillomavirus (HPV), on the basis of increased expression of the transmembrane protein alpha6 integrin in the latter. Here, the cells to be tested are bound to a microchannel floor coated with anti alpha6 integrin antibodies. In an appropriate flow rate range, normal cells are washed away while HPV-infected cells remain bound. The model allows interpolation between data points to choose the optimal flow rate and provides insight into interaction of cell mechanical properties and the flow-induced detachment mechanism. Notably, the results suggest a significant influence of cell elastic response on detachment.

Gold and silver nanoparticles in sensing and imaging: sensitivity of plasmon response to size, shape, and metal composition.
Lee KS, El-Sayed MA.
J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2006 Oct 5;110(39):19220-5.
[ expand abstract ]

Plasmonic metal nanoparticles have great potential for chemical and biological sensor applications, due to their sensitive spectral response to the local environment of the nanoparticle surface and ease of monitoring the light signal due to their strong scattering or absorption. In this work, we investigated the dependence of the sensitivity of the surface plasmon resonance (frequency and bandwidth) response to changes in their surrounding environment and the relative contribution of optical scattering to the total extinction, on the size and shape of nanorods and the type of metal, that is, Au vs Ag. Theoretical consideration on the surface plasmon resonance condition revealed that the spectral sensitivity, defined as the relative shift in resonance wavelength with respect to the refractive index change of surrounding materials, has two controlling factors: first the bulk plasma wavelength, a property dependent on the metal type, and second on the aspect ratio of the nanorods which is a geometrical parameter. It is found that the sensitivity is linearly proportional to both these factors. To quantitatively examine the dependence of the spectral sensitivity on the nanorod metal composition and the aspect ratio, the discrete dipole approximation method was used for the calculation of optical spectra of Ag-Au alloy metal nanorods as a function of Ag concentration. It is observed that the sensitivity does not depend on the type of the metal but depends largely on the aspect ratio of nanorods. The direct dependence of the sensitivity on the aspect ratio becomes more prominent as the size of nanorods becomes larger. However, the use of larger nanoparticles may induce an excessive broadening of the resonance spectrum due to an increase in the contribution of multipolar excitations. This restricts the sensing resolution. The insensitivity of the plasmon response to the metal composition is attributable to the fact that the bulk plasma frequency of the metal, which determines the spectral dispersion of the real dielectric function of metals and the surface plasmon resonance condition, has a similar value for the noble metals. On the other hand, nanorods with higher Ag concentration show a great enhancement in magnitude and sharpness of the plasmon resonance band, which gives better sensing resolution despite similar plasmon response. Furthermore, Ag nanorods have an additional advantage as better scatterers compared with Au nanorods of the same size.

Enzyme-nanoparticle functionalization of three-dimensional protein scaffolds.
Hill RT, Shear JB.
Anal Chem. 2006 Oct 1;78(19):7022-6.
[ expand abstract ]

Various surface modification techniques have been developed for patterning functional biomolecules in two dimensions, allowing enzymes, antibodies, and other compounds to be localized for applications in bioanalysis and bioengineering. Here, we report a strategy for extending high-resolution patterning of biomolecules to three dimensions. In this approach, three-dimensional protein scaffolds are created by a direct-write process in which multiphoton excitation promotes photochemical cross-linking of protein molecules from aqueous solution within specified volume elements. After scaffold fabrication, protein microstructures are functionalized with enzyme-gold nanoparticle conjugates via a targeting process based in part on electrostatic attraction between the low-isoelectric-point enzyme and the microstructure, fabricated from high-isoelectric-point proteins. High signal-to-background ratios ( approximately 20:1) are demonstrated for fluorescent product streams created by dephosphorylation of the fluorogenic compound, fluorescein diphosphate, at microstructures decorated with alkaline phosphatase-gold nanoparticle conjugates. We also demonstrate feasibility for using such structures to quantify substrate concentrations in flowing streams with low-micromolar detection limits and to create sensor suites based on both enzyme-nanoparticle functionalization and intrinsic enzymatic activity of protein scaffolds. These topographically complex sensors and dosing sources have potential applications in microfluidics, sensor array fabrication, and real-time chemical modification of cell culture environments.

Nanoparticle probes with surface enhanced Raman spectroscopic tags for cellular cancer targeting.
Kim JH, Kim JS, Choi H, Lee SM, Jun BH, Yu KN, Kuk E, Kim YK, Jeong DH, Cho MH, Lee YS.
Anal Chem. 2006 Oct 1;78(19):6967-73.
[ expand abstract ]

We have developed biocompatible, photostable, and multiplexing-compatible surface-enhanced Raman spectroscopic tagging material (SERS dots) composed of silver nanoparticle-embedded silica spheres and organic Raman labels for cellular cancer targeting in living cells. SERS dots showed linear dependency of Raman signatures on their different amounts, allowing their possibility for the quantification of targets. In addition, the antibody-conjugated SERS dots were successfully applied to the targeting of HER2 and CD10 on cellular membranes and exhibited good specificity. SERS dots demonstrate the potential for high-throughput screening of biomolecules using vibrational information.

Nanoscale dielectrophoretic spectroscopy of individual immobilized mammalian blood cells.
Lynch BP, Hilton AM, Simpson GJ.
Biophys J. 2006 Oct 1;91(7):2678-86.
[ expand abstract ]

Dielectrophoretic force microscopy (DEPFM) and spectroscopy have been performed on individual intact surface-immobilized mammalian red blood cells. Dielectrophoretic force spectra were obtained in situ in approximately 125 ms and could be acquired over a region comparable in dimension to the effective diameter of a scanning probe microscopy tip. Good agreement was observed between the measured dielectrophoretic spectra and predictions using a single-shell cell model. In addition to allowing for highly localized dielectric characterization, DEPFM provided a simple means for noncontact imaging of mammalian blood cells under aqueous conditions. These studies demonstrate the feasibility of using DEPFM to monitor localized changes in membrane capacitance in real time with high spatial resolution on immobilized cells, complementing previous studies of mobile whole cells and cell suspensions.

Sensitive Immunoassay of a Biomarker Tumor Necrosis Factor-alpha Based on Poly(guanine)-Functionalized Silica Nanoparticle Label.
Wang J, Liu G, Engelhard MH, Lin Y.
Anal Chem. 2006 Oct 1;78(19):6974-9.
[ expand abstract ]

A novel electrochemical immunosensor for the detection of tumor necrosis factor-alpha (TNF-alpha) based on poly(guanine)-functionalized silica nanoparticles (NPs) label is presented. The detection of mouse TNF-alpha via immunological reaction is based on a dual signal amplification: (1) a large amount of guanine residues introduced on the electrode surface through sandwich immunoreaction and poly(guanine)-functionalized silica NP label; (2) Ru(bpy)(3)(2+)-induced catalytic oxidation of guanine, which results in great enhancement of anodic current. The synthesized silica NP conjugates were characterized with atomic force microscopy, X-ray photoelectron spectroscopy, and electrochemistry. These experiments confirmed that poly(guanine) and avidin were immobilized on the surface of silica NPs. The performance of the electrochemical immunosensor was evaluated and some experiment parameters (e.g., concentration of Ru(bpy)(3)(2+), incubation time of TNF-alpha, etc.) were optimized. The detection limit for TNF-alpha is found to be 5.0 x 10(-)(11) g mL(-)(1) (2.0 pM), which corresponds to 60 amol of TNF-alpha in 30 muL of sample. This immunosensor based on the poly(guanine)-functionalized silica NP label offers great promise for rapid, simple, cost-effective analysis of biological samples.

A bio-barcode assay for on-chip attomolar-sensitivity protein detection.
Goluch ED, Nam JM, Georganopoulou DG, Chiesl TN, Shaikh KA, Ryu KS, Barron AE, Mirkin CA, Liu C.
Lab Chip. 2006 Oct;6(10):1293-9.
[ expand abstract ]

Functionalized nanoparticles hold great promise in realizing highly sensitive and selective biodetection. We report a single disposable chip which is capable of carrying out a multi-step process that employs nanoparticles--a bio-barcode assay (BCA) for single protein marker detection. To illustrate the capability of the system, we tested for the presence of prostate specific antigen (PSA) in buffer solution and goat serum. Detection was accomplished at PSA concentrations as low as 500 aM. This corresponds to only 300 copies of protein analytes using 1 microL total sample volume. We established that the on-chip BCA for PSA detection offers four orders of magnitude higher sensitivity compared to commercially available ELISA-based PSA tests.

Miniaturized platforms for the detection of single-nucleotide polymorphisms.
Ng JK, Liu WT.
Anal Bioanal Chem. 2006 Oct;386(3):427-34.
[ expand abstract ]

Conventional methods for detecting single-nucleotide polymorphisms (SNPs), the most common form of genetic variation in human beings, are mostly limited by their analysis time and throughputs. In contrast, advances in microfabrication technology have led to the development of miniaturized platforms that can potentially provide rapid high-throughput analysis at small sample volumes. This review highlights some of the recent developments in the miniaturization of SNP detection platforms, including microarray-based, bead-based microfluidic and microelectrophoresis-based platforms. Particular attention is paid to their ease of fabrication, analysis time, and level of throughput.

Merging microfluidics with microarray-based bioassays.
Situma C, Hashimoto M, Soper SA.
Biomol Eng. 2006 Oct;23(5):213-31.
[ expand abstract ]

Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins (antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes (micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as, reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and clinical applications.

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip.
Li YJ, Bi LJ, Zhang XE, Zhou YF, Zhang JB, Chen YY, Li W, Zhang ZP.
Anal Bioanal Chem. 2006 Sep 28; [Epub ahead of print] .
[ expand abstract ]

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin-streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K (D) approximately 4.01x10(-7). All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.

Design of a highly sensitive and specific nucleotide sensor based on photon upconverting particles.
Zhang P, Rogelj S, Nguyen K, Wheeler D.
J Am Chem Soc. 2006 Sep 27;128(38):12410-1.
[ expand abstract ]

No abstract available

Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies.
Tholouli E, Hoyland JA, Di Vizio D, O'Connell F, Macdermott SA, Twomey D, Levenson R, Yin JA, Golub TR, Loda M, Byers R.
Biochem Biophys Res Commun. 2006 Sep 22;348(2):628-36.
[ expand abstract ]

Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.

Multiple label-free detection of antigen-antibody reaction using localized surface plasmon resonance-based core-shell structured nanoparticle layer nanochip.
Endo T, Kerman K, Nagatani N, Hiepa HM, Kim DK, Yonezawa Y, Nakano K, Tamiya E.
Anal Chem. 2006 Sep 15;78(18):6465-75.
[ expand abstract ]

In this research, a localized surface plasmon resonance (LSPR)-based bioanalysis method for developing multiarray optical nanochip suitable for screening bimolecular interactions is described. LSPR-based label-free monitoring enables to solve the problems of conventional methods that require large sample volumes and time-consuming labeling procedures. We developed a multiarray LSPR-based nanochip for the label-free detection of proteins. The multiarray format was constructed by a core-shell-structured nanoparticle layer, which provided 300 nanospots on the sensing surface. Antibodies were immobilized onto the nanospots using their interaction with Protein A. The concentrations of antigens were determined from the peak absorption intensity of the LSPR spectra. We demonstrated the capability of the array measurement using immunoglobulins (IgA, IgD, IgG, IgM), C-reactive protein, and fibrinogen. The detection limit of our label-free method was 100 pg/mL. Our nanochip is readily transferable to monitor the interactions of other biomolecules, such as whole cells or receptors, with a massively parallel detection capability in a highly miniaturized package. We anticipate that the direct label-free optical immunoassay of proteins reported here will revolutionize clinical diagnosis and accelerate the development of hand-held and user-friendly point-of-care devices.

Direct DNA Hybridization at Disposable Graphite Electrodes Modified with Carbon Nanotubes.
Erdem A, Papakonstantinou P, Murphy H.
Anal Chem. 2006 Sep 15;78(18):6656-6659.
[ expand abstract ]

The performance of glassy carbon (GCE) and graphite pencil electrodes (PGE) modified with multiwalled carbon nanotubes (CNTs) are compared, based on the direct electrochemical detection of nucleic acids. This is accomplished by monitoring the differential pulse voltammetry changes of the guanine signal. CNT-modified PGE compares favorably to that of the commonly used CNT-modified GCE owing to the intrinsic improved performance of the supporting PGE. The better intrinsic characteristics of the PGE are related to its composite structure and higher level of porosity compared to GCE. The performance characteristics of the direct DNA hybridization on the disposable CNT-modified PGE are studied in terms of optimum analytical conditions such as probe concentration, target concentration, hybridization time, and selectivity. The new DNA biosensor described here has shown some important advantages such being inexpensive, sensitive, selective, and able to generate reproducible results using a simple and direct electrochemical protocol.

Lab-on-a-chip technology for determination of protein isoform profiles.
Lonnberg M, Carlsson J.
J Chromatogr A. 2006 Sep 15;1127(1-2):175-82.
[ expand abstract ]

A novel lab-on-a-chip technique for rapid (<15 min) and quantitative isoform-profile determination is presented. Ion-exchange chromatographic separation of protein-isoforms and a sensitive immunoassay detection are combined in a porous monolith chip. Thin lines of immobilized antibodies are used for specific capturing of target molecules, which can be detected by the reaction with antibodies bound to carbon black nano-strings. The bound carbon black is quantified by the use of an image scanner. As demonstrated with transferrin isoforms, differing only by 0.1 pH unit in their pI, this technology can distinguish minor differences in protein carbohydrate structure and enable specific determination of proteins in a complex environment, requiring only a few picogram of isoform for detection.

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9.
Hestekin CN, Jakupciak JP, Chiesl TN, Kan CW, O'connell CD, Barron AE.
Electrophoresis. 2006 Sep 14; [Epub ahead of print] .
[ expand abstract ]

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.

Ag/SiO(2) core-shell nanoparticle-based surface-enhanced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools.
Gong JL, Liang Y, Huang Y, Chen JW, Jiang JH, Shen GL, Yu RQ.
Biosens Bioelectron. 2006 Sep 11; [Epub ahead of print] .
[ expand abstract ]

A simple, sensitive and highly specific immunoassay has been developed based on surface-enhanced Raman scattering for human alpha-fetoprotein (AFP), a tumor marker for the diagnosis of hepatocellular carcinoma. This strategy combines the Ag/SiO(2) core-shell nanoparticles embedded with rhodamine B isothiocyanate dye molecules as Raman tags and the amino group modified silica-coated magnetic nanoparticle as immobilization matrix and separation tool. In the proposed system, a sandwich-type immunoassay was performed between polyclonal antibody functionalized Ag/SiO(2) nanoparticle-based Raman tags and monoclonal antibody modified silica-coated magnetic nanoparticles. The presence of the analyte and the reaction between the antigen and antibody can be monitored by the Raman spectra of the Ag/SiO(2) tags. Compared to the previous surface-enhanced Raman immunoassays, the main advantage of this strategy lies in two aspects. One is the high stability of Raman tags derived from the silica shell-coated silver core-shell nanostructure. The other is the use of silica-coated magnetic nanoparticles as immobilization matrix and separation tool, thus avoiding complicated pretreatment and washing steps. We have studied in detail the experimental parameters such as the effects of the antibody concentration modified on the Raman tags and on the magnetic particles, and the immunoreaction time. Using this strategy, concentration of human AFP up to 0.12mug/ml was detected with a detection limit of 11.5pg/ml.

2D aggregation and selective desorption of nanoparticle probes: A new method to probe DNA mismatches and damages.
Charrier A, Candoni N, Liachenko N, Thibaudau F.
Biosens Bioelectron. 2006 Sep 5; [Epub ahead of print].
[ expand abstract ]

A 2D colorimetric DNA sensor is reported based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes resulting from the molecular hybridization between these latest and their complementary single stranded DNA targets. To increase their mobility the nanoparticles are adsorbed on a fluid lipid bilayer, itself supported on a substrate. The hybridization between the target and the mobile nanoparticle probes creates links between the nanoparticles resulting in the formation of nanoparticle aggregates in the plane of the substrate. This aggregation is detected using a new method based on the selective desorption of non-aggregated nanoparticles. The addition of dextran sulfate induces the substitution of non-aggregated gold nanoparticles while aggregated ones are stable on the substrate. We show that this detection method is highly specific and allows the detection of DNA mismatches and damages.

Anomalous resonance in a nanomechanical biosensor.
Gupta AK, Nair PR, Akin D, Ladisch MR, Broyles S, Alam MA, Bashir R.
Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13362-7.
[ expand abstract ]

The decrease in resonant frequency (-Deltaomega(r)) of a classical cantilever provides a sensitive measure of the mass of entities attached on its surface. This elementary phenomenon has been the basis of a new class of bio-nanomechanical devices as sensing components of integrated microsystems that can perform rapid, sensitive, and selective detection of biological and biochemical entities. Based on classical analysis, there is a widespread perception that smaller sensors are more sensitive (sensitivity approximately -0.5omega(r)/m(C), where m(C) is the mass of the cantilever), and this notion has motivated scaling of biosensors to nanoscale dimensions. In this work, we show that the response of a nanomechanical biosensor is far more complex than previously anticipated. Indeed, in contrast to classical microscale sensors, the resonant frequencies of the nanosensor may actually decrease or increase after attachment of protein molecules. We demonstrate theoretically and experimentally that the direction of the frequency change arises from a size-specific modification of diffusion and attachment kinetics of biomolecules on the cantilevers. This work may have broad impact on microscale and nanoscale biosensor design, especially when predicting the characteristics of bio-nanoelectromechanical sensors functionalized with biological capture molecules.

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode.
Li A, Yang F, Ma Y, Yang X.
Biosens Bioelectron. 2006 Sep 5; [Epub ahead of print].
[ expand abstract ]

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles

Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots.
Fountaine TJ, Wincovitch SM, Geho DH, Garfield SH, Pittaluga S.
Mod Pathol. 2006 Sep;19(9):1181-91.
[ expand abstract ]

Determination of the expression and spatial distribution of molecular epitopes, or antigens, in patient tissue specimens has substantially improved the pathologist's ability to classify disease processes. Certain disease pathophysiologies are marked by characteristic increased or decreased expression of developmentally controlled antigens, defined as Cluster of Differentiation markers, that currently form the foundation for understanding lymphoid malignancies. While chromogens and organic fluorophores have been utilitized for some time in immunohistochemical analyses, developments in synthetic, inorganic fluorophore semiconductors, namely quantum dots, offer a versatile alternative reporter system. Quantum dots are stable fluorophores, are resistant to photobleaching, and are attributed with wide excitation ranges and narrow emission spectra. To date, routinely processed, formalin-fixed tissues have only been probed with two quantum dot reporters simultaneously. In the present study, streptavidin-conjugated quantum dots with distinct emission spectra were tested for their utility in identifying a variety of differentially expressed antigens (surface, cytoplasmic, and nuclear). Slides were analyzed using confocal laser scanning microscopy, which enabled with a single excitation wavelength (488 nm argon laser) the detection of up to seven signals (streptavidin-conjugated quantum dots 525, 565, 585, 605, 655, 705 and 805 nm) plus the detection of 4'6-DiAmidino-2-PhenylIndole with an infra-red laser tuned to 760 nm for two photon excitation. Each of these signals was specific for the intended morphologic immunohistochemical target. In addition, five of the seven streptavidin-conjugated quantum dots tested (not streptavidin-conjugated quantum dots 585 or 805 nm) were used on the same tissue section and could be analyzed simultaneously on routinely processed formalin-fixed, paraffin-embedded sections. Application of this multiplexing method will enable investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously.

An integrated digital microfluidic chip for multiplexed proteomic sample preparation and analysis by MALDI-MS.
Moon H, Wheeler AR, Garrell RL, Loo JA, Kim CJ.
Lab Chip. 2006 Sep;6(9):1213-9.
[ expand abstract ]

To realize multiplexed sample preparation on a digital microfluidic chip for high-throughput Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), several fluidic functions need to be integrated. These include the generation of multiple droplets from a reservoir and parallel in-line sample purification. In this paper, we develop two critical new functions in handling protein solutions and standard proteomic reagents with electrowetting-on-dielectric (EWOD) actuation, leading to an integrated chip for multiplexed sample preparation for MALDI-MS. The first is a voltage sequence designed to generate a series of droplets from each of the three reservoirs-proteomic sample, rinsing fluid, and MALDI reagents. It is the first time that proteomic reagents have been dispensed using EWOD in an air (as opposed to oil) environment. The second is a box-in-box electrode pattern developed to allow droplet passing over dried sample spots, making the process of in-line sample purification robust for parallel processing. As a result, parallel processing of multiple sample droplets is demonstrated on the integrated EWOD-MALDI-MS chip, an important step towards high-throughput MALDI-MS. The MS results, collected directly from the integrated devices, are of good quality, suggesting that the tedious process of sample preparation can be automated on-chip for MALDI-MS applications as well as other high-throughput proteomics applications.

Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization.
Edwards KA, Baeumner AJ.
Anal Bioanal Chem. 2006 Aug 31; [Epub ahead of print] .
[ expand abstract ]

A novel protocol for the synthesis of dye-encapsulating liposomes tagged with DNA oligonucleotides at their outer surface was developed. These liposomes were optimized for use as signal enhancement agents in lateral-flow sandwich-hybridization assays for the detection of single-stranded RNA and DNA sequences. Liposomes were synthesized using the reverse-phase evaporation method and tagged with oligonucleotides by adding cholesteryl-modified DNA probes to the initial lipid mixture. This resulted in a greatly simplified protocol that provided excellent control of the probe coverage on the liposomes and cut the preparation time from 16 hours to just 6 hours. Liposomes were prepared using probe concentrations ranging from 0.00077 to 0.152 mol% of the total lipid, several hydrophobic and polyethylene glycol-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 208 nm to 365 nm. The liposomes were characterized by dynamic light scattering, visible spectroscopy, and fluorescence spectroscopy. Their signal enhancement functionality was compared by using them in lateral-flow optical biosensors for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.013 mol%, liposome diameter of 315 nm, and liposome optical density of 0.4-0.6 at 532 nm were found. The spacer length between the cholesteryl anchor and the probe showed no significant effect on the signals in the lateral-flow assays. The results presented here provide important data for the general use of liposomes as labels in analytical assays, with specific emphasis on nucleic acid detection via lateral flow assays.

Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers.
Yu X, Munge B, Patel V, Jensen G, Bhirde A, Gong JD, Kim SN, Gillespie J, Gutkind JS, Papadimitrakopoulos F, Rusling JF.
J Am Chem Soc. 2006 Aug 30;128(34):11199-205.
[ expand abstract ]

We describe herein the combination of electrochemical immunosensors using single-wall carbon nanotube (SWNT) forest platforms with multi-label secondary antibody-nanotube bioconjugates for highly sensitive detection of a cancer biomarker in serum and tissue lysates. Greatly amplified sensitivity was attained by using bioconjugates featuring horseradish peroxidase (HRP) labels and secondary antibodies (Ab(2)) linked to carbon nanotubes (CNT) at high HRP/Ab(2) ratio. This approach provided a detection limit of 4 pg mL(-)(1) (100 amol mL(-)(1)), for prostate specific antigen (PSA) in 10 microL of undiluted calf serum, a mass detection limit of 40 fg. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. PSA was quantitatively measured in prostate tissue samples for which PSA could not be differentiated by the gold standard immunohistochemical staining method. These easily fabricated SWNT immunosensors show excellent promise for clinical screening of cancer biomarkers and point-of-care diagnostics.

Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system.
Sajonz P, Gong X, Leonard WR Jr, Biba M, Welch CJ.
Chirality. 2006 Aug 11; [Epub ahead of print] .
[ expand abstract ]

The Eksigent Express 800 8-channel microfluidic HPLC system was investigated for carrying out multiparallel screening and development of fast normal phase chiral separations. In contrast to the familiar automated sequential chiral method development approaches that often afford a next day result, the multiparallel approach offers the exciting possibility of near "real time" method development, often affording an optimized method in less than 1 h. In this study, four column types (300 mum i.d.) with two different mobile phases are screened using a universal standard gradient approach. Interestingly, parallel method optimization following initial screening was shown to sometimes lead to surprising and unanticipated outcomes, emphasizing the value of the multiparallel screening approach. A variety of standard test racemates were analyzed, with optimized separation methods for most in the 1- to 2-min range. These results compare favorably with results obtained on a single channel conventional HPLC system using 4.6-mm i.d. columns. In addition, isocratic methods developed on the microbore columns are readily translated to the larger column format.

Functional Antibody Arrays through Metal Ion-Affinity Templates.
Vega RA, Maspoch D, Shen CK, Kakkassery JJ, Chen BJ, Lamb RA, Mirkin CA.
Chembiochem. 2006 Aug 8; [Epub ahead of print] .
[ expand abstract ]

No abstract available

Microfluidic liquid chromatography system for proteomic applications and biomarker screening.
Lazar IM, Trisiripisal P, Sarvaiya HA.
Anal Chem. 2006 Aug 1;78(15):5513-24.
[ expand abstract ]

A microfluidic liquid chromatography (LC) system for proteomic investigations that integrates all the necessary components for stand-alone operation, i.e., pump, valve, separation column, and electrospray interface, is described in this paper. The overall size of the LC device is small enough to enable the integration of two fully functional separation systems on a 3 in. x 1 in. glass microchip. A multichannel architecture that uses electroosmotic pumping principles provides the necessary functionality for eluent propulsion and sample valving. The flow rates generated within these chips are fully consistent with the requirements of nano-LC platforms that are routinely used in proteomic applications. The microfluidic device was evaluated for the analysis of a protein digest obtained from the MCF7 breast cancer cell line. The cytosolic protein extract was processed according to a shotgun protocol, and after tryptic digestion and prefractionation using strong cation exchange chromatography (SCX), selected sample subfractions were analyzed with conventional and microfluidic LC platforms. Using similar experimental conditions, the performance of the microchip LC was comparable to that obtained with benchtop instrumentation, providing an overlap of 75% in proteins that were identified by more than two unique peptides. The microfluidic LC analysis of a protein-rich SCX fraction enabled the confident identification of 77 proteins by using conventional data filtering parameters, of 39 proteins with p < 0.001, and of 5 proteins that are known to be cancer-specific biomarkers, demonstrating thus the potential applicability of these chips for future high-throughput biomarker screening applications.

Microfluidic isolation of leukocytes from whole blood for phenotype and gene expression analysis.
Sethu P, Moldawer LL, Mindrinos MN, Scumpia PO, Tannahill CL, Wilhelmy J, Efron PA, Brownstein BH, Tompkins RG, Toner M.
Anal Chem. 2006 Aug 1;78(15):5453-61.
[ expand abstract ]

Technologies that enable the isolation of cell subtypes from small samples of complex populations will greatly facilitate the implementation of proteomics and genomics to human diseases. Transcriptome analysis of blood requires the depletion of contaminating erythrocytes. We report an automated microfluidic device to rapidly deplete erythrocytes from whole blood via deionized water lysis and to collect enriched leukocytes for phenotype and genomic analyses. Starting with blood from healthy subjects, we demonstrate the utility of this microfluidic cassette and lysis protocol to prepare unstimulated leukocytes, and leukocytes stimulated ex vivo with Staphylococcal enterotoxin B, which mimics some of the cellular effects seen in patients with severe bacterial infections. Microarrays are used to assess the global gene expression response to enterotoxin B. The results demonstrate that this system can isolate unactivated leukocytes from small blood samples without any significant loss, which permits more information to be obtained from subsequent analysis, and will be readily applicable to clinical settings.

Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry.
Chattopadhyay PK, Price DA, Harper TF, Betts MR, Yu J, Gostick E, Perfetto SP, Goepfert P, Koup RA, De Rosa SC, Bruchez MP, Roederer M.
Nat Med. 2006 Aug;12(8):972-7.
[ expand abstract ]

Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses.

Bio-assay based on single molecule fluorescence detection in microfluidic channels.
Hollars CW, Puls J, Bakajin O, Olsan B, Talley CE, Lane SM, Huser T.
Anal Bioanal Chem. 2006 Aug;385(8):1384-8.
[ expand abstract ]

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.

Carbon nanotube DNA sensor and sensing mechanism.
Tang X, Bansaruntip S, Nakayama N, Yenilmez E, Chang YL, Wang Q.
Nano Lett. 2006 Aug;6(8):1632-6.
[ expand abstract ]

We report the fabrication of single-walled carbon nanotube (SWNT) DNA sensors and the sensing mechanism. The simple and generic protocol for label-free detection of DNA hybridization is demonstrated with random sequence 15mer and 30mer oligonucleotides. DNA hybridization on gold electrodes, instead of on SWNT sidewalls, is mainly responsible for the acute electrical conductance change due to the modulation of energy level alignment between SWNT and gold contact. This work provides concrete experimental evidence on the effect of SWNT-DNA binding on DNA functionality, which will help to pave the way for future designing of SWNT biocomplexes for applications in biotechnology in general and also DNA-assisted nanotube manipulation techniques.

Detection of DNA using cationic polyhedral oligomeric silsesquioxane nanoparticles as the probe by resonance light scattering technique.
Zou QC, Yan QJ, Song GW, Zhang SL, Wu LM.
Biosens Bioelectron. 2006 Jul 31; [Epub ahead of print] .
[ expand abstract ]

A novel cationic polyhedral oligomeric silsesquioxane nanoparticle (cationic POSS) was synthesized and successfully used as a new probe for the detection of DNA by resonance light scattering technique (RLS). It was found that the electrostatic interaction of cationic POSS and DNA could obviously enhance the RLS signal, the enhanced RLS intensity at 360nm was proportional to the concentration of nucleic acids within the range of 0.35-42.82mugml(-1) for calf thymus DNA, the determination limit (3sigma) was 0.32ngml(-1). The results showed this method was very sensitive, convenient, rapid and reproducible.

DNA detection method based on the two-dimensional aggregation and selective desorption of nanoparticle probes.
Charrier A, Candoni N, Thibaudau F.
J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2006 Jul 6;110(26):12896-900.
[ expand abstract ]

A label-free two-dimensional colorimetric DNA sensor is reported. This sensor is based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes induced by the molecular hybridization of single-stranded oligonucleotide probes and their complementary single-stranded DNA targets. To detect the aggregation, we have developed a new detection method based on the selective desorption of nonaggregated nanoparticles. We will show here that this detection method is highly specific and allows the quantification of the DNA targets.

Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes.
Stoeva SI, Lee JS, Smith JE, Rosen ST, Mirkin CA.
J Am Chem Soc. 2006 Jul 5;128(26):8378-9.
[ expand abstract ]

We have developed the chemistry for preparing a universal probe and the appropriate nano- and microparticle labels that can be used to do highly selective multiplexed detection of three protein cancer markers at low-femtomolar concentration in buffer and serum media. The approach relies on a new multiplexed version of the biobarcode amplification method and offers new opportunities for studying multiple protein markers in a single sample. This could lead to new forms of disease diagnosis and monitoring disease recurrence in a variety of settings.

Selective Photoelectrochemical Detection of DNA with High-Affinity Metallointercalator and Tin Oxide Nanoparticle Electrode.
Liu S, Li C, Cheng J, Zhou Y.
Anal Chem. 2006 Jul 1;78(13):4722-4726.
[ expand abstract ]

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)(2)dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)(2)dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)(2)dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-)(10) M in solution.

Multiplexed molecular detection using encoded microparticles and nanoparticles.
Li Y, Luo D.
Expert Rev Mol Diagn. 2006 Jul;6(4):567-74.
[ expand abstract ]

Signal-encoded microparticles and nanoparticles have been used to label many reactions simultaneously for target identification in assays, and thus are an indispensable part of multiplex technologies. With the increasing demand for multiplexed molecular detection, encoded particles have evolved from pattern encoding to signal-intensity encoding, and also from signal-molecule encapsulation to signal-molecule tagging. The fabrication and utilization of such nano- and microparticles should advance multiplexed analysis. This short review focuses on how these encoded particles work and briefly touches on their applications in multiplexed molecular detection.

Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes.
Neuzil P, Zhang C, Pipper J, Oh S, Zhuo L.
Nucleic Acids Res. 2006 Jun 28;34(11):e77.
[ expand abstract ]

We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the system's thermal constant of 0.27 s, we have achieved a heating rate of 175 degrees C s(-1) and a cooling rate of -125 degrees C s(-1). A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput.

Bio-assay based on single molecule fluorescence detection in microfluidic channels.
Hollars CW, Puls J, Bakajin O, Olsan B, Talley CE, Lane SM, Huser T.
Anal Bioanal Chem. 2006 Jun 27; [Epub ahead of print].
[ expand abstract ]

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.

Sensitivity enhancement of DNA microarray on nano-scale controlled surface by using a streptavidin-fluorophore conjugate.
Sunkara V, Hong BJ, Park JW.
Biosens Bioelectron. 2006 Jun 23; [Epub ahead of print].
[ expand abstract ]

High throughput analysis of DNA in low concentration and small volume is an important issue and a continuing challenge in the field of DNA microarray and sensor. Recently, we have demonstrated that the DNA microarray on nano-scale controlled surface provides ample space for hybridization resulting in the best discrimination efficiency for SNP analysis. Here, we report the utility of the nano-scale controlled surface in conjunction with a multiply tagged protein. Application of streptavidin-fluorophore conjugates in combination with the highly controlled surface that suppresses non-specific binding of DNA allows highly sensitive detection of DNA while maintaining superior SNP discrimination efficiency comparable to our earlier results. The sensitivity of DNA microarray on the mesospaced surface is two orders of magnitude higher than that of the generic surface when a streptavidin-fluorophore conjugate was employed, and the detection limit on the former surface was found to be 50fM of 15-mer target DNA. Various streptavidin-fluorophore conjugates including streptavidin-Cy3, streptavidin-Cy5, streptavidin-Alexa Flour 555 and streptavidin-phycoerythrin were examined.

Thermotropic phase behavior of DPPC liposome systems in the presence of the anti-cancer agent 'Ellipticine'.
Cavalcanti LP, Torriani IL.
Eur Biophys J. 2006 Jun 23; [Epub ahead of print].
[ expand abstract ]

This letter presents our first results on the structural changes in DPPC multilamellar vesicles dispersed in water in the presence of the anti-cancer agent Ellipticine. The thermotropic phase transitions of the lamellar packing inside lipid vesicles were characterized in situ by small angle X ray diffraction. The results lead to the determination of a critical concentration value for drug loading on the vesicle system around 4% molar fraction of Ellipticine, an indication of the localization of the drug in the alkyl chains and the influence of the drug on the decreasing rate of the bilayer period after the main phase transition.

Emerging implications of nanotechnology on cancer diagnostics and therapeutics.
Cuenca AG, Jiang H, Hochwald SN, Delano M, Cance WG, Grobmyer SR.
Cancer. 2006 Jun 22; [Epub ahead of print].
[ expand abstract ]

Nanotechnology is multidisciplinary field that involves the design and engineering of objects <500 nanometers (nm) in size. The National Cancer Institute has recognized that nanotechnology offers an extraordinary, paradigm-changing opportunity to make significant advances in cancer diagnosis and treatment. In the last several decades, nanotechnology has been studied and developed primarily for use in novel drug-delivery systems (e.g. liposomes, gelatin nanoparticles, micelles). A recent explosion in engineering and technology has led to 1) the development of many new nanoscale platforms, including quantum dots, nanoshells, gold nanoparticles, paramagnetic nanoparticles, and carbon nanotubes, and 2) improvements in traditional, lipid-based nanoscale platforms. The emerging implications of these platforms for advances in cancer diagnostics and therapeutics form the basis of this review. A widespread understanding of these new technologies is important, because they currently are being integrated into the clinical practice of oncology. Cancer 2006. (c) 2006 American Cancer Society.

A label-free electrochemical immunoassay for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and nonconductive polymer film.
Tang H, Chen J, Nie L, Kuang Y, Yao S.
Biosens Bioelectron. 2006 Jun 22; [Epub ahead of print].
[ expand abstract ]

A simple and sensitive label-free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) has been developed. CEA antibody (CEAAb) was covalently attached on glutathione (GSH) monolayer-modified gold nanoparticle (AuNP) and the resulting CEAAb-AuNP bioconjugates were immobilized on Au electrode by electro-copolymerization with o-aminophenol (OAP). Electrochemical impedance spectroscopy and cyclic voltammetry studies demonstrate that the formation of CEA antibody-antigen complexes increases the electron transfer resistance of [Fe(CN)(6)](3-/4-) redox pair at the poly-OAP/CEAAb-AuNP/Au electrode. The use of CEA antibody-AuNP bioconjugates and poly-OAP film could enhance the sensitivity and anti-nonspecific binding of the resulting immunoassay electrode. The preliminary application of poly-OAP/CEAAb-AuNP/Au electrode for detection of CEA was also evaluated.

Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots.
Fountaine TJ, Wincovitch SM, Geho DH, Garfield SH, Pittaluga S.
Mod Pathol. 2006 Jun 16; [Epub ahead of print].
[ expand abstract ]

Determination of the expression and spatial distribution of molecular epitopes, or antigens, in patient tissue specimens has substantially improved the pathologist's ability to classify disease processes. Certain disease pathophysiologies are marked by characteristic increased or decreased expression of developmentally controlled antigens, defined as Cluster of Differentiation markers, that currently form the foundation for understanding lymphoid malignancies. While chromogens and organic fluorophores have been utilitized for some time in immunohistochemical analyses, developments in synthetic, inorganic fluorophore semiconductors, namely quantum dots, offer a versatile alternative reporter system. Quantum dots are stable fluorophores, are resistant to photobleaching, and are attributed with wide excitation ranges and narrow emission spectra. To date, routinely processed, formalin-fixed tissues have only been probed with two quantum dot reporters simultaneously. In the present study, streptavidin-conjugated quantum dots with distinct emission spectra were tested for their utility in identifying a variety of differentially expressed antigens (surface, cytoplasmic, and nuclear). Slides were analyzed using confocal laser scanning microscopy, which enabled with a single excitation wavelength (488 nm argon laser) the detection of up to seven signals (streptavidin-conjugated quantum dots 525, 565, 585, 605, 655, 705 and 805 nm) plus the detection of 4'6-DiAmidino-2-PhenylIndole with an infra-red laser tuned to 760 nm for two photon excitation. Each of these signals was specific for the intended morphologic immunohistochemical target. In addition, five of the seven streptavidin-conjugated quantum dots tested (not streptavidin-conjugated quantum dots 585 or 805 nm) were used on the same tissue section and could be analyzed simultaneously on routinely processed formalin-fixed, paraffin-embedded sections. Application of this multiplexing method will enable investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously.

Properties of Polysaccharides Grafted on Nanoparticles Investigated by EPR.
Bertholon I, Hommel H, Labarre D, Vauthier C.
Langmuir. 2006 Jun 6;22(12):5485-5490.
[ expand abstract ]

The in vivo fate of nanoparticles developed as drug delivery systems is influenced by the surface characteristics of the colloidal particles. In the present work, surface characteristics of a series of poly(isobutylcyanoacrylate) nanoparticles prepared by redox radical emulsion polymerization with polysaccharides of different molecular weight and nature were characterized by EPR. To this aim, a spin label was grafted on the polysaccharide chains after synthesis of the nanoparticles. The percentage of label showing fast movements was evaluated from EPR spectra which were analyzed according to the Kivelson theory. The results showed that mobility depended on temperature, type, and molecular weight of the polysaccharides. Differences between nanoparticles appeared with low-molecular-weight polysaccharides, while over a defined molecular weight which depended on the nature of the polysaccharide, the spin label behaved almost the same way in the different types of nanoparticles. Paradoxically, the percentage of fast moving label was the highest when linked to the shortest chitosan, which was the most rigid polysaccharide tested in this study. Thus, it was concluded that the apparent mobility of the polysaccharide evaluated by the EPR method depended on the capacity of the polysaccharide chains to fold making possible hydrophobic interactions between the label and the nanoparticle core. The transition between the unfolded-folded regiment depended on the molecular weight and on the nature of the polysaccharide. Results of this study may be useful to improve the understanding of the nanoparticle interactions with blood proteins and complement which in turn influence the in vivo fate of nanoparticles used as drug delivery systems.

Silver nanoparticle-based ultrasensitive chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms.
Liu CH, Li ZP, Du BA, Duan XR, Wang YC.
Anal Chem. 2006 Jun 1;78(11):3738-44.
[ expand abstract ]

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which the DNA targets are first hybridized to the captured oligonucleotide probes immobilized on polystyrene microwells and then the silver nanoparticles modified with alkylthiol-capped oligonucleotides are used as probes to monitor the presence of the specific target DNA. After being anchored on the hybrids, silver nanoparticles are dissolved to Ag(+) in HNO(3) solution and sensitively determined by a coupling CL reaction system (Ag(+)-Mn(2+)-K(2)S(2)O(8)-H(3)PO(4)-luminol). The combination of the remarkable sensitivity of the CL method with the large number of Ag(+) released from each hybrid allows the detection of specific sequence DNA targets at levels as low as 5 fM. The sensitivity increases 6 orders of magnitude greater than that of the gold nanoparticle-based colorimetric method and is comparable to that of surface-enhanced Raman spectroscopy, which is one of the most sensitive detection approaches available to the nanoparticle-based detection for DNA hybridization. Moreover, the perfectly complementary DNA targets and the single-base mismatched DNA strands can be evidently differentiated through controlling the temperature, which indicates that the proposed CL assay offers great promise for single-nucleotide polymorphism analysis.

Resonance Rayleigh-scattering method for the determination of proteins with gold nanoparticle probe.
Liu S, Yang Z, Liu Z, Kong L.
Anal Biochem. 2006 Jun 1;353(1):108-16; Epub 2006 Mar 27.
[ expand abstract ]

Gold nanoparticles with a 12-nm diameter were used as probes for the determination of proteins by resonance Rayleigh-scattering techniques. In weak acidic solution, large amounts of citrate anions will self-assemble on the surface of positively charged gold nanoparticles to form supermolecular compounds with negative charges. Below the isoelectric point, proteins with positive charges such as human serum albumin (HSA), bovine serum albumin (BSA), and ovalbumin (Ova) can bind gold nanoparticles to form larger volume products (the diameter of the binding product of gold nanoparticles with HSA is 23 nm.) through electrostatic force, hydrogen bonds, and hydrophobic effects, which can result in a red shift of the maximum absorption wavelength, the remarkable enhancement of the resonance Rayleigh-scattering intensity (RRS), and the appearance of the RRS spectra. At the same time, the second-order-scattering (SOS) and frequency-doubling-scattering (FDS) intensities are also enhanced. The binding products of gold nanoparticles with different proteins have similar spectral characteristics and the maximum wavelengths are located near 303 nm for RRS, 540 nm for SOS, and 390 for FDS, respectively. The scattering enhancement (DeltaI) is directly proportional to the concentration of proteins. Among them, the RRS method has the highest sensitivity and the detection limits are 0.38 ng/ml for HSA, 0.45 ng/ml for BSA, and 0.56 ng/ml for Ova, separately. The methods have good selectivity. A new RRS method for the determination of trace proteins using a gold nanoparticle probe has been developed. Because gold nanoparticle probes do not need to be modified chemically in advance, the method is very simple and fast.

Real-time monitoring of DNA polymerase activity using molecular beacon.
Ma C, Tang Z, Wang K, Tan W, Li J, Li W, Li Z, Yang X, Li H, Liu L.
Anal Biochem. 2006 Jun 1;353(1):141-3; Epub 2006 Feb 28.
[ expand abstract ]

No abstract available

Derivatized Nanoparticle Coated Capillaries for Purification and Micro-extraction of Proteins and Peptides.
Bakry R, Gjerde D, Bonn GK.
J Proteome Res. 2006 Jun;5(6):1321-31.
[ expand abstract ]

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe(3+) or Ni(2+) for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.

Gold nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentration-a prelude of gel electrophoresis.
Wang A, Wu CJ, Chen SH.
J Proteome Res. 2006 Jun;5(6):1488-92.
[ expand abstract ]

Protein enrichment is essential for biological samples that contain low protein concentrations, especially for proteomic studies that require sufficient quantities for subsequent MS analysis. Traditional precipitation methods, however, are limited in the sample volume and protein concentration required to cause efficient precipitations. We showed that gold nanoparticles (Au-NPs) can be easily applied to concentrate proteins from more than 15 mL of human urine, in which the total protein concentration is less than 1.4 ppm. Moreover, Au-NP-aggregated proteins can be directly applied to gel electrophoresis for Au-NP-protein dissociation followed by free protein separation as well as for the subsequent in-gel digestion and protein identification by mass spectrometry. We compared this method with trichloroacetic acid (TCA) precipitation method, one of the most common precipitation methods, and TCA method showed no enrichment effect for protein samples with large volumes (>2 mL) or with low protein concentrations (4 ppm). Therefore, Au-NP aggregation is not only a simple and efficient method for enriching a broad range of proteins, it is also particularly useful for concentrating proteins from a relatively large volume of dilute biological fluids, under which TCA method is ineffective.

Highly Sensitive Biomolecular Fluorescence Detection Using Nanoscale ZnO Platforms.
Dorfman A, Kumar N, Hahm JI.
Langmuir. 2006 May 23;22(11):4890-5.
[ expand abstract ]

Fluorescence detection is currently one of the most widely used methods in the areas of basic biological research, biotechnology, cellular imaging, medical testing, and drug discovery. Using model protein and nucleic acid systems, we demonstrate that engineered nanoscale zinc oxide structures can significantly enhance the detection capability of biomolecular fluorescence. Without any chemical or biological amplification processes, nanoscale zinc oxide platforms enabled increased fluorescence detection of these biomolecules when compared to other commonly used substrates such as glass, quartz, polymer, and silicon. The use of zinc oxide nanorods as fluorescence enhancing substrates in our biomolecular detection permitted sub-picomolar and attomolar detection sensitivity of proteins and DNA, respectively, when using a conventional fluorescence microscope. This ultrasensitive detection was due to the presence of ZnO nanomaterials which contributed greatly to the increased signal-to-noise ratio of biomolecular fluorescence. We also demonstrate the easy integration potential of zinc oxide nanorods into periodically patterned nanoplatforms which, in turn, will promote the assembly and fabrication of these materials into multiplexed, high-throughput, optical sensor arrays. These zinc oxide nanoplatforms will be extremely beneficial in accomplishing highly sensitive and specific detection of biological samples involving nucleic acids, proteins and cells, particularly under detection environments involving extremely small sample volumes of ultratrace-level concentrations.

Multiplexed DNA Detection with Biobarcoded Nanoparticle Probes.
Stoeva SI, Lee JS, Thaxton CS, Mirkin CA.
Angew Chem Int Ed Engl. 2006 May 12;45(20):3303-3306.
[ expand abstract ]

No abstract available

Qdot Nanobarcodes for Multiplexed Gene Expression Analysis.
Eastman PS, Ruan W, Doctolero M, Nuttall R, de Feo G, Park JS, Chu JS, Cooke P, Gray JW, Li S, Chen FF.
Nano Lett. 2006 May 10;6(5):1059-1064.
[ expand abstract ]

We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of </=10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4-fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods.

