L ab-on-a-chip for drug development Bernhard H. …L ab-on-a-chip for drug development Bernhard H. Weigl , Ron L. Bardell , Catherine R. Cabreraa, a b,c* aMicronics Inc., 8463 154th

Advanced Drug Delivery Reviews 55 (2003) 349–377www.elsevier.com/ locate/addr

L ab-on-a-chip for drug developmenta , a b,c*Bernhard H. Weigl , Ron L. Bardell , Catherine R. Cabrera

aMicronics Inc., 8463 154th Ave. NE, Redmond, WA 98052,USAbBioengineering, University of Washington, Seattle, WA 98095,USA

cLincoln Laboratory, Lexington, MA 02420,USA

Abstract

Significant advances have been made in the development of micro-scale technologies for biomedical and drug discoveryapplications. The first generation of microfluidics-based analytical devices have been designed and are already functional.Microfluidic devices offer unique advantages in sample handling, reagent mixing, separation, and detection. We introduceand review microfluidic concepts, microconstruction techniques, and methods such as flow-injection analysis, electrokinesis,and cell manipulation. Advances in micro-device technology for proteomics, sample preconditioning, immunoassays,electrospray ionization mass spectrometry, and polymerase chain reaction are also reviewed. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Microfluidic; Lab-on-a-chip; MicroTAS; Electrokinetic

Contents

1 . Introduction to micro-scale phenomena..................................................................................................................................... 3501 .1. Characterization of fluid flow............................................................................................................................................ 3501 .2. Why use microfluidic technology?..................................................................................................................................... 351

2 . Methods of moving fluid in microfluidic devices ....................................................................................................................... 3522 .1. Pressure-driven flow......................................................................................................................................................... 3522 .2. Electroosmotic flow ......................................................................................................................................................... 353

2 .2.1. Electrokinetic flow control ...................................................................................................................................... 3542 .2.2. Electrokinetic mixing ............................................................................................................................................. 354

2 .3. Summary of fluid handling ............................................................................................................................................... 3543 . Micro-construction techniques.................................................................................................................................................. 354

3 .1. Traditional lithographic techniques.................................................................................................................................... 3543 .2. Soft lithography ............................................................................................................................................................... 3553 .3. Laminate technologies ...................................................................................................................................................... 356

4 . Applications of microfluidic devices ......................................................................................................................................... 3574 .1. Laminar fluid diffusion interface ....................................................................................................................................... 3574 .2. Flow injection analysis ..................................................................................................................................................... 3604 .3. Cell manipulation and sensing........................................................................................................................................... 360

4 .3.1. Flow cytometry...................................................................................................................................................... 360

*Corresponding author.E-mail addresses: [email protected](B.H. Weigl), http: / /www.micronics.net(B.H. Weigl).

0169-409X/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.doi:10.1016/S0169-409X(02)00223-5

mailto:[email protected]

http://www.micronics.net




350 B.H. Weigl et al. / Advanced Drug Delivery Reviews 55 (2003) 349–377

4 .3.2. Living cells on self-assembled layers ....................................................................................................................... 3614 .4. Electrokinetic techniques .................................................................................................................................................. 361

4 .4.1. Electrophoresis ...................................................................................................................................................... 3614 .4.2. Isoelectric focusing ................................................................................................................................................ 3634 .4.3. Dielectrophoresis ................................................................................................................................................... 363

4 .5. Proteomics....................................................................................................................................................................... 3644 .6. Sample preconditioning .................................................................................................................................................... 3644 .7. Immunoassays ................................................................................................................................................................. 3654 .8. Integrated multi-step systems ............................................................................................................................................ 3664 .9. Sample preparation for mass spectrometry ......................................................................................................................... 3684 .10. Polymerase chain reaction............................................................................................................................................... 3694 .11. Novel concepts and applications ...................................................................................................................................... 370

5 . Advances in modeling and analysis .......................................................................................................................................... 3706 . Future directions ..................................................................................................................................................................... 371References .................................................................................................................................................................................. 371

1 . Introduction to micro-scale phenomena on chip directly from normal, complex and heteroge-neous clinical samples, such as whole blood.

