Electrophoresis 2003, 24, 3563-3576 3563

Review

Samuel K. SlaGeorge M. Whitesides

1 introduction 3563

2 PDMS: properties and fabrication 35643 Fluids in microchannels: the toolbox 35643,1 Fluid flow 3565

3.2 Fluid switching: valves 3566

3,3 Multiple fluid streams: laminar flow 3567

3.4 Multiple fluid streams: mixers 35674 PDMS-based microfluidic devices for

biological studies 3567

4,1 Detection using microfluldic immunoassays 3568

4.1.1 Immunoassays 3568

4.1.2 Multiplexing 3569

4.2 Separation of proteins and DNA 3569

4.2.1 PDMS open channels: capillaryelectrophoresis 3569

4.2.2 PDMS as stationary phase 3570

4.2,3 Interfacing PDMS microchannels and massspectrometry 3571

4.3 Sorting and manipulation of cells 3571

43.1 Cell sorting by flow cytometry 3571

4.3.2 Magnetic sorting 3572

Department of Chemistryand Chemical Biology,Harvard University,Cambridge, MA, USA

Contents

Correspondence: Dr. George M. Whitesides, Department ofChemistry and Chemical Biology, Harvard University 12 OxfordSt, Cambridge, MA 02138, USAE-mail: gwhitesides@gmwgroup.haivard.eduFax: +617-495-9857

Introduction

This review describes microfluidic systems in poly(dimethylsiloxane) (PDMS) for bio-logical studies. Properties of PDMS that make it a suitable platform for miniaturizedbiological studies, techniques for fabricating PDMS microstructures, and methods forcontrolling fluid flow in microchannels are discussed. Biological procedures that havebeen miniaturized into PDMS-based microdevices includejmnun,,qwsays, legationof proteins and DNA, sorting and manipulation of cells, studies of cells in micro-

channels exposed to laminarkonatitkille, .ensiJargezscaje,scpbinatorlel screening,The review emphasizes the advantages of miniaturization for biological analysis, suchas efficiency of the device and special insights into cell biology.

Microfluidic devices fabricated inpoly(dimethylsiloxane) for biological studies

Abbreviations: HIM human immunodeficiency virus; PDMS,poly(dimethylsiloxane)

C) 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Keywords; Biological assays/ Microfluidics I Miniaturization / Poly(dimethylsiloxane)/ ReviewDOI 10.1002/elps.200305584

4.3.3 Sorting and manipulation of motile cells „ 35724,4 Cell biology using laminar flow 35724.4.1 Laminar flow over single cells 35724.4.2 Molecular gradients 35734.5 Combinatorial screening 35745 Conclusions 35746 References 3575

Microfluidic systems provide a powerful platform for bio-logical assays 0-31 In microfluidics, small volumes ofsolvent, sample, and reagents are moved through micro-channels embedded in a chip. Examples of bioassaysand biological procedures that have been miniaturizedinto a chip format includepak.amtenoing, pplyrneraaechain reaction (INF.% elestr2pActrasia, 9.N.ek §,ppargtio,

eli.41:14.tAgpwp, immyriops.says, cell cowling, celtsort-

ing, and cell culture [4-6]. Miniaturized versions of bio-assays offer many advantages, including small restuire-

,mentsjoLs.- olvents reagents, a.,91.99Alpritical for valu-

able samples and for high-throughput screening), short—reaction times, portatfly, low .cost, low consumption

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versatility in design, and potential farfiaratio1

9atrillon end for integration with otliefryilniaturized de-vices.

In this review, we briefly describe the properties of poly(climethylsiloxane) (PDMS) and techniques for the fabrica-tion of PDMS microstructures, with an emphasis on the

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advantages of using PDMS for miniaturized bioassays,We then discuss the panel of PDMS-based componentsavailable for microfluidics, paying attention to the chal-lenges posed by the special physics of fluid flows in smallchannels, and the technologies developed to addressand exploit these flows, Finally, we describe PDMS-based miniaturized bioassays that integrate componentsinto functional devices. Whenever possible, we highlightthe advantages of miniaturization, such as the efficiencyof the device and special insights into biology that it pro-vides.

2 PDMS: properties and fabrication

The use of PDMS elastomer for miniaturized bioassays

has numerous advantages over 'Dior andvlass,,PDMSaLa material is inexpensive, flexible, and-opt* tOw"Ra!eicf 230 nm (and therefore compatible withmany optical methods for detection). It is compatiblewith biological studies because it is impermeable towater, nontoxic to cells, and permeable to gases. A final,major advantage of PDMS over glass and silicon is theease with which it can be fabricated and bonded to other

surfaces. For the development of bioassays, where manydesigns may need to be tested, the ease of rapid proto-typing in PDMS is a Critical advantage.

Procedures for the fabrication of PDMS structures formicrofluidics have been described in detail elsewhere

[7-9]. Briefly, the design of the microstructures is made in

a computer-aided desf9n,(CAD) pregtorh. Using commer-

cial services, the CA1)oneratad_patternaaraprinted-ontraasparancies (these services have overnight turnaroundtimes), Lateral resolutions of 25 ign can be routinely

achieved with image setters operating at 5080. do,tper

lach,and can be extended to 8 pm LISing'ichOttOtterSoperating at 20 000_d9te per, 1110. [10]. (For featuresbeyond 8 pm, Zhrome masks can be used, but they takelonger to fabricate commercially, and are more expensivethan transparencies.) The transparency is then used as aphotomask in UV- • hotolithii sr, • hy to generate a mas-ter. In this procedure, a thin layer o p o (resist (for

example, theowalikiee2LyIi, SU-8 is spin-coatedonto a acon_wkir, Using different types of SU-8 of var-ious viscosities, thicknesses of 1-300 prIl can be reliablyspin-coated. The photoresist is exposed to UV lightthrough the photomask, and a developing reagent isused to dissolve the unexposed regions. Thafesulting

bas-relief structuremr_vtiLsLmeter for fabricatingPDMS ffirak-ls.To create the PDMS mold, the surface of _the_silicon/photoresist master is treated ;-‘,$-Itti-fluorin,a,Vd silanes

