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ANALYSIS

The interest in microfabricated fluidic (lab-on-a-chip) devices for performing chemicaland biochemical assays appears to be in anexponential phase of growth. A handful ofresearchers began investigating the possibili-ties of using microfabrication tools to buildminiature devices that perform conventional“benchtop” chemical and biochemical exper-iments in the late 1980s. Now there are sever-al dozen laboratories across the worldinvolved in such investigations. In this issue,Quake and colleagues1 report on a newachievement in miniaturization—a dispos-able microfabricated fluorescence-activatedcell sorter that can function independently,or as a component of a microanalytical chip.

Researchers developing such microfluidicsystems have a well-established model to fol-low: the remarkable evolution of the micro-electronics and information technology sec-tor. This is illustrated in Figure 1, beginningwith a photograph of what is generally con-sidered to be the first successful computer,the ENIAC. The ENIAC was first operated in1946 and employed 18,000 vacuum tubes toperform “hard-wired” calculations. Thoughimpressive for its time, it occupied a hugeamount of space and was inflexible in solvingvarying problems. A year after the ENIACdemonstration, the transistor was developedat the Bell Telephone Laboratories, and thisin turn led to the development of integratedcircuits. The transistor permitted a reductionby orders of magnitude in the size, weight,and power requirements of computers—andthe rest is history. Modern hand-held com-puters are tremendously more powerful thanearlier models which filled entire rooms.

Clearly the microelectronics industry hashad an immense impact on global economicsand information technology. Likewise, tech-nology that greatly reduces the time and costof acquiring chemical and biochemical infor-mation also will have far-reaching economicramifications, since virtually all industrialproducts rely on chemical information tosome extent. The pharmaceutical industry,for example, relies heavily on the acquisitionof chemical information. Considering thenumber of human gene products (100,000)and the literally millions of compounds, suchas oligopeptide sequences, which can be test-ed for gene-specific activity, the total numberof experiments for potentially useful drugs is

enormous. For such large-scale experi-mental endeavors to be tractable, a techno-logical shift away from the present-day lab-oratory may be necessary. In fact, micro-fabricated fluidics may represent theenabling technology capable of providingthe necessary quantum leap in experimen-tal processing power.

One parallel in the development ofmicrofluidics and microelectronics is theirfabrication: photolithographic patterningtools that originated in the microelectronicsindustry are often used to produce microflu-idic devices. And although the “carriers” in amicrofluidic system are liquid reagents flow-ing through a fluidic circuit, rather thanelectrons flowing through a semiconductorcircuit, the miniature scale of both systemsoffers analogous advantages. For instance, inmultifluidic circuits, molecules travel short-er distances just as electrons do in microcir-cuits, thereby speeding up processing. This isa particular advantage in chemical separa-tion devices2 and microreactors, wherereagents have to be mixed3,4. The smalldimensions also reduce the amounts of car-riers (reagents) necessary to conduct achemical process: for a single experiment,volumes are often in the nanoliter to picol-iter range rather than the microliter range orlarger in conventional experiments.

In addition, the photolithographic fab-rication of lab-on-a-chip devices potentiallycan provide a number of economic advan-tages as well as experimental. Devices contain-ing highly replicated channel structures canbe fabricated with small incremental cost perchannel, allowing for massively parallel chem-ical analysis for high-throughput experimen-tation. Such economy of scale will allow man-ufacture of inexpensive disposable devices formany applications such as research and clini-cal diagnostics. Photolithographic patterningalso enables low dead volume fluidic connec-tions within the device’s substrate, allowingthe integration of serial chemical processes orautomation of sample processing at the nano-liter volume scale.

Lab-on-a-chip devices that integrate allthe necessary chemical processing steps togenerate chemical information from a “raw”sample will be quite powerful. Sophisticatedchemistry and biochemistry procedures canbe incorporated into the device at the pointof manufacture, allowing novices to performcomplex assay procedures much as comput-ers permit the use of advanced design andanalysis tools without intricate understand-ing of underlying theory.