Integration of microcolumns and microfluidic fractionators on multitasking centrifugal microfluidic platforms for the analysis of biomolecules.
Moschou EA, Nicholson AD, Jia G, Zoval JV, Madou MJ, Bachas LG, Daunert S.
Anal Bioanal Chem. 2006 May 9; [Epub ahead of print].
[ expand abstract ]

This work demonstrates the development of microfluidic compact discs (CDs) for protein purification and fractionation integrating a series of microfluidic features, such as microreservoirs, microchannels, and microfluidic fractionators. The CDs were fabricated with polydimethylsiloxane (PDMS), and each device contained multiple identical microfluidic patterns. Each pattern employed a microfluidic fractionation feature with operation that was based on the redirection of fluid into an isolation chamber as a result of an overflow. This feature offers the advantage of automated operation without the need for any external manipulation, which is independent of the size and the charge of the fractionated molecules. The performance of the microfluidic fractionator was evaluated by its integration into a protein purification microfluidic architecture. The microfluidic architecture employed a microchamber that accommodated a monolithic microcolumn, the fractionator, and an isolation chamber, which was also utilized for the optical detection of the purified protein. The monolithic microcolumn was polymerized "in situ" on the CD from a monolith precursor solution by microwave-initiated polymerization. This technique enabled the fast, efficient, and simultaneous polymerization of monoliths on disposable CD microfluidic platforms. The design of the CD employed allows the integration of various processes on a single microfluidic device, including protein purification, fractionation, isolation, and detection.

Nucleic acid aptamers and enzymes as sensors.
Navani NK, Li Y.
Curr Opin Chem Biol. 2006 May 3; [Epub ahead of print].
[ expand abstract ]

The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology.

Aptamer-conjugated nanoparticles for selective collection and detection of cancer cells.
Herr JK, Smith JE, Medley CD, Shangguan D, Tan W.
Anal Chem. 2006 May 1;78(9):2918-24.
[ expand abstract ]

We have developed a method for the rapid collection and detection of leukemia cells using a novel two-nanoparticle assay with aptamers as the molecular recognition element. An aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for CCRF-CEM acute leukemia cells that, when applied in this method, allows for specific recognition of the cells from complex mixtures including whole blood samples. Aptamer-modified magnetic nanoparticles were used for target cell extraction, while aptamer-modified fluorescent nanoparticles were simultaneously added for sensitive cell detection. Combining two types of nanoparticles allows for rapid, selective, and sensitive detection not possible by using either particle alone. Fluorescent nanoparticles amplify the signal intensity corresponding to a single aptamer binding event, resulting in improved sensitivity over methods using individual dye-labeled probes. In addition, aptamer-modified magnetic nanoparticles allow for rapid extraction of target cells not possible with other separation methods. Fluorescent imaging and flow cytometry were used for cellular detection to demonstrate the potential application of this method for medical diagnostics.

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions.
Li Y, Wark AW, Lee HJ, Corn RM.
Anal Chem. 2006 May 1;78(9):3158-64.
[ expand abstract ]

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.

Multiplexed DNA Detection with Biobarcoded Nanoparticle Probes.
Stoeva SI, Lee JS, Thaxton CS, Mirkin CA.
Angew Chem Int Ed Engl. 2006 Apr 7; [Epub ahead of print] .
[ expand abstract ]

No abstract available.

A gold nanoparticles/sol-gel composite architecture for encapsulation of immunoconjugate for reagentless electrochemical immunoassay.
Chen J, Tang J, Yan F, Ju H.
Biomaterials. 2006 Apr;27(10):2313-21.
[ expand abstract ]

A highly hydrophilic, non-toxic and conductive colloidal gold nanoparticle/titania sol-gel composite membrane with a low contact angle was prepared on a glassy carbon electrode via a vapor deposition method. With human chorionic gonadotrophin (hCG) as a model antigen and encapsulation of horseradish peroxidase-labeled hCG antibody (HRP-anti-hCG) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. It displayed a porous and homogeneous composite architecture without the aggregation of the immobilized protein molecules. The presence of gold nanoparticles provided a congenial microenvironment for adsorbed biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of immunoconjugate by a simple one-step immunoreaction between hCG in sample solution and the immobilized HRP-anti-hCG introduced a barrier of direct electrical communication between the immobilized HRP and the electrode surface. Under optimal conditions, the hCG analyte could be determined in two linear ranges from 0.5 to 5.0mIU/mL and 5.0 to 30mIU/mL with a relatively low detection limit of 0.3mIU/mL at 3sigma. The hCG immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. This composite membrane could be used efficiently for the entrapment of different biomarkers and clinical applications.

Fast DNA Sequencing via Transverse Electronic Transport.
Lagerqvist J, Zwolak M, Di Ventra M.
Nano Lett. 2006 Apr;6(4):779-82.
[ expand abstract ]

A rapid and low-cost method to sequence DNA would usher in a revolution in medicine. We propose and theoretically show the feasibility of a protocol for sequencing based on the distributions of transverse electrical currents of single-stranded DNA while it translocates through a nanopore. Our estimates, based on the statistics of these distributions, reveal that sequencing of an entire human genome could be done with very high accuracy in a matter of hours without parallelization, that is, orders of magnitude faster than present techniques. The practical implementation of our approach would represent a substantial advancement in our ability to study, predict, and cure diseases from the perspective of the genetic makeup of each individual.

Homogenous rapid detection of nucleic acids using two-color quantum dots.
Zhang CY, Johnson LW.
Analyst. 2006 Apr;131(4):484-8; [Epub 2006 Jan 13].
[ expand abstract ]

We report a homogenous method for rapid and sensitive detection of nucleic acids using two-color quantum dots (QDs) based on single-molecule coincidence detection. The streptavidin-coated quantum dots functioned as both a nano-scaffold and as a fluorescence pair for coincidence detection. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary target DNA through a sandwich hybridization reaction. The DNA hybrids were first caught and assembled on the surface of 605 nm-emitting QDs (605QDs) through specific streptavidin-biotin binding. The 525 nm-emitting QDs (525QDs) were then added to bind the other end of DNA hybrids. The coincidence signals were observed only when the presence of target DNA led to the formation of 605QD/DNA hybrid/525QD complexes. In comparison with a conventional QD-based assay, this assay provided high detection efficiency and short analysis time due to its high hybridization efficiency resulting from the high diffusion coefficient and no limitation of temperature treatment. This QD-based single-molecule coincidence detection offers a simple, rapid and ultra sensitive method for genomic DNA analysis in a homogenous format.

Detection of protein analytes via nanoparticle-based bio bar code technology.
Bao YP, Wei TF, Lefebvre PA, An H, He L, Kunkel GT, Muller UR.
Anal Chem. 2006 Mar 15;78(6):2055-9.
[ expand abstract ]

We describe a new format for the recently introduced bio bar code technology, which improves the dose response over 10,000-fold and thereby makes this technique analytically useful. Unlike other ultrasensitive protein detection methods, such as immuno-PCR or immuno-RCA, the bio bar code technique does not employ any enzymes to achieve detection limits in the attomolar range. By sandwiching a target between a magnetic bead and an amplifier nanoparticle, a multiplicity of bar code oligonucleotides are released for each captured target analyte. These surrogate bar code targets are then hybridized to microarrays and detected with silver-amplified gold nanoparticle probes. Using PSA detection as a model, we demonstrate a linear dose response over at least 4 orders of magnitude in both target concentration and concomitant signal and a 1000-fold improvement in detection limit compared to the best ELISA system.

Au nanoparticle conjugation for impedance and capacitance signal amplification in biosensors.
Wang J, Profitt JA, Pugia MJ, Suni II.
Anal Chem. 2006 Mar 15;78(6):1769-73.
[ expand abstract ]

Amplification of the electrochemical impedance and capacitance signals in a biosensor is demonstrated for the model fluorescein/anti-fluorescein system. Following immobilization of fluorescein onto Au through formation of a self-assembled monolayer, goat anti-fluorescein conjugated with 10-nm Au nanoparticles is introduced into the system. This results in an increase in the capacitance of approximately 400 nF/cm(2), whereas no change can be observed for goat anti-fluorescein without the Au nanoparticle conjugate. An even greater sensitivity is obtained by introduction of a redox probe, [Fe(CN)(6)](3)(-)(/4)(-), whereby the charge-transfer resistance (R(ct)) is reduced to approximately 25% of its original value. This allows construction of high-sensitivity electrochemical impedance biosensors at a single low frequency, where the signal is sensitive to the interfacial R(ct). This change in the electrochemical impedance signal upon binding to goat anti-fluorescein conjugated with Au nanoparticles can be attributed to the much higher electrochemical activity of Au surfaces relative to the underlying organic layer.

Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with composite nanoparticles of CdS/PAA.
Chen H, Xu F, Hong S, Wang L.
Spectrochim Acta A Mol Biomol Spectrosc. 2006 Mar 7; [Epub ahead of print].
[ expand abstract ]

This paper describes the development of composite nanoparticles. A novel composite nanoparticle has been prepared by an in situ polymerization method. The nano-CdS has been prepared, then the polymerization of acrylic acid (AA) was carried out by initiator potassium persulfate (KPS) under ultrasonic irradiation. The surface of the composite nanoparticles was covered with abundant carboxylic groups (-COOH). The nanoparticles are water-soluble, stable and biocompatible. Reaction of the composite nanoparticles with proteins results in an enhanced resonance light scattering (RLS) at 380nm. Based on this, a new resonance light-scattering (RLS) method was developed for the determination of proteins including BSA, HSA and human gamma-IgG. Under the optimum conditions, the enhanced RLS intensity is linearly proportional to the concentration of proteins. The liner range is 0.1-15mugmL(-1) for HSA, 0.2-20mugmL(-1) for BSA and 0.1-50.0mugmL(-1) for human gamma-IgG, respectively. The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are in good agreement with those reported by the hospital. This method proved to be very sensitive, rapid, simple and tolerant of most interfering substances.

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Homogeneous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis.
Yeh HC, Ho YP, Shih IeM, Wang TH.
Nucleic Acids Res. 2006 Mar 3;34(5):e35.
[ expand abstract ]

This report describes a new genotyping method capable of detecting low-abundant point mutations in a homogeneous, separation-free format. The method is based on integration of oligonucleotide ligation with a semiconductor quantum dot (QD)-mediated two-color fluorescence coincidence detection scheme. Surface-functionalized QDs are used to capture fluorophore-labeled ligation products, forming QD-oligonucleotide nanoassemblies. The presence of such nanoassemblies and thereby the genotype of the sample is determined by detecting the simultaneous emissions of QDs and fluorophores that occurs whenever a single nanoassembly flows through the femtoliter measurement volume of a confocal fluorescence detection system. The ability of this method to detect single events enables analysis of target signals with a multiple-parameter (intensities and count rates of the digitized target signals) approach to enhance assay sensitivity and specificity. We demonstrate that this new method is capable of detecting zeptomoles of targets and achieve an allele discrimination selectivity factor >10(5).

Non-cross-linking gold nanoparticle aggregation for sensitive detection of single-nucleotide polymorphisms: Optimization of the particle diameter.
Sato K, Onoguchi M, Sato Y, Hosokawa K, Maeda M.
Anal Biochem. 2006 Mar 1;350(1):162-4.
[ expand abstract ]

No abstract available

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Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots.
Bocsi J, Lenz D, Mittag A, Varga VS, Molnar B, Tulassay Z, Sack U, Tarnok A.
Cytometry A. 2006 Mar;69(3):131-4.
[ expand abstract ]

BACKGROUND: Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8). AIMS: We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM. METHODS: Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM. RESULTS: Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM. CONCLUSION: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.

Electrochemical microfluidic biosensor for the detection of nucleic acid sequences.
Goral VN, Zaytseva NV, Baeumner AJ.
Lab Chip. 2006 Mar;6(3):414-21.
[ expand abstract ]

A microfluidic biosensor with electrochemical detection for the quantification of nucleic acid sequences was developed. In contrast to most microbiosensors that are based on fluorescence for signal generation, it takes advantage of the simplicity and high sensitivity provided by an amperometric and coulorimetric detection system. An interdigitated ultramicroelectrode array (IDUA) was fabricated in a glass chip and integrated directly with microchannels made of poly(dimethylsiloxane) (PDMS). The assembly was packaged into a Plexiglas((R)) housing providing fluid and electrical connections. IDUAs were characterized amperometrically and using cyclic voltammetry with respect to static and dynamic responses for the presence of a reversible redox couple-potassium hexacyanoferrate (ii)/hexacyanoferrate (iii) (ferri/ferrocyanide). A combined concentration of 0.5 microM of ferro/ferricyanide was determined as lower limit of detection with a dynamic range of 5 orders of magnitude. Background signals were negligible and the IDUA responded in a highly reversible manner to the injection of various volumes and various concentrations of the electrochemical marker. For the detection of nucleic acid sequences, liposomes entrapping the electrochemical marker were tagged with a DNA probe, and superparamagnetic beads were coated with a second DNA probe. A single stranded DNA target sequence hybridized with both probes. The sandwich was captured in the microfluidic channel just upstream of the IDUA via a magnet located in the outside housing. Liposomes were lysed using a detergent and the amount of released ferro/ferricyanide was quantified while passing by the IDUA. Optimal location of the magnet with respect to the IDUA was investigated, the effect of dextran sulfate on the hybridization reaction was studied and the amount of magnetic beads used in the assay was optimized. A dose response curve using varying concentrations of target DNA molecules was carried out demonstrating a limit of detection at 1 fmol assay(-1) and a dynamic range between 1 and 50 fmol. The overall assay took 6 min to complete, plus 15-20 min of pre-incubation and required only a simple potentiostat for signal recording and interpretation.

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Detection of DNA hybridization using the near-infrared band-gap fluorescence of single-walled carbon nanotubes.
Jeng ES, Moll AE, Roy AC, Gastala JB, Strano MS.
Nano Lett. 2006 Mar;6(3):371-5.
[ expand abstract ]

We demonstrate the optical detection of DNA hybridization on the surface of solution suspended single-walled carbon nanotubes (SWNTs) through a SWNT band gap fluorescence modulation. Hybridization of a 24-mer oligonucleotide sequence with its complement produces a hypsochromic shift of 2 meV, with a detection sensitivity of 6 nM. The energy shift is modeled by correlating the surface coverage of DNA on SWNT to the exciton binding energy, yielding an estimated initial fractional coverage of 0.25 and a final coverage of 0.5. Hybridization on the nanotube surface is confirmed using Forster resonance energy transfer of fluorophore-labeled DNA oligonucleotides. This detection is enabled through a new technique to suspend SWNTs using adsorption of single-stranded DNA and subsequent removal of free DNA from solution. While the kinetics of free DNA hybridization are relatively fast (<10 min), the kinetics of the process on SWNTs are slower under comparable conditions, reaching steady state after 13 h at 25 degrees C. A second-order kinetic model yields a rate constant of k = 4.33 x 10(5) (M h)(-1). This optical, selective detection of specific DNA sequences may have applications in the life sciences and medicine as in vitro or in vivo detectors of oligonucleotides.

Integrated microfluidic biochips for DNA microarray analysis.
Liu RH, Dill K, Fuji HS, McShea A.
Expert Rev Mol Diagn. 2006 Mar;6(2):253-61.
[ expand abstract ]

A fully integrated and self-contained microfluidic biochip device has been developed to automate the fluidic handling steps required to perform a gene expression study of the human leukemia cell line (K-562). The device consists of a DNA microarray semiconductor chip with 12,000 features and a microfluidic cartridge that consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. A single-color transcriptional analysis of K-562 cells with a series of calibration controls (spiked-in controls) was performed to characterize this new platform with regard to sensitivity, specificity and dynamic range. The device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than 3 orders of magnitude. Experiments also demonstrated that chip-to-chip variability was low, indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis.

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Multicolor quantum dots for molecular diagnostics of cancer.
Smith AM, Dave S, Nie S, True L, Gao X.
Expert Rev Mol Diagn. 2006 Mar;6(2):231-44.
[ expand abstract ]

In the pursuit of sensitive and quantitative methods to detect and diagnose cancer, nanotechnology has been identified as a field of great promise. Semiconductor quantum dots are nanoparticles with intense, stable fluorescence, and could enable the detection of tens to hundreds of cancer biomarkers in blood assays, on cancer tissue biopsies, or as contrast agents for medical imaging. With the emergence of gene and protein profiling and microarray technology, high-throughput screening of biomarkers has generated databases of genomic and expression data for certain cancer types, and has identified new cancer-specific markers. Quantum dots have the potential to expand this in vitro analysis, and extend it to cellular, tissue and whole-body multiplexed cancer biomarker imaging.

Labeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates.
Chu TC, Shieh F, Lavery LA, Levy M, Richards-Kortum R, Korgel BA, Ellington AD.
Biosens Bioelectron. 2006 Feb 20; [Epub ahead of print].
[ expand abstract ]

Aptamers that bind to prostate specific membrane antigen (PSMA) were conjugated to luminescent CdSe and CdTe nanocrystals for cell-labeling studies. The aptamer-nanocrystal conjugates showed specific targeting of both fixed and live cells that overexpressed PSMA. More importantly, aptamers were able to label cells dispersed in a collagen gel matrix simulating tissue. The specific binding abilities and synthetic accessibility of aptamers combined with the photostability and small size of semiconductor nanocrystals offers a powerful and general tool for cellular imaging.

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Counting single native biomolecules and intact viruses with color-coded nanoparticles.
Agrawal A, Zhang C, Byassee T, Tripp RA, Nie S.
Anal Chem. 2006 Feb 15;78(4):1061-70.
[ expand abstract ]

Nanometer-sized particles such as semiconductor quantum dots and energy-transfer nanoparticles have novel optical properties such as tunable light emission, signal brightness, and multicolor excitation that are not available from traditional organic dyes and fluorescent proteins. Here we report the use of color-coded nanoparticles and dual-color fluorescence coincidence for real-time detection of single native biomolecules and viruses in a microfluidic channel. Using green and red nanoparticles to simultaneously recognize two binding sites on a single target, we demonstrate that individual molecules of genes, proteins, and intact viruses can be detected and identified in complex mixtures without target amplification or probe/target separation. Real-time coincidence analysis of single-photon events allows rapid detection of bound targets and efficient discrimination of excess unbound probes. Quantitative studies indicate that the counting results are remarkably precise when the total numbers of counted molecules are more than 10. The use of bioconjugated nanoparticle probes for single-molecule detection is expected to have important applications in ultrasensitive molecular diagnostics, bioterrorism agent detection, and real-time imaging and tracking of single-molecule processes inside living cells.

Fluoroimmunoassay for antigen based on fluorescence quenching signal of gold nanoparticles.
Ao L, Gao F, Pan B, He R, Cui D.
Anal Chem. 2006 Feb 15;78(4):1104-6.
[ expand abstract ]

A unique, sensitive, and highly specific fluoroimmunoassay system for antigen detection using gold and magnetic nanoparticles has been developed. The assay is based on the fluorescence quenching of fluorescein isothiocyanate caused by gold nanoparticles coated with monoclonal antibody. To demonstrate its analytical capabilities, the magnetic nanoparticles were coated with anti-alpha-fetoprotein polyclonal antibodies, which specifically bound with alpha-fetoprotein. Gold nanoparticles coated with anti-alpha-fetoprotein monoclonal antibodies could sandwich the alpha-fetoprotein captured by the magnetic nanoparticle probes. The sandwich-type immunocomplex was formed on the surface of magnetic nanoparticles and could be separated by a magnetic field. The supernatant liquid, which contained the unbound gold nanoparticle probes, was used to quench the fluorescence, and the fluorescence intensity of fluorescein isothiocyanate at 516 nm was proportional to the alpha-fetoprotein concentration. The result showed that the limit of detection of alpha-fetoprotein was 0.17 nM. This new system can be extended to detect target molecules with matched antibodies and has broad potential applications in immunoassay and disease diagnosis.

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Parallel analysis of biomolecules on a microfabricated capillary array chip.
Shen Z, Liu X, Long Z, Liu D, Ye N, Qin J, Dai Z, Lin B.
Electrophoresis. 2006 Feb 7; [Epub ahead of print] .
[ expand abstract ]

This paper focused on a self-developed microfluidic array system with microfabricated capillary array electrophoresis (mu-CAE) chip for parallel chip electrophoresis of biomolecules. The microfluidic array layout consists of two common reservoirs coupled to four separation channels connected to sample injection channel on the soda-lime glass substrate. The excitation scheme for distributing a 20 mW laser beam to separation channels in an array is achieved. Under the control of program, the sample injection and separation in multichannel can be achieved through six high-voltage modules' output. A CCD camera was used to monitor electrophoretic separations simultaneously in four channels with LIF detection, and the electropherograms can be plotted directly without reconstruction by additional software. Parallel multichannel electrophoresis of series biomolecules including amino acids, proteins, and nucleic acids was performed on this system and the results showed fine reproducibility.

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Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics.
Bhattacharyya A, Klapperich CM.
Anal Chem. 2006 Feb 1;78(3):788-92.
[ expand abstract ]

A polymeric microfluidic device for solid-phase extraction (SPE)-based isolation of nucleic acids is demonstrated. The plastic chip can function as a disposable sample preparation system for different biological and diagnostic applications. The chip was fabricated in a cyclic polyolefin by hot-embossing with a master mold. The solid phase consisted of a porous monolithic polymer column impregnated with silica particles. The extraction was achieved due to the binding of nucleic acids to the silica particles in the monolith. The solid phase was formed within the channels of the device by in situ photoinitiated polymerization of a mixture of methacrylate and dimethacrylate monomers, UV-sensitive free-radical initiator, and porogenic solvents. The channel surfaces were pretreated via photografting to covalently attach the monolith to the channel walls. The solid phase prepared by this method allowed for successful extraction and elution of nucleic acids in the polymeric microchip.

Parallel picoliter rt-PCR assays using microfluidics.
Marcus JS, Anderson WF, Quake SR.
Anal Chem. 2006 Feb 1;78(3):956-8.
[ expand abstract ]

The development of microfluidic tools for high-throughput nucleic acid analysis has become a burgeoning area of research in the post-genome era. Here, we have developed a microfluidic chip to perform 72 parallel 450-pL RT-PCRs. We took advantage of Taqman hydrolysis probe chemistry to detect RNA templates as low as 34 copies. The device and method presented here may enable highly parallel single cell gene expression analysis.

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Nanotechnologies for biomolecular detection and medical diagnostics.
Cheng MM, Cuda G, Bunimovich YL, Gaspari M, Heath JR, Hill HD, Mirkin CA, Nijdam AJ, Terracciano R, Thundat T, Ferrari M.
Curr Opin Chem Biol. 2006 Feb;10(1):11-9.
[ expand abstract ]

Nanotechnology-based platforms for the high-throughput, multiplexed detection of proteins and nucleic acids in heretofore unattainable abundance ranges promise to bring substantial advances in molecular medicine. The emerging approaches reviewed in this article, with reference to their diagnostic potential, include nanotextured surfaces for proteomics, a two-particle sandwich assay for the biological amplification of low-concentration biomolecular signals, and silicon-based nanostructures for the transduction of molecular binding into electrical and mechanical signals, respectively.