Microfluidic devices are often described as minia- Other companies active in the microfluidics areature versions of their macro-scale counterparts. While (e.g., Micronics, Cepheid, Fluidigm, and Nanos-this analogy is true for some aspects of microfluidic tream) have tried to address this issue by their choicedevices, many phenomena do not simply scale of chip materials, structure, and dimensions, bylinearly from large to small implementations. Exam- selecting a fluid transport method that is compatibleples include the following: increased surface area-to- with biological fluids, and by constructively allowingvolume ratio and the omnipresence of laminar flow. for the handling of blood and cell-laden streams on

The first commercial microfluidic lab-on-a-chip- microchips.based systems were introduced for life science In 2001, Kricka [1] surveyed the range of microapplications less than three years ago. Since that time analytical devices, from microchips and gene chipsthe field has also seen the formation of a number of to bioelectronic chips, and their impact on diagnosticdiverse microfluidics companies; the publication of testing. He predicted a move of clinical testing frommore than two thousand scientific papers on mi- central laboratory to non-laboratory settings with acrofluidics; and development of several additional positive impact on healthcare costs.microfluidics-based products. MIT Technology Re-view named microfluidics one of ten technologies 1 .1. Characterization of fluid flowthat will change the world and one of the many areasin which this will be seen is in the life sciences Fluid flow is typically characterized as one of twosector. regimes: laminar or turbulent. These two different

To date, several lab-on-a-chip companies, includ- regimes are characterized by the relative importanceing Aclara, Caliper, and Orchid Biosciences, have of inertial to viscous forces, which is usually de-developed microfluidic technologies that work for scribed by the Reynolds number [2–4], the ratio ofhighly predictable and homogeneous samples that are inertial and viscous forces on fluid flowing in acommon in the drug discovery process, whether in channel, i.e. a ratio of the momentum of the fluid tocompound screening, genomic analysis or the friction force imparted by the channel walls.proteomics [1–5]. The first generations of many of Originally proposed by Osborne Reynolds in 1883these systems address the non-FDA-regulated life [5], it describes the character of the flow.sciences research market. One of the primary chal- A low Reynolds-number flow is a laminar, orlenges for homogenous sample-based lab chip pro- layered, flow in which fluid streams flow parallel toviders, however, is their inability to perform analysis each other and mix only through convective and

B.H. Weigl et al. / Advanced Drug Delivery Reviews 55 (2003) 349–377 351

Fig. 1. A glacier illustrates laminar flow. No mixing occurs between the two side-by-side streams of ice.

molecular diffusion (see Fig. 1). Laminar flow is 1 .2. Why use microfluidic technology?dominated by viscous forces; fluid velocity at alllocations is invariant with time when boundary Microfluidic devices present unique advantages forconditions are constant. There is convective mass sample handling, reagent mixing, separation, andtransport only in the direction of fluid flow. A high detection. This technology is ideal for handlingReynolds-number flow is a turbulent flow in which costly and difficult-to-obtain samples and reagents.inertial forces dominate and various-size parcels of Typical microfluidic structures require betweenfluid exhibit motions that are simultaneously random 100 nl and 10ml of sample and reagents.in both space and time. Significant convective mass Microfluidic channel dimensions typically rangetransport occurs in all directions. The transition from 1 to 1000mm in width and height. Frequently,between laminar and turbulent flow typically occurs only one channel dimension is within that range; thisabove Re52000 in internal flows. The Reynolds is sufficient to provide laminar flow characteristicsnumber is defined as following Eqs. (1) and (2). In addition to obvious

advantages that are associated with smaller sample,reagent, and waste volumes, several phenomenav DH