Impressive progress in development ofmicrofluidic lab-on-a chip devices has beenmade in the last decade. A number of basicfluidic components have been assembled indiverse ways to perform various chemicalmeasurements. Many of these are based onelectrokinetic transport principles, andinclude valves, mixing structures, chemicalreactors, and chemical separation channels.In addition, chemical separation mecha-nisms have been miniaturized, includingfree-solution and gel electrophoresis, solventprogrammed chromatography, isoelectricfocusing, isotachophoresis, and two-dimen-sional separations based on liquid chro-matography and free-solution electrophore-sis. Surface interactions have been exploitedfor solid-phase extraction to process samplesand for hybridization of target DNAs, andnanoliter-scale reactors have been demon-strated for continuous flow, stopped flow,and thermal cycling reactions.

Miniaturized devices for cellular and par-ticle manipulation, such as the fluorescence-activated flow cytometer developed by Quake

The burgeoning power of the shrinking laboratory

J. Michael Ramsey

J. Michael Ramsey is corporate research fellowat the Oak Ridge National Laboratory, OakRidge, TN, 37831-6142. (ramseyjm@ornl.gov).

Figure 1. A pictorial history of the developmentof microelectronics and microfluidics. On theleft: the path leading to today’s digitalcomputers, from the ENIAC, to the transistorand the modern day laptop computer. On theright: The biochemist’s laboratory. Willmicrofabricatred fluidic devides lead to a similarexpansion in experimental power?

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homologous recombination that were devel-oped by the groups of Smithies2 andCapecchi3, the Cre-loxP recombination sys-tem developed by Sternberg4 (originally iso-lated from the bacteriophage P1), and thetetracycline control switch developed byBujard5 (composed of components of thebacterial tetracycline repressor and the her-pesvirus transcription element VP16). Lastly,DNA control elements that have the capacityto direct gene expression to specific cell typeshave been identified over the past decade.

Why do we need sophisticated geneswitches? The mouse is the organism of choiceto study mammalian development, physiolo-gy, and cancer. Analyses of genetic pathwaysin the mouse, through the deletion of individ-ual genes by conventional homologousrecombination, is powerful but can run intoroadblocks. Researchers who wish to study aparticular gene in a specific organ oftenencounter problems. First, the deletion of agene from every cell in a mouse (the standardknockout technique) can result in embryoniclethality, and thus the tissue is not availablefor analysis. For example, mutations in theBRCA1 gene predispose women to breast can-cer, but its analysis in mouse mammary tissuewas accomplished only recently through a tis-sue-specific gene deletion6. Second, even if thedeletion of a gene by conventional homolo-gous recombination leads to the expectedphenotype, it still needs to be determinedwhether the defect is cell autonomous or theresult of systemic or cell–cell interactions.

Individually, cell-specific Cre-loxP medi-ated recombination5 and a cell-specific tetra-cycline-dependent time switch7,8 have beenused successfully in mice. Early efforts to fuse

The right time and place formolecular scissors

Lothar Hennighausen and Priscilla A. Furth

The development of genetic tools has been andwill continue to be a driving force in biologicaldiscovery. In particular, the ability to mutategenes within the mouse genome has beeninstrumental in the identification and under-standing of genetic pathways controllingorganogenesis and tumorigenesis. Now, Wen-Hwa Lee and colleagues1 report experimentsthat take the application of molecular tools tonew heights. They combined the generic Cre-loxP recombination system with a tetracycline-dependent switch and topped it off with tissue-specific control elements. This technical voyagewill enable biologists to precisely manipulateindividual genes in specific tissues and at pre-determined time points.

What are the fundamentals of advancedgene switches? During the past decade engi-neers in many molecular workshops aroundthe world have searched through the toolboxes of prokaryotes and turned up gadgetsand gismos that greatly aided molecular engi-neers in manipulating the mouse genomewith an ever-increasing sophistication.Included in our repertoire are the tools of

Lothar Hennighausen is chief of the laboratoryof Genetics and Physiology, National Instituteof Diabetes, Digestive and Kidney Diseases,National Institutes of Health, Bldg. 8, Rm. 101,Bethesda, MD 20892 (mammary@nih.gov) or(http://mammary.nih.gov/lgp). Priscilla A.Furth is associate professor of medicine andphysiology at the Institute of Human Virology,departments of medicine and physiology,University of Maryland Medical School, 725West Lombard Street, Room 545, Baltimore,MD 21201-1192 (furth@umbi.umd.edu).

