Riding the Microfluidic Wave

Photo: Courtesy of Eksigent Technologies Eksigent Technologies' electrokinetic high-flow-rate EKPump These days, miniaturization is king. In the emerging field of microfluidics, routine laboratory analyses are shrinking to the microliter, nanoliter, or even picoliter level. The result: a vast reduction in sample and reagent consumption, decreased waste generation, dramatically faster operation, and an incredible potential for the automation and massive, parallel processing of laboratory procedures. Best of all, these benefits come bundled with greater resolution in separations, exquisite control over mixing, and the capacity for expediting chemical reactions within highly controlled microenvironments. Microfluidic approaches lend themselves to a versatile, cost-efficient model of operation, in which a single, central instrument processes numerous disposable, application-specific, microfluidic devices. The marketplace already supports a diverse array of applications, and researchers and microfluidics engineers alike are keen to miniaturize and integrate many more. To develop these systems, companies are sampling from a growing palate of microfluidic technologies, materials, and fluid-control strategies. Advances in this field will have a wide impact. Among the potential benefits are streamlined pharmaceutical research and development, improved clinical diagnostics, enhanced drug delivery, improved tissue engineering,1 and better bioweapons sensors. "We view the National Institutes of Health and US military as the primary drivers behind some of the most exciting and near-term focused microfluidic and nanotech product-development programs," notes Karen Hedine, president of Redmond, Wash.-based Micronics. The field has matured since our last review.2 Several new companies, including BioTrove and CelTor, have entered the microfluidics arena, while a few, such as Molecular Sensing, have left it. And many of the companies discussed in last year's roundup--Gyros, for example--have now moved from the development stage to product delivery. What follows is an alphabetical roster of the companies dealing in microfluidics. MICROFLUIDIC MACROCOSM Mountain View, Calif.-based ACLARA Biosciences offers plastic, card-like microfluidic consumables dubbed LabCard™ devices. Microliter volumes of samples and reagents are mixed, incubated, and separated according to their sizes and charges "on-card," followed by fluorescence-based quantification of relevant reaction products. One potential application: high-throughput screening (HTS) of drugs and drug targets. Photo: Courtesy of Gyros The Gyrolab MALDI SP1 CD for multiplexed sample processing Codeveloped with Caliper Technologies (see below), Palo Alto, Calif.-based Agilent Technologies' Agilent 2100 Bioanalyzer employs Caliper's planar LabChip devices. For molecular separation assays, the Bioanalyzer uses electrokinetic forces to drive samples through the chips' microchannels, where components are resolved and detected in real time. When used with a separate kit containing the needed pressure cartridge and cell fluorescence software, the Bioanalyzer is capable of pressure-driven sample movement for flow-cytometric analyses. Agilent offers several LabChip devices, including those for analyzing DNA, RNA, proteins, and cells. Salt Lake City-based BioMicro Systems' microfluidic devices exploit the properties of hydrophobic materials. The company's passive fluid control (PFC™) technology relies on the natural repulsion between hydrophilic and hydrophobic entities--in this case, aqueous samples and capillary wells, respectively. The fluid flow is controlled by pressure in conjunction with "passive valves" formed by narrowed microcapillary segments, and different configurations of microfluidic modules can be combined for different applications. "These biochips can be rapidly prototyped using our proprietary laser ablation technology, and then inexpensively manufactured in quantity using traditional injection molding processes," says Bill Pagels, BioMicro Systems' vice president of business development and marketing. The company's Microarray User Interface (MAUI™) hybridization system--which relies on PFC--mixes low-microliter volumes of concentrated samples on standard microscope slide-based microarrays, thereby enhancing the hybridization procedure. The company also plans to integrate its microfluidics technologies into the hybridization platform: The target nucleic acids can be fluorescently labeled or otherwise modified before transfer to the hybridization chamber, and the hybridization conditions can be readily optimized. Cambridge, Mass.-based BioTrove offers microfluidics-based HTS with its Living Chip™ devices, which contain high-density microchannel arrays in a microtiter plate format. Up to roughly 100,000 nanoliter-scale reactions can be simultaneously performed and monitored, with reactions initiated by aligning and stacking sets of adjoining plates. Scientists can currently use this technology to screen small-molecule libraries using homogenous fluorescence, enzyme-linked immunosorbent assay (ELISA), and luminescent formats. The company is also developing miniaturized genetic testing solutions. "Having a very high-density screening platform is pointless if it takes days or weeks to reformat samples from conventional plates," comments Tanya Kanigan, BioTrove's director of chip technology. "We have made it a point to develop rapid sample-handling schemes for introducing and retrieving samples into and out of our Living Chip platform." Photo: Courtesy of Agilent Technologies The Agilent 2100 Bioanalyzer, together with its Cell Assay Extension, can now perform simple flow-cytometric analysis. Caliper Technologies of Mountain View, Calif. has developed a variety of disposable microfluidic devices under its LabChip® product line, which comprises two broad classes of LabChip devices: "sipper" chips for HT applications, and planar chips for the lower-throughput