Nanoparticles: potential biomarker harvesters.
Geho DH, Jones CD, Petricoin EF, Liotta LA.
Curr Opin Chem Biol. 2006 Feb;10(1):56-61.
[ expand abstract ]

A previously untapped bank of information resides within the low molecular weight proteomic fraction of blood. Intensive efforts are underway to harness this information so that it can be used for early diagnosis of diseases such as cancer. The physicochemical malleability and high surface areas of nanoparticle surfaces make them ideal candidates for developing biomarker harvesting platforms. Given the variety of engineering strategies afforded through nanoparticle technologies, a significant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, sequestering them for later study using high sensitivity proteomic tests. To date, applications of nanoparticles have largely focused on imaging systems and drug delivery vectors. As such, biomarker harvesting is an underutilized application of nanoparticle technology and is an area of nanotechnology research that will likely undergo substantial growth.

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Fluorescent labels for proteomics and genomics.
Waggoner A.
Curr Opin Chem Biol. 2006 Feb;10(1):62-6.
[ expand abstract ]

Fluorescent labeling reagents are an essential component of a huge industry built on sensitive fluorescence detection. This technology has grown over 30 years and is in some ways mature. Excellent labeling reagents with close to maximum theoretical brightness are available in many different colors. Large fluorescent proteins like phycobiliproteins are also widely used that are exceedingly bright. Other fluorescent proteins like the GFP family can be obtained for creating genetically encoded protein labels in living cells. A new 'solid state' quantum dot technology is being exploited for large-scale multiparameter labeling. This technology provides the 'ultimate' photostable labeling reagent. Still, there are advances to be made. Not available is the ultimate tool kit of low molecular weight, strongly light absorbing, photostable labels with narrow emission bands ranging from the UV to the IR.

Integration of a surface acoustic wave biosensor in a microfluidic polymer chip.
Lange K, Blaess G, Voigt A, Gotzen R, Rapp M.
Biosens Bioelectron. 2006 Jan 31; [Epub ahead of print] .
[ expand abstract ]

SAW devices based on horizontally polarized surface shear waves (HPSSW) enable label-free, sensitive and cost-effective detection of biomolecules in real time. It is known that small sampling volumes with low inner surface areas and minimal mechanical stress arising from sealing elements of miniaturized sampling chambers are important in this field. Here, we present a new approach to integrate SAW devices with sampling chamber. The sensor device is encapsulated within a polymer chip containing fluid channel and contact points for fluidic and electric connections. The chip volume is only 0.9mul. The polymeric encapsulation was performed tailor-made by Rapid Micro Product Development 3Dimensional Chip-Size-Packaging (RMPD((R)) 3D-CSP), a 3D photopolymerisation process. The polymer housing serves as tight and durable package for HPSSW biosensors and allows the use of the complete chips as disposables. Preliminary experiments with these microfluidic chips are shown to characterise the performance for their future applications as generic bioanalytical micro devices.

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Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents.
Souza GR, Christianson DR, Staquicini FI, Ozawa MG, Snyder EY, Sidman RL, Miller JH, Arap W, Pasqualini R.
Proc Natl Acad Sci U S A. 2006 Jan 31;103(5):1215-20.
[ expand abstract ]

Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au-phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au-phage-imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.

Affinity capillary electrophoresis with a DNA-nanoparticle conjugate as a new tool for genotyping.
Adachi K, Noda N, Nakashige M, Tsuneda S, Kanagawa T.
J Chromatogr A. 2006 Jan 27; [Epub ahead of print] .
[ expand abstract ]

We have developed a novel method for genotyping based on free solution affinity capillary electrophoresis. We prepared DNA-nanoparticle conjugates by mixing biotin-modified DNA and NeutrAvidin-modified polystyrene nanoparticles; this mixture was then injected into a capillary. Subsequently, we injected the fluorescent-labeled sample DNAs into the capillary, applied the voltage, increased its temperature after 7min, and detected the fluorescence at its anodic end. This novel method was applied for genotyping human c-K-ras, and the three genotypes were definitely distinguishable with high reproducibility. This method can be easily automated, and it is useful for high-throughput gene mutation analysis.

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Electrocatalytical properties of polymethylferrocenyl dendrimers and their applications in biosensing.
Armada MP, Losada J, Zamora M, Alonso B, Cuadrado I, Casado CM.
Bioelectrochemistry. 2006 Jan 26; [Epub ahead of print] .
[ expand abstract ]

The electrochemical characterization of polymethylferrocenyl dendrimers deposited onto a platinum electrode and their applications as hydrogen peroxide and glucose sensor are described. The redox dendrimers consist of flexible poly(propileneimine) dendrimer cores functionalised with octamethylferrocenyl units. Amperometric biosensors for glucose were prepared by immobilization of glucose oxidase onto these modified electrodes. The influence of the dendrimer generation and the thickness of the dendrimer layer, the effect of the substrate concentration, and the interferences and reproducibility on the response of the sensors were investigated.

Development of nanostructured biomedical micro-drug testing device based on in situ cellular activity monitoring.
Prasad S, Quijano J.
Biosens Bioelectron. 2006 Jan 15;21(7):1219-29.
[ expand abstract ]

Integration of micro and nanofabrication techniques with biotechnology has resulted in the development of in vitro analytical and diagnostic tools for biomedical applications. The focus of such technology has primarily been on therapeutic and sensing applications. The long-term integration of cells with inorganic materials provides the basis for novel sensing platforms. This paper describes the creation of, nanoporous, biocompatible, alumina membranes as a platform for incorporation into a cell based device targeted for in situ recording of cellular electrical activity variations due to the changes associated with the surrounding microenvironments more specifically due to the effect of therapeutic drugs. Studies described herein focus on the interaction of nanoporous alumina substrates embedded in silicon, patterned with cells of interest. The cells that have been used to develop the in vitro test platform are primary hippocampal neurons. Demonstrated here, is the fidelity of such a system in terms of determination of cell viability, proliferation, and functionality. The response of the cells to the "drug" molecules is electro-optically characterized in an in situ manner. The capability of such, micro fabricated nanoporous membranes as in vitro drug testing platforms, is first theoretically estimated using two dimensional finite element modeling of the diffusion of the molecules of interest through the nanoporous substrate using CFDRC. It is then experimentally established, using glucose and immunoglobulin G (IgG).

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A highly sensitive fluorescent immunoassay based on avidin-labeled nanocrystals.
Sin KK, Chan CP, Pang TH, Seydack M, Renneberg R.
Anal Bioanal Chem. 2006 Jan 5;1-7.
[ expand abstract ]

Nanocrystals of the fluorogenic precursor fluorescein diacetate (FDA) were applied as labels in order to improve on the assay sensitivity achieved in our previous studies. Each FDA nanocrystal can be converted into approximately 2.6x10(6) fluorescein molecules, which is useful for improving immunoassay sensitivity and limits of detection. NeutrAvidin was simply adsorbed onto the surface of the FDA nanocrystals, which were coated with distearoylglycerophosphoethanolamine (DSPE) modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. This can be applied to detect different kinds of analytes that are captured by corresponding biotinylated biomolecules in different bioanalytical applications. The applicability of the NeutrAvidin-labeled nanocrystals was demonstrated in an immunoassay using the labeled avidin-biotin technique. Biotinylated antibody and FDA-labeled avidin were applied to the assay sequentially. The performance was compared with the traditional sandwich-type assay for mouse immunoglobulin G detection. Following the immunoreaction, the nanocrystals were released by hydrolysis and dissolution instigated by adding a large volume of organic solvent/sodium hydroxide mixture. The limit of detection was lower (by a factor of 2.5-21) and the sensitivity was (3.5-30-fold) higher than immunoassays using commercial labeling systems (FITC and peroxidase). This study shows that using fluorescent nanocrystals in combination with the avidin-biotin technique can enhance assay sensitivity and provide a lower limit of detection without requiring long incubation times as in enzyme-based labels.

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Protein Recognition via Surface Molecularly Imprinted Polymer Nanowires.
Li Y, Yang HH, You QH, Zhuang ZX, Wang XR.
Anal Chem. 2006 Jan 1;78(1):317-20.
[ expand abstract ]

In this paper, we present a technique for the preparation of polymer nanowires with the protein molecule imprinted and binding sites at surface. These surface imprinting nanowires exhibit highly selective recognition for a variety of template proteins, including albumin, hemoglobin, and cytochrome c. This recognition may be through a multistep adsorption, with the specificity conferred by hydrogen bonding and shape selectivity. Due to the protein imprinted sites are located at, or close to, the surface; these imprinted nanowires have a good site accessibility toward the target protein molecules. Furthermore, the large surface area of the nanowires results in large protein molecule binding capacity of the imprinted nanowires.

Uncoated, Broad Fluorescent, and Size-Homogeneous CdSe Quantum Dots for Bioanalyses.
Zhelev Z, Bakalova R, Ohba H, Jose R, Imai Y, Baba Y.
Anal Chem. 2006 Jan 1;78(1):321-330.
[ expand abstract ]

In the present study, we describe the synthesis of highly luminescent uncoated water-soluble CdSe quantum dots (QDs) possessing the following characteristics: approximately 2 nm in diameter, with very good size distribution (in 95% homodispersed) accompanied by a broad-band photoluminescent spectrum. The synthetic procedure is simple, is conducted at room temperature, in the absence of the most popular coordinating ligands (as TOPO or HDA), and is highly reproducible. The obtained CdSe core QDs possessed a comparatively long fluorescence half-life ( approximately 30-90 ns, depending on the emission wavelength) detected by time-resolved spectroscopy. These QDs were further conjugated with antibodies and applied in several biochemical analyses.

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Optical Readout of Gold Nanoparticle-Based DNA Microarrays without Silver Enhancement.
Blab GA, Cognet L, Berciaud S, Alexandre I, Husar D, Remacle J, Lounis B.
Biophys J. 2006 Jan;90(1):L13-5.
[ expand abstract ]

We present a novel readout scheme for gold nanoparticle-based DNA microarrays relying on "Laser-Induced Scattering around a NanoAbsorber". It provides direct counting of individual nanoparticles present on each array spot and stable signals, without any silver enhancement. Given the detection of nanometer-sized particles, which minimize the steric hindrance, the linear dynamic range of the method is particularly large and well suited for microarray detection.

DNA mutation detection and analysis using miniaturized microfluidic systems.
Handal MI, Ugaz VM.
Expert Rev Mol Diagn. 2006 Jan;6(1):29-38.
[ expand abstract ]

Identification of genetic sequence variations occurring on a population-wide scale is key to unraveling the complex interactions that are the underlying cause of many medical disorders and diseases. A critical need exists, however, for advanced technology to enable DNA mutation analysis to be performed with significantly higher throughput and at significantly lower cost than is currently attainable. Microfluidic systems offer an attractive platform to address these needs by combining the ability to perform rapid analysis with a simplified device format that can be inexpensively mass-produced. This paper will review recent progress toward developing these next-generation systems and discuss challenges associated with adapting these technologies for routine laboratory use.

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Engineering novel diagnostic modalities and implantable cytomimetic nanomaterials for next-generation medicine.
Ho D, Fung AO, Montemagno CD.
Biol Blood Marrow Transplant. 2006 Jan;12(1 Suppl 1):92-9.
[ expand abstract ]

The advent of 21st century medicine will be based on a comprehensive approach to achieving the highly sensitive and specific detection of diseases, as well as the development of novel materials and devices based on biotic-abiotic interfacing as interventional modalities. Novel technologies that enable early identification of physiological changes will serve as a gateway tool for the proper treatment of these disorders. Toward the realization of these technologies, microfabrication and nanofabrication methods have been applied to biomedical systems that allow scientists to interact with cellular and molecular systems on their native size scales. Future enabling systems will build on the foundation composed of such devices. With respect to the envisioned fruition of biofunctional nanomaterials and systems, foundational studies of biological systems and molecules, as well as their interfacing with biocompatible materials, have produced a domain of components that can be integrated and engineered toward eventual cytomimetic materials for transplantation. In addition, the potential underscoring of their future applications in nanoscale medicine is based on the ability to engineer and design intelligent membrane/protein self-assembling and organization phenomena that are typically found in nature into these artificial composite systems. These devices will provide a powerful suite of solutions with broad applicabilities in nanomedicine, for example, (1) the use of concomitant protein functionality toward energy production and the powering of medical implants and (2) replacement of damaged cells (e.g., heart and neuron) with implantable biologically intelligent engineered materials. This work will examine key advances in the areas of diagnostics and synthetic biology that have led to visionary contributions to next-generation medicine. Furthermore, we present 2 devices that will contribute to the realization of compelling biosensing and biofunctional material technologies. These systems include advanced diagnostic platforms for whole-cell detection, as well as copolymeric materials that have been functionalized by the coupled activity of their embedded membrane proteins. They are envisioned to successfully bridge the gap between foundational scientific progress and the realization of rapid point-of-care disease assessment and biofunctional devices with higher-order behavior.

Gold nanoparticle probes for the detection of nucleic acid targets.
Thaxton CS, Georganopoulou DG, Mirkin CA.
Clin Chim Acta. 2006 Jan;363(1-2):120-6.
[ expand abstract ]

BACKGROUND: Advances in nanoscience are having a significant impact on many scientific fields and are resulting in the development of a variety of important technologies. This impact is particularly large in the field of biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection, with DNA- or protein-functionalized gold nanoparticles used as the target-specific probes. METHODS: Assays provide an analysis of the unique biophysical properties displayed by gold nanoparticles and have advantages over conventional detection methods (e.g., molecular fluorophores, real-time polymerase chain reaction, RT-PCR, enzyme linked immunosorbent assays, ELISAs, gel electrophoresis, and microarray technologies). CONCLUSION: Some of the advantages include the assays' PCR-like sensitivity, their selectivity for target sequences, their capacity for massive multiplexing, their time efficiency, and most importantly, their ability to be performed at the point of care.

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A disposable microfluidic cassette for DNA amplification and detection.
Wang J, Chen Z, Corstjens PL, Mauk MG, Bau HH.
Lab Chip. 2006 Jan;6(1):46-53.
[ expand abstract ]

A pneumatically driven, disposable, microfluidic cassette comprised of a polymerase chain reaction (PCR) thermal cycler, an incubation chamber to label PCR amplicons with up-converting phosphor (UPT) reporter particles, conduits, temperature-activated, normally closed hydrogel valves, and a lateral flow strip, was constructed and tested. The hydrogel valves, which were opened and closed with the aid of electrically controlled thermoelectric units, provided a simple means to seal the PCR reactor and suppress bubble formation. The hydrogel-based flow control was electronically addressable, leakage-free, and biocompatible. To test the device, a solution laden with genomic DNA isolated from B. cereus was introduced into the microfluidic cassette and a specific 305 bp fragment was amplified. The PCR amplicons were labelled with the phosphor (UPT) reporter particles, applied to the lateral flow strip, bound to pre-immobilized ligands, and detected with an IR laser that scanned the lateral flow strip and excited the phosphor (UPT) particles that, in turn, emitted light in the visible spectrum. The UPT particles do not bleach, they provide a permanent record, and they readily facilitate the filtering of background noise. The cassette described herein will be used for rapid testing at the point of care.

2005

Microwave-Accelerated Metal-Enhanced Fluorescence: Platform Technology for Ultrafast and Ultrabright Assays.
Aslan K, Geddes CD.
Anal Chem. 2005 Dec 15;77(24):8057-8067.
[ expand abstract ]

We describe an exciting assay platform technology that promises to fundamentally address two underlying physical constraints of modern assays and immunoassays, namely, assay sensitivity and rapidity. By combining the use of metal-enhanced fluorescence with low-power microwave heating, we can indeed significantly increase the sensitivity of surface assays as well as >95 % kinetically complete the assay within a few seconds. Subsequently, this new technology promises to fundamentally change the way we currently employ immunoassays in clinical medicine. This new model platform system can be potentially applied to many other important assays, such as to the clinical assessment of myoglobin, where both assay speed and sensitivity is paramount for the assessment and treatment of acute myocardial infarction. To demonstrate the utility of microwave-accelerated metal-enhanced fluorescence (MAMEF), we show that a simple protein-based assay system can be optically amplified approximately 10-fold by using silver nanostructures, while being kinetically complete in less than 20 s. This new platform approach is subsequently over 10-fold more sensitive and approximately 90 times faster than a control assay that operates both at room temperature and without the use of metal-enhanced fluorescence. Finally, we show that low-power heating by microwaves in our model system does not denature proteins, as evidenced by no protein structural changes, probed by fluorescence resonance energy transfer.

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Isothermal DNA amplification coupled with DNA nanosphere-based colorimetric detection.
Tan E, Wong J, Nguyen D, Zhang Y, Erwin B, Van Ness LK, Baker SM, Galas DJ, Niemz A.
Anal Chem. 2005 Dec 15;77(24):7984-92.
[ expand abstract ]

We present a simple, rapid method for detecting short DNA sequences that combines a novel isothermal amplification method (EXPAR) with visual, colorimetric readout based on aggregation of DNA-functionalized gold nanospheres. The reaction is initiated by a trigger oligonucleotide, synthetic in nature for this proof-of-principle study, which is exponentially amplified at 55 degrees C and converted to a universal reporter oligonucleotide capable of bridging two sets of DNA-functionalized gold nanospheres. This reaction provides >10(6)-fold amplification/conversion in under 5 min. When combined with a solution containing DNA nanospheres, the bridging reporter causes nanosphere aggregation. The resulting color change from red to dark purple or blue is enhanced through spotting the solution onto a C18 reversed-phase thin-layer chromatography plate. The reaction can easily be adapted for detection of different trigger oligonucleotides using the same set of DNA nanospheres. It permits detection of as low as 100 fM trigger oligonucleotide in under 10 min total assay time, with minimal reagent consumption and requirement for instrumentation. We expect that combining this simple, versatile assay with trigger generation from a genomic target DNA sequence of interest will be a powerful tool in the development of rapid and simple point-of-care molecular diagnostic applications.

A Bio-Bar-Code Assay Based upon Dithiothreitol-Induced Oligonucleotide Release.
Thaxton CS, Hill HD, Georganopoulou DG, Stoeva SI, Mirkin CA.
Anal Chem. 2005 Dec 15;77(24):8174-8.
[ expand abstract ]

The recently developed bio-bar-code assay for the PCR-less detection of protein and nucleic acid targets has been shown to be extraordinarily sensitive, exhibiting low attomolar sensitivity for protein targets and high zeptomolar sensitivity for nucleic acid targets. In the case of DNA detection, the original assay relies on three distinct oligonucleotide strands on a single nanoparticle for target identification and signal amplification. Herein, we report the development of a new nanoparticle probe that can be used in the bio-bar-code assay, which requires only one thiolated oligonucleotide strand. This new assay relies on the ability to liberate the adsorbed thiolated oligonucleotides from the gold nanoparticle surface with dithiothreitol (DTT), which simplifies the assay and increases its quantitative capabilities. The utility of this new DTT-based system is demonstrated by detecting a mock mRNA target using both fluorescent and scanometric assay readouts. When the scanometric readout is used, the sensitivity of the assay is 7 aM and quantification can be accomplished over the low-attomolar to the mid-femtomolar concentration range.

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Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method.
Wang H, Qian WJ, Mottaz HM, Clauss TR, Anderson DJ, Moore RJ, Camp DG 2nd, Khan AH, Sforza DM, Pallavicini M, Smith DJ, Smith RD.
J Proteome Res. 2005 Dec 12;4(6):2397-2403.
[ expand abstract ]

Challenges associated with the efficient and effective preparation of micro- and nanoscale (micro- and nanogram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein, we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic cosolvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally sized proteomic samples (>100 mug protein content), based on both sample recovery and numbers of peptide/protein identifications. The effectiveness of this protocol for micro- and nanoscale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples ( approximately 30 mug total protein content) and also for samples of approximately 5000 MCF-7 human breast cancer cells ( approximately 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.

Electrochemical biosensors: Towards point-of-care cancer diagnostics.
Wang J.
Biosens Bioelectron. 2005 Dec 1; [Epub ahead of print].
[ expand abstract ]

Wide-scale point-of-care diagnostic systems hold great promise for early detection of cancer at a curable stage of the disease. This review discusses the prospects and challenges of electrochemical biosensors for next-generation cancer diagnostics. Electrochemical biosensors have played an important significant role in the transition towards point-of-care diagnostic devices. Such electrical devices are extremely useful for delivering the diagnostic information in a fast, simple, and low cost fashion in connection to compact (hand-held) analyzers. Modern electrochemical bioaffinity sensors, such as DNA- or immunosensors, offer remarkable sensitivity essential for early cancer detection. The coupling of electrochemical devices with nanoscale materials offers a unique multiplexing capability for simultaneous measurements of multiple cancer markers. The attractive properties of electrochemical devices are extremely promising for improving the efficiency of cancer diagnostics and therapy monitoring. With further development and resources, such portable devices are expected to speed up the diagnosis of cancer, making analytical results available at patient bedside or physician office within few minutes.

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Highly reproducible hybridization assay of zeptomole DNA based on adsorption of nanoparticle-bioconjugate.
Mo Z, Wang H, Liang Y, Liu F, Xue Y.
Analyst. 2005 Dec;130(12):1589-94.
[ expand abstract ]

A nanoparticle-bioconjugate was formed by homogeneous hybridization of one polynucleotide target with two oligonucleotide probes labelled by thiol and a nanoparticle, respectively. Deposition of the nanoparticle-bioconjugate on a gold surface by thiol-gold reaction was monitored in situ by quartz crystal microbalance (QCM) and applied for flow analysis of zeptomole amounts of polynucleotide. The formation in solution and adsorption of thiolated conjugates on gold could be fast, uniform and effective, and has been successfully exploited to construct a highly reproducible and sensitive platform for detection of target sequences. Being more rapid, reproducible, sensitive and amenable to automation than previously reported microgravimetric hybridization assays, this technology has great promise for practical applications in molecular diagnostics.

Enhancing the efficiency of a PCR using gold nanoparticles.
Li M, Lin YC, Wu CC, Liu HS.
Nucleic Acids Res. 2005 Nov 27;33(21):e184.
[ expand abstract ]

We found that the PCR could be dramatically enhanced by Au nanoparticles. With the addition of 0.7 nM of 13 nm Au nanoparticles into the PCR reagent, the PCR efficiency was increased. Especially when maintaining the same or higher amplification yields, the reaction time could be shortened, and the heating/cooling rates could be increased. The excellent heat transfer property of the nanoparticles should be the major factor in improving the PCR efficiency. Different PCR systems, DNA polymerases, DNA sizes and complex samples were compared in this study. Our results demonstrated that Au nanoparticles increase the sensitivity of PCR detection 5- to 10-fold in a slower PCR system (i.e. conventional PCR) and at least 10(4)-fold in a quicker PCR system (i.e. real-time PCR). After the PCR time was shortened by half, the 100 copies/microl DNA were detectable in real-time PCR with gold colloid added, however, at least 10(6) copies/microl of DNA were needed to reach a detectable signal level using the PCR reagent without gold colloid. This innovation could improve the PCR efficiency using non-expensive polymerases, and general PCR reagent. It is a new viewpoint in PCR, that nanoparticles can be used to enhance PCR efficiency and shorten reaction times.

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Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy.
Amemiya Y, Tanaka T, Yoza B, Matsunaga T.
J Biotechnol. 2005 Nov 21;120(3):308-14.
[ expand abstract ]

A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100 pg/ml. The minimum streptavidin detection limit using this technique is 1 pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.