]]Re5 (1) occur differently at these dimensions than in larger-mscale traditional devices:

where v is the average velocity of the fluid in the • All flow in such channels tends to be laminar,channel, m is the viscosity of the fluid, and the thus allowing the parallel flow of several layershydraulic diameterD is ‘‘four times the cross-H of fluid. This enables the design of separationsectional area divided by the wetted perimeter’’, and detection devices based on laminar fluidwhich leads to diffusion interfaces, which will be discussed

later.d pipe diameter • At these dimensions, diffusion becomes a viable2h slot height method to move particles, mix fluids, andD 5 (2)H 2 control reaction rates. For example, Cephradine5]]] channel height and width1/h 1 1/w (MW 349), a small antibiotic, diffuses about

14.3 mm/s at room temperature in aqueousdepending on the type of channel, e.g. round pipe, solutions. This allows the establishment ofwide slot with height /width ratio less than 0.1, or controlled concentration gradients in flowingchannel with rectangular cross-section. systems, as well as complete equilibration of the


molecule across a 100mm channel in less than include, but certainly are not limited to, the follow-1 min without any active mixing elements. ing issues:

• Sedimentation at standard gravity, unaided bycentrifugation, becomes a viable means to sepa- • Interfaces between the macroscopic and micro-rate dispersed particles by density across small scopic environments. This area includes samplechannel dimensions. For example, cells in introduction and extraction as well as sampleslightly-diluted whole blood sediment in a 100 interrogation.mm deep channel in about 1 min, and generate a • Mixing of fluids without turbulence in a timely50 mm layer of slightly-diluted plasma in the fashion.process. • Fluid transport. Two main methods are current-

• Active particle transportation and separation ly in use (see following section) electroosmoticmethods, such as capillary electrophoresis, show pumping and pressure-driven flow. Each meth-greatly enhanced separation performance in od has advantages and disadvantages; neithersmall channels. one provides a generic solution to the fluid

• In microfluidic channels, the diffusion distance transport problem.can be made extremely small, particularly iffluid streams are hydrodynamically focused.Thus diffusion-controlled chemical reactions 2 . Methods of moving fluid in microfluidicoccur more rapidly than in comparable macro- devicesscopic reaction vessels. For example, Hatch etal. [6] have shown a microfluidic immunoassay One of the most critical elements of any mi-that was completed in less than 25 s, as opposed crofluidic system is its fluidic transport system.to more typical immunoassay reaction times of Depending on the size of the fluidic channel net-10 min or more. work, such fluid drivers need to be capable of

generating flow rates ranging from hundreds ofmicroliters per minute for high-volume-throughputIn addition to these unique phenomena, there areapplications such as flow cytometry [7] down toadditional reasons to pursue micro-scale technology:picoliters per second for applications using micron orsubmicron-sized channels [8].

• Plastic microfluidic structures can be mass-pro- Numerous different fluid driving methods forduced at very low unit cost, allowing them to be microfluidics have been reported. Electroosmotic,made disposable. gas-pressure, positive displacement, micro-peristal-

• Micro devices are amenable to high throughput tic, thermal, and many other pumping modes, orby processing several assays in parallel. combinations thereof, all have their advantages and

• Micro devices require only small volumes of disadvantages. However, the two fluid driving modessample and reagents, and produce only small most commonly used are electroosmosis and hy-amounts of waste, which can often be contained draulic pressure, and they will therefore be discussedwithin the disposable device. in more detail in the following subsections.