(which prevents irreversible bonding to PDMS), and a

C) 2003 WILEYNCH Vedag GmbH & Co. KGaA, Weinheim

Electrophoresis 2003,24,3563-3576

liquid PDMS prepolymer (in a mixture of 1:10 base poly-mer:curing agen is poured onto it. The PDMS is curia-at70T-leir 1 War more anar)dakitioff the master, producing

the final replica bearing the designed microstructures.Small holes are drilled into the PDMS using a borer to pro-duce inlets and outlets. Finally, PDMS can seal to itselfand other fiat surfaces reversibly by conformal contact(via van der Waals forces), or irreversibly if both surfacesare Si-based materials and have been oxidized by plasmabefore contact (a process that forms a covalent 0-S1-0

bond) Saals_are_wayi,rt_ ight and can be formed under

phient_snaciitiops (ortigt-sj, Ofele7TE,E1-70-K7(67: which

teForgge§....iar_ adhesives) If

desired, many PDIVTB-lifilicas can be made from m-aiiiigle

master, This procedure of producing the PDMS structurefrom the silicon master, called molding, can becarried out under normal laboratory conditions withoutan expensive clean room, and can replicate certain typesof features with dimensions down to 10 nal. Replicamolding, along with procedures such as microcontactprinting, casting, injection molding and embossing, com-prise a set of techniques for manipulating elastomericstructures called soft lithography [11]. Although we focuson PDMS in this review, soft lithography has been demon-strated for other elastomers (such as polyurethane andepoxy).

PDMS consists of repeating-OSACK) - units; the CH3-groups make 1_21mi-1_1921y std2tiol_iic„ This hydropho-

iicity results in poor wettabilixtvith aqueous solvents,renders rh191)91-sinelazo,,,seplitlejILttittanira of

air bubbles, and malle_q_th, surface_ prone to nonspeci-17eTil

made hydrophilic by exposure toan air plasma 9.5)._a

plasma cleaner lasma oxidizes the sur-

'gt-ffpi-s;tu), The plasiiii:Rialfffd"itilea-a

remains hydrophilic if it stays in contact with water.In air, rearrangements occur within 30 min, which bringhydrophobic groups to the surface to lower the sur-face free energy. The surface of oxidized PDMS canbe modified further by treatment with functionalizedsilanes.

3 Fluids in miorochannels: the toolbox

Fluid flow in microchannels exhibits a number of charac-teristic features, the most important of which is laminar flow. We describe components such as valves and mix-ers, which have been developed to handle fluid flow inmicrochannels, as well as devices such as gradient gen-erators, which exploit the special physics of microfluidics.We focus on techniques that have been shown to workin PDMS-based devices.

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Electrophoresis 2003, 24, 3563-3576

3.1 Fluid flow

There are two main methods for driving the flow of fluids in

microchannels: zrasze-driyzi andolzglstatm, Bothmethods are used in PDMS devices, In pressure-drivenflow (also called hydrodynamic flow) (Fig, 1A), the flowrate 0 (rn3/s) is given by Q = AP/R, where AP is the pres-

sure drop across the channel (Pa), and R is the channelresistance (Pa. s/m3), The pressure drop can be created

either by opening the inlet to atmospheric pressure andapplying a vacuum at the outlet, or by applying positivepressure at the inlet (e.g., via a syringe pump) and open-

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Microfluidic devices fabricated in PDMS for biological studies 3565

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ing the outlet to atmospheric pressure. Both methodswork well, although for syringe pump-driven flow, it isnecessary to form an irreversible seal for PDIVIS devices( reversibl sealed structures can withstand easures of30-50 pa confor Ay sealed structures canwithstand pressures of 5 psi). For vacuum-driven flow,CoTI; irreveiSibie an-ct seals can be used.For pressure-driven flow, the other determinant of flowrate is the channel resistance R. For a circular channel,

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Figure 1. Toolbox for PDMS-based microfluidic bioassays. (A) Methods for driving fluid flow. (i) Pres-sure-driven flow using a vacuum at the outlet or a syringe pump at the inlet. (ii) Electrokinetic flowusing a voltage applied across the microchannel. (B) Switches and valves for control of fluid move-ment. (I) Pneumatically actuated monolithic valve. When pressure is applied above, flow in the bottomrounded channel stops [14 (ii) Channel crossing in which the fluid flow can be switched, When airpressure is applied above and below the crossing, the fluid turns 90' instead of flowing straight. Fig-ure adapted from [20]. (C) Gradient generator using laminar flow. A solution or surface-bound molec-ular gradient is generated perpendicular to the direction of fluid flow in the microchanna Figureadapted from [3i]. (D) Chaotic mixer. Neighboring streams of fluids are passively mixed. Homogene-ity is observed after 16 cycles of the staggered herringbone structure. Figure adapted from [321

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3566 S. K. Sia and G. M. Whitesides

g is the fluid viscosity (Pa* s), L isthe length (m), risthe radius(m), h is the height (m), and w is the width of the channel(m). (See[S] for the formula fora rectangular microchannelwith an intermediate aspect ratio.) A long narrow channeltherefore exhibits high fluidic resistance, and a short widechannel exhibits low fluidic resistance. Pressure-driven

flow has the key advantages that it is effective for solventsWith a wide range of compositions (e.g., including sol-vents that are not electrically conductive), and for chan-nels made of a wide range of materials (e.g., even if elec-trically conductive, such as silicon). Pressure-driven flow,however, requires an external pump or a vacuum source.

Theimethod also,p9ffels in ass_ey_s_regL. high:-resolu-tioration because the velocity profile of a cross-section is parabolic, and samples in the form of pitgs

unrilsgp_axial dispersion arIdWak broadening. Finally,because of the relations A cc 1 /r4 or 1/wh3, high pressure

drops are needed to drive fluid flow in small microchan-nels.