environment. In the former, a fused-silica glass capillary tube is used to transfer a few nanoliters of each sample or reagent from microplate wells to the channels of a chip-like microfluidic device, where mixing and a series of application-dependent processing steps take place. In contrast, pipettes are used to load samples and reagents for lower-throughput applications into the reservoirs of planar chips. Caliper's sipper-chip-based LabChip HTS system can perform tens of thousands of experiments per chip. The latest model, the 250 HTS system, performs fluorogenic and mobility-shift assays for multiple target classes, including kinases, proteases, and phosphatases; cellular assays are in development. The sipper-chip-based AMS 90 SE automates the analysis of DNA samples stored in microplates--from adding internal calibration standards and fluorescently staining the nucleic acids, to electrophoretic separation and laser-induced fluorescence detection. Caliper plans to expand its HT product line to include a high-density, dry-reagent storage system, and is also developing an HT single nucleotide polymorphism (SNP) genotyping system. Santa Clara, Calif.-based CelTor Biosystems, a drug discovery company, uses its microfluidic and imaging platform, the CytoChip™ system, for real-time, phenotypic screening of partner companies' drug candidate libraries. CelTor's UniqueFlow™ microfluidic chip technology uses hydrodynamic focusing to precisely position cells and reagents in assays that simulate the biological environment associated with cell-mediated diseases. Based in Sunnyvale, Calif., Cepheid plans to introduce its GeneXpert™ technology platform for automated DNA extraction and sequence detection next year. GeneXpert instruments will combine disposable-cartridge-based sample preparation along with the Cepheid's I-CORE® modules for DNA amplification and real-time detection. This system is a hybrid of sorts, integrating macroscale and microfluidic technologies, with sample volumes in the microliter range. Developed with portability and flexibility in mind, the units will be compact, with low power requirements. Bar-coded fluidic cartridges with different internal configurations will be used for different classes of assays, and linking additional cartridge-processing blocks to the system will allow scientists to analyze multiple samples simultaneously. Cepheid and its collaborators are currently evaluating the possibility of adapting GeneXpert technology to meet certain national security and public safety needs, including the de- tection of biological warfare agents in mail-sorting systems,3 and development of rapid diagnostic tests for tuberculosis and other infectious diseases in children. Livermore, Calif.-based Eksigent Technologies' electrokinetic flow-based EKPumps have a range of applications. They can measure and deliver reagents in drug-discovery platforms and portable medical instruments, but they can also be used as circulators that protect electronic equipment from overheating. The company's NanoLC system performs HT and nanoscale analyses, as well as high-performance liquid chromatography (HPLC), with applications in proteomics and drug discovery. South San Francisco, Calif.-based Fluidigm is commercializing multilayer soft-lithography (MSL™) techniques developed by Steven Quake, professor of applied physics and physics at the California Institute of Technology. Separately cast, individual layers of soft elastomer are imprinted and bound together to form complex microfluidic networks, complete with integral pumps and valves. Fluidigm is currently developing an MSL-based system to miniaturize and integrate the often expensive and time-consuming procedure of optimizing protein crystallization conditions. The current prototype can screen 144 different crystallization conditions using less than three microliters of protein sample. GenoMEMS of Woburn, Mass., has partnered with Kyoto, Japan-based Shimadzu Biotech to commercialize a microfluidic DNA sequencer. Daniel Ehrlich, director of the BioMEMS Laboratory of the Whitehead Institute for Biomedical Research in Cambridge, Mass., and colleagues developed the fully automated BioMEMS-768. The system employs two relatively large chips (30 x 50 cm) housing microfluidic networks through which up to 768 samples are electrophoretically separated. According to Ehrlich, though the system uses picoliter volumes for separation and analysis, it requires nanoliter-scale injection volumes. "We hope to miniaturize upstream steps in future generation instruments," he says. Expect to see the BioMEMS in mid-2003. Grosserkmannsdorf/Dresden,Germany-based GeSiM offers custom manufacturing and packaging of microfluidic chips, employing components made from silicon, glass, metals, plastics, and ceramics. Applications include nanoliter dispensing/ mixing, noncontact microarraying, and single-cell manipulation. Gyros of Uppsala, Sweden, develops application-specific microfluidic devices in a plastic-based, compact disc (CD)-like format. Each microfluidic CD contains many identical microstructures for multiplexed sample processing. Microliter or nanoliter volumes of samples are loaded near the center of the CD, and application-specific treatments occur as centrifugal force, achieved by controlled spinning, drives the fluids through different zones en route to the disc's periphery. This past March, Gyros launched its first commercialized microfluidic CD, the Gyrolab MALDI SP1, along with the Gyrolab Workstation instrument platform. The MALDI SP1 streamlines sample preparation prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Up to 96 protein digests can be concentrated, desalted, and crystallized directly onto target areas on the disc, which is then transferred to a mass spectrometer for sample analysis. The Gyrolab Workstation can accommodate up to five CDs per batch, and will be able to utilize other application-specific CDs when available. The company is currently developing microfluidic CDs for immunoassays. Micronics entered the microfluidics field with versatile, thin-film plastic laminate-based microfluidic devices that harness laminar flow differential diffusion rates for separating and detecting molecules, cells, and particles. The company has now expanded its repertoire to include custom-designed lab cards for both "active" (instrument driven) and "passive" applications, as well as micro-pumps and valves that can be incorporated into existing hardware systems or designed as interface solutions. "We have found that while the interest in microfluidics remains strong, our primary challenge today is to provide the means for integrating currently existing macro systems, such as [HPLC] and MS, with the miniature domain of microfluidics," notes Hedine. The company is currently working with Plymouth, Minn.