• The micro scale allows development of total-analysis systems (Micro-TAS) capable of hand- 2 .1. Pressure-driven flowling all steps of the analysis on-chip, fromsampling, sample processing, separation and One of the more common methods of generatingdetection steps to waste handling. This integra- pressure for fluid flow in microfluidic circuits istion makes complex analyses simpler to per- positive displacement pumping [9]. The main advan-form. tage of positive displacement is that such circuits

have very little compliance, which makes controllingAs might be expected in a field less than 30 years the exact amount of pumped fluid, and knowing the

old, significant technical challenges remain. These exact location of a fluid meniscus much easier. Some


disadvantages are mechanical complexity, size, andfrequently the fact that no such pumps are commer-cially available for lower flow rates. Ultra-precisesyringe pumps often are used for this purpose [10–13]. Caliper Technologies has developed a platformthat provides both electrokinetic and pressure-drivenfluid flow [14]. Frequently, however, even the mostprecise commercially available syringe pumps do notprovide a flow that is smooth enough for manymicrofluidic applications. Micronics (Redmond,WA), in conjunction with Honeywell (Minneapolis,MN) has developed a flow-sensor-calibrated ultra-precise syringe pump unit as used by Jandik et al.[15].

An additional complication is that pressure-drivenflow in a channel exhibits a non-uniform velocityprofile, which is pseudo-parabolic (depending on theaspect ratio of the channel), maximal in the center of

Fig. 2. Schematic of electroosmotic flow.the channel and decreasing to zero velocity immedi-ately proximal to the channel walls. The fluiddynamics of this phenomenon are well-understoodand are modeled by the Navier–Stokes equations is the dielectric constant of the fluid,F is the electricwith proper boundary conditions [4,3]. The implica- field strength, andm is the viscosity of the fluid.tions of this behavior for microfluidic assays remain EOF-driven flow is clearly dependent on thean area of significant research [13,16]. interaction between the channel wall surfaces and the

enclosed fluid. EOF-driven flow is particularly effi-cient in channels of less than 0.1 mm in diameter2 .2. Electroosmotic flow[19]. EOF-driven flow has been successfully im-plemented with a range of channel materials, mostA second method of moving fluids in microfluidiccommonly glass [20], although EOF has also beendevices takes advantage of the well-known phenom-used in polymeric devices [21,22]. Capillary electro-enon of electroosmotic flow (EOF). EOF occurs inphoresis devices are driven almost exclusively bydevices with channel walls made of materials that areEOF [20]. Glass devices with EOF-driven flow havecharged under experimental conditions. The fluidbeen used successfully to mix different solutions,comprising the double-layer proximal to the chargedperform chemical reactions, and immunological as-surface will not be neutral, but rather will contain asays [23]. Various groups have experimented withhigher-than-bulk fluid concentration of counter-ions,different surface modifications to change the wallresulting in a charged fluid (see Fig. 2). If an electricsurface charge, and therefore the velocity of the fluidpotential is applied parallel to the channel walls, thisundergoing EOF, via either covalent modification ofcharged fluid will be attracted to the electrode ofthe surface or through additives in the fluid [24–27].opposite charge, resulting in convective fluid flowThe electroosmotic mobility (EOM) of polydi-with a blunt (uniform) velocity profile [17]. The bulkmethylsiloxane (PDMS) and its ability to supportvelocity v can be calculate using the followingelectroosmotic flow were investigated [28]. EOMequation from Sherbet [18]:was increased four-fold in low ionic-strength solu-z e Fr

]] tions at neutral pH in oxidized PDMS channels, butv 5 4p mhigh ionic-strength buffers at neutral pH compatible

where v is the average velocity of the fluid in the with living eukaryotic cells greatly diminished thischannel,z is the zeta potential at the channel wall,e effect.r


Bruin [29] reviewed microfluidic separation de- carbonate and laser-modified to create a series ofvices in which the flow is electrokinetically driven. slanted wells at the junction, was reported to mixExamples were given of microchip layouts, func- two streams within 0.44 mm instead of 23.0 mm astional elements, use of alternative materials to glass, required using diffusion only [35]. An alternateand multiple detection methods. Bousse [30] dis- strategy to intersecting channels is the use ofcussed applications of electrokinetically-driven mi- sinusoidally-oscillating electroosmotic channel flowscrodevices, including: DNA separations, enzyme [36]. The optimal mixing technique for each applica-assays, immunoassays, and polymerase chain re- tion depends on the particular microdevice, but theaction (PCR) amplification integrated with microfl- fundamental physical basis for mixing in the micro-uidic assays. scale is diffusion.