•g.12....30zolinetiglow is based onlistraomementatincl_s-cules in an electric field due to their charges CFI IAThere are two components to ifeefrovineirajw:itectre,-

) whicb_results-frem-theteramatie..,t9ileskalga.ot, . olocul ' tdc field balancedby the frictionglormartel electroosmosis which crealesa uniform pluOlke flow of fluid down the channel. InericTre7olEnotic flow in glass capillaries, a layer of fluidenriched In solvated cations forms at the surface ofnegatively charged sill groups of the channel wall;

,s- an electric field drives the layer of cations towards the

61' ^ negatively charged cathode, and by viscous drag, trans-

fers the motion to the rest of the liquid (given a suffi-ciently small cross-section in the microchannel). PDMS-based channels (normally uncharged at the surface) canbe made to support electroosmotic flow effectively byplasma oxidation immediately before the addition of buf-fer; this oxidation generates silanol groups at the chan-nel surface.

For electrokinetic flow, small channels have the advan-

tage of a high surface-to-volume ratio, and thus theydissipate heat more efficiently than large channels. Also,elgottgamottgAgyi,,results in IteLvelccity-pudiles, and

ialls_e_!2.212..!_apel_ts and high resolution separations

in capillary electrophoresip. Another advantage of electro-kinetic flow Is that fluid flows in a microfluidic network

can be controlled easily by switching voltages on and off;this control circumvents the rieid -16? valves. Never-theless, elecfrokinetic flow has important drawbacks for

bioassays, including buffer incompatibility,gicay buffersof .apatopriate pH and ionic strength are_compatible), theneed for an off-Efil1515iikei-645010-req- uent changes of

voltage settings (due to ion depletion, and to compensatefor pressure and resistive imbalances in the channels),

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Electrophoresis 2003, 24, 3663-3576

eleslrottlq_bAllejorznation, and eyap oration of solvent4tte_toltettlilg. Also, electrophoretic demixing -the sepa-ration of components in a heterogeneous mixture due todifferent electrophoretic mobilities - is unfavorable inbioassays requiring a uniform flow for all species.

Fluid flow in microchannels using other principles hasbeen described. Delamarche at a/. [12] used capillaryaction in plasma-oxidized PDMS to deposit immuno-globulins onto a surface. Centrifugal force was used todrive fluid flow in PDMS channels on a plastic disk, onwhich enzymatic assays were performed [13]. In non-PDMS-based systems, fluid flow was directed using gra-dients in surface pressure due to redox-active surfactants[141, gradients in temperature [15], patterning of self-assembled monolayers with different surface free ener-gies [16], and capillary action [17].

3.2 Fluid switching: valves

In electrokinetic flow, fluid flow can be controlled byapplying voltages to electrodes integrated in microchan-nels. A more general strategy for manipulating fluid flowis the use of valves to open and close microchannels.The elastomeric property of PDMS can be exploited tomake a mechanical valve. Quake at ai. [18, 19] used across-channel architecture made of PDMS to fabricate a

pneumatically actuated valve. In this design, pressure isapplied to the upper channel, deflecting a thin PDMSmembrane downward; this deflection closes the tower,rounded channel and stops fluid flow (Fig. 1B). We de-monstrated an elastomeric switch in a PDMS system fea-turing two crossing channels, each in a different layer(Fig. 1B) [201. Application of an external pressure aboveand below the crossing of the channel decreases theaspect ratio at the crossing, such that the fluid turns intothe other channel due to lower fluidic resistance, insteadof flowing straight through the crossing. Finally, pneuma-tically actuated PDMS valves can also be combined withglass microfluidic channels [21]. Advantages of pneuma-tically actuated valves include ease of fabrication (bymultistep lithography), rapid response time, avoidanceof air bubbles, and wide fluid compatibility. In the future,the integration of valves in microlluldics, although addingto the complexity of the system, will become more preva-lent, especially for devices featuring large numbers of in-dependent channels [22].

In another approach for constructing valves, Beebe at al.[5, 231 used pH-sensitive hydrogels. Although stimuli-responsive hydrogels have a slow response time, theyare intriguing because they are autonomous, responsiveonly to the environment in the microchannel, and requireno external control. Other strategies for the fabrication of

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Electrophoresis 2003, 24, 3563-3576

valves include electrochemically generated microbubbles[24], and thermally induced expandable microspheres[25],

3.3 Multiple fluid streams: laminar flow

Parallel streams of liquids can exhibit either laminar flow,where the streams flow parallel along each Other and mix-ing occurs only by diffusion, or turbulent flow, where tur-bulence mixes the streams. The parameter that indicateswhether flow is laminar or turbulent is the Reynolds num-ber (dimensionless): Re = v/p/p, where v is the velocityof the fluid (m/s), / is the cross-sectional dimension (m),p is the density of the fluid (for water, 1000 kg/m3), andp Is the viscosity of the fluid (for water, 10-3 kg/(w s)).For aqueous solutions, p and II are fixed parameters(characteristics of the fluid), and the rate of fluid flow vand channel dimension / are changeable. Under typicalmicrofluidic conditions of small channels (< 100 pm) anda low rate of fluid flow (1 cm/s), Re is almost always low(Re < 1), a value that correlates with laminar flow behav-ior (with Re above —2000, fluid usually exhibits turbulentflow).

The prevalence of laminar flow in microfluidics enablesnew technologies. For laminar flow, parallel streams offluid mix only by diffusion at their boundaly. Yager etal.126, 271 used diffusion at the boundary as the basis foran immunoassay. We have demonstrated membranelesselectrochemistry using the slowly diffusing boundary as abarrier t28], and microfabrication at the boundary usingmultiphase laminar flow patterning [29]. In another tech-nique, we use controlled diffusive mixing of laminar flowfluids to generate stable molecular gradients perpen-dicular to the direction of flow (Fig. 1C) [30, 31]. Themethod is based on repeated splitting, mixing and re-combination of neighboring fluid streams. The gradientscan be generated in solution and on surfaces, and theyare spatially and temporally stable. Moreover, we cangenerate gradients of complex shapes by using multiplemicrofluidic networks 131]. The use of solution and sur-face gradients for studying cell biology is described laterin this review.

3.4 Multiple fluid streams: mixers

Diffusive mixing is a slow process. For example, the timefor diffusion in one dimension is given by t d2/2D, where

d is the distance a particle moves (in cm) in a time t (in s),and D is the diffusion coefficient (for most proteins, be-tween 10-6 and 10-7 cm2s-1). Thus, a globular protein

Ci 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Microfluidic devices fabricated in PDMS for biological studies 3567

of 70 kDa needs only 1 s to diffuse 10 pm, but more than10 days to diffuse 1 cm; the distance along the channelrequired for the mixing of the contents in two neighboringstreams can be prohibitively long (» 1 cm; estimated byv12/D) [32].

