NCI Alliance for Nanotechnology in Cancer  |  Monthly Feature  |  August 2005
Download as PDF Microfluidics: The Flow of Innovation Continues While some may think that doing thousands of experiments on a microchip the size of a dime is pure science fiction, scientists are actually using these tiny devices now, for everything from single molecule monitoring to gene sequencing to understanding cell communication. Nano.Cancer.Gov News reviews some of the innovative experimentation that is occurring at nanoscale in microfluidic devices and how it will impact cancer research.

Harold Craighead, Ph.D., a pioneer of nanotechnology, used to spend much of his time thinking about heavy metal. As a professor of Applied and Engineering Physics at Cornell University, he designed nanoscale devices for use in industrial applications, such as electronic equipment. Then, several years ago, while serving as the director of the National Nanofabrication Facility at Cornell University, he noticed that an increasing number of requests were for nanomachinery with application to biomedical sciences.

"The applications of nanotechnology to electronics are mature and very focused," he says. "It progresses in a set manner. The use of nanostructures in biological research and biomedicine, however, is very young, and there are still so many ways in which nanotechnology can be utilized. It was this breadth of potential that inspired me to push on the interface between biology and nanotechnology."

Redirecting his focus to applying nanotechnology to basic biomedical research, Craighead and his group have since developed multiple small-scale devices for understanding biological matter at the level of a single molecule. From a recent breakthrough in creating fluorescent probes for single molecule detection to creating devices that allow DNA analysis, Craighead's machinery is designed to enable rapid, cost-effective experimentation on small quantities of biological material.

Many of the devices built in his lab are based on microfluidics, the study of liquid flow in very small volumes. Nanotechnology plays a critical role in the engineering of components of microfluidics devices. Designed at the nanometer scale, the major components of these devices include valves for directing fluid flow and nanochannels that direct small fractions of fluid and individual cells to nanodetectors.

To better appreciate the scale of nanotechnology and microfluidics, consider a tennis ball (see Figure 1). A cancer cell, at 10 to 100 microns (1000 nanometers = 1 micron), is 1000 to 10,000 times smaller than a tennis ball. Many microfluidics devices are built with channels just wide enough to accommodate single cells, such as the device described below for watching cell-cell communication. Others, such as a device for separating DNA pieces by size, also described below, have nanometer-wide channels. Nanometer-size particles, comparable in size to large proteins such as antibodies or small pieces of DNA, are between 100 and 10,000 times smaller than the average cancer cell. Nanoparticles are small enough that they can be joined to biomolecules such as proteins, and because of their small size, can potentially enter and exit cells readily.

Figure 1. Nanoscale materials are used to build microfluidic devices, which can be used for studying DNA, proteins and whole cells. The average cancer cell is between 10 and 100 microns and many nanomaterials are between 100 and 10,000 times smaller than a cell.

Many microfluidic devices used in biology are built on microchips with channels that conduct liquid under pressure or with an applied electrical current (see Figure 2). There is little to no turbulence in water flow at the nanoscale level, so particles suspended in the liquid are very easy to track as they move through channels. In the case of biology, the liquid may contain small particles with fluorescent labels, which are measured with high-powered laser microscopes. Particles can also be attached to microscopic beads and are also monitored by microscope. These particles could be proteins, DNA, or even single cells, allowing researchers to monitor the changes that occur from cell to cell during the development of disease. The devices are easily manipulated, as researchers have the capability to modify the width and depth of channels, to best suit the needs of a particular experiment.
(See April 2005 and May 2005 Monthly Features for more details.)

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Figure 2. Microfluidic devices, some only slightly bigger than a dime, can run thousands of experiments at once, and will be useful in defining the genetic basis of cancer. Courtesy: Stephen Quake, Ph.D., Stanford University

The potential of microfluidics to address major questions in cancer biology is considerable, according to Craighead. "Early in my lifetime, cancer was a death sentence," he says. "But now with advances in nanotechnology and microfluidic technology, there are numerous opportunities to create less invasive procedures and testing methods that will show us early evidence of disease, or the ability to understand the circumstances that foster disease. Microfluidics will change how we treat cancer."

In a world of biomolecules, finding one of interest in a disease-gene hunt is sometimes similar to finding a needle in a haystack. Craighead has developed a means for finding that needle, by adding fluorescent molecules that help the target stand out from the crowd.