2 .2.1. Electrokinetic flow control 2 .3. Summary of fluid handlingAn electroosmotically-induced hydraulic mi-

cropump was developed that consists of a tee-junc- EOF provides a blunt flow profile but is verytion with one inlet held at high voltage and one sensitive to variations in both fluid composition andoutlet at ground [31]. The remaining outlet has no channel wall coatings, thus limiting its use as aelectric potential and experiences pressure-inducedgeneric pumping solution. Pressure-driven flow isflow when electroosmosis in the ground channel is significantly less sensitive to such variations (fluidreduced by a viscous polymer coating. Anions can be viscosity being a notable exception) but results in aswitched between outlets by changing the flow non-uniform flow profile, which is rarely a benefit.resistance in the field-free channel relative to the The optimal choice of fluid transport system remainsground channel. In another electroosmotically- application-specific.pumped device gaseous electrolysis products wereremoved though a permeable polymeric membrane[32]. The electroosmotically-induced fluid velocities

3 . Micro-construction techniqueswere independent of the field strength and length ofthe electroosmotically-pumped region, but dependent

There are many different ways to manufactureon applied voltage.microfluidic devices. Kovacs [37] provides a goodField-effect control was achieved on a microfluidicreview of traditional lithographic methods.system fabricated from PDMS on a silicon wafer

base that was electrically-grounded and had anelectrically-insulating layer of silicon dioxide [33]. 3 .1. Traditional lithographic techniquesVarying the polarity and the magnitude of theresulting radial electric potential gradient across the Micro-device construction techniques began as asilicon dioxide layer gave direct control of the zeta offshoot of the computer chip processing industry, inpotential and the resulting electroosmotic flow. which hard substrates, most commonly silicon, are

used as the primary construction material. The basic2 .2.2. Electrokinetic mixing paradigm of standard lithographic techniques in-

Because microfluidic flows are typically laminar, volves the use of electromagnetic radiation, typicallycomplete mixing cannot be achieved by the typical ultraviolet (UV) light, to transfer a pattern to amacro-scale techniques. Techniques that employ surface, such as silicon, covered with photoresist.micro-scale physics are being investigated. A 100 The ‘‘mask’’ that contains the pattern can be aspicoliter static mixer using multiple intersecting simple as an overhead with the desired designchannels and driven by electroosmotic flow was printed on it or more elaborate, such as a chromemicrofabricated, tested, and theoretically simulated mask. The pattern is transferred to the photoresist-[34]. The extent of mixing was determined by covered surface by shining electromagnetic radiationconfocal microscopy and CCD detection. Another through the mask onto the surface. Typically ultra-microdevice, a T-microchannel imprinted in poly- violet (UV) light is used, although electromagnetic


waves with narrower wavelengths, such as X-rays, could be used to measure extent of mixing inhave been used to achieve a finer resolution. The electrokinetically-driven flows.photoresist is then developed such that the areas These methods are amenable to parallel processingexposed to the electromagnetic radiation behave in and can produce mechanically strong andan opposite manner to unexposed areas; one set of chemically-resistant devices, with feature sizes asareas polymerizes and remains on the surface while small as 0.1 m. However, these methods are alsothe other set is washed away. Now that the mask has expensive, require significant chemical processingbeen transferred to the surface, any one or a number equipment outlays, and often require toxic chemicals.of surface modification techniques, or a sequence of In addition, the processes are often time-consumingtechniques, can be deployed. Chemical etchants, in and not amenable to rapid design iterations. Newerwhich exposed surfaces are ‘‘eaten away’’ while methods of MEMS fabrication address many of theseprotected surfaces remain, are one example of sur- concerns.face modification techniques. Glass and silicon arethe two most typical substrates used for fabricating 3 .2. Soft lithographymicrofluidic devices with these methods. A recentreview by McCreedy [17] offers a detailed summary Recent developments in MEMS fabrication tech-of the majority of these techniques. niques have moved away from the mask/etch