We designed a mixer that uses asymmetric grooves onthe floor of the channel to introduce a transverse com-

ponent to the flow (Fig. 1D) [32]. Using this structure,fluid elements are twisted and folded into one another;this folding increases the contact area between the twostreams, and thus the rate of diffusive mixing. Neigh-boring streams of fluids mixed efficiently in a micro-channel containing staggered grooves of different geo-metries (for two streams of protein-containing solutions,a microchannel of 1 cm length could produce nearlycomplete mixing). We believe that this design, which iseasily fabricated by two-step lithography and compati-ble with steady pressure-driven flow, will find manyapplications is bioassays that require the mixing offluids.

Other mixers have been demonstrated in PDMS-based

systems. Quake etal. [33] fabricated a rotary, pneumati-

cally actuated pump that actively mixes fluids from differ-ent inlets. Crooks et al. [34] built a device that achievedefficient mixing (> 90%) by flowing fluid streams intothe small spaces between the microbeads; this bed in-creased the interfacial area of the fluid elements and the

rate of diffusive mixing. Ismagilov etal. [35] developed amixer that initially flowed the reactants as laminar streamsin a microchannel; injection of a water-immiscible phase(perfluorodecaline) generated uniform plugs, inside whichthe reactants mixed by chaotic advection. Other mixershave also been described in non-PDMS-based systems,using a serpentine channel [36], a 1-channel [37], andintersecting channels [381.

4 PDMS-based microfluidic devices forbiological studies

To build a functional microfluidic bioassay or a "Iab-on-a-chip", one must effectively integrate componentssuch as pumps, valves, and reservoirs. This sectiondescribes examples of functional microfluidic devicesfor applications in biology. We focus on PDMS-basedsystems, for which substantial progress has been madeon the integration of components, because they bothallow rapid prototyping and serve as final functional de-vices. Microfluidic components have been integratedusing other materials to build impressive devices forbioassays [39].

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4.1 Detection using microtluidic immunoassays

4.1.1 immunoassays

Immunoassay is widely used to detect analytes usingantibodies. Most immunoassays are heterogeneous: theantigen-antibody complex is bound to a solid substrate,and free antibodies are removed by washing. In homoge-neous immunoassays, the free and bound antibodies donot need to be separated via a solid substrate. Thesetypes of procedures minimize washing steps and fluidhandling, but they require that the free and antigen-boundantibodies exhibit different electrophoretic mobilities.Miniaturization of homogeneous immunoassays offersadvantages [26, 40], but more work has been done onthe miniaturization of heterogeneous immunoassays thanof homogeneous immunoassays.

A significant disadvantage of heterogeneous immuno-assays (such as enzyme-linked immunosorbent assay.or ELISA) in microtiter wells is that they require a longtime to perform. Incubation times of hours are required

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Figure 2. Detection of blomole-cules using microffuldics. (A) Im-munoassay employing a micro-dilutor network. The microdilutor

gp41 network uses chaotic mixers tomix neighboring streams of fluids,serially diluting the sample withbuffers. Anti-HIV antibodies froma patient are serially diluted anddetected using two antigens(gp120 and gp41) in parallel. Fig-ure adapted from [43]. (13) Two-dimensional microfluidic arrays.

Microwell system where thechannel crossings are separatedby twoporous membranes.and athin PDMS membrane with em-bedded microwells. Reactionstake place in the microwells,which produce a fluorescent sig-nal. In this example, the coloredchamber corresponds to a re-action between the fluorescentdye, fluo-3, and Ca24. Figure

adapted from [44]. (ii)Two-dimen-sional immunoassay (451. In thefirst dimension, parallel antigenstripes are patterrned onto a sub-strate using microfluidic delivery.In the second dimension, a PDMS

stamp with parallel channels are placed onto the substrate at right angles to theantigen stripes, and samples are flowed through the channels. An antibody-anti-gen binding event generates a signal at a crossing.

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to allow diffusion of the analyte from the solution to thesurface. Miorofluidics can shorten the incubation times

needed for surface events by minimizing the diffusiondistance in microchannels, and by replenishing the diffu-sion layer with a fixed concentration of molecules. In onestudy, an immunoassay detecting immunoglobutin G(IgG) was performed in a PDMS microchannel, requiringincubation times of only 1-6 min Pill Also, ELISA wasperformed on a microchip of polyethylene microchannelsfeaturing 5 min incubation times; this assay was able todetect about 1 nm o-dimer, a protein used as a negativeIndicator for deep vein thrombosis [42].

In a typical microwell ELISA assay for detecting serumantibodies, serial dilutions of the sample are accom-plished manually, and the assay repeated for each anti-gen to be tested. Thus, the analysis of a single sampletypically requires many microwells. We developed amicrofluidic immunoassay that automatically seriallydiluted the sample and presented multiple antigens onthe surface for analysis (Fig. 2A) [43]. The device em-

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Bectrophoresis 2003, 24, 3563-3576

Electrophoresis 2003, 24, 3563-3576

pioyed a microdilutor network that mixed the samplewith buffer using a chaotic mixer. Each mixing achieveda dilution factor of 2; ten mixing steps resulted in a dilu-tion factor of 213 103. The serially diluted samples then

flowed over a polycarbonate membrane, onto whichstripes of antigens have been patterned. Using a fluo-rescently labelled secondary antibody, we demon-strated the detection of anti-human deficiency virus(anti-HIV) antibodies in HIV+ serum with an automatedserial dilution profile, using two different HIV antigensin parallel.

