Jerry Radich is looking for a needle in a haystack, and he's counting on a microfluidics device to help him find it. Every year, a small fraction (about 4%) of leukemia patients develop resistance to Gleevec and related drugs. Radich and his group at the Fred Hutchinson Cancer Research Center in Seattle would like to detect resistance-causing mutations at their earliest stages, before the drugs stop working and patients stop getting better. This means detecting a singly mutated mRNA molecule against a background of 10,000 or more wild-type copies, a detection level virtually impossible using conventional methods.
So Radich is testing a so-called lab-on-a-chip, or microfluidic device made by Fluidigm of South San Francisco, Calif., containing hundreds of tiny channels that intersect to form thousands of nano-liter-scale reaction chambers. Partitioning a blood sample into these tiny chambers separates RNA molecules into small groups of one to 10 molecules, effectively enriching individual mutant sequences relative to wild type and greatly enhancing chances of detection. Testing is still preliminary, but Radich says he thinks it's going to work. "If it does it will be phenomenal."
Radich's endeavor is an example of the enabling potential of microfluidics devices – also called microTAS (micro-total analysis systems) or bioMEMS (biological micro-electro-mechanical systems). He and others agree the technology's power will be manifested not only in its ability to make experiments smaller, faster, and cheaper, but also in its ability to perform previously impossible experiments and answer previously "unaskable" questions.
"Microfluidics allows a whole new realm of experiments," says J. Michael Ramsey, a University of North Carolina, Chapel Hill, chemistry professor who was involved in the founding of Hopkinton, Mass.-based industry leader, Caliper Life Sciences. Ramsey is one of a group of scientists who are designing chips to measure proteins, nucleic acids, or metabolites from single cells or even organelles. Others, like Klavs Jensen at the Massachusetts Institute of Technology, are designing devices that perform a multitude of tiny parallel reactions, for synthesis of drug candidates or highly complex biological molecules, for instance.
Though routine use of such chips still lies in the future, even now microfluidics is having an impact in the lab, and in the clinic, by allowing detection and isolation of rare cells, nucleic acids, and proteins; speeding protein crystallization trials; providing faster detection of pathogens and biohazards; and accelerating clinical diagnostics.
Andrea Chow, Caliper's vice president for microfluidics, draws parallels between the lab-on-a-chip and computer industries. "The semiconductor industry did not grow up overnight," she says. "This industry is still in its first decade." Chow predicts that, just as microcomputers have completely changed how we process information, microfluidic devices will change how life-science research operates. This change will be "much more profound than just cheaper," she adds.
SILICON VALLEY MEETS BIOLOGY
Chow's comparison to the computer industry is apt, as the initial idea for microfluidics came from integrated circuit technology. Thirty or so years ago, Stanford University researchers began applying semiconductor fabrication techniques to bioanalytical devices to improve their sensitivity, producing a miniature gas chromatograph in 1979.1 But, this instrument chilled rather than stimulated the emerging field. "It didn't work very well," says Ramsey.
"It instigated a lot of thought, but it also put up a barrier," he explains; so much so that when he and others, nearly 20 years later, proposed putting fluids through microchannels, they encountered resistance. "I tried this idea out on some of my colleagues and they kind of snickered at me," Ramsey recalls. His first grant proposal was rejected, but by 1992 he obtained funding, and two years later produced a capillary electrophoresis chip.
Though Ramsey's patent searches turned up nothing resembling his work, he wasn't alone in the field. Others, including Andreas Manz and Jed Harrison at Ciba-Geigy in Basel, Switzerland, were also working on the idea, as was Allen Northrup, of Pleasanton, Calif.-based Microfluidic Systems.
Northrup was using microreaction chambers produced at the University of California, Berkeley's state-of-the-art fabrication facility, when "it dawned on me that the volumes in PCR are in microliters," he says. He went to Cetus, where PCR was largely invented, and proposed the idea of putting the reaction on a microfluidics device. "They were pretty excited about the idea," he recalls. Collaborations ensued between Berkeley, Cetus, and Lawrence Livermore National Laboratory, where Northrup did his postdoctoral work. Launching the technology was mostly a matter of "establishing collaborations with people on the leading edge," he says.