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and colleagues, are important elements toadd to the lab-on-a-chip tool kit. Their workrepresents the first demonstration of a micro-fabricated fluidic device for cell sorting that isbased on a fluorescence signature. Instead ofbreaking the fluid stream into droplets andseparating the cells by electrostatic deflection,this device electrokinetically directs cells froman analysis channel into one of two possibleoutput channels by simply selecting whichoutput channel is connected to the drivingvoltage. Using this system, the workers wereable to achieve enrichments of 30- to 100-folddepending on the experimental details, andfor cells, 20% viability after sorting. Thethroughput was lower than conventional flu-orescence-activated cell sorting (10–20 Hz),but the sorting speed is likely to be improvedby using higher electric field strengths andparallel sorting structures.

In the device described in this study, thecells were confined laterally by the use of nar-row channels so that they were forced to transitthrough the probing laser beam. However inanother variation of a miniature flow cytome-ter reported last month in AnalyticalChemistry, electrokinetic focusing (analogousto hydrodynamic focusing used in convention-al cell sorters) was used for spatial confine-ment, allowing the use of broader channels5.

The ultimate goal of microfluidics is tointegrate as many different chemical process-es as necessary to solve a given measurementproblem on a single device. On this front,there have been some successes with low lev-els of integration, including mixing ofreagents to induce a chemical reaction, fol-lowed by chemical separations 6,7. Furtherdegrees of integration will likely be demon-strated in the near future.

One of the visions of developers of lab-on-a-chip devices is to bring the plug & play ver-satility of a video game to laboratory experi-mentation. Like a video game, you will have ageneric controller that accepts microfluidicchips rather than game cartridges.Microfluidics chips would be designed to per-form different assays, but all would be accept-ed and operated by the generic controller. Theresult could be a versatile and powerfulresearch tool. In fact this goal is now a com-mercial reality in applications focusing onchemical separations8. More complex applica-tions will surely be addressed by such plug &play strategies as higher degrees of microflu-idic integration are demonstrated.

Lab-on-a-chip devices have progressedrapidly, but there are remaining obstaclesthat must be overcome to achieve the fullpower that many envision. One area requiredfor high-throughput processing and analysisthat needs improvement is the ability tobring samples to devices in rapid succession;we call this the “world-to-chip” interface.Other areas that will become quite valuable

are fabrication processes that will allow viablemanufacturing of three-dimensional fluidicsdevices and spatially heterogeneous pattern-ing of surface chemistries, and sensitivedetection systems in addition to optical fluo-rescence and mass spectrometry. Finally,computer-aided design tools that include flu-idics, chemistry, and biochemistry will benecessary to rapidly design the future genera-tions of fluidics microchips, much as micro-electronics chips are designed today.

The future for microfabricated fluidicsdevices—or the lab-on-a-chip—looks quitepromising. We can only imagine what type ofmicrofluidic systems will emerge in the coming

decades to replace the question mark in Figure1, but they are sure to intrigue, and indeed,may transform the way we conduct research.

1. Fu, A.Y. et al. Nat. Biotechnol. 17, 1109–1111 (1999).2. Jacobson, S.C., Culbertson, C.T., Daler, J.E. &

Ramsey, J.M. Anal. Chem. 70, 3476–3480 (1998).3. Ermakov, S.V., Jacobson, S.C. & Ramsey, J.M.

Anal. Chem. 70, 4494–4504 (1998).4. He, B. & Regnier, F. J. Pharm. Biomed. Anal. 17,

925–932 (1998).5. Schrum, D.P., Culbertson, C.T., Jacobson, S.C. &

Ramsey, J.M. Anal. Chem. 71, 4173–4177 (1999).6. Jacobson, S.C., Hergenröder, R., Moore, A.W. Jr. &

Ramsey, J.M. Anal. Chem. 66, 4127–4132 (1994).7. Chiem, N.H. & Harrison, D.J. Clin. Chem. 44,

591–598 (1998).8. Hewlett-Packard HP 2100 Bioanalyzer.

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