4.1.2 Multiplexing

Microfluidic systems have the potential to perform a largenumber of biochemical assays in parallel, and enablelarge-scale combinatorial processes. An intriguing ap-proach is a two-dimensional array where two sets ofmicrofluidic channels are crossed at right angles. In theseapproaches, the screening of a library of N samplesagainst a library of M reagents requires only a singlechip, instead of N chips for conventional arrays of the titerwell format. We fabricated a three-dimensional systemwhere two PDMS molds of crossing channels are placedorthogonally to each other, separated either by a porouspolycarbonate membrane, or by two polycarbonatemembranes and a microwell (Fig, 213) [44]. The wholesystem is conformally sealed. The membranes allow fordiffusion of the reactants and provide a high resistanceto convective flow through the crossing, thereby minimiz-ing cross-contamination between the crossing channels.We showed that a variety of biochemical reactions can beperformed in this system, such as enzymatic reactionsand detection of Staphylococcus aureus by bead aggluti-nation. The system, however, is more difficult to fabricatethan a microtiter plate, and requires pressure balancingto control the flow across the membrane that mixes thereactants.

DeLamarche at al. [45] demonstrated a different imple-mentation of a two-dimensional immunoassay (Fig. 213).In this method, parallel stripes of antigens are first pat-terned onto the surface using PDMS microfluidic chan-nels. The channel system is demounted, and a secondsystem of parallel microfluidic channels is placed ontothe patterned antigens at right angles. Samples contain-ing the analytes are caused to flow onto the patternedantigens. The method was effective in detecting anti-bodies using either a sandwich EUSA or fluorescentiylabelled secondary antibodies. Compared to conven-tional ELISA assays, this method required only nanolitervolumes and took only minutes to complete.

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Microfluidic devices fabricated in PDMS for biological studies 3569

4.2 Separation of proteins and DNA

4.2.1 PDMS open channels: capillaryelectrophoresis

Techniques for separating proteins and DNA - such ascapillary electrophoresis and liquid chromatography -can be performed on a microfluidic chip. Advantages ofminiaturization include reduced cost and analysis time,and potential for high-throughput analysis and for inte-gration with other microfluidic components (for example,sample filtration and extraction). The ease with whichfluid flow can be controlled electrokinetically has madecapillary electrophoresis a popular technique for minia-turization onto a chip. In comparison, the difficulty inminiaturizing high-pressure systems for driving fluidflow in packed columns has limited the work on miniatur-izing liquid chromatography (see [46] for a discussion ofrecent work).

PDMS can be easily molded to form channels for theseparation of biological molecules. It has the added ad-vantage thatple_sma_ol_ddation of its,.surfase_ganerates.ailanol.aroups that arq„negativsly_ charged.at neutral or

basic pH; this _charged surface enables electroosploitc,„'tow towards the,negabvely,.charpat catgaa [if. In an

Riffildemonstration of capillary electropnoreiii in PDMSmicrochannels, Effenhauser at al. [47] achieved efficientseparation of DNA fragments in native PDMS channelsusing electrokinetic flow in a sieving matrix. Joule heatingwas effectively dissipated by PDMS for field strengthsless than 1 kV/cm. We demonstrated capillary zone elec-trophoresis in plasma-oxidized PDMS channels, whichsupported uniform electroosmotic flow (Fig. 3) [7]. The de-vice efficiently separated amino acids and protein chargeladders, and, in the presence of a sieving matrix, DNAfragments. Harrison et al. [48] showed that native PDMScould also support a reproducible and stable electroos-motic flow (the origin of the surface charge may stemfrom silica fillers in the polymer). The ability of oxidizedand native PDMS to support electroosmotic flow maydepend on the ionic strength of the buffer 1491.

One-dimensional sodium dodecyl sulfate (SDS) capillarygel electrophoresis (CGE) has been performed in a micro-channel [50]. The microchannel-based SDS/CGE sepa-rated a six-protein mixture with greater efficiency andspeed than a conventional capillary-based SDS/CGE. Toseparate components in complex mixtures of proteinssuch as cell lysates, two-dimensional (2-D) gel electro-phoresis is often used. In this method, the first dimensionis isoelectric focusing (1EF) and the second dimension isSDS get electrophoresis. Compared to a slab gel, a min-iaturized format of 2-D gel electrophoresis would require

3570 S. K, Sia and G. M. Whitesides

PDNIS

--;- -di 80

60c

.5_ 40

202

0

openchannel

capillaryelectropkoreala

impuritiesOin,Pkri

150 200 250 300

time (sec)

less sample and may exhibit less heat-induced peakbroadening due to more efficient heat dissipation (from ahigh surface area-to-volume ratio).

Previous methods to miniaturize 2-0 gel electrophoresis(and other 2-0 separations) have focused on the injectionof effluent from the first dimension into a second dimen-

sion. This process is slow and serial. In an initial demon-stration, we have built a PDMS-based channel system toperform all separations in the second dimension in paral-lel, similar to conventional 2-D gel electrophoresis [51].PDMS was a particularly appropriate material for thisdesign because of convenient procedures for fabricating3-D microfluidic channels, and the facility with whichPDMS-based systems can be assembled and disas-sembled. After separation using IEF in the first dimension,we disassembled the channel system and connected theIEF gel (filled with the partially separated protein mixture)to a 3-D channel for SDS gel electrophoresis. As a proofof concept, we demonstrated the separation of three pro-teins in a mixture. This demonstration is at an early stageand does not represent a practical method of separation.Higher efficiency of separation may be achieved by opti-mizing the design of the channel system.

PDMS channels can also be used to separate DNA. SinceDNA fragments of different sizes exhibit similar charge-to-mass ratios, they separate poorly in an open channel.

eaplllpnjelectrochivilatography

© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

PomS columns

0 200 400 600

time (sec)

Electrophoresis 2003, 24, 3583-3576

Figure 3. Separation of bio-molecules using PDMS-basedmicrofluidic devices. One-di-mensional separation in aPDMS-based microchip. (Left)In an open channel, capillaryzone electrophoresis was usedto separate a mixture of RTC-labeled amino acids (figureadapted from [7]). The sepa-ration voltage was 5 kV, andlaser-induced fluorescence wasdetected by a photomultipliertube. (Right) In a channel withPDMS monolithic posts, capil-lary electrochromatography wasused to separate a trypticdigest of FITC-BSA (reprintedfrom [55], with permission). Theseparation voltage was 1 kV,and laser-induced fluorescencewas detected by a photomulti-oiler tube.