Identification of peptides using gold nanoparticle-assisted single-drop microextraction coupled with AP-MALDI mass spectrometry.
Sudhir PR, Wu HF, Zhou ZC.
Anal Chem. 2005 Nov 15;77(22):7380-5.
[ expand abstract ]

A novel technique, gold nanoparticle-assisted single-drop microextraction (SDME) combined with atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) for the identification of peptides has been described. The SDME of peptides from aqueous solution was achieved using gold nanoparticles prepared in toluene as the acceptor phase. A simple phenomenon of isoelectric point (pI) of the peptides has been utilized successfully to extract the peptides into a single drop of nanogold in toluene. After extraction, a single-drop nano gold solution was directly spotted onto the target plate with an equal volume of matrix, proportional, variant-cyanohydroxy cinnamic acid ( proportional, variant-CHCA) and analyzed in AP-MALDI-MS. The parameters, such as solvent selection, extraction time, agitation rate, and pH effect, were optimized for the SDME technique. Using this technique, in aqueous solution, the lowest concentration detected for Met- and Leu-enkephalin peptides was 0.2 and 0.17 microM, respectively. In addition, the application of this technique to obtain the signal for the selected peptides in a mass spectrum in the presence of matrix interferences such as 1% Triton X-100 and 6.5 M urea has been showed. The application was extended to identify the peptides spiked into urine.

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Rapid detection of ssDNA and RNA using multi-walled carbon nanotubes modified screen-printed carbon electrode.
Ye Y, Ju H.
Biosens Bioelectron. 2005 Nov 15;21(5):735-41.
[ expand abstract ]

A method for rapid sensitive detection of DNA or RNA was designed using a composite screen-printed carbon electrode modified with multi-walled carbon nanotubes (MWNTs). MWNTs showed catalytic characteristics for the direct electrochemical oxidation of guanine or adenine residues of signal strand DNA (ssDNA) and adenine residues of RNA, leading to indicator-free detection of ssDNA and RNA concentrations. With an accumulation time of 5min, the proposed method could be used for detection of calf thymus ssDNA ranging from 17.0 to 345mugml(-1) with a detection limit of 2.0mugml(-1) at 3sigma and yeast tRNA ranging from 8.2mugml(-1) to 4.1mgml(-1). AC impedance was employed to characterize the surface of modified electrodes. The advantages of convenient fabrication, low-cost detection, short analysis time and combination with nanotechnology for increasing the sensitivity made the subject worthy of special emphasis in the research programs and sources of new commercial products.

Three-layer composite magnetic nanoparticle probes for DNA.
Stoeva SI, Huo F, Lee JS, Mirkin CA.
J Am Chem Soc. 2005 Nov 9;127(44):15362-3.
[ expand abstract ]

A method for synthesizing composite nanoparticles with a gold shell, an Fe(3)O(4) inner shell, and a silica core has been developed. The approach utilizes positively charged amino-modified SiO(2) particles as templates for the assembly of negatively charged 15 nm superparamagnetic water-soluble Fe(3)O(4) nanoparticles. The SiO(2)-Fe(3)O(4) particles electrostatically attract 1-3 nm Au nanoparticle seeds that act in a subsequent step as nucleation sites for the formation of a continuous gold shell around the SiO(2)-Fe(3)O(4) particles upon HAuCl(4) reduction. The three-layer magnetic nanoparticles, when functionalized with oligonucleotides, exhibit the surface chemistry, optical properties, and cooperative DNA binding properties of gold nanoparticle probes, but the magnetic properties of the Fe(3)O(4) inner shell.

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Label-Free Detection of Peptide Nucleic Acid-DNA Hybridization Using Localized Surface Plasmon Resonance Based Optical Biosensor.
Endo T, Kerman K, Nagatani N, Takamura Y, Tamiya E.
Anal Chem. 2005 Nov 1;77(21):6976-84.
[ expand abstract ]

The development of label-free optical biosensors for DNA and other biomolecules has the potential to impact life sciences as well as screening in medical and environmental applications. In this report, we developed a localized surface plasmon resonance (LSPR) based label-free optical biosensor based on a gold-capped nanoparticle layer substrate immobilized with peptide nucleic acids (PNAs). PNA probe was designed to recognize the target DNA related to tumor necrosis factor. The nanoparticle layer was formed on a gold-deposited glass substrate by the surface modified silica nanoparticles using silane-coupling reagent. The optical properties of gold-capped nanoparticle layer substrate were characterized through monitoring the changes in the absorbance strength, as the thickness of the biomolecular layer increased with hybridization. The detection of PNA-DNA hybridization with target oligonucleotides and PCR-amplified real samples were performed with a limit of detection value of 0.677 pM target DNA. Selective discrimination against a single-base mismatch was also achieved. Our LSPR-based biosensor with the gold-capped nanoparticle layer substrate is applicable to the design of biosensors for monitoring of the interaction of other biomolecules, such as proteins, whole cells, or receptors with a massively parallel detection capability in a highly miniaturized package.

Colorimetric bio-barcode amplification assay for cytokines.
Nam JM, Wise AR, Groves JT.
Anal Chem. 2005 Nov 1;77(21):6985-8.
[ expand abstract ]

The bio-barcode amplification assay has become a powerful tool in detecting tens to hundreds of biological targets such as proteins and nucleic acids in the entire sample. However, current bio-barcode detection schemes still require many experimental steps including microarrayer-based immobilization of oligonucleotides on a glass chip, silver enhancement of immobilized gold nanoparticles on a chip, and light-scattering measurement. Here, we report a colorimetric bio-barcode method that minimizes the above requirements while detecting 30 aM concentrations of cytokines ( approximately 3 orders of magnitude more sensitive than conventional nonenzymatic cytokine detection assays). The assay is based on porous microparticles, which enable loading of a large number of barcode DNA per particle, and gold nanoparticle-based colorimetric barcode detection method.

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Silver/Dendrimer nanocomposites as biomarkers: fabrication, characterization, in vitro toxicity, and intracellular detection.
Lesniak W, Bielinska AU, Sun K, Janczak KW, Shi X, Baker Jr JR, Balogh LP.
Nano Lett. 2005 Nov;5(11):2123-30.
[ expand abstract ]

We have synthesized water-soluble, biocompatible, fluorescent, and stable silver/dendrimer nanocomposites that exhibit a potential for in vitro cell labeling. Amino-, hydroxyl-, and carboxyl-terminated ethylenediamine core generation 5 poly(amidoamine) dendrimers were utilized to prepare aqueous silver(I)-dendrimer complexes (with the molar ratio of 25 Ag(+) per dendrimer) at the biologic pH of 7.4. Conversion of silver(I)-dendrimer complexes into dendrimer nanocomposites was achieved by irradiating the solutions with UV light to reduce the bound Ag(+) cations to zerovalent Ag(0) atoms, which were simultaneously trapped in the dendrimer network, resulting in the formation of {(Ag(0))(25)-PAMAM_E5.NH(2)}, {(Ag(0))(25)-PAMAM_E5.NGly}, and {(Ag(0))(25)-PAMAM_E5.NSAH} dendrimer nanocomposites (DNC), respectively. The silver-DNCs were characterized by means of UV-vis, fluorescence spectroscopy, dynamic light-scattering, zeta potential measurements, high-resolution transmission electron microscopy, X-ray energy dispersive spectroscopy, and selected area electron diffraction. The cytotoxicity of dendrimers and related silver nanocomposites was evaluated using an XTT colorimetric assay of cellular viability. The cellular uptake of nanoparticles was examined by transmission electron and confocal microscopy. Results indicate that {(Ag(0))(25)-PAMAM_E5.NH(2)}, {(Ag(0-))(25)-PAMAM_E5.NGly}, and {(Ag(0))(25)-PAMAM_E5.NSAH} form primarily single particles with diameters between 3 and 7 nm. The dendrimer nanocomposites are fluorescent, and their surface charge, cellular internalization, toxicity, and cell labeling capabilities are determined by the surface functionalities of dendrimer templates. The {(Ag(0))(25)-PAMAM_E5.NH(2)} and {(Ag(0))(25)-PAMAM_E5.NSAH} nanocomposites exhibit potential application as cell biomarkers.

A lab-on-a-chip for spectrophotometric analysis of biological fluids.
Minas G, Wolffenbuttel RF, Correia JH.
Lab Chip. 2005 Nov;5(11):1303-9.
[ expand abstract ]

This paper reports a lab-on-a-chip for application in clinical analysis, especially in the spectrophotometric analysis of biological fluids. It is composed of three parts: (1) a microfluidic system die containing the microchannels fabricated using SU-8 techniques; (2) an optical filtering system based on highly selective Fabry-Perot optical resonators using a stack of CMOS process compatible thin-film layers; (3) a detection and readout system fabricated in a CMOS microelectronic process. The system enables low-cost and selective measurement of the concentration of several biomolecules in biological fluids. Operation is based on optical absorption in a well-defined part of the visible spectrum, defined by the reaction of a specific reagent with a specific biomolecule. Signals proportional to the intensity of the light transmitted through the biological fluid are available at the output in the form of bit streams, which allows simple computer interfacing. Moreover, the optical filtering system enables the measurement using white light illumination, thus avoiding the use of a wavelength dependent light source. This characteristic makes the lab-on-a-chip portable and ensures that the analysis can be performed at any location with instantaneous results, without the use of complex and expensive analysis systems. The quantitative measurement of uric acid and total protein in urine is demonstrated.

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Emerging tools for real-time label-free detection of interactions on functional protein microarrays.
Ramachandran N, Larson DN, Stark PR, Hainsworth E, Labaer J.
FEBS J. 2005 Nov;272(21):5412-25.
[ expand abstract ]

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions - yeast two-hybrid and immunoprecipitation with mass spectrometric detection - have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.

Quantum-Dot Aptamer Beacons for the Detection of Proteins.
Levy M, Cater SF, Ellington AD.
Chembiochem. 2005 Oct 27; [Epub ahead of print].
[ expand abstract ]

No abstract available

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A colorimetric method for point mutation detection using high-fidelity DNA ligase.
Li J, Chu X, Liu Y, Jiang JH, He Z, Zhang Z, Shen G, Yu RQ.
Nucleic Acids Res. 2005 Oct 27;33(19):e168.
[ expand abstract ]

The present study reported proof-of-principle for a genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) through the gold nanoparticle assembly and the ligase reaction. By incorporating the high-fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation-based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold nanoparticle-tagged probes to hybrid with the target DNA strand, a ligase reaction that generates the ligation between perfectly matched probes while no ligation occurred between mismatched ones and a thermal treatment at a relatively high temperature that discriminate the ligation of probes. When the reaction mixture was heated to denature the formed duplex, the purple color of the perfect-match solution would not revert to red, while the mismatch gave a red color as the assembled gold nanoparticles disparted. The present approach has been demonstrated with the identification of a single-base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild-type and mutant type were successfully scored. To our knowledge, this was the first report concerning SNP detection based on the ligase reaction and the gold nanoparticle assembly. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in practical clinical diagnosis of gene-mutant diseases.

Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.
Pilarski LM, Lauzon J, Strachan E, Adamia S, Atrazhev A, Belch AR, Backhouse CJ.
J Immunol Methods. 2005 Oct 20;305(1):94-105.
[ expand abstract ]

Human cancer is inherently heterogeneous, so the ability to monitor individual cancer cells at every clinic visit would be a valuable tool. This work describes the first step towards developing handheld and automated devices for molecular and phenotypic analysis of cancer cells. Here, we show that use of capillary electrophoresis to detect PCR product amplified from either transcripts (high abundance template) or genomic DNA (low abundance template) encoding clonotypic immunoglobulin heavy chain VDJ of plasma cells from patients with multiple myeloma. High abundance IgH VDJ transcripts amplified in conventional systems or by capillary electrophoresis through channels on microfluidic chips or, alternatively, PCR product amplified from individual myeloma plasma cells in a single stage RT-PCR reaction was readily detectable on microfluidic chips. For low abundance templates, a nested PCR strategy was needed to detect PCR product by any method. Using microfluidic chips, PCR products amplified from genomic IgH VDJ DNA were detected in six out of eight plasma cells. Comparison of the ABI3100 and the microfluidic chip indicates that approximately 20 times more sample is injected into the ABI 3100 capillary than for the microfluidics chip. Overall, for high and low abundance template in individual cells, the microfluidic separation/detection system is at least as sensitive as the ABI 3100. In the future, integrated microfluidic platforms that incorporate both PCR cycling and product detection on the same chip are likely to exceed conventional systems in sensitivity and speed of genetic analysis by RT-PCR or PCR.

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An adaptable microvalving system for on-chip polymerase chain reactions.
Pilarski PM, Adamia S, Backhouse CJ.
J Immunol Methods. 2005 Oct 20;305(1):48-58.
[ expand abstract ]

On-chip genetic analysis systems are beginning to provide a viable alternative to conventional gene profiling and amplification devices, through minimal reagent use, high detection resolution, and the potential for high-throughput parallel testing of the genetic material, even from single cells. Despite the advantages, there are many difficulties inherent in creating an integrated microfluidic diagnostic platform. One major challenge is the accurate control and manipulation of fluid, and particularly the immobilization of reaction mixtures during heating phases of polymerase chain reactions (PCR). In this paper we present a pumping and valving system based on the use of three servomotor-controlled valve fingers that actuate microchannels within a poly-dimethylsiloxane (PDMS) fluidic chip. We characterize the valving ability of the system in terms of fluid loss and show the successful fluid retention of the system over 35-cycle PCR runs at temperatures of up to approximately 96 degrees C. In addition, we demonstrate the system's ability to perform PCR by successfully amplifying a sample of beta2 microglobulin transcript obtained from the peripheral blood of a patient with multiple myeloma. This work has proven to be a successful approach to multi-use valving and a viable method of alleviating the fluid control difficulties inherent in performing a PCR reaction in an on-chip environment. In addition, it opens the door for further automation and integration with other chip-based genetic analysis platforms.

Microfluidic-based diagnostics for cervical cancer cells.
Du Z, Colls N, Cheng KH, Vaughn MW, Gollahon L.
Biosens Bioelectron. 2005 Oct 18; [Epub ahead of print].
[ expand abstract ]

The use of biomarkers has facilitated the detection of specific tumor cells. However, the technology to apply these markers in a clinical setting has not kept pace with their increasing availability. In this project, we use an antibody-based microfluidics platform to recognize and capture cervical cancer cells. Because HPV-16 infection of cervical cells and up-regulation of alpha6-integrin cell surface receptors are correlated, we utilized alpha6-integrin as a capture antibody bound to the channel surface. Normal human glandular epithelial cells (HGEC), human cervical stromal cells (HCSC) and cervical cancer cells (HCCC) were suspended in PBS and flowed through the system. Greater than 30% of the cancer cells were captured while the capture of the normal cell types was less than 5%. The technique is sensitive and accurate. It is potentially useful in the detection of cervical cancer at all stages, as well as other of cancers with similar characteristics of cell surface antigen expression.

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Magnetically trigged direct electrochemical detection of DNA hybridization using au(67) quantum dot as electrical tracer.
Pumera M, Castaneda MT, Pividori MI, Eritja R, Merkoci A, Alegret S.
Langmuir. 2005 Oct 11;21(21):9625-9.
[ expand abstract ]

A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.

Gold nanoparticle probes for the detection of nucleic acid targets.
Thaxton CS, Georganopoulou DG, Mirkin CA.
Clin Chim Acta. 2005 Oct 5; [Epub ahead of print].
[ expand abstract ]

BACKGROUND: Advances in nanoscience are having a significant impact on many scientific fields and are resulting in the development of a variety of important technologies. This impact is particularly large in the field of biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection, with DNA- or protein-functionalized gold nanoparticles used as the target-specific probes. METHODS: Assays provide an analysis of the unique biophysical properties displayed by gold nanoparticles and have advantages over conventional detection methods (e.g., molecular fluorophores, real-time polymerase chain reaction, RT-PCR, enzyme linked immunosorbent assays, ELISAs, gel electrophoresis, and microarray technologies). CONCLUSION: Some of the advantages include the assays' PCR-like sensitivity, their selectivity for target sequences, their capacity for massive multiplexing, their time efficiency, and most importantly, their ability to be performed at the point of care.

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Toward an ICPMS-Linked DNA Assay Based on Gold Nanoparticles Immunoconnected through Peptide Sequences.
Merkoci A, Aldavert M, Tarrason G, Eritja R, Alegret S.
Anal Chem. 2005 Oct 1;77(19):6500-6503.
[ expand abstract ]

Gold nanoparticles modified with anti-mouse IgG have been used to trace oligonucleotides carrying a c-myc peptide. Two strategies, a dot-blot format as well as inductively coupled plasma mass spectrometry (ICPMS) have been used to detect the nanoparticle tracer. For both cases, oligonucleotide-peptide conjugates were first applied to a nitrocellulose membrane using a manifold attached to a suction device. After immobilization of the oligonucleotide by UV radiation, the samples were incubated with an anti-c-myc monoclonal antibody. In the case of the dot-blot format strategy, it was followed by incubation with a secondary antibody conjugated to horseradish peroxidase and development with luminol as chemiluminescent substrate. In the case of ICPMS strategy, it was followed by incubation with the secondary antibody (anti-mouse IgG) conjugated to gold nanoparticles and their ICPMS detection after dissolving. The nonspecific adsorptions were found to be around zero. The limit of detection for peptide-modified DNA was 0,2 pmol. The method may have significant potential as an important ICPMS-based nonradioactive DNA detection method for the simultaneous determination of various sequences by labeling different kinds of inorganic nanoparticles.

Microfluidic tool box as technology platform for hand-held diagnostics.
Pugia MJ, Blankenstein G, Peters RP, Profitt JA, Kadel K, Willms T, Sommer R, Kuo HH, Schulman LS.
Clin Chem. 2005 Oct;51(10):1923-32.
[ expand abstract ]

BACKGROUND: Use of microfluidics in point-of-care testing (POCT) will require on-board fluidics, self-contained reagents, and multistep reactions, all at a low cost. Disposable microchips were studied as a potential POCT platform. METHODS: Micron-sized structures and capillaries were embedded in disposable plastics with mechanisms for fluidic control, metering, specimen application, separation, and mixing of nanoliter to microliter volumes. Designs allowed dry reagents to be on separate substrates and liquid reagents to be added. Control of surface energy to +/-5 dyne/cm(2) and mechanical tolerances to </=1 mum were used to control flow propulsion into adsorptive, chromatographic, and capillary zones. Fluidic mechanisms were combined into working examples for urinalysis, blood glucose, and hemoglobin A(1c) testing using indicators (substances that react with analyte, such as dyes, enzyme substrates, and diazonium salts), catalytic reactions, and antibodies as recognition components. Optical signal generation characterized fluid flow and allowed detection. RESULTS: We produced chips that included capillary geometries from 10 to 200 mum with geometries for stopping and starting the flow of blood, urine, or buffer; vented chambers for metering and splitting 100 nL to 30 muL; specimen inlets for bubble-free specimen entry and containment; capillary manifolds for mixing; microstructure interfaces for homogeneous transfer into separation membranes; miniaturized containers for liquid storage and release; and moisture vapor barrier seals for easy use. Serum was separated from whole blood in <10 s. Miniaturization benefits were obtained at 10-200 mum. CONCLUSION: Disposable microchip technology is compatible with conventional dry-reagent technology and allows a highly compact system for complex assay sequences with minimum manual manipulations and simple operation.

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Microcantilever biosensors.
Hansen KM, Thundat T.
Methods. 2005 Sep 28; [Epub ahead of print].
[ expand abstract ]

Biosensors are sensors in which biomolecular interactions are used as sensing reactions. Biomolecular interactions, when combined with a microcantilever platform, can produce an extremely powerful biosensing design. The resonance frequency of a microcantilever shifts sensitively due to mass loading from molecular interaction as in the case of any acoustic sensors. In addition, the microcantilevers also undergo bending if the molecular adsorption is confined to a single surface of a microcantilever. This cantilever bending is due to a differential surface stress caused by the forces involved in the adsorption process and is amplified by making the cantilever surfaces chemically different. Lack of specificity, the main disadvantage of the cantilevers, can be overcome by using the extremely selective biochemical reactions such as receptor-ligand, antibody-antigen, or enzyme-substrate reactions. Here we review the microcantilever technology and discuss a number of highly sensitive biochemical sensor applications based on microcantilevers.

Nanoprobe-based affinity mass spectrometry for selected protein profiling in human plasma.
Chou PH, Chen SH, Liao HK, Lin PC, Her GR, Lai AC, Chen JH, Lin CC, Chen YJ.
Anal Chem. 2005 Sep 15;77(18):5990-7.
[ expand abstract ]

In recent decades, magnetic nanoparticles have emerged as a promising new platform in biomedical applications, particularly bioseparations. We have developed an immunoassay using antibody-conjugated magnetic nanoparticles as an efficient affinity probe to simultaneously preconcentrate and isolate targeted antigens from biological media. We combined this probe with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI MS) to profile proteins in diluted human plasma. The nanoparticles were designed to detect several disease-associated proteins and could be used directly in MALDI MS without an elution step, thereby facilitating multiple antigen screening and the characterization of antigen variants. Plasma antigens bound rapidly (approximately 10 min) to the antibody-conjugated nanoparticles, allowing the assay to be performed within 20 min. With sensitivity of detection in the femtomole range, the nanoscale immunoassay is superior to assays using microscale particles. We applied our method to comparative protein profiling of patients with gastric cancer and healthy individuals and found differential protein expression levels associated with the disease as well as individuals. Given the flexibility of manipulating functional groups on the nanoprobes, their low cost, robustness, and simplicity of the assay, our approach shows promise for targeted proteome profiling in clinical settings.

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DNA biosensor based on chitosan film doped with carbon nanotubes.
Li J, Liu Q, Liu Y, Liu S, Yao S.
Anal Biochem. 2005 Sep 15; [Epub ahead of print].
[ expand abstract ]

A biosensor based on chitosan doped with carbon nanotube (CNT) was fabricated to detect salmon sperm DNA. Methylene blue (MB) was employed as a DNA indicator. It was found that CNTs can enhance the electroactive surface area threefold (0.28+/-0.03 and 0.093+/-0.06cm(2) for chitosan-CNT- and chitosan-modified electrodes, respectively) and can accelerate the rate of electron transfer between the redox-active MB and the electrode. A low detection limit of 0.252nM fish sperm DNA was achieved, and no interference was found in the presence of 5mug/ml human serum albumin. The differential pulse voltammetry signal of MB was linear over the fish sperm DNA concentration range of 0.5-20nM.

Automatic bio-sampling chips integrated with micro-pumps and micro-valves for disease detection.
Wang CH, Lee GB.
Biosens Bioelectron. 2005 Sep 15;21(3):419-25.
[ expand abstract ]

The present study reports a microfluidic system using the concept of membrane-movement to design and fabricate micro-pneumatic valves and pumps to form a bio-sensing diagnostic chip. The automatic bio-sampling system includes a micro-diagnostic chip fabricated by using MEMS (micro-electro-mechanical systems) technology and an automatic platform comprising of a control circuit, a compressed air source and several electromagnetic valve switches. The control circuit is used to regulate the electromagnetic valve switches, causing thin PDMS membranes to deflect pneumatically by the compressed air and generate valving and pumping effects. The micro-diagnostic chip allows for the quick detection of diseases. Compared to large-scale systems, the new microfluidic system uses smaller amounts of samples and reagents and performs fast diagnosis in an automated format. Instead of using traditional pneumatic micro-pumps, the current study adopts a new design called "spider-web" micro-pumps to increase the pumping rate, and more importantly, improve the uniformity of flow rates inside multiple micro-channels. Experimental data show that for disease diagnosis, the bio-sensing chips integrated with the micro-pneumatic valves and the peristaltic micro-pumps could successfully perform diagnosis tests. Small amounts of samples and reagents could be injected into the diagnosis chips using the micro-pumps and the micro-pneumatic valves could effectively control the movement of the samples and reagents. In order to demonstrate the functionality of the developed device, detection of hepatitis C virus (HCV) and syphilis has been performed using the bio-sampling chips. Experimental data show that fluorescence signals from the microfluidic system were comparable to the ones using conventional testing methods. The developed chip could be easily extended for multiple disease detection. The automatic bio-sensing chips could provide a useful tool for fast disease detection and be crucial for a micro-total-analysis system.

