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.
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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.
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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.
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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.
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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.
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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] .
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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.
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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.
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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