Doyle et al. [52] demonstrated the use of a stationaryphase consisting of a self-assembled magnetic matrixfor separating DNA in a PDMS channel. Large DNA frag-ments (10-50 kbp) were effectively separated in this de-vice. In another approach, PDMS was used as an inter-mediate layer between a high-voltage source and theseparation channel; the hybrid POMS-glass microchipeffectively separated DNA samples [53]. Finally, PDMSwas used as a cover slip on nanochannels (as small as150 by 180 nm) fabricated in silicon [541 The electro-phoretic behavior of individual k-DNA molecules wasstudied in the nanochannels.

4.2.2 PDMS as stationary phase

Whereas PDMS open channels have been well studied,less focus has been placed on using PDMS as a station-ary phase for separations of proteins and of DNA. Thereare numerous advantages to this approach, the mostsignificant one being that PDMS microstructures can beprecisely and inexpensively fabricated. In this way, dif-ferent microtabricated patterns of stationary phase canbe rapidly prototyped and precisely controlled. The highdegree of control allows for high channel homogeneityand total control of channel dimensions and geometry,compared to conventional packed columns (which haveinhomogeneous beds).

Electropho rests 2003, 24, 3563-3576 Microfluidic devices fabricated in PDMS for biological studies 3571

Regnier etal. [55] fabricated a microcolumn consisting ofPDMS support structures of 10 gm dimensions; thesestructures covered over 60% of the surface area of the

separation section of the device (Fig. 3). Operating in thecapillary electrochromatography mode, the device sepa-rated peptides from a tryptic digest of bovine albumin.As hydrophobic stationary phases are most effective forpeptide separations in high-performance liquid chroma-tography, PDMS support structures derivatized withhydrophobic moities (silanes containing phenyl groupsor 08-C18 alkyl groups) gave rise to better separationthan native PDMS, which is only moderately effective asa hydrophobic support (it is roughly the equivalent of a Clphase). An important consideration in using this methodis the nonspecific interaction of analytes with the PDMSwalls,

4.2.3 interfacing PDMS microchannels withmass spectrometry

Mass spectrometry is a powerful tool for postcolumn anal-ysis of peptides, proteins, and small molecules. Severalapproaches have been taken to connect PDMS micro-channels to electrospray ionization-mass spectrometry(ESI-MS). Aebersold et at [56] connected a fused-silicacapillary to the outlet of a prefabricated PDMS channel;the other end of the capillary was connected to the ESI-Ma Interfaces between PDMS and silica capillary can beformed with minimal dead volumes by taking advantage ofthe molding properties of PDMS. For example, PDMS wascast directly on a fused-silica capillary; after curing thePDMS, removal of a part of the embedded capillary gener-ated a PDMS microchannel that formed a smooth interface

with the remaining embedded capillary [57]. In anotherstudy, PDMS was cast on a metal wire inserted into a silicacapillary; removal of the metal wire generated a PDMSmicIdchannel that connected to the silica capillary withno dead volume [58].

Alternatively, the PDMS-capillary interfaces can be elimi-nated altogether by fabricating PDMS microchannels withtapered ends; these ends functioned as ESI emitters [59].This device employed pressure-driven flow for sampleinfusion. In another study, ESI was obtained by directspraying from PDMS microchannels using electrokineticflow [60].

4.3 Sorting and manipulation of cells

The two most common methods for sorting and enrichingcell populations are the fluorescence activated cell sorterand magnetic filtration. Both methods can be miniatur-ized to devices that are sensitive, cost-effective, and

10 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

easy-to-operate. This section also describes PDMS-based systems for manipulating and culturing repro-ductive cells.

4.3.1 Cell sorting by flow cytometry

Using soft lithography, Quake et al. [61] microfabricateda fluorescence activated cell sorter (FAGS) driven byeleotrokinetic flow. The sample was introduced into aT-shaped junction, and upon detection of fluorescencenear the junction, voltages were switched to divert thesample to the collection channel. For a sample containingfluorescent and nonfluorescent Escherichia coil, the cellsorter enriched fluorescent E. colt by 30-fold, and 20%of the recovered cells were viable. In a subsequent study,the microfabricated cell sorter was modified with valves

and pumps to use pressure-driven flow instead of electro-osmosis (Fig. 4A) [62]. The pressure-driven device exhib-

Awaste

Nolvos(ttaNO thkuVel)

r"-A"

toReetionr

&Mee kw.window

htita

— Oen

10 gm

Figure 4. Miniaturized sorting devices. (A) Microlabri-cated fluorescence-activated cell sorter. Samples con-taining fluorescently labelled molecules flow from theinput well to the detection window. Detection of fluores-cence triggers pneumatically actuated switch valves thatforce the cells to flow to the collection well [62]. (B) Mag-netic filtration device. Next to a permanent magnet, thenickel posts create a large local magnetic field gradientand capture 4.5 rAm superparamagnetio beads (colored)from a mixture also containing nonmagnetic beads (notcolored). Figure adapted from [64].

8572 S. K. Sia and G. M. Whitesides

ited higher cell viability and sorting accuracy than theelectrokinetically driven sorter. Takayama et al. [63]fabri-cated a PDMS-based flow cytometer using pressure-driven flow. In this device, the use of air as the sheathfluid (instead of liquid in conventional FAGS) to focus thesample flow stream eliminated the need of large liquidreservoirs, and may allow for higher flow rates and higherthroughput than systems that do not use sheath fluids.Microfabricated cell sorters exhibit a lower cost than

benchtop systems, and they have the potential for sin-gle-cell studies and integration with other microfluidiccomponents. Disadvantages of microtabricated cell sor-ters compared to conventional FAGS include the lowthroughput of sorting (less than 100 cells per second,compared to thousands of cells per second for conven-tional PACS), and a low recovery of viable cells.

4.3.2 Magnetic sorting

Magnetic cell sorting is a technique commonly used forenriching one cell population from a mixture of cells. Inthis technique, target cells are labelled with antibody-coated superparamagnetic beads (50 nm or 3 gm). Themixture is then passed through a separation column, typi-cally containing ferromagnetic collection elements to actas field concentrators, in the presence of a strong mag-netic field; the labelled cells are retained on the column,the column is washed, the magnetic field is removed,and the retained cells are eluted.