DOLLARS AND SENSE
Once these pioneers had laid the initial groundwork, the field developed rapidly. Commercialization followed closely with the founding of Caliper in 1995. According to Chow, the company chose to start with capillary electrophoresis ("low-hanging fruit," as she calls it), for faster time-to-market and earlier revenue potential. The strategy seems to have paid off: Caliper today is the only lab-on-a-chip company with significant revenues from product sales rather than grants and collaborations, according to Marlene Bourne, an industry analyst with EmTech Research, Ann Arbor, Mich.
Moving beyond the low-hanging fruit
Though many companies have delivered microfluidics products, they still have barely scratched the surface of the technology's potential. To move beyond that point, says David Beebe of the University of Wisconsin-Madison, engineers must get a better handle on the biological problems. As Mike Ramsey of the University of North Carolina puts it, microfluidics designers must address the nontrivial experiments, "not the low-hanging fruit."
Some time back, Beebe became disenchanted with the engineers' penchant for building what he calls "widgets," devices that incorporate a pet technology but fail to solve any concrete problem. So he went back to school to study cell biology firsthand, in order to identify real questions needing answers.
So far, Beebe has learned at least one thing from his sojourns in colleagues' cell biology labs: "Pipetting is a really stupid way to do cell biology." So, he is looking for better ways to deliver fluids to cells, trying to improve the "macro-to-micro interface."
"In order to move forward with genomics, there has to be a better way of doing things," says Beebe. Another project: designing devices that mimic the cell's natural microenvironment, "inverting the microfluidic paradigm" with nonflowing systems. Now he is studying how mammary gland cells grow and interact in these microenvironments, hoping thereby to better understand breast cancer.
Chris Luft is one of four assay developers at Amphora Discovery Corp. of Research Triangle Park, NC, a company whose scientific strategy hinges on Caliper technology. Together, these four scientists have developed 150 protease, kinase, and phosphatase assays for drug screening in only two years, due to the speed and data quality microfluidics provides, he says. Amphora researchers can test an entire 130,000-compound library in a week, generally using just one vial of enzyme, for $5,000 to $7,000 per screen. "For a minimal amount, you can get a bucket load of information," he points out.
But those savings must be balanced against the cost of the hardware. Amphora has 24 Caliper instruments, which cost nearly half a million dollars each. The chips themselves cost on the order of $100 apiece. Chow says this cost will drop as more companies adopt the technology, predicting economies of scale. "It's a chicken and egg phenomenon," she notes. "Most new products are expensive [inhibiting adoption], but you must get to high volume to make them cheaper."
In the meantime, "just putting a Caliper machine in your lab is not a guarantee of success," Luft says. Amphora's engineers worked for nearly a year to optimize the instruments' operating parameters and software to achieve the necessary data quality.
Several companies have followed Caliper's lead, placing other bioanalytical separation techniques onto microfluidics chips. Nanostream of Pasadena, Calif., and Eksigent of Livermore, Calif., have developed chip-based high-performance liquid chromatography (HPLC) systems, primarily for use in adsorption, metabolism, and toxicology studies of drug candidates. Palo Alto-based Agilent Technologies has introduced an integrated HPLC-mass spectroscopy chip for proteomics applications. And a Dutch company named Concept to Volume, has developed a new miniaturized gas chromatograph for remote, rapid detection of specific biothreats.
Also pursuing "homeland security" applications are Microfluidic Systems, which is developing automated PCR systems for forensics and biothreat detection, and Cepheid of Sunnyvale, Calif. "Extremely fast PCR really requires microfluidics," says Northrup, owing to the unique physical properties of very small volumes. Cepheid makes PCR-based threat-detection devices installed at mail processing facilities around the United States. But the company also has its sights set on clinical diagnostics, hoping to develop chips that can distinguish among different types of cancers or infectious agents.
Meanwhile, Biotrove of Woburn, Mass., has developed a high-density, PCR-based chip for genotyping and RNA analysis, designed for use in pharmacogenomics. Costing $300 to $350 each, these chips use 64-fold less polymerase and three- to four-times less sample than conventional methods, says chief technology officer Colin Brenan, and are currently being used for biomarker identification at Johnson & Johnson Pharmaceutical Research and Development in Rahway, NJ.