Many variations on these techniques have been paradigm inherited from the computer micropro-reported. A lateral percolation filter was created by cessor industry and instead exploit the properties ofdeep reactive-ion etching 1.5310 micron channels in polymers. One common method is to use traditionalthe bottom of quartz microchannels [38]. Flow lithographic techniques to create a template in a thickthrough the filter was driven by electroosmosis layer of photoresist, (see Fig. 3). This template cantriggered by silanol groups on the walls and was free then be filled with a heat- or UV-curable polymerof particles of sizes from dust to cells. Manufacture and used to cast numerous replicas of the desiredof a microfabricated electrophoretic device with 40 device. Dr. George Whitesides’ group at MIT [44–cm long channels was announced [39] for human 46] and Dr. David Beebe’s group at University ofgenome sequencing. It achieves an average of 800 Wisconsin [47] have developed many soft lithog-base reads compared to 550 with capillary array raphic techniques using polydimethylsiloxaneelectrophoresis. A microfabricated nozzle with a (PDMS) and UV-curable polymers. Some examplesvolume of less than 25 picoliters was developed for of the use of these soft lithographic methods areelectrospray of liquids for analysis by mass spec- described below.trometry [40]. It showed up to three times more Fabrication with hydrogels facilitates buildingsensitivity and approximately half the signal vari- device components that respond to chemical factors,ation compared to a pulled capillary. A glass-based such as pH, in the local environment [48,49]. Onemultireflection absorbance cell for microchip-based group has created pH-sensitive values that directcapillary electrophoresis was photolithographically fluid flow depending on the pH of the fluid [50].fabricated with aluminum mirrors positioned above Two imprinting techniques were used to fabricateand below the flow channel [41]. The device demon- microfluidic devices on poly methyl methacrylatestrated higher absorbance sensitivity than previous (PMMA) substrates: creating an impression with aplanar devices. Powderblasting was presented as a wire in heated substrate and imprinting with a moldtechnique for direct etching of microchannels [42]. A micromachined on a silicon waver [51]. Reproduc-universal conductivity detector thus fabricated was ible electrophoretic injections and an immunoassayable to detect micromolar concentrations of charged were demonstrated. Analysis of DNA fragments byspecies. Conductivity detectors have been reported to another wire-imprinted PMMA device was demon-have a lower relative standard deviation than the strated [52].current monitoring method for measuring electro- Methods are described for fabrication of free-osmotic flow [43]. In addition, electrolyte conduc- standing, non-cylindrical three-dimensional micro-tivity is linear with electrolyte concentration and structures from two-dimensional patterns [53]. Mi-


Fig. 3. One example of soft lithography. (a) Using silicon as substrate, generate layer of UV-curable photoresist; (b) use mask to selectivelydevelop sections of photoresist, rinse away undeveloped photoresist to create template; (c) pour curable (heat, UV, chemical) polymer overtemplate, cure, and remove from template.

croelectrodeposition adds strength to the thin metal microfluidic networks, but its hydrophobicity resultsdesigns. Another method described as the ‘‘mem- in uncontrolled adsorption on channel walls. A three-brane sandwich’’ was developed to produce topo- layer biotin–neutravidin surface coating was de-logically complex three-dimensional microfluidic veloped that decreases adsorption without inhibitingchannel systems in PDMS [44]. An example created electroosmotic flow [58]. The modified surface canwas a square coiled channel that surrounds, but does be patterned with biochemical probes to enablenot connect to, a straight channel. affinity binding assays.