In a miniaturized format, a permanent magnet can beplaced next to the microchannel to effect magnetic sepa-ration. We developed a magnetic filtration system con-sisting of 15 gm diameter nickel posts which act as mag-netic field concentrators in the presence of an externalmagnetic field (Fig. 4B) [64]. The device separated 4.5 gmparamagnetic from diamagnetic beads with 95% effi-ciency. We believe that this system can be extended toseparate and sort magnetically tagged cells, although ithas not so far been used for this purpose.

4,3.3 Manipulation of motile cells

Takayama of at. [65] built a PDMS-based device thatsorted motile sperm from nonmotile sperm, a procedureimportant for choosing viable sperm for in vitro fertiliza-tion. The microscale sperm sorter made use of the abilityof motile sperm to cross streamlines under laminar flowconditions. Nearly 100% of the sorted sperm was motile.Compared to conventional sorting methods (such ashand sorting), the microscale sperm sorter was quicker,simpler to use, and produced a comparable yield (theratio of the number of motile sperm sorted and the total

2003 WILEY-VCH Vedag GmbH & Co. KGaA, Weinheim

Electrophoresis 2003, 24, 3563-3576

number of sperm in the sample). Beebe et al. [66] havealso developed a microfluidic device for transporting ovaand sperm in microchannels and for culturing embryos.This device has the potential to automate in vitro fertiliza-tion and to increase its rate of success. The technology isnow being commercialized (Table 1).

Table 1. PDMS-based microfluidic devices in the privatesector

Company UHL PDMS-based product

Cellectricon

Fluidigm

Surface Lop

%Medic

www.cellectricon.se

www.fluidigm.com

www.surfaceloglx.com

www.vitaelic.com

*Parallel patch clamp, ionchannel drug screening

*Screen for proteincrystallization

*Biosystems for drugdiscovery

Microfluidics for assistedreproduction

' Product is on the market.

We list only companies that use PDMS in their final prod-ucts (many companies use PDMS for prototyping). For ageneral summary of microfluidic products in the privatesector (fabricated in any material), see (1, 811.

4.4 Cell biology using laminar flow

4.4.1 Laminar flow over single cells

The flow of fluids in a microchannel is normally laminarWe have used laminar flow in PDMS microchannels to

deposit proteins and cells onto a solid substrate [67](in general, microfluldic channels can be used to depositproteins and cells onto substrates [12, 68, 69]). Usinglaminar flow in microchannels, we can also deliver mole-cules to different regions of live cells with subcellular pre-cision [70]. Specifically, we placed a PDMS microchannelover live capillary endothelial cells, and controlled the flowin a way that caused solutions containing mitochondria'dyes of different colors to contact different parts of a cell;sub-populations of mitochondria inside the cell werelabelled with different dyes. It was possible to follow themovement of the different populations of mitochondria;they mixed throughout the cell after 2.5 h. Also, by flowinga membrane-permeable, actin-disrupting molecule overspecific locations, we disrupted actin filaments at tar-geted portions inside a cell.

As a tool to study subcellular biology, this method has theadvantages over microiniection and microperfusion inthat it involves no complicated micromanipulation, andthat it works for any type of cell that can be grown on solid

Electropharesis 2003, 24, 3563-3576

support. Laminar flow was used to address the funda-mental question of how signaling is transmitted in a cellafter stimulation by a ligand [71). Specifically, epidermalgrowth factor (EGF) receptors in localized areas of aCOS cell were stimulated by a flow stream containingEGF, and the propagation of EGF signals was followedby fluorescent indicators (using genetically engineeredproteins that fluoresced upon tyrosine phosphorylationor Ras activation). The authors concluded that the signalsspread over the entire cell in cells overexpressing the EGFreceptor, but that the signals were localized to the stimu-lated regions in cells expressing only a basal level ofEGF receptors.

4.4.2 Molecular gradients

Laminar flow can be used to generate stable gradients ofmolecules in solution and bound to the surface (Fig. 'IC).This technology enables the quantitative investigation ofcellular phenomena involving molecular gradients over adistance of several hundred gm. In one study, Toner eta!,(72] used a PDMS microfluidic gradient generator to pro-duce stable solution gradients of IL-8, an important cyto-kine in inducing chemotaxis of neutrophils to the site ofinfection during the inflammatory response (Fig. 5A). Flowof IL-8 gradients over surface-bound neutrophils madepossible the measurement of the migratory response ofthe neutrophils as a function of the steepness and shapeof the gradient. With gradual gradients, neutrophilsmigrated past the area of maximum chemoattractantbefore reversing direction; with a steep gradient, the neu-trophils halted migration at the boundary. Overall, thetechnique provided an assay to study the behavior of che-motactic cells with a quantitative precision and controlnot possible in earlier studies.

Neurons are another important class of cells that re-spond to extracellular gradients. In particular, gradientsof solution and surface-bound chemoattractants and

chemorepellants are important in determining the be-havior of axon growth, a key step in brain development.We fabricated a network of PDMS microchannels to

generate linear gradients over hundreds of microns ofsurface-bound laminin (Fig. 5B) [73). Hippocampal neu-rons were cultured on the immobilized laminin gradients,and the growth of processes from the neurons followedby microscopy. Of the several processes formed fromneurons in the first days of culture, one process (de-signated the axon) elongates much more rapidly thanthe others. We found that after 24 h in culture, gradientsof surface-bound iaminin meeting a threshold slopeoriented axonal specification in the direction of increas-ing laminin concentration for 60% of the neurons, com-

© 2003 W1LEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Microlluidic devices fabricated in PDMS for biological studies 3573

buffer

A 11-8: 0 nglmL 50 ngimt

BSA

IL-8Solutiongradient

lam▪ inin

surfacegradient

IL-8

laminin 20 pm

200 pm

1, removePDMS slab

2. cultureneurons

1 = 90 min

Figure 5. Studying the responses of cells to moleculargradients in microchannels. (A) Study of chemotaxis ofneutrophils in a solution gradient of 1L-8. In a linear gradi-ent of IL-8 from 0 ngtmL to 50 ng/mL, neutrophilsmigrated to the region of highest IL-8. Figure adaptedfrom 1721 (B) Dependence of axonal specification ofhippocampal neurons on surface-bound gradients oflaminin. A surface-bound gradient of laminin is formedfrom a solution gradient. Neurons cultured on the gradi-ents exhibit preferential axonal specification in the direc-tion of increasing laminin concentration. Figure adaptedfrom [731

pared to 33% (random orientation) in the absence ofthe gradient. Cramer et al. [74] used a simple laminarflow setup in a PDMS microchannel to create a Solutiongradient of chemoeffectors for bacterial chemotaxis.The microfluidic assay for chemotaxis showed greatersensitivity than conventional capillary assays; for exam-

3574 S. K. Sia and G. M. Whitesides Electrophoresis 2003,24, 3563-3576

pie, the micrafluidic assay showed that E. call chemo-taxis was sensitive to a chemoattractor, L-Asp, at con-centrations three orders of magnitude lower than pre-viously reported.