In a recent study,¹ Craighead has identified an exact combination of two fluorescent labels, in green and red, needed to distinguish a single molecule in a microfluidic channel under a microscope. This study looked at DNA as the single molecule in question, but the results, he notes, are applicable to other single molecules, such as proteins or antibodies. The experimental design allowed for DNA molecules with several combinations of red or green tags to flow through a microfluidic channel that was uniformly affected by a high-powered laser microscope. In this way, the researchers could observe molecules as they passed through the field of view, and test whether individual DNA molecules were distinguishable when excited by a laser. Using these tags in a microfluidic device, Craighead's group was able to distinguish single DNA molecules linked to the combination of three red and one green fluorophore from other single DNA molecules labeled with three green and one red fluorophore.

The potential applications of such probe technology to cancer research are extensive. Researchers can use these fluorescent probes to study gene expression differences between cancer cells and healthy cells to identify potential cancer biomarkers, or to screen small compound libraries to discover new drugs to treat cancer. In addition, the probes can be applied to clinical diagnostics, in which the presence or absence of a cancer biomarker could be rapidly identified in patient tissue.

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In a series of studies,² Craighead and his laboratory developed microfluidic devices that separate DNA based on size, improving and miniaturizing a technique commonly used in laboratories for everything from gene identification to mutation analysis (see Figure 3). In one such device, Craighead takes advantage of changes in physical properties such as entropy (the amount of energy in a system that is available to do work) that occur at nanoscale. An electrical field is applied briefly to DNA of varying lengths in a microfluidic channel with grooves in the base. The grooves, which confine DNA, create an unfavorable energetic climate. The larger DNA molecules, in essence, become claustrophobic, and when the energy field is removed, the molecules recoil, or push out of the groove. The smaller pieces remain trapped, and the electrical field is applied again. Eventually, at the end of the experiment, the largest pieces of DNA, the ones that keep recoiling out of the grooves, can be isolated from the smaller pieces that remain trapped.

Figure 3. Microfluidic devices can be used to separate DNA of different sizes, a technique commonly used to identify gene mutations. In this technique, DNA is added to the chip (a), and an electrical current is added (b). At the current's highest voltage, the DNA strands enter the chambers of the device (c). Once the electrical current is discontinued (d), larger pieces of DNA (green) that are not fully inside the chambers will eventually recoil, or exit the chamber (e). The smaller pieces (red) remain trapped.

In one such device, Craighead takes advantage of changes in physical properties such as entropy (the amount of energy in a system that is available to do work) that occur at nanoscale. An electrical field is applied briefly to DNA of varying lengths in a microfluidic channel with grooves in the base. The grooves, which confine DNA, create an unfavorable energetic climate. The larger DNA molecules, in essence, become claustrophobic, and when the energy field is removed, the molecules recoil, or push out of the groove. The smaller pieces remain trapped, and the electrical field is applied again. Eventually, at the end of the experiment, the largest pieces of DNA, the ones that keep recoiling out of the grooves, can be isolated from the smaller pieces that remain trapped.

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For discovering cancer-causing genes, this DNA separation technique could effectively describe gene mutations. Because of the device's small size, it could be scaled for high-throughput gene mutation screens in tumors.

One of the most faithful methods of identifying genetic mutations is through gene sequencing. A single mutation in a gene can radically affect either the production or function of the protein that is made from it. Either the loss of a specific protein, or an altered function can affect the biochemical balance of a cell, triggering the early changes that lead to cancer. Identifying genetic mutations is important for determining disposition to disease, as well as in drug discovery.

For the consensus "healthy" genome that was sequenced in the Human Genome Project, a considerable amount of time, reagents and money were needed. For large-scale genome comparison projects, such as sequencing the genome of various cancers or within a single tumor, the current methodology could be highly cost- and time-intensive.

Enter nanotechnology. Microfluidic devices that miniaturize sequencing projects are already in use, and are being optimized to attempt large-scale genome sequencing projects.

Stephen Quake, Ph.D. at Stanford University, a recipient of the 2004 NIH Director's Pioneer Award for his innovative research in bringing nanotechnology to biomedical science, has created such a sequencing device for DNA sequencing-by-synthesis. In the sequencing-by-synthesis method, single molecules of DNA that need to be sequenced are immobilized on a microchip. The chemical mixture for determining the sequence—which contains, among other things, enzymes, buffers, and fluorescently tagged DNA building blocks—is added, and the DNA sequence is determined as fluorescence is given off from the building blocks getting used in the sequencing process.