THE NEXT GENERATION
Though borne of the semiconductor industry, the industry's traditional materials of glass and silicon are not always the best choice for lab-on-a-chip devices. Fluid valves require a soft seat to seal, like the rubber washer in a faucet. In 1998, Harvard University chemist George Whitesides introduced "soft lithography," a technique for fabricating microchannels in rubber-like materials such as polydimethylsiloxane. Besides providing better seals, these soft materials can be fabricated faster, more cheaply, and in smaller dimensions than their silicon and glass counterparts.
How has the lab-on-a-chip transformed the life sciences: Brought the advantages of miniaturization to the life sciences
How long has it been around: Since the 1970's
What are the main applications: Take your pick: electrophoresis, chromatography, PCR, crystallography trials, sample prep, and more
Pros: Fast, small sample sizes, highly parallel, minimal waste
Cons: High equipment and chip costs
Key reference: A. Manz et al., "Miniaturized total analysis systems: A novel concept for chemical sensors,"
How will it transform medicine: Lowers drug development costs; enables new class of medical tests
Steve Quake, a California Institute of Technology bioengineer, refined Whitesides' method by introducing "multilayer soft lithography," which enables design of very small micromechanical valves. Quake patented his method in 1999 and founded Fluidigm. Since then, company researchers have designed chips with ever-higher valve densities, like the dynamic array chips Radich is testing. The newest chip allows testing of 48 samples under 48 different reaction conditions, for a total 2,304 experiments. Such an experiment would be highly impractical using microplates, says Marc Unger, a Fluidigm scientist.
Such massive parallellization will also have an impact on the production of drug candidates, says Jensen, who is developing microfluidics-based chemical reactors at MIT. Jensen's chips allow difficult, expensive syntheses to be tried under many conditions in parallel, on an otherwise impractical scale, while their use of very tiny volumes improves safety and reduces hazardous waste. "It puts the chemist back into the role of discovery scientist rather than spending a lot of time synthesizing chemicals. It has the potential to revolutionize drug discovery," he says.
And it makes once-unthinkable experiments possible. Ramsey, for instance, is using labs-on-a-chip to profile single cells. Cell populations that appear uniform on a macroscopic scale can be quite heterogeneous, confounding analysis of signaling pathways, for example. He and Nancy Allbritton of University of California, Irvine, are collaborating on high-throughput chips that can analyze the individual contents of hundreds of cells per minute.2 Among other applications, such chips could facilitate detection of extremely rare abnormal cells for cancer diagnosis.
But beyond the benefits of microscopic scale and massive parallelization, researchers point to integration as a key asset of future lab-on-a-chip devices. "My guess is that it will very much follow the way the MEMS field is going ... more integration of multiple technologies," says David Beebe, a biomedical engineer at the University of Wisconsin-Madison. Caliper's Chow agrees. "It is the integration of different processes on one platform that has made integrated circuits so powerful." Such processing power will be needed to take full advantage of genomics and offer personalized medicine in the future, she says. Amphora founder, Bill Janzen, adds: "We are in the forefront of a new paradigm for drug discovery."
Miniature gas chromatograph fabricated on a silicon wafer at Stanford University
Andreas Manz proposes "MicroTAS" architecture, envisioning sample preparation, separation, and detection on a single chip
Manz introduces miniature liquid chromatograph
Development of electrokinetics to move fluids on chips
Capillary electrophoresis devices introduced by Manz and Jed Harrison at Ciba-Geigy and Michael Ramsey at Oak Ridge National Labs
Allen Northrup develops PCR-on-a-chip
Mixing of substrate, inhibitor, and enzyme by electrokinetic flow
George Whitesides (below) introduces soft lithography for microfluidics
Steve Quake develops multilayer soft lithography and designs active valves
Caliper and Agilent launch first commercial lab-on-a-chip system, the 2100 Bioanalyzer
Fluidigm launches first product, a protein crystallization chip
Agilent sells its one-millionth Caliper chip for 2100 Bioanalyzer