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Complementary detection of prostate-specific antigen using in(2)o(3) nanowires and carbon nanotubes.
Li C, Curreli M, Lin H, Lei B, Ishikawa FN, Datar R, Cote RJ, Thompson ME, Zhou C.
J Am Chem Soc. 2005 Sep 14;127(36):12484-5.
[ expand abstract ]

We report complementary detection of prostate-specific antigen (PSA) using n-type In(2)O(3) nanowires and p-type carbon nanotubes. Our innovation involves developing an approach to covalently attach antibodies to In(2)O(3) NW surfaces via the onsite surface synthesis of phosphonic acid-succinylimide ester. Electronic measurements under dry conditions revealed complementary response for In(2)O(3) NW and SWNT devices after the binding of PSA. Real-time detection in solution has also been demonstrated for PSA down to 5 ng/mL, a benchmark concentration significant for clinical diagnosis of prostate cancer, which is the most frequently diagnosed cancer.

Gold nanoparticle-based competitive colorimetric assay for detection of protein-protein interactions.
Tsai CS, Yu TB, Chen CT.
Chem Commun (Camb). 2005 Sep 14;(34):4273-5.
[ expand abstract ]

A gold nanoparticle-based competitive colorimetric assay uses the ensemble of Concanavalin (ConA) and mannopyranoside-encapsulated gold nanoparticles (Man-GNPs) to identify the binding partners for ConA and the binding constants are determined based on the wavelength shifts.

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Complementary detection of prostate-specific antigen using in(2)o(3) nanowires and carbon nanotubes.
Li C, Curreli M, Lin H, Lei B, Ishikawa FN, Datar R, Cote RJ, Thompson ME, Zhou C.
J Am Chem Soc. 2005 Sep 14;127(36):12484-5.
[ expand abstract ]

We report complementary detection of prostate-specific antigen (PSA) using n-type In(2)O(3) nanowires and p-type carbon nanotubes. Our innovation involves developing an approach to covalently attach antibodies to In(2)O(3) NW surfaces via the onsite surface synthesis of phosphonic acid-succinylimide ester. Electronic measurements under dry conditions revealed complementary response for In(2)O(3) NW and SWNT devices after the binding of PSA. Real-time detection in solution has also been demonstrated for PSA down to 5 ng/mL, a benchmark concentration significant for clinical diagnosis of prostate cancer, which is the most frequently diagnosed cancer.

Protease-activated quantum dot probes.
Chang E, Miller JS, Sun J, Yu WW, Colvin VL, Drezek R, West JL.
Biochem Biophys Res Commun. 2005 Sep 9;334(4):1317-21.
[ expand abstract ]

We have developed a novel nanoparticulate luminescent probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This construct may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Release of AuNPs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 h of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker.

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Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.
Pilarski LM, Lauzon J, Strachan E, Adamia S, Atrazhev A, Belch AR, Backhouse CJ.
J Immunol Methods. 2005 Sep 5; [Epub ahead of print].
[ expand abstract ]

Human cancer is inherently heterogeneous, so the ability to monitor individual cancer cells at every clinic visit would be a valuable tool. This work describes the first step towards developing handheld and automated devices for molecular and phenotypic analysis of cancer cells. Here, we show that use of capillary electrophoresis to detect PCR product amplified from either transcripts (high abundance template) or genomic DNA (low abundance template) encoding clonotypic immunoglobulin heavy chain VDJ of plasma cells from patients with multiple myeloma. High abundance IgH VDJ transcripts amplified in conventional systems or by capillary electrophoresis through channels on microfluidic chips or, alternatively, PCR product amplified from individual myeloma plasma cells in a single stage RT-PCR reaction was readily detectable on microfluidic chips. For low abundance templates, a nested PCR strategy was needed to detect PCR product by any method. Using microfluidic chips, PCR products amplified from genomic IgH VDJ DNA were detected in six out of eight plasma cells. Comparison of the ABI3100 and the microfluidic chip indicates that approximately 20 times more sample is injected into the ABI 3100 capillary than for the microfluidics chip. Overall, for high and low abundance template in individual cells, the microfluidic separation/detection system is at least as sensitive as the ABI 3100. In the future, integrated microfluidic platforms that incorporate both PCR cycling and product detection on the same chip are likely to exceed conventional systems in sensitivity and speed of genetic analysis by RT-PCR or PCR.

A DNA assay protocol in a lab-on-valve meso-fluidic system with detection by laser-induced fluorescence.
Chen X, Wang W, Wang J.
Analyst. 2005 Sep;130(9):1240-4.
[ expand abstract ]

An automatic protocol for in-situ assay of dsDNA is presented by employing a micro-sequential injection lab-on-valve meso-fluidic system, which facilitates precise fluidic handling at the 0.1-10 microl level. Sub-nano-liter to a few micro-liters of DNA sample and ethidium bromide (EB) solutions were introduced into the meso-fluidic system, where EB binding onto DNA takes place and an intercalated DNA-EB adduct was formed, which was afterwards excited in the flow cell of the LOV by a 473 nm laser beam, and the emitted fluorescence was monitored in-situvia optical fibers. The experimental variables, i.e., pH of the buffer solution, the concentration and volume of EB solution, the reaction time and the fluid flow rates, were investigated. By loading 600 nl sample and 1.0 microl EB solution, a linear calibration graph was obtained within 0.03-3.0 microg ml(-1)(dsDNA), and a detection limit (3sigma) of 0.009 microg ml(-1) was achieved, along with a sampling frequency of 60 h(-1) and a precision of 1.9% at the 1.0 microg ml(-1) level. The detection limit was further improved to 0.006 microg ml(-1) by increasing the sample volume to 2.0 microl. Plasmid DNA in E. Coli extraction and lambda-DNA/Hind III in four synthetic samples were assayed by using this procedure. For the plasmid DNA, a good agreement with the documented UV method was obtained, while spiking recoveries for the synthetic samples were 95.6-103.4%.

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A system for micro/nano fluidic flow diagnostics.
Nath P, Roy S, Conlisk T, Fleischman AJ.
Biomed Microdevices. 2005 Sep;7(3):169-77.
[ expand abstract ]

A system for flow measurement in micro/nano fluidic components is presented. Microfabricated arrays of straight channels with noncircular cross-sections were used for flow rate measurement. The calculated flow rates in these channels were determined using a finite difference approximation method. A pneumatic pumping system was utilized to control the pressure drop across the channels and flow rates were measured by collecting the fluids on a sensitive balance. The experimental setup was validated using long narrow circular tubes that mimic the range of flow resistances characteristic of micro/nano fluidic devices. Two types of channels cross-section were investigated. The first type contained an array of channels that were approximately trapezoidal (microchannels, approximately 6.5 mu m deep) in cross-section and exhibited flow rates of 27.7-119.4 mu L/min within a pressure range of 64.1-277.1 kPa (9.3-40.2 psi). The second type contained an array of channels that were approximately arc-shaped (nanochannels, approximately 600 nm deep) and generated flow rates of 0.29-0.99 mu L/min within a pressure range of 137.2-334.4 kPa (19.9-48.5 psi). The flow rates calculated by the finite difference approximation method were within 5.5% and 19.68% of the average experimental flow rates in the microchannels and nanochannels, respectively.

Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy.
Amemiya Y, Tanaka T, Yoza B, Matsunaga T.
J Biotechnol. 2005 Aug 16; [Epub ahead of print].
[ expand abstract ]

A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100pg/ml. The minimum streptavidin detection limit using this technique is 1pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.

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Fluorescence detection of enzymatic activity within a liposome based nano-biosensor.
Vamvakaki V, Fournier D, Chaniotakis NA.
Biosens Bioelectron. 2005 Aug 15;21(2):384-8.
[ expand abstract ]

The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel

Analysis of mitochondrial DNA in microfluidic systems.
Taylor P, Manage DP, Helmle KE, Zheng Y, Glerum DM, Backhouse CJ.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Aug 5;822(1-2):78-84.
[ expand abstract ]

Abnormalities in mitochondrial function play a major role in many human diseases. It is often of critical importance to ascertain what proportion of the mitochondria within a cell, or cells, bear a given mutation (the mitochondrial "demographics"). In this work, a rapid, novel, on-chip procedure was used, in which a restriction enzyme was employed to excise a mitochondrial DNA (mtDNA) sequence from plasmid DNA that acted as a prototypical mitochondrial genome. The DNA was then denatured, reassembled to form duplexes, fluorescently labelled and analysed. This method was able to differentiate between a homogeneous population and a heterogeneous population. Using a microfluidic chip, the method could be performed in about 45min, even without robotics or multiplexed operation, whereas conventional methods of analysis require days to perform. This method may ultimately form the basis for a means of characterizing the mitochondrial demographics of a single cell.

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Simultaneous Multiple Immunoassays in a Compact Disc-Shaped Microfluidic Device Based on Centrifugal Force.
Honda N, Lindberg U, Andersson P, Hoffmann S, Takei H.
Clin Chem. 2005 Aug 4; [Epub ahead of print].
[ expand abstract ]

BACKGROUND: We explored the potential of a microfluidic device based on centrifugal force as an immunoassay platform by examining the imprecision of assays carried out with 200 nL of sample and use of the method to determine affinities of reagent antibodies. METHODS: Biotinylated antibodies against alpha-fetoprotein (AFP), interleukin-6 (IL-6) and carcinoembryonic antigen [CEA; 0.1 g/L in 15 mmol/L phosphate-buffered saline (PBS) containing 0.1 mL/L Tween 20] were attached to a microcolumn packed with streptavidin-coated particles. A 200-nL sample was then allowed to pass through the microcolumn for 240 s, followed by Alexa 647-labeled detection antibody (7.5 mg/L in 15 mmol/L PBS containing 10 g/L bovine serum antigen). The flow rate was controlled by altering the rotational speed. Up to 104 sandwich type immunoassays were completed within 50 min. RESULTS: For AFP, IL-6, and CEA the detection limits were, respectively, 0.15, 1.25 and 1.31 pmol/L. Inter- and intraassay imprecisions (CVs) were <10% and <20%, respectively, for analyte concentrations >5 pmol/L. The CEA antibody had the lowest affinity according to fluorescence image analysis of the microcolumn region. The result was confirmed in a comparative study using BIAcore 3000. CONCLUSIONS: Day-to-day (total) imprecision (CV) of immunoassays on the compact disc-shaped device are <20%. Analysis of fluorescence images allows rapid ranking of antibodies according to their affinities.

Nanotechnology in clinical laboratory diagnostics.
Jain KK.
Clin Chim Acta. 2005 Aug;358(1-2):37-54.
[ expand abstract ]

Nanotechnology-the creation and utilization of materials, devices, and systems through the control of matter on the nanometer-has been applied to molecular diagnostics. This article reviews nanobiotechnologies that are clinically relevant and have the potential to be incorporated in clinical laboratory diagnosis. Nanotechnologies enable the diagnosis at single cell and molecule level and some of these can be incorporated in the current molecular diagnostics such as biochips. Nanoparticles, such as gold nanoparticles and quantum dots, are the most widely used but various other nanotechnologies for manipulation at nanoscale as well as nanobiosensors are reviewed. These technologies will extend the limits of current molecular diagnostics and enable point-of-care diagnosis as well as the development of personalized medicine. Although the potential diagnostic applications are unlimited, most important current applications are foreseen in the areas of biomarker research, cancer diagnosis and detection of infectious microorganisms.

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Developing implantable optical biosensors.
Ziegler KJ.
Trends Biotechnol. 2005 Jul 22; [Epub ahead of print].
[ expand abstract ]

Nanobiotechnologists are developing devices that can measure specific enzymes and proteins. These devices are expected to detect single enzyme or protein molecules accurately, providing highly sensitive biosensing applications. A recent study by Strano and co-workers shows that single-walled carbon nanotubes (SWNTs) hold great promise as implantable biosensors. Although most researchers have focused on substrate-oriented biosensors, Strano and colleagues have shown that the inherent fluorescent properties of suspended individual SWNTs can be used for solution-phase beta-d-glucose sensing.

Separation of DNA fragments for fast diagnosis by microchip electrophoresis using programmed field strength gradient.
Kang SH, Park M, Cho K.
Electrophoresis. 2005 Jul 22; [Epub ahead of print].
[ expand abstract ]

We evaluated a novel strategy for fast diagnosis by microchip electrophoresis (ME), using programmed field strength gradients (PFSG) in a conventional glass double-T microfluidic chip. The ME-PFSG allows for the ultrafast separation and enhanced resolving power for target DNA fragments. These results are based on electric field strength gradients (FSG) that use an ME separation step in a sieving gel matrix poly-(ethylene oxide). The gradient can develop staircase or programmed shapes FSG over the time. The PFSG method could be easily used to increase separation efficiency and resolution in ME separation of specific size DNA fragments. Compared to ME that uses a conventional and constantly applied electric field (isoelectrostatic) method, the ME-PFSG achieved about 15-fold faster analysis time during the separation of 100 bp DNA ladder. The ME-PFSG was also applied to the fast analysis of the PCR products, 591 and 1191 bp DNA fragments from the 18S rRNA of Babesia gibsoni and Babesia caballi.

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Restriction mapping in nanofluidic devices.
Riehn R, Lu M, Wang YM, Lim SF, Cox EC, Austin RH.
Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10012-6.
[ expand abstract ]

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg(2+) and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of approximately 1.5 kbp in 1 min using single DNA molecules.

Multiple enzyme layers on carbon nanotubes for electrochemical detection down to 80 DNA copies.
Munge B, Liu G, Collins G, Wang J.
Anal Chem. 2005 Jul 15;77(14):4662-6.
[ expand abstract ]

Signal amplification using enzyme multilayers on carbon nanotube (CNT) templates is shown to yield a remarkably sensitive electrochemical detection of proteins and nucleic acids. The electrostatic layer-by-layer (LBL) self-assembly onto CNT carriers maximizes the ratio of enzyme tags per binding event to offer the greatest amplification factor reported to date. Absorption spectroscopy, TEM, and electrochemical characterization confirm the formation of LBL enzyme nanostructures on individual CNT carriers. The enzymatic activity is found to increase with the number of enzyme layers. The new protocol is illustrated for monitoring sandwich hybridization and antibody-antigen interactions in connection with alkaline phosphatase tracers. Factors affecting the enzyme loading and the analytical performance have been optimized. Such amplified bioelectronic assays allow detection of DNA and proteins down to 80 copies (5.4 aM) and 2000 protein molecules (67 aM), respectively. Given the enormous amplification afforded by the new CNT-LBL biolabel, such route offers great promise for ultrasensitive detection of infectious agents and disease markers.

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Layer-by-layer self-assembled multilayer films of carbon nanotubes and platinum nanoparticles with polyelectrolyte for the fabrication of biosensors.
Yang M, Yang Y, Yang H, Shen G, Yu R.
Biomaterials. 2005 Jul 15; [Epub ahead of print].
[ expand abstract ]

Platinum nanoparticle-doped chitosan (CHIT) solution can be easily prepared by treating the CHIT solution with aqueous H(2)PtCl(6) solution followed by chemical reduction of Pt(IV) with NaBH(4). Multiwalled carbon nanotubes (MWCNT) are then dispersed in the nanoparticle-doped solution. The resulting Pt-CNT-CHIT material brings new capabilities for electrochemical devices by using the synergistic action of Pt nanaoparticles and CNT. Positively charged Pt-CNT-CHIT solution and negatively charged poly(sodium-p-styrenesulfonate) salt (PSS) have been employed to fabricate stable ultrathin multilayer films on gold electrode and quartz glass slides in a layer-by-layer fashion. Cyclic voltammetric and UV-vis adsorption spectroscopy confirms the consecutive growth of the multilayer films. The modified gold electrode allows low-potential detection of hydrogen peroxide with high sensitivity and fast response time. With the immobilization of cholesterol oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to cholesterol has been constructed. In pH 6.98 phosphate buffer, almost interference free determination of cholesterol has been realized at 0.1V vs. SCE with a linear range from 0.01 to 3mM and response time<30s. With the immobilization of another cholesterol esterase enzyme layer, the biosensor was used to determine total cholesterol in serum samples with satisfactory results.

Present and future applications of carbon nanotubes to analytical science.
Valcarcel M, Simonet BM, Cardenas S, Suarez B.
Anal Bioanal Chem. 2005 Jul 9; [Epub ahead of print].
[ expand abstract ]

his article reviews the impact of carbon nanotubes on analytical science, and the main current and future applications of carbon nanotubes in this field. Given that it is necessary to solubilize carbon nanotubes for many applications, we consider the procedures developed to achieve this. The use of carbon nanotubes in analytical chemistry as a target analyte and as an analytical tool is also discussed. Chromatographic and electrophoretic methods used to separate and characterize carbon nanotubes are presented. The use of carbon nanotubes as an analytical tool in filters and membranes, as sorbent material for solid phase extraction, in electrochemical (bio)sensors, and in separation methods is discussed. It is clear that while nanotubes are being tested for use in many different fields, their truly enormous potential has yet to be realized in analytical chemistry.

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Restriction mapping in nanofluidic devices.
Riehn R, Lu M, Wang YM, Lim SF, Cox EC, Austin RH.
Proc Natl Acad Sci U S A. 2005 Jul 6; [Epub ahead of print].
[ expand abstract ]

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg(2+) and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of approximately 1.5 kbp in 1 min using single DNA molecules.

Electrochemical sensing based on redox mediation at carbon nanotubes.
Zhang M, Gorski W.
Anal Chem. 2005 Jul 1;77(13):3960-5.
[ expand abstract ]

An electrochemical sensing platform was developed based on the integration of redox mediators and carbon nanotubes (CNT) in a polymeric matrix. To demonstrate the concept, a redox mediator Azure dye (AZU) was covalently attached to polysaccharide chains of chitosan (CHIT) and interspersed with CNT to form composite films for the amperometric determination of beta-nicotinamide adenine dinucleotide (NADH). The incorporation of CNT into CHIT-AZU matrix facilitated the AZU-mediated electrooxidation of NADH. In particular, CNT decreased the overpotential for the mediated process by an extra 0.30 V and amplified the NADH current by approximately 35 times (at -0.10 V) while reducing the response time from approximately 70 s for CHIT-AZU to approximately 5 s for CHIT-AZU/CNT films. These effects were discussed in terms of the AZU/CNT synergy, which improved charge propagation through the CHIT-AZU/CNT matrix. The concept of CNT-facilitated redox mediation in polymeric matrixes has a potential to be of general interest for expediting redox processes in electrochemical devices such as sensors, biosensors, and biological fuel cells and reactors.

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Real-time detection of virus particles and viral protein expression with two-color nanoparticle probes.
Agrawal A, Tripp RA, Anderson LJ, Nie S.
J Virol. 2005 Jul;79(13):8625-8.
[ expand abstract ]

Respiratory syncytial virus (RSV) mediates serious lower respiratory tract illness in infants and young children and is a significant pathogen of the elderly and immune compromised. Rapid and sensitive RSV diagnosis is important to infection control and efforts to develop antiviral drugs. Current RSV detection methods are limited by sensitivity and/or time required for detection. In this study, we show that antibody-conjugated nanoparticles rapidly and sensitively detect RSV and estimate relative levels of surface protein expression. A major development is use of dual-color quantum dots or fluorescence energy transfer nanobeads that can be simultaneously excited with a single light source.

PDMS microfludic device for optical detection of protein immunoassay using gold nanoparticles.
Luo C, Fu Q, Li H, Xu L, Sun M, Ouyang Q, Chen Y, Ji H.
Lab Chip. 2005 Jul;5(7):726-9.
[ expand abstract ]

A simple but highly specific immunoassay system for goat anti-human IgG has been developed using gold nanoparticles and microfluidic techniques. The assay is based on the deposition of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies to microfluidic channel surface. The effects of time accumulation, the flow velocity, and the concentration of antibodies to the red light absorption percentage (RAP) of deposition were investigated with an ordinary optical microscope. By controlling the reaction time and flow velocity, a dynamic range of 3 orders of magnitude and a detection sensitivity of 10 ng ml(-1) of goat anti-human IgG were achieved. Because of its simplicity and flexibility, this new technique should be useful for fast, highthroughput screening of antibodies in clinical diagnostic applications.

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Molecular beacons for bioanalytical applications.
Tan L, Li Y, Drake TJ, Moroz L, Wang K, Li J, Munteanu A, James Yang C, Martinez K, Tan W.
Analyst. 2005 Jul;130(7):1002-5.
[ expand abstract ]

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties. They act like switches and are normally in a closed state, when the fluorophore and the quencher are brought together to turn "off" the fluorescence. When prompted to undergo conformational changes that open the hairpin structure, the fluorophore and the quencher are separated, and fluorescence is turned "on." This Education will outline the principles of MBs and discuss recent bioanalytical applications of these probes for in vitro RNA and DNA monitoring, biosensors and biochips, real-time monitoring of genes and gene expression in living systems, as well as the next generation of MBs for studies on proteins, the MB aptamers. These important applications have shown that MBs hold great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.

Microfluidic systems and proteomics: Applications of the electrocapture technology to protein and peptide analysis.
Astorga-Wells J, Vollmer S, Bergman T, Jornvall H.
Anal Biochem. 2005 Jun 30; [Epub ahead of print].
[ expand abstract ]

No abstract available.

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A modular microfluidic architecture for integrated biochemical analysis.
Shaikh KA, Ryu KS, Goluch ED, Nam JM, Liu J, Thaxton CS, Chiesl TN, Barron AE, Lu Y, Mirkin CA, Liu C.
Proc Natl Acad Sci U S A. 2005 Jun 28; [Epub ahead of print].
[ expand abstract ]

Microfluidic laboratory-on-a-chip (LOC) systems based on a modular architecture are presented. The architecture is conceptualized on two levels: a single-chip level and a multiple-chip module (MCM) system level. At the individual chip level, a multilayer approach segregates components belonging to two fundamental categories: passive fluidic components (channels and reaction chambers) and active electromechanical control structures (sensors and actuators). This distinction is explicitly made to simplify the development process and minimize cost. Components belonging to these two categories are built separately on different physical layers and can communicate fluidically via cross-layer interconnects. The chip that hosts the electromechanical control structures is called the microfluidic breadboard (FBB). A single LOC module is constructed by attaching a chip comprised of a custom arrangement of fluid routing channels and reactors (passive chip) to the FBB. Many different LOC functions can be achieved by using different passive chips on an FBB with a standard resource configuration. Multiple modules can be interconnected to form a larger LOC system (MCM level). We demonstrated the utility of this architecture by developing systems for two separate biochemical applications: one for detection of protein markers of cancer and another for detection of metal ions. In the first case, free prostate-specific antigen was detected at 500 aM concentration by using a nanoparticle-based bio-bar-code protocol on a parallel MCM system. In the second case, we used a DNAzyme-based biosensor to identify the presence of Pb(2+) (lead) at a sensitivity of 500 nM in <1 nl of solution.