4.5 Combinatorial screening

An exciting application of microfluidics is combinatorialscreening by the use of many microchannels on asingle chip. Quake at al. [75] built a PDMS-based micro-fluidic device that rapidly screened conditions for pro-tein crystallization. Because PDMS is gas-permeable,large numbers of microchanneis in a complex architec-ture could be filled with solutions with no trapped airbubbles [76]. The chip consumed less than 3 tit of pro-tein sample, and tested for 144 different crystallizationconditions in parallel using free interface diffusion. Com-pared to the conventional vapor diffusion method usinga sparse matrix for sampling crystallization conditions,the microfluidic chip detected more conditions thatgenerated crystals (of a variety of qualities), and con-sumed two orders of magnitude less protein sample. Anumber of different protein samples were crystallized(including the bacterial 705 ribosome, a large protein-RNA complex), and crystals extracted from the chip dif-fracted X-rays. The technology is now commercialized(Table 1).

Temperature is another important variable in biochem-ical assays. The temperatures in different parts of aPDMS-based chip can be varied by using heating de-vices. For example, by placing tungsten heaters at differ-ent parts of a circular channel, a PCR device consumingonly 12 nL of sample was constructed [77]. Cremer et al.[78] fabricated a device that exhibited a temperature

gradient either parallel to or perpendicular to the micro-channels. The temperature gradient was used to con-struct a melting curve of double-stranded DNA, whichcould distinguish perfectly complementary DNA strandsfrom those containing single mismatches. The tempera-ture gradient can also be combined with another vari-able (such as sample concentration) in a 2-D format, inorder to screen for optimal conditions for bioassays(such as protein crystallization, biochemical reactions,or cell behavior).

Large-scale microfluidic chips have many biological ap-plications. Quake of al. [22] constructed a two-dimen-sional micralluidic array of 256 individually addressablechambers by integrating thousands of micromechanicalvalves. In one biological application of this array, E. coilexpressing cytochrome c oxidase was identified in eachchamber and noninvasively purged from the microfluidicchip. Potential uses of large-scale microfluidic arrays

0 2003 WILEY-VCH Vedag GmbH & Co. KGaA, Weinheim

include high-throughput analysis of proteins and DNA,and manipulation of cells (such as high-throughput cellfusion [79]).

5 Conclusions

Microfluidics offers a set of exciting tools for studyingbiology. It reduces the time and cost of common bio-

anayliseteem&and les to sJog_____Litil y_92!itin detail. As the material of choice for microfluidic sys-tems, polymers such as PDMS exhibit adve_nteassveL

2!1_15,or_lenc_l_Ageet because t.hey.._aLe_,--e:aiy jakigple,

and co m e67—",nauirementaptimenx12i9meayse

[80]. PDMS-based microffuldic systems can be used as auseful step to test new designs, or as a final product, asshown by a number of functional devices developed inacademic institutions and private companies (see Table 1

for work done In the private sector). Some disadventages of PDMS include: hydrophobicity of its surface, whichresists wettinalmas_KLous solutions and is prone to non-specific protein adsorption (necessitatinglh some casessurface modification); incompegbiitty, with _high cpneen-trations of some organic solvents, whicilrnay otherwisebe_useful Isayi fp71:,:190,51_0r991M9graP136

a.lt_p_yi2rAillstkirlyalitstionallisighnig.99me-

tjlts_gp.±.51sity ro its he aspeotKatipe_pithe feetures

due to shrinking or sagging).

The technology for microlluidics is growing but still imma-ture. Several key challenges must be met in order to builda functional "lab-on-a-chip". These challenges includethe following: building a seamless world-to-chip inter-face; developing methods for pretreating "real-world"

'samples (from the laboratcgApdy _f_pr handling..fithds on-chip ..

due to small particles of dust or sample precipitation;IgTaling multiple microfluidic components and assays

(each with different requirements foLlauftecand „Imamonditins Alsb-, the use of small amounts of

AlY1111.4 .1rtl.T.A11.11911Y.MAY,§1..P..1.9 !?r1.9.0.A.PmEt..C.1.!PaclY„P.97

tages:, it. reqyires a .sanaltive, method ctdetecon .(wp!pn

iriibases a significant limitation for dilute samples) andhas a very limited capacity ;for preparative yark,. None-tiiéIe, applications are many, including min-iaturized bioanalytical instruments, clinical diagnostics,and perhaps, high-throughput methods for drug screen-ing, gene-expression profiling, proteomics and combina-torial assays. We believe that polymeric systems will playan important role in the developments of miniaturizedmicrofluidic devices, by lowering the barrier to entry fornew researchers through simple fabrication procedures,and maintaining high compatibility with bioassay require-ments.

Poc,4-t7-C. -Cave Tec-Vp ( Po C, i)

h,ect, -kkl-,--La cut 0-r vlect r

s':tt a: r>ct/t k.rvt cavt

Electrophoresis 2003, 24, 3563-3576 Microfluidic devices fabricated in PDMS for biological studies 3575

Financial support was provided by A111-1 (GM 65364).thank Wncent Linder for helpful discussions.

Received June 19, 2003

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From: Ralf Seemann et al. Droplet based http://corefacilities.systemshiology.netifoswildimierolluidics. 2012 Rep. Prog. Phys. 75 016601 biniview/Mierofluidics/Protocols

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