In 2000, Becker [54] reviewed a wide variety ofmicrofabrication methods for polymer substrates, 3 .3. Laminate technologiesincluding: hot embossing, injection molding andcasting, molding-master fabrication, laser ablation, Laminate fabrication methods are based on alayering techniques, layer bonding, and dicing. different paradigm than either of the two methodolo-

Fabrication of microfluidic channels with poly- gies described above. As with the other technologies,electrolyte multilayers to alter surface charge to device design begins with designing patterns. How-control flow direction was reported [55,56]. Flow in ever, in laminate fabrication the device consists ofopposite directions in the same channel was achieved layers of material, lamina, that have been laser-cut orby using oppositely-charged polyelectrolytes on op- stamp-cut into the desired shape and then heldposite walls and measured by fluorescence imaging together with some form of adhesive, most common-and particle velocimetry. ly pressure-sensitive or thermally-activated adhesive.

PDMS-glass microchips with eight parallel capil- Mylar is presently the most commonly-used material,lary-electrophoresis channels were designed with although other materials such as glass and PDMScross and tee injectors that have narrow sample have also been successfully incorporated into lami-channels instead of uniform channel width and depth nate devices. Laminate fabrication has been used toat the injection intersection [57]. Improved resolu- build devices ranging in complexity from a simpletion, column efficiency, and sensitivity were demon- single-component module [59,60] to complicatedstrated. fluidic circuits used to perform hematology analyses.

PDMS is an excellent material for construction of Micronics’ microfluidic circuits [61] comprise


Fig. 4. A blow-up view showing the individual layers that are stacked up to form a laminate card.

laminates built of several layers of individually cut into the design (such as electrodes, filter membranes,or stamped fluidic circuits. While each layer can be sensors, etc.).manufactured very easily and inexpensively, thelamination process yields complex 3-dimensionalmicrofluidic structures. This allows the design, forexample, of 3D hydrodynamic focusing channels for 4 . Applications of microfluidic devicescell analysis, or of multiple separate circuits withcrossing channels on a single card. Figs. 4 and 5 4 .1. Laminar fluid diffusion interfaceshow an example of a Micronics disposable.

While other processes such as hot embossing, One of the great advantages of microfluidic flow ismicro-injection molding, and, in particular, silicon or that it allows the flow and interdiffusion of multipleglass lithographic techniques yield significantly bet- streams in parallel in a single channel. This methodter dimensional tolerances [9], the plastic-laminate has been discussed in the literature as Laminar Fluidmethod has its major advantages in turn-around time, Diffusion Interface (LFDI) technologycost, and the ease of generating 3-dimensional [9,10,13,6,62]. This method relies heavily on con-structures, as well as incorporating hybrid elements trolled and reproducible introduction of several fluids

into one channel, and generally requires eitherpositive displacement pumping or pressurized flowcoupled with a feedback flow sensor to provide thedesired flow velocity and rate. Other pumping meth-ods tend to be unreliable for this purpose, especiallywhen samples having variable viscosity or ionicbackground are used.

The LFDI enables diffusion-based separation anddetection, which allows the parallel flow of two ormore individual streams in a single microfluidicchannel. Under microfluidic conditions, fluids usual-ly flow in a very predictable, laminar fashion,thereby allowing miscible fluids (e.g., whole bloodand phosphate buffered saline) to flow next to eachother without turbulent mixing or the need for

Fig. 5. Micronics disposables are typically credit-card-sized, and physical separation by a membrane. Diffusion natu-most structural elements on these cards have dimensions ranging

rally occurs under these conditions. Smaller particlesfrom about 100mm to a few millimeters using a low-cost rapid(such as ions, small proteins, many drug molecules,prototyping process that allows the design and testing of new

microfluidic structures in 24 h or less. etc.) tend to diffuse quickly across the boundary


layer, whereas large molecules and particles, such as from whole blood without prior use of a filter orcells, tend to diffuse only minimally. centrifuge for sample preparation or provides a