Analysis of mitochondrial DNA in microfluidic systems.
Taylor P, Manage DP, Helmle KE, Zheng Y, Glerum DM, Backhouse CJ.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Jun 27; [Epub ahead of print].
[ expand abstract ]

Abnormalities in mitochondrial function play a major role in many human diseases. It is often of critical importance to ascertain what proportion of the mitochondria within a cell, or cells, bear a given mutation (the mitochondrial "demographics"). In this work, a rapid, novel, on-chip procedure was used, in which a restriction enzyme was employed to excise a mitochondrial DNA (mtDNA) sequence from plasmid DNA that acted as a prototypical mitochondrial genome. The DNA was then denatured, reassembled to form duplexes, fluorescently labelled and analysed. This method was able to differentiate between a homogeneous population and a heterogeneous population. Using a microfluidic chip, the method could be performed in about 45min, even without robotics or multiplexed operation, whereas conventional methods of analysis require days to perform. This method may ultimately form the basis for a means of characterizing the mitochondrial demographics of a single cell.

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Detection of single DNA molecules by multicolor quantum-dot end-labeling.
Crut A, Geron-Landre B, Bonnet I, Bonneau S, Desbiolles P, Escude C.
Nucleic Acids Res. 2005 Jun 20;33(11):e98.
[ expand abstract ]

Observation of DNA-protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA-protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.

Advances in polymerase chain reaction on microfluidic chips.
Roper MG, Easley CJ, Landers JP.
Anal Chem. 2005 Jun 15;77(12):3887-93.
[ expand abstract ]

No abstract available.

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Nanoparticle labels in immunosensing using optical detection methods.
Seydack M.
Biosens Bioelectron. 2005 Jun 15;20(12):2454-69.
[ expand abstract ]

Efforts to improve the performance of immunoassays and immunosensors by incorporating different kinds of nanostructures have gained considerable momentum over the last decade. Apart from liposomes, which will not be discussed here, most groups focus on artificial, particulate marker systems, both organic and inorganic. The underlying detection procedures may be based either on electro-magnetical or optical techniques. This review will be confined to the latter only, comprising nanoparticle applications generating signals as diverse as static and time-resolved luminescence, one- and two-photon absorption, Raman and Rayleigh scattering as well as surface plasmon resonance and others. In general, all endeavors cited are geared to achieve one or more of the following goals: lowering of detection limits (if possible, down to single-molecule level), parallel integration of multiple signals (multiplexing), signal amplification by several orders of magnitude and prevention of photobleaching effects with concomitant maintenance of antigen binding specificity and sensitivity. Inorganic nanoparticle labels based on noble metals, semiconductor quantum dots and nanoshells appear to be the most versatile systems for these bioanalytical applications of nanophotonics.

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Nanoparticle PCR: Nanogold-Assisted PCR with Enhanced Specificity.
Li H, Huang J, Lv J, An H, Zhang X, Zhang Z, Fan C, Hu J.
Angew Chem Int Ed Engl. 2005 Jun 8; [Epub ahead of print].
[ expand abstract ]

No abstract available

Contamination-free continuous flow microfluidic polymerase chain reaction for quantitative and clinical applications.
Dorfman KD, Chabert M, Codarbox JH, Rousseau G, de Cremoux P, Viovy JL.
Anal Chem. 2005 Jun 1;77(11):3700-4.
[ expand abstract ]

We present a method for performing polymerase chain reaction (PCR) using isolated droplets flowing in an immiscible fluorinated solvent system. Thanks to an optimized control of interfacial properties, we could achieve in this capillary-based system reproducible amplification factors, without any detectable contamination between neighboring droplets. The system is readily amenable to further miniaturization and automation and serves as the first step toward a clinically viable, high-throughput, quantitative continuous flow PCR apparatus.

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A 768-lane microfabricated system for high-throughput DNA sequencing.
Aborn JH, El-Difrawy SA, Novotny M, Gismondi EA, Lam R, Matsudaira P, McKenna BK, O'neil T, Streechon P, Ehrlich DJ.
Lab Chip. 2005 Jun;5(6):669-74.
[ expand abstract ]

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm [times] 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor. A total of greater than 172000 bases, 99% accuracy (corresponding to quality score 20) is achieved for each iteration of a 384 lane plate. At current operating conditions, this implies a system throughput exceeding 4 megabases of raw sequence (Phred 20) per day on the new platform. Standard operation is at "1/32[times]" Sanger chemistry, equal to typical genome center operation on mature capillary array machines, and a 16-fold improvement in scaling relative to previous microfabricated devices. Experiments provide evidence that sample concentration can be further reduced to 1/256[times] Sanger chemistry in the microdevice. Life-testing indicates a usable life of >150 hours (more than 50 runs) for the 384 lane plates. The combined advances, particularly those in read length and sample requirement, directly address the cost model requirements for adaptation of the new technology as the next step beyond capillary array instruments.

New reagents for phosphatidylserine recognition and detection of apoptosis.
Hanshaw RG, Smith BD.
Bioorg Med Chem. 2005 May 21; [Epub ahead of print].
[ expand abstract ]

The phospholipid bilayer surrounding animal cells is made up of four principle phospholipid components, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These four phospholipids are distributed between the two monolayers of the membrane in an asymmetrical fashion, with PC and SM largely populating the extracellular leaflet and PE and PS restricted primarily to the inner leaflet. Breakdown in this transmembrane phospholipid asymmetry is a hallmark of the early to middle stages of apoptosis. The consequent appearance of PS on the extracellular membrane leaflet is commonly monitored using dye-labeled Annexin V, a 36kDa, Ca(2+)-dependent PS binding protein. Substitutes for Annexin V are described, including small molecules, nanoparticles, cationic liposomes, and other proteins that can recognize PS in a membrane surface. Particular attention is given to the use of these reagents for detecting apoptosis

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Nanotechnology in clinical laboratory diagnostics.
Jain KK.
Clin Chim Acta. 2005 May 9; [Epub ahead of print].
[ expand abstract ]

Nanotechnology-the creation and utilization of materials, devices, and systems through the control of matter on the nanometer-has been applied to molecular diagnostics. This article reviews nanobiotechnologies that are clinically relevant and have the potential to be incorporated in clinical laboratory diagnosis. Nanotechnologies enable the diagnosis at single cell and molecule level and some of these can be incorporated in the current molecular diagnostics such as biochips. Nanoparticles, such as gold nanoparticles and quantum dots, are the most widely used but various other nanotechnologies for manipulation at nanoscale as well as nanobiosensors are reviewed. These technologies will extend the limits of current molecular diagnostics and enable point-of-care diagnosis as well as the development of personalized medicine. Although the potential diagnostic applications are unlimited, most important current applications are foreseen in the areas of biomarker research, cancer diagnosis and detection of infectious microorganisms.

Investigation of Molecular Beacon Aptamer-Based Bioassay for Platelet-Derived Growth Factor Detection.
Vicens MC, Sen A, Vanderlaan A, Drake TJ, Tan W.
Chembiochem. 2005 May 6;6(5):900-907.
[ expand abstract ]

This report describes studies on the use of a molecular-beacon aptamer (MBA) as a synthetic high-affinity DNA probe that exhibits fluorescence resonance energy transfer (FRET) in response to a specific protein biomarker, platelet-derived growth factor (PDGF). As a step toward the application of the MBA in a fluorescence-based assay for biological specimens, we examined the influence of certain physical and chemical parameters of incubation that would affect DNA conformation and DNA-backbone modification, and thus improve nuclease resistance. This bioassay is compatible with pH, temperature, and monovalent cation levels typically encountered in biological samples, and phosphorothioate backbone-modified MBA is able to exhibit specific FRET. With minimal sample processing and without assay optimization, the MBA is able to detect as little as 10 ng PDGF per mug of serum proteins from cell-culture media. We also show that different sets of known fluorophore-quencher pairs can be successfully used in the MBA for sensitive detection of the PDGF target. It should, therefore, be possible to develop multiplex bioassays that monitor either quenching or enhancement for the simultaneous detection of several biomarkers by using MBAs created from high-affinity DNA ligands for the desired protein targets. Interestingly, we observed that, with a DNA ligand with multiple binding sites for a standard multimeric protein target, the FRET bioassay could be accomplished by using a mixture of two individually labeled DNAs-one carrying the fluorophore and the other with the matching quencher. This observation has significant implications in the future design of more selective DNA-based FRET bioassays that use more than one ligand for the same protein target.

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Electrochemical bioassay utilizing encapsulated electrochemical active microcrystal biolabels.
Mak WC, Cheung KY, Trau D, Warsinke A, Scheller F, Renneberg R.
Anal Chem. 2005 May 1;77(9):2835-41.
[ expand abstract ]

A new approach to perform electrochemical immunoassay based on the utilization of encapsulated microcrystal was developed. The microcrystal labels create a "supernova effect" upon exposure to a desired releasing agent. The microcrystal cores dissolve, and large amounts of signal-generating molecules diffuse across the capsule wall into the outer environment. Layer-by-Layer (LbL) technology was employed for the encapsulation of electrochemical signal-generating microcrystals (ferrocene microcrystals). The encapsulated microcrystals were conjugated with antibody molecules through the adsorption process. The biofunctionalized microcrystals were utilized as a probe for immunoassays. The microcrystal-based label system provided a high-signal molecule to antibody (S/P) ratio of 10(4)-10(5). Microcrystal biolabels with different antibody surface coverage (1.60-5.05 mg m(-2)) were subjected to a solid-phase immunoassay for the detection of mouse immunoglobulin G (M-IgG) molecules. The microcrystal-based immunoassay for the detection of M-IgG performed with microcrystals having antibody surface coverage of 5.05 mg m(-2) showed a sensitivity of 3.93 nA microg(-1) L(-1) with a detection limit of 2.82 microg L(-1).

Polymerase chain reaction of nanoparticle-bound primers.
Shen HB, Hu M, Wang YB, Zhou HQ.
Biophys Chem. 2005 May 1;115(1):63-6.
[ expand abstract ]

Using one or two primers respectively bound to the surface of Au nanoparticles (AuNPs) or magnetic nanoparticles (MNPs), polymerase chain reaction (PCR) based on nanoparticles was systemically studied, agarose gel electrophoresis and atomic force microscopy (AFM) were respectively used to detect and observe the PCR product. The results obtained indicated that with either one or two primers respectively bound to the nanoparticle surface, PCR can proceed successfully under optimized condition and is subject to certain rules, consequently a symmetric PCR technique and an asymmetric PCR technique based on nanoparticles have been developed. A kind of nanostructured aggregates can be constructed by a symmetric PCR using two nanoparticle-bound primers.

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Technologies for nanofluidic systems: top-down vs bottom-up-a review.
Mijatovic D, Eijkel JC, van den Berg A.
Lab Chip. 2005 May;5(5):492-500.
[ expand abstract ]

This paper gives an overview of the most commonly used techniques for nanostructuring and nanochannel fabrication employed in nanofluidics. They are divided into two large categories: top-down and bottom-up methods. Top-down methods are based on patterning on large scale while reducing the lateral dimensions to the nanoscale. Bottom-up methods arrange atoms and molecules in nanostructures. Here, we review the advantages and disadvantages of those methods and give some future perspectives. It is concluded that technology in the region of 1-10 nm is lacking and potentially can be covered by using the pulsed-laser deposition method as a controlled way for thin film deposition (thickness of a few nanometers) and further structuring by the top-down method.

Integrated microfabricated systems including a purification module and an on-chip nano electrospray ionization interface for biological analysis.
Carlier J, Arscott S, Thomy V, Camart JC, Cren-Olive C, Le Gac S.
J Chromatogr A. 2005 Apr 15;1071(1-2):213-22.
[ expand abstract ]

We report here on an integrated microfabricated device dedicated to the preparation of biological samples prior to their on-line analysis by electrospray ionization-mass spectrometry (ESI-MS). This microfluidic device is fabricated using the negative photoresist SU-8 by microtechnology techniques. The device includes a chromatographic module plus an ESI interface for MS. The chromatographic module is dedicated to sample purification and is based on a polymer monolithic phase which includes hydrophobic moieties. The ESI interface is integrated onto the chip and is based on a capillary slot. We present here the integration of these different modules onto a single system that is fabricated via a SU-8-based microtechnology route. We present also their testing for the purification of peptide samples. This started with a partial integration step with the combination of at least two of the modules (microsystem + monolith; microsystem + nib) and their test before the fabrication and testing of fully integrated microsystems.

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Novel electrical detection of label-free disease marker proteins using piezoresistive self-sensing micro-cantilevers.
Wee KW, Kang GY, Park J, Kang JY, Yoon DS, Park JH, Kim TS.
Biosens Bioelectron. 2005 Apr 15;20(10):1932-8.
[ expand abstract ]

We report an electro-mechanical biosensor for electrical detection of proteins with disease markers using self-sensing piezoresistive micro-cantilevers. Electrical detection, via surface stress changes, of antigen-antibody (Ag-Ab) specific binding was accomplished through a direct nano-mechanical response of micro-fabricated self-sensing micro-cantilevers. A piezoresistive sensor measures the film resistance variation with respect to surface stress caused by biomolecules specific binding. When specific binding occurred on a functionalized Au surface, surface stress was induced throughout the cantilever, resulting in cantilever bending and resistance change of the piezoresistive layer. The cantilever biosensors were used for the detection of prostate specific antigen (PSA) and C-reactive proteins (CRP), which are a specific marker of prostate cancer and cardiac disease. From the above experiment, it was revealed that the sensor output voltage was proportional to the injected antigen concentration (without antigen, 10 ng/ml, 100 ng/ml, 1 microg/ml). PSA and CRP antibodies were found to be very specific for their antigens, respectively. This indicated that the self-sensing micro-cantilever approach is beneficial for detecting disease markers, and our piezoresistive micro-cantilever sensor system is applicable to miniaturized biosensor systems.

Protein biosensors based on biofunctionalized conical gold nanotubes.
Siwy Z, Trofin L, Kohli P, Baker LA, Trautmann C, Martin CR.
J Am Chem Soc. 2005 Apr 13;127(14):5000-1.
[ expand abstract ]

There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the alpha-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mechanical and chemically robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not observed. Instead, the protein analyte binds to a biochemical molecular-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different molecular-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/immunoglobulin, and (iii) an antibody to the protein ricin with ricin as the analyte.

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Impedance spectroscopy flow cytometry: On-chip label-free cell differentiation.
Cheung K, Gawad S, Renaud P.
Cytometry A. 2005 Apr 11; [Epub ahead of print].
[ expand abstract ]

BACKGROUND: The microfabricated impedance spectroscopy flow cytometer used in this study permits rapid dielectric characterization of a cell population with a simple microfluidic channel. Impedance measurements over a wide frequency range provide information on cell size, membrane capacitance, and cytoplasm conductivity as a function of frequency. The amplitude, opacity, and phase information can be used for discrimination between different cell populations without the use of cell markers. METHODS: Polystyrene beads, red blood cells (RBCs), ghosts, and RBCs fixed in glutaraldehyde were passed through a microfabricated flow cytometer and measured individually by using two simultaneously applied discrete frequencies. The cells were characterized at 1,000 per minute in the frequency range of 350 kHz to 20 MHz. RESULTS: Cell size was easily measured with submicron accuracy. Polystyrene beads and RBCs were differentiated using opacity. RBCs and ghosts were differentiated using phase information, whereas RBCs and fixed RBCs were differentiated using opacity. RBCs fixed using increasing concentrations of glutaraldehyde showed increasing opacity. This increased opacity was linked to decreased cytoplasm conductivity and decreased membrane capacitance, both resulting from protein cross-linking. CONCLUSIONS: This work presents label-free differentiation of cells in an on-chip flow cytometer based on impedance spectroscopy, which will be a powerful tool for cell characterization.

Microautosamplers for discrete sample injection and dispensation.
Huang CW, Lee GB.
Electrophoresis. 2005 Apr 5; [Epub ahead of print].
[ expand abstract ]

Microfluidic systems show considerable potential for use in the continuous reaction and analysis of biosamples for various applications, such as drug screening and chemical synthesis. Typically, microfluidic chips are externally connected with large-scale autosamplers to inject specific volumes of discrete samples in the continuous monitoring and analysis of multiple samples. This paper presents a novel microelectromechanical system (MEMS)-based autosampler capable of performing the discrete injection and dispensation of variable-volume samples. This microdevice can be integrated with other microfluidic devices to facilitate the continuous monitoring and analysis of multiple biosamples. By means of electroosmotic focusing and switching controlled by the direct application of electric sources on specific fluid reservoirs, a precise sample volume can be injected into the specified outlet port. Fluorescence dye images verify the performance of the developed device. An injection-and-washing scheme is developed to prevent cross-contamination during the continuous injection of different samples. This approach renders feasible the injection of several discrete samples using a single microchip. Compared to its large-scale counterparts, the developed microautosampler is compact in size, has low fabrication costs, is straightforward to control, and most importantly, is readily integrated with other microfluidic devices (e.g., microcapillary electrophoresis chips) to form a microfluidic system capable of the continuous monitoring and analysis of bioreactions. The proposed microautosampler could be promising towards realizing the micrototal analysis system (mu-TAS) concept.

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Investigation of Molecular Beacon Aptamer-Based Bioassay for Platelet-Derived Growth Factor Detection.
Vicens MC, Sen A, Vanderlaan A, Drake TJ, Tan W.
Chembiochem. 2005 Apr 5; [Epub ahead of print].
[ expand abstract ]

This report describes studies on the use of a molecular-beacon aptamer (MBA) as a synthetic high-affinity DNA probe that exhibits fluorescence resonance energy transfer (FRET) in response to a specific protein biomarker, platelet-derived growth factor (PDGF). As a step toward the application of the MBA in a fluorescence-based assay for biological specimens, we examined the influence of certain physical and chemical parameters of incubation that would affect DNA conformation and DNA-backbone modification, and thus improve nuclease resistance. This bioassay is compatible with pH, temperature, and monovalent cation levels typically encountered in biological samples, and phosphorothioate backbone-modified MBA is able to exhibit specific FRET. With minimal sample processing and without assay optimization, the MBA is able to detect as little as 10 ng PDGF per mug of serum proteins from cell-culture media. We also show that different sets of known fluorophore-quencher pairs can be successfully used in the MBA for sensitive detection of the PDGF target. It should, therefore, be possible to develop multiplex bioassays that monitor either quenching or enhancement for the simultaneous detection of several biomarkers by using MBAs created from high-affinity DNA ligands for the desired protein targets. Interestingly, we observed that, with a DNA ligand with multiple binding sites for a standard multimeric protein target, the FRET bioassay could be accomplished by using a mixture of two individually labeled DNAs-one carrying the fluorophore and the other with the matching quencher. This observation has significant implications in the future design of more selective DNA-based FRET bioassays that use more than one ligand for the same protein target.

Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets.
Wittig R, Salowsky R, Blaich S, Lyer S, Maa JS, Muller O, Mollenhauer J, Poustka A.
Electrophoresis. 2005 Apr 5; [Epub ahead of print].
[ expand abstract ]

Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA.

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Nanobiotechnology: the promise and reality of new approaches to molecular recognition.
Fortina P, Kricka LJ, Surrey S, Grodzinski P.
Trends Biotechnol. 2005 Apr;23(4):168-73.
[ expand abstract ]

Nanobiotechnology is the convergence of engineering and molecular biology that is leading to a new class of multifunctional devices and systems for biological and chemical analysis with better sensitivity and specificity and a higher rate of recognition. Nano-objects with important analytical applications include nanotubes, nanochannels, nanoparticles, nanopores and nanocapacitors. Here, we take a critical look at the subset of recent developments in this area relevant to molecular recognition. Potential benefits of using nano-objects (nanotubes, quantum dots, nanorods and nanoprisms) and nanodevices (nanocapacitors, nanopores and nanocantilevers) leading to an expanded range of label multiplexing are described along with potential applications in future diagnostics. We also speculate on further pathways in nanotechnology development and the emergence of order in this somewhat chaotic, yet promising, new field.

Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with beta-cyclodextrin as a fluorescence probe.
Wang L, Bian G, Wang L, Dong L, Chen H, Xia T.
Spectrochim Acta A Mol Biomol Spectrosc. 2005 Apr;61(6):1201-5.
[ expand abstract ]

A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/beta-cyclodextrin(beta-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/beta-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15mugmL(-1) for calf thymus DNA (ct-DNA) and 0.3-12mugmL(-1) for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01mugmL(-1) for ct-DNA and 0.02mugmL(-1) for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0mugmL(-1) ct-DNA and 1.4% for 2.0mugmL(-1) fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

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Chip-based microfluidic devices coupled with electrospray ionization-mass spectrometry.
Sung WC, Makamba H, Chen SH.
Electrophoresis. 2005 Mar 30; [Epub ahead of print].
[ expand abstract ]

We present the current status of the development of microfluidic devices fabricated on different substrates for coupling with electrospray ionization-mass spectrometry (ESI-MS). Until now, much success has been gained in fabricating the ESI chips, which show better performances due to miniaturization when compared with traditional methods. Integration of multiple steps for sample preparation and ESI sample introduction, however, remains a great challenge. This review covers the main technical development of electrospray device that were published from 1997 to 2004. This article does not attempt to be exclusive. Instead, it focuses on the publications that illustrated the breath of the development and applications of microchip devices for MS-based analysis.

Chemical detection with a single-walled carbon nanotube capacitor.
Snow ES, Perkins FK, Houser EJ, Badescu SC, Reinecke TL.
Science. 2005 Mar 25;307(5717):1942-5.
[ expand abstract ]

We show that the capacitance of single-walled carbon nanotubes (SWNTs) is highly sensitive to a broad class of chemical vapors and that this transduction mechanism can form the basis for a fast, low-power sorption-based chemical sensor. In the presence of a dilute chemical vapor, molecular adsorbates are polarized by the fringing electric fields radiating from the surface of a SWNT electrode, which causes an increase in its capacitance. We use this effect to construct a high-performance chemical sensor by thinly coating the SWNTs with chemoselective materials that provide a large, class-specific gain to the capacitance response. Such SWNT chemicapacitors are fast, highly sensitive, and completely reversible.

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Inhibition assay of biomolecules based on fluorescence resonance energy transfer (FRET) between quantum dots and gold nanoparticles.
Oh E, Hong MY, Lee D, Nam SH, Yoon HC, Kim HS.
J Am Chem Soc. 2005 Mar 16;127(10):3270-1.
[ expand abstract ]

An inhibition assay method was developed based on the modulation in the FRET efficiency between quantum dots (QDs) and gold nanoparticles (AuNPs) in the presence of the molecules which inhibit the interactions between QD- and AuNP-conjugated biomolecules. For the functionalization, AuNPs were first stabilized by chemisorption of n-alkanethiols and then capped with the first generation polyamidoamine (G1 PAMAM) dendrimers. By employing a streptavidin-biotin couple as a model system, avidin was quantitatively analyzed as an inhibitor by sensing the change in photoluminescence (PL) quenching of SA-QDs by biotin-AuNPs. The detection limit for avidin was about 10 nM. It is anticipated that the PL quenching-based sensing system can be used for the quantitative analysis and high throughput screening of molecules which inhibit the specific biomolecular interactions.

Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs
Fortier MH, Bonneil E, Goodley P, Thibault P
Anal Chem 2005 Mar 15;77(6):1631-40
[ expand abstract ]

The present investigation describes the analytical performances of a microfluidic device comprising an enrichment column, a reversed-phase separation channel, and a nanoelectrospray emitter embedded altogether in polyimide layers. This configuration minimizes transfer lines and connections and reduces postcolumn peak broadening and dead volumes. This compact and versatile modular nanoLC-chip system was interfaced to both ion trap and time-of-flight mass spectrometers, and its analytical potentials were evaluated in the context of proteomics applications. The figures of merit of this system in terms of peak capacity, reproducibility, sensitivity, and linear dynamic range of peptide detection were determined using tryptic digests of complex protein extracts including albumin- and immunoglobulin-depleted rat plasma samples. The analysis of peak profiles for more than 600 peptide ions reproducibly detected across replicate nanoLC-chip-MS runs (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity with RSD values of less than 0.5 and 9.1%, respectively. Variation in peptide abundance as low as 2-fold changes was identified for spiked tryptic digests present at levels of 2-5 fmol in plasma samples. Sensitivity measurements were performed on dilution series of protein digests spiked into rat plasma samples and provided a detection limit of 1-5 fmol. The modular concept of the microfluidic systems also facilitated the integration of two-dimensional chromatography (strong cation exchange/C18) thereby increasing the sample loading and selectivity of the nanoLC-chip-MS system. The application of this integrated device was evaluated for complex rat plasma samples to compare the number of protein identifications obtained using one- and two-dimensional nanoLC-chip-MS/MS.