-based Honeywell Research Laboratories to develop a miniaturized blood-cell-counting instrument for military personnel, and is also subcontracting on a number of other microfluidic biodetection systems for the military. Micronit of Enschede, Netherlands, designs, prototypes, and develops glass- and silicon-based microfluidic devices for a variety of applications, including capillary electrophoresis, flow cells for blood analysis, micropipettes for clamping DNA molecules, and microneedles for blood collection. Nanostream of Pasadena, Calif., develops microfluidic tools for drug discovery and development applications, with an emphasis on products that can be readily integrated with procedures already in use by pharmaceutical companies. Nanostream's Snap-n-Flow™ platform offers a considerable selection of microfluidic modules that scientists can mix and match as needed. Scandinavian Micro Biodevices of Lyng-by, Denmark, offers contract development, manufacture, and supply of microsystems for the life sciences, including microfluidic polymer biochips for a variety of applications, and hybrid systems for cell testing and manipulation. Brighton, Mass.-based Surface Logix develops high-end bioassays for the drug discovery and development markets; the company's microfluidic know-how is provided by George Whitesides, professor of chemistry and chemical biology, Harvard University. Whitesides' team has pioneered a manufacturing approach in which nanoscale to microscale components are fashioned from a rubber-like polymer. Known as soft lithography, this technology employs "masters" made of the transparent elastomer polydimethylsiloxane (PDMS) to rapidly--and inexpensively--mold, imprint, or emboss the specified features. With soft lithography, scientists can craft a wide variety of components, including diffraction gratings, three-dimensional structures, and microfluidic networks. Surface Logix's devices employ microstructures of various shapes and sizes, such as wells or channels, tailored for the intended application. The microstructure surfaces are engineered using different proprietary chemistries for capturing particular biomolecules or controlling the cellular environment. Zurich, Switzerland-based Tecan's LabCD™ microfluidic product line--like Gyros'--employs a CD-like disposable component. Initial efforts have been geared towards developing HT adsorption, distribution, metabolism, excretion, and toxicity assays for pharmaceutical research. Tecan recently launched its LabCD applications for cytochrome P450 inhibition and serum protein-binding assays for evaluating potential drug candidates. Finally, Watertown, Mass.-based Teragenics helps other companies design, develop, and implement custom-designed microfluidic systems. A diverse array of services, software, and equipment for the design and manufacture of microfluidic systems is also provided by companies specializing in the broader sector of microelectromechanical systems, including Corning Intellisense of Wilmington, Mass., Coventor of Carry, NC, Innovative Micro Technology of Santa Barbara, Calif., Intelligent Micropatterning of St. Petersburg, Fla., Kionix of Ithaca, NY, Micralyne of Edmonton, Alberta, Nanostructures of Santa Clara, Calif., Seyonic of Neuchâtel, Switzerland, and thinXXS of Mainz/Zweibrücken, Germany. MICROFLUIDIC OUTLOOK Microfluidics advances could fundamentally change the way research is done, yet the industry is young, and in such fields, initial product development efforts often stumble as technical obstacles arise. "Because of the exceptional promise of microfluidic approaches, and fueled by the early success of the [Agilent] Bioanalyzer ... expectations were set much too high for the ensuing generation of microfluidic deliverables," says BioMicro's Pagels. "The gleam in the eyes of potential investors and corporate partners was diminished due to this early gap between expectation and reality, with the unfortunate result that successive, promising product entries have been met with severe scrutiny." Nevertheless, microfluidics researchers continue to press forward with new ideas and discoveries, while others pursue "nanofluidics"--fluid manipulation at near the molecular scale. In such a climate, new innovations are inevitable, but not necessarily immediately realized--or accepted. Predicts Pagels: "Instead of the microfluidics field and its general reception exploding with highly-touted successes, I believe that we will see an evolution of acceptance as more and more microfluidics applications prove their value." Deborah A. Fitzgerald (biotechnology@consultant.com) is a freelance writer in Gulf Breeze, Fla. References 1. W.M. Saltzman, W.L. Olbricht, "Building drug delivery into tissue engineering," Nature Reviews Drug Discovery, 1:177-86, 2002. 2. D.A. Fitzgerald, "Making every nanoliter count," The Scientist, 15[21]:22, Oct. 29, 2001. 3. J. Kling, "USPS, Biotech forge alliance," The Scientist, 16[13]:36, June 24, 2002.

Riding the Microfluidic Wave

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Photo: Courtesy of Eksigent Technologies
Eksigent Technologies' electrokinetic high-flow-rate EKPump