The LFDI effect can be used to both separate desalting step as part of DNA sample preparation.particles by size, and to extract components from The result is reduction in reagent usage, equipmentsamples. This principle has been shown, for exam- costs and time.ple, with the H-Filter� platform (see Fig. 6), which The LFDI effect has been used by Kenis et al. [63]allows a serum equivalent to be generated directly to form channel walls within microstructures by

precipitation at the fluid interface.Micronics’ T-Sensor technology (see Fig. 7) is

based on a related principle, and allows the samplemolecules to diffuse into a parallel stream containinga reagent and/or an indicator, thus allowing quali-tative and quantitative detection. A T-Sensor lab cardpermits the parallel flow of small quantities of both asample, a reagent, and a control solution simul-taneously in one microfluidic channel. These threeflowing solutions naturally form diffusion interactionzones between them. Typically, a fluorescent orabsorption indicator may be added to the reagent.The sample or control analyte molecules then diffuseinto the reagent stream, where they react with theindicator and form detectable diffusion interactionzones. These zones can be either visually interpreted(i.e., by comparison of width and color to a chart—similar to a test strip) for qualitative or semi-quan-titative assays, or they may be monitored withFig. 6. Micronics liquid–liquid extraction platform, which uses

laminar fluid diffusion interfaces to separate particles by size. optical systems such as CCD cameras, linear diode

Fig. 7. T-Sensor schematic and a physical realization that is absorption-driven.


arrays, or scanning lasers for quantitative results. By well as electroosmotically injected sample plugsdetermining the ratio of an optical property of these [30]. For example, a 10 times longer detectiontwo zones (using chemiluminescence, fluorescence, time would enable the detector to capture 10absorption), an essentially calibration-free concen- times more photons, and to increase the sen-tration measurement can be derived. With T-Sensor sitivity of the assay accordingly.devices, and using standard chemistries developed • In a self-referencing T-Sensor, a control orfor serum analysis, the quantitation can take place reference solution containing a known or con-directly in whole blood—omitting time-consuming stant concentration of sample analyte can becentrifugation steps. T-Sensor devices have been analyzed in parallel within the same channel asused for numerous types of assays including kinetic, the sample. This allows real-time referencingprotein, enzyme, immunoassays, and electrolyte as- and control determination. Therefore, it is pos-says. sible to compensate for effects such as varia-

Some of the advantages of T-Sensors include: tions in flow cell geometry, temperature depen-dent reaction kinetics, light source stability,

• Analyte concentrations can be determined opti- instabilities in the optical system and detectioncally in turbid and strongly colored solutions, electronics, as well as fluid parameters such assuch as blood, without the need for preproces- turbidity, color, concentration of detectionsing such as blood cell removal by centrifuga- chemistry, viscosity, and flow speed.tion. • Measuring the concentration of the analyte at

• Many standard optically based (fluorescent or several locations along the channel may provideabsorption) and electrochemical analytical an additional means to compensate for residualmethods may be performed within the T-Sensor. cross-sensitivities.

• In many cases, indicator-dye cross-sensitivity to • Monitoring signal intensities along the T-Sensorlarger sample particulates can be avoided in the detection channel (in flow direction) provides aT-Sensor by making use of the size-based means for looking at the kinetics of a reaction,diffusion separation feature of T-Sensors. thus allowing kinetic diagnostic reactions to be

• In a T-Sensor, the indicator dye and/or the measured not as a function of time but ofdetection chemistry can be kept in a solution distance from the starting point of the diffusionformulated to display optimal characteristics of interaction.the detection reaction without affecting the • It is possible to flow more than three separatechemical composition and equilibrium in the streams through a single T-Sensor, allowing, forsample solution. For example, cross-sensitivities example, real-time determination of a sample,to pH or ionic strength can be suppressed by as well as a low and high control.using strongly buffered solutions. • It is possible to determine two or more different

• The location of the diffusion boundaries yields sample analyte concentrations in one single T-information about flow speed, sample viscosity, Sensor, allowing, for example, the determina-sample concentration, dye diffusivity, analyte tion of ionized calcium in whole blood whilediffusivity, and dye dynamic range. simultaneously