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Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets
He M, Edgar JS, Jeffries GD, Lorenz RM, Shelby JP, Chiu DT
Anal Chem 2005 Mar 15;77(6):1539-44
[ expand abstract ]

This paper describes a method, which combines optical trapping and microfluidic-based droplet generation, for selectively and controllably encapsulating a single target cell or subcellular structure, such as a mitochondrion, into a picoliter- or femtoliter-volume aqueous droplet that is surrounded by an immiscible phase. Once the selected cell or organelle is encased within the droplet, it is stably confined in the droplet and cannot be removed. We demonstrate in droplet the rapid laser photolysis of the single cell, which essentially "freezes" the state that the cell was in at the moment of photolysis and confines the lysate within the small volume of the droplet. Using fluorescein di-beta-d-galactopyranoside, which is a fluorogenic substrate for the intracellular enzyme beta-galactosidase, we also assayed the activity of this enzyme from a single cell following the laser-induced lysis of the cell. This ability to entrap individual selected cells or subcellular organelles should open new possibilities for carrying out single-cell studies and single-organelle measurements.

Electrically assisted sampling across membranes with electrophoresis in nanometer inner diameter capillaries
Woods LA, Gandhi PU, Ewing AG
Anal Chem 2005 Mar 15;77(6):1819-23
[ expand abstract ]

A nondestructive method for sampling from ultrasmall environments has been developed utilizing electrophoresis in nanometer inner diameter capillaries and etched electrochemical detection. The desire to study increasingly smaller biological environments such as mammalian cells has led to the need for capillary electrophoresis techniques with subpicoliter volume sampling capabilities. This sampling technique involves the fabrication of a microinjector at the tip of a 770-nm-inner diameter capillary and the use of electroporation for insertion through the membrane. Separations of catecholamines sampled from the interior of intact liposomes have been achieved. A separation of a cytoplasmic sample taken from an intact mammalian cell has also been obtained.

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Gold nanoparticle-modified etched capillaries for open-tubular capillary electrochromatography
Yang L, Guihen E, Holmes JD, Loughran M, O'sullivan GP, Glennon JD
Anal Chem 2005 Mar 15;77(6):1840-6
[ expand abstract ]

The use of gold nanoparticles in conjunction with etched capillary-based open-tubular capillary electrochromatography (OTCEC) to improve the efficiency of separation and the selectivity between selected solutes is described. The fused-silica capillaries (50-microm i.d.) were etched with ammonium hydrogen difluoride, followed by prederivatization of the new surface with (3-mercaptopropyl)trimethoxysilane (MPTMS) for the immobilization of dodecanethiol gold nanoparticles, for OTCEC. The electrochromatography of a "reversed-phase" test mixture and of selected polycylic aromatic hydrocarbons was investigated, and efficient separations and high theoretical plate numbers per meter were obtained. The electroosmotic flow characteristics of the etched gold nanoparticle capillary, unetched gold nanoparticle capillary, bare capillary, and etched bare capillary were studied by varying the percentage of organic modifier in buffer, buffer pH, and separation voltage. Optical microscopy and scanning electron microscopy were used to examine the process of etching and modification and the surface features of the etched gold nanoparticle capillary. The results confirm that dodecanethiol gold nanoparticles bonded on the etched inner wall of the fused-silica capillary can provide sufficient solute-bonded phase interactions to obtain OTCEC separations with reproducible retention, as well as characteristic reversed-phase behavior, even with the inner diameter of the capillary of 50 microm.

Single-walled carbon nanotubes displaying multivalent ligands for capturing pathogens.
Gu L, Elkin T, Jiang X, Li H, Lin Y, Qu L, Tzeng TR, Joseph R, Sun YP.
Chem Commun (Camb). 2005 Feb 21;(7):874-6. Epub 2004 Dec 17.
[ expand abstract ]

A single-walled carbon nanotube was exploited for its semi-flexible pseudo-one-dimensional nanostructure as a unique scaffold to display multivalent carbohydrate ligands, with a specific demonstration showing that galactosylated carbon nanotubes were effective in the capturing of pathogenic Escherichia coli in solution.

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Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion.
Seok HJ, Hong MY, Kim YJ, Han MK, Lee D, Lee JH, Yoo JS, Kim HS.
Anal Biochem. 2005 Feb 15;337(2):294-307.
[ expand abstract ]

The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100 fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1 ng/mL. The use of antibodies against the proteins involved in the metabolic pathway of L-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates.

SNP identification in unamplified human genomic DNA with gold nanoparticle probes.
Bao YP, Huber M, Wei TF, Marla SS, Storhoff JJ, Muller UR.
Nucleic Acids Res. 2005 Jan 19;33(2):e15.
[ expand abstract ]

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the 'point of care'.

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Aggregation of DNA-modified gold nanoparticles in a non-cross-linking configuration has extraordinary selectivity against terminal mismatch of the surface-bound duplex. In this paper, we demonstrate the utility of this selectivity for detection of single-base substitutions. The samples were prepared through standard protocols: DNA extraction, PCR amplification and single-base primer extension. Oligonucleotide-modified nanoparticles correctly responded to the unpurified products from the primer extension: aggregation for the full match and dispersion for all the mismatches. Applicability of this method to genomic DNA was tested with five human tumor cell lines, and verified by conventional technologies: mass spectrometry and direct sequencing. Unlike the existing methods for single-base substitution analysis, this method does not need specialized equipments, and opens up a new possibility of point-of-care diagnosis for single-nucleotide polymorphisms.

Electrochemical biosensors for DNA analysis.
Ju H, Zhao H.
Front Biosci. 2005 Jan 1;10:37-46.
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Over the past few years, progress in electrochemical biosensors for DNA analysis is outstanding. This article briefly reviewed the principals of such biosensors, followed by a selection of topics that are of particular current interest. Selectivity and sensitivity are the two major challenges in DNA analysis. The former can be accomplished using peptide nucleic acids as probes, or relying on the DNA-mediated electron transfer. Special emphasis has been given to the efforts for high sensitivity, involving combination with polymerase chain reaction techniques, enzyme-labeled methods, direct label-free detection and nano-based techniques.

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Direct fluorimetric determination of gamma-globulin in human serum with organic nanoparticle biosensor.
Wang L, Wang L, Dong L, Bian G, Xia T, Chen H.
Spectrochim Acta A Mol Biomol Spectrosc. 2005 Jan 1;61(1-2):129-33.
[ expand abstract ]

This paper describes the development of organic fluorescence nanoparticles. The nanoparticles have a narrow, tunable, symmetric emission spectrum and a broad, continuous excitation spectrum. The nanoparticles have high room-temperature fluorescence quantum yields and long fluorescence lifetime. They are also photochemically stable and water-soluble. They were used as fluorescence biosensor in the determination of proteins, which was proved to be a simple, rapid and specific method. In comparison with single organic fluorephores, these nanoparticles are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimal conditions, the linear ranges of the calibration curves were 0.1-4.5 microg ml(-1) for human serum albumin (HSA), 0.2-3.5 microg ml(-1) for bovine serum albumin (BSA) and 0.04-0.8 microg ml(-1) for gamma globulin (gamma-IgG), respectively. The detection limits were 0.062 microg ml(-1) for HSA, 0.036 microg ml(-1) for BSA and 0.022 microg ml(-1) for gamma-IgG, respectively. However, when the content of HSA is lower than 0.8 microg ml(-1), HSA makes little contribution to the fluorescence quenching. So, the method was applied to direct selective quantification of gamma-IgG in human blood serum without separation of HSA. The results were in good agreement with these reported by the hospital, indicating that the method presented here is not only sensitive, selective and simple, but also reliable and suitable for practical applications.

Reagentless electrochemical biosensor based on the multi-wall carbon nanotubes and nanogold particles composite film.
Zhu JJ, Xu JZ, Hu Z, Chen HY.
Front Biosci. 2005 Jan 1;10:521-9.
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A novel method was used to prepare the nano-composite by assembling nanogold (NG) particles on the multiwall carbon nanotubes (MWNTs) surface. The nano-composite could be immobilized on a glassy carbon (GC) electrode to get a novel modified electrode. The electrode can easily immobilize the horseradish peroxidase (HRP) molecules to construct a reagentless biosensor. The NG particles in the composite film have a good biological compatibility. And due to the existence of quinone groups on the MWNTs surface, the MWNTs can promote the electron transfer between enzymes and electrode surface. The biosensor shows a good stability and responds to H2O2 in the range from 2.0 microM to 3.5 mM with a detection limit of 1.0 microM.

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Large-scale fabrication and characterization of Cd-doped ZnO nanocantilever arrays.
Zhou SM, Meng XM, Zhang XH, Fan X, Zou K, Wu SK, Lee ST.
Micron. 2005;36(1):55-9.
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We demonstrate bulk synthesis of highly crystalline Cd-doped ZnO nanocantilever arrays (CZNAs) using Cd and Zn powders at 600 degrees C, which is characterized via scanning electron microscopy, powder X-ray diffraction, X-ray photoelectron spectroscopy, transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy, selected area electron diffraction, and high resolution TEM. The results show that the as-prepared CZNAs have diameters of about 15-50nm, and lengths up to 400nm and the corresponding process of growth is suggested for conventional vapor solid mechanism.

2004

DNA biosensors based on self-assembled carbon nanotubes.
Wang SG, Wang R, Sellin PJ, Zhang Q.
Biochem Biophys Res Commun. 2004 Dec 24;325(4):1433-7
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DNA biosensors based on self-assembled multi-walled carbon nanotubes (MWNTs) were described in this paper, in which the probe DNA oligonucleotides were immobilized by forming covalent amide bonds between carboxyl groups at the nanotubes and amino groups at the ends of the DNA oligonucleotides. Hybridization between the probe and target DNA oligonucleotides was confirmed by the changes in the voltammetric peak of the indicator of methylene blue. Our results demonstrate that the DNA biosensors based on self-assembled MWNTs had a higher hybridization efficiency compared to those based on random MWNTs. In addition, the developed DNA biosensors also had a high selectivity of hybridization detection.

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Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria.
Dwarakanath S, Bruno JG, Shastry A, Phillips T, John A, Kumar A, Stephenson LD.
Biochem Biophys Res Commun. 2004 Dec 17;325(3):739-43.
[ expand abstract ]

CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates.

Pt based enzyme electrode probes assembled with Prussian Blue and conducting polymer nanostructures.
Curulli A, Valentini F, Orlanduci S, Terranova ML, Palleschi G.
Biosens Bioelectron. 2004 Dec 15;20(6):1223-32.
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Conductive polymer nanotubules of 1,2-diaminobenzene (1,2-DAB) were prepared using a porous polycarbonate membrane template, placed on a Pt foil and used to support the polymer, then, the electropolymerisation was performed by chronocoulometry. The obtained conductive polymer nanostructures were then placed on Pt electrode and used to support highly dispersed prussian blue (PB), which acts as the active component for H(2)O(2) detection. The observed good stability of PB as catalyst of H(2)O(2) was related to the presence of organic non-conventional conducting polymers in a composite nanostructured film. These nanostructured polymer/PB composite films were also characterised by scanning electron microscopy (SEM) and Raman spectroscopy. The non-conventional conducting polymer nanotubules/PB modified Pt electrodes were tested by cyclic voltammeter for stability at different pH values, then, by amperometry, for hydrogen peroxide, ascorbic acid, acetaminophen, uric acid and acetylcholine. Glucose oxidase (GOD), lactate oxidase (LOD), l-aminoacid oxidase (l-AAOD), alcohol oxidase (AOD), glycerol-3-phosphate oxidase (GPO), lysine oxidase (LyOx), and choline oxidase (ChOx) were immobilised on PB layer supported on 1,2-diaminobenzene (1,2-DAB) nanotubules onto the Pt electrodes. Different strategies for enzyme immobilisation were performed and used. Analytical parameters such as reproducibility, interference rejection, response time, storage and operational stability of the sensors have been studied and optimised. Results provide a guide to design high sensitive, stable and interference-free biosensors. The glucose biosensors assembled with nanostructured poly(1,2-DAB) showed a detection limit of 5 x 10(-5)moll(-1), a wide linearity range (5 x 10(-5) to 5 x 10(-3)moll(-1)), a high selectivity, a stability of 3 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical parameters and stability were also studied for l-(+)-lactic acid, l-leucine, ethanol, glycerol-3-phosphate, lysine, and choline biosensors.

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Miniaturised nucleic acid analysis.
Auroux PA, Koc Y, Demello A, Manz A, Day PJ.
Lab Chip. 2004 Dec;4(6):534-46. Epub.
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The application of micro total analysis systems has grown exponentially over the past few years, particularly diversifying in disciplines related to bioassays. The primary focus of this review is to detail recent new approaches to sample preparation, nucleic acid amplification and detection within microfluidic devices or at the microscale level. We also introduce some applications that have as yet to be explored in a miniaturised environment, but should benefit from improvements in analytical efficiency and functionality when transferred to planar-chip formats. The studies described in this review were published in commonly available journals as well as in the proceedings of three major conferences relevant to microfluidics (Micro Total Analysis Systems, Transducers and The Nanotechnology Conference and Trade Show). Although an emphasis has been placed on papers published since 2002, pertinent articles preceding this publication year have also been included.

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles.
Lian W, Litherland SA, Badrane H, Tan W, Wu D, Baker HV, Gulig PA, Lim DV, Jin S.
Anal Biochem. 2004 Nov 1;334(1):135-44.
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Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.

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A novel fluorescent label based on organic dye-doped silica nanoparticles for HepG liver cancer cell recognition.
He X, Duan J, Wang K, Tan W, Lin X, He C.
J Nanosci Nanotechnol. 2004 Jul;4(6):585-9.
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In this paper, we report a method for the recognition of HepG liver cancer cells with the use of a novel fluorescent label based on organic dye-doped fluorescent silica nanoparticles. The novel organic dye-doped silica nanoparticles are prepared with a water-in-oil microemulsion technique. The silica network is produced by the controlled synchronous hydrolysis of tetraethoxysilane and 3-amino-propyltriethoxysilane (APTES). The organic dye fluorescein isothiocyanate is doped inside as a luminescent signaling element, through covalent bonding to the amino group of APTES. The organic dye-doped core-shell nanoparticles are highly luminescent and exhibit minimal dye leaching and excellent photostability. A novel fluorescent label method based on biological fluorescent nanoparticles has been developed. The dye-doped fluorescent silica nanoparticles are covalently immobilized with anti-human liver cancer monoclonal antibody HAb18. We have used antibody-labeled fluorescent nanoparticles to recognize HepG liver cancer cells. It has been observed that the bioassay based on the organic dye-doped nanoparticles can identify the target cells selectively and efficiently. The fluorescent nanoparticle label also exhibits high photostability.

Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye.
Choi NS, Yoo KH, Yoon KS, Maeng PJ, Kim SH.
J Biochem Mol Biol.2004 May 31;37(3):298-303
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In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID-TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

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Applications of biochips: from diagnostics to personalized medicine.
Jain KK.
Curr Opin Drug Discov Devel. 2004 May;7(3):285-9.
[ expand abstract ]

This review examines the role of advances in biochip and microarray technologies in the development of personalized medicine. Biochips (eg, GeneChip, CYP450, electrochemical biochips, protein biochips, microfluidic biochips and nanotechnology-based biochips) are assuming an important role in molecular diagnostics, and their application in point-of-care diagnosis is expected to facilitate the development of personalized medicine. Gene expression profiling by microarrays should advance the progress of personalized cancer treatment based on the molecular classification of subtypes. Refinements in biochip miniaturization with the advent of nanotechnology will further contribute to molecular diagnostics and the development of personalized medicine.

Optical sensor for the detection of caspase-9 activity in a single cell.
Kasili PM, Song JM, Vo-Dinh T.
J Am Chem Soc. 2004 Mar 10;126(9):2799-806.
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We demonstrate for the first time, the application and utility of a unique optical sensor having a nanoprobe for monitoring the onset of the mitochondrial pathway of apoptosis in a single living cell by detecting enzymatic activities of caspase-9. Minimally invasive analysis of single live MCF-7 cells for caspase-9 activity is demonstrated using the optical sensor which employs a modification of an immunochemical assay format for the immobilization of nonfluorescent enzyme substrate, Leucine-GlutamicAcid-Histidine-AsparticAcid-7-amino-4-methylcoumarin (LEHD-AMC). LEHD-AMC covalently attached on the nanoprobe tip of an optical sensor is cleaved during apoptosis by caspase-9 generating free AMC. An evanescent field is used to excite cleaved AMC and the resulting fluorescence signal is detected. By quantitatively monitoring the changes in fluorescence signals, caspase-9 activity within a single living MCF-7 cell was detected. By comparing of the fluorescence signals from apoptotic cells induced by photodynamic treatment and nonapoptotic cells, we successfully detected caspase-9 activity, which indicates the onset of apoptosis in the cells.

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Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping.
Kim S, Lim YT, Soltesz EG, De Grand AM, Lee J, Nakayama A, Parker JA, Mihaljevic T, Laurence RG, Dor DM, Cohn LH, Bawendi MG, Frangioni JV.
Nat Biotechnol. 2004 Jan;22(1):93-7. Epub 2003 Dec 07.
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The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm(2). Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.

Emerging biomarker technologies.
Gunn L, Smith MT.
IARC Sci Publ. 2004;(157):437-50.
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New technology offers great potential for advances in cancer biomarker research. Here, we describe a number of new technologies and discuss their potential for use in molecular cancer epidemiology. The successful sequencing of the human genome has revealed several new insights, including the fact that the human genome consists of only 40,000 genes and is highly variable, with approximately 60,000 functional polymorphisms. High-throughput genomic technologies continue to facilitate the identification and analysis of mutations and polymorphisms in key genes and expand the spectrum of available genomic biomarkers. The next major challenge is the identification of novel proteins and understanding the structure, function and interaction of proteins and other molecules--information that cannot be obtained from genomics alone. Emerging technologies including arrays, proteomics and nanotechnology provide new platforms for high-throughput, highly sensitive, functional assays. These technologies will complement existing and emerging genomic technologies and result in the identification of new biomarkers of cancer risk. They will, however, require extensive validation in epidemiological studies.

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2003

Nanotechnologies and microchips in genetic diseases.
J Nephrol.2003 Jul-Aug;16(4):597-602
[ expand abstract ]

Microarrays or microchips represent a new area of high technology, which will completely change the methodological approach to basic research and clinical diagnostics. This technology can be used for genotyping, expression profiling and proteome analysis. Genetics and molecular medicine have an expanding need for rapid genotyping, mutational analysis and DNA re-sequencing technologies, i.e. microarrays that have a clear potential for miniaturization, parallelization, and automation and enable high-throughput screening. Expression profiling technology is a new tool for investigating expression patterns, identifying new disease genes either for monogenic disorders or for complex traits, identifying new functional and cellular relationships and identifying new pathways and possible related drugs. This technology has been successfully applied to the study of complex traits, i.e. cardiovascular diseases, cancer and type II diabetes, providing new insights into possible pathogenetic mechanisms and new therapeutical approaches. Finally, microarray can further improve proteome analysis. This review discusses these points.

A whole blood immunoassay using gold nanoshells.
Hirsch LR, Jackson JB, Lee A, Halas NJ, West JL.
Anal Chem. 2003 May 15;75(10):2377-81.
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A rapid immunoassay capable of detecting analyte within complex biological media without any sample preparation is described. This was accomplished using gold nanoshells, layered dielectric-metal nanoparticles whose optical resonance is a function of the relative size of its constituent layers. Aggregation of antibody/nanoshell conjugates with extinction spectra in the near-infrared was monitored spectroscopically in the presence of analyte. Successful detection of immunoglobulins was achieved in saline, serum, and whole blood. This system constitutes a simple immunoassay capable of detecting sub-nanogram-per-milliliter quantities of various analytes in different media within 10-30 min.

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Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots.
Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP.
Nat Biotechnol. 2003 Jan;21(1):41-6. Epub 2002 Dec 02.
[ expand abstract ]

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.

Discovery of ovarian cancer biomarkers in serum using NanoLC electrospray ionization TOF and FT-ICR mass spectrometry.
Bergen HR 3rd, Vasmatzis G, Cliby WA, Johnson KL, Oberg AL, Muddiman DC.
Dis Markers. 2003-2004;19(4-5):239-49.
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Treatment of cancer patients is greatly facilitated by detection of the cancer prior to metastasis. One of the obstacles to early cancer detection is the lack of availability of biomarkers with sufficient specificity. With modem differential proteomic techniques, the potential exists to identify high specificity cancer biomarkers. We have delineated a set of protocols for the isolation and identification of serum biomarkers for ovarian cancer that exist in the low molecular weight serum fraction. After isolation of the low molecular weight fraction by ultrafiltration, the potential biomarkers are separated by reversed phase nano liquid chromatography. Detection via TOF or FT-ICR yields a data set for each sample. We compared stage III/IV ovarian cancer serum with postmenopausal age-matched controls. Using bioinformatics tools developed at Mayo, we normalized each sample for intensity and chromatographic alignment. Normalized data sets are subsequently compared and potential biomarkers identified. Several candidate biomarkers were found. One of these contains the sequence of fibrinopeptide-A known to be elevated in many types of cancer including ovarian cancer. The protocols utilized will be examined and would be applicable to a wide variety of cancers or disease states.

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2002

Bioassays based on molecular nanomechanics.
Majumdar A.
Dis Markers. 2002;18(4):167-74.
[ expand abstract ]

Recent experiments have shown that when specific biomolecular interactions are confined to one surface of a microcantilever beam, changes in intermolecular nanomechanical forces provide sufficient differential torque to bend the cantilever beam. This has been used to detect single base pair mismatches during DNA hybridization, as well as prostate specific antigen (PSA) at concentrations and conditions that are clinically relevant for prostate cancer diagnosis. Since cantilever motion originates from free energy change induced by specific biomolecular binding, this technique is now offering a common platform for label-free quantitative analysis of protein-protein binding, DNA hybridization DNA-protein interactions, and in general receptor-ligand interactions. Current work is focused on developing "universal microarrays" of microcantilever beams for high-throughput multiplexed bioassays.

Nanotechnology: Emerging Developments and Early Detection of Cancer. A two-day workshop sponsored by the National Cancer Institute and the National Institute of Standards and Technology, August 30-31 2001, on the National Institute of Standards and Techno.
Zullo SJ, Srivastava S, Looney JP, Barker PE.
Dis Markers. 2002;18(4):153-8.
[ expand abstract ]

A recent meeting jointly sponsored by the National Cancer Institute (NCI) and National Institute of Standards and Technology (NIST) brought together researchers active in nanotechnology and cancer molecular biology to discuss and evaluate the interface between disciplines. Emerging areas where nanotechnologies may impact cancer prevention and early cancer detection were elaborated by key researchers who catalyzed interdisciplinary dialogue aimed at fostering cross-discipline communications and future collaboration.

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