In the microfluidic sequencing chip Quake created, there is the possibility of doing multiple sequencing runs simultaneously, with very rapid assay times, and a minimum of required reagents.³ The amount of DNA needed for an experiment is 10 times less than what is used in other techniques. In addition, the scale of the experiment allows for simultaneous reproducibility, which is key in declaring an experiment successful. The ability to sequence in nanoscale preserves time, money and precious biological resources.

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Other labs have contributed significantly to optimizing the science behind nanoscale sequencing. Jingyue Ju, Ph.D. at Columbia University, has engineered DNA building blocks with fluorescent molecules on one end and caps on the other. The fluorescent tag helps identify which building block was used in the sequencing reaction, and is rapidly removed after the signal has been gathered. The cap on the other end pauses the sequencing reaction long enough for unambiguous flourescent readout, and is removed for the sequencing process to continue.⁴ In previous sequencing-by-synthesis methods, the sequencing reaction normally happens very quickly, and sometimes the fluorescent signal can be missed, meaning that data that is gathered is incomplete. Ju's simple changes to the building blocks improve the output of the experiment in two ways. The fluorescent tag does not get in the way of the subsequent sequencing reaction, improving the overall completion of the sequencing steps, and the cap allows for the unambiguous identification of each sequence step before the reaction can continue, ensuring accurate data.

In another recently published study,⁵ a microfluidic sequencing device that can perform 1.6 million individual sequencing reactions has been created. This device, and the machinery associated with it, was validated for efficiency and accuracy by reproducing the sequence of the bacterium Mycoplasma genitalium. This sequencing experiment is the first report of a microfluidic-enabled whole organism genome sequence. The research team, led by Jonathan Rothberg, Ph.D. of 454 Sciences Corporation in Branford, Connecticut, was able to simultaneously read hundreds of thousands of sequences, at 99.96 percent accuracy in just four hours, at a substantially reduced cost because it was conducted at nanoscale.

Such innovations, along with many others, have pushed nanoscale sequencing to the forefront as the most promising way to address large-scale, multifaceted genome sequencing projects. According to Quake, these innovations are vital to sequencing success. "The prevailing technology for creating the consensus genome sequence was good, but for addressing sequence diversity, which one would want to do in a cancer genome sequencing project of any sort, you need better technology to push the basic research forward," says Quake. "Single molecule sequencing offers the highest possible throughput and is going to be the leading technique for affordable sequencing projects."

While cancer is a genetic disease, it is also a disease of bad communication. Cells are connected to each other, and the way that cells interact with their neighbors can greatly influence the outcome of nearly any developmental process in nature, be it during normal cell growth, or in tumor development. Tumor development hinges on the ability of cancer cells to send altering signals to one another, and even to the healthy cells nearby. Breaking down these lines of communication at the most basic level is a major target of drug discovery efforts, but to identify the culprit molecular signals, the ability to monitor single cells and two cells "talking" is essential.

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In a collaborative effort among several laboratories at the University of California Berkeley and the University of California San Francisco, investigators have created a microfluidic device that allows for the capture of cell pairs, facilitating observation of communication through gap junctions that connect them.⁶ This device is set with channels just wide enough to allow cells to flow from one side to another in opposing directions, with another set of channels that alters fluid flow just enough to allow pairs to form when cells pass by each other. Observing individual pairs of cells, the researchers were able to see the transfer of small amounts of dye from one cell to the other, showing that the cells were capable of passing biological material from one to the other. Watching the movement of cancer-causing molecules from one cell to the other will be possible in great detail using a microfluidic device such as this one. Determining which molecules promote cancer formation through this device will also allow the testing of small molecules for drug development, enabling the creation of therapies that "stop the bad-talk."

The rapid rate of technology development in the nano-community is poised to have a major impact on cancer, from determining its genetic origins to developing improved therapeutics. While the many branches of nanotechnology will affect cancer discovery in the future, the scope and diversity of discovery via microfluidics and single molecule monitoring is advancing the field now, providing valuable genetic and proteomic information at the single-cell level for the fight against cancer.

Indeed, microfluidics is rapidly becoming a key tool in understanding cancer, confirms Greg Downing, D.O., Ph.D., director of the Office of Technology and Industrial Relations at the National Cancer Institute. "Because cancer is a complex disease, we need to be able to rapidly identify mutations that might predispose someone to cancer, and learn how cancer cells communicate with each other to cause disease," says Downing. "Nanotechnology can help us with tools that will allow rapid, high-throughput single molecule analysis, be it protein or DNA. Microfluidic devices can reduce both the time needed and the amount of precious biological sample needed to conduct a large number of experiments, creating the promise of greater understanding of cancer and improved therapeutic options."

— Megha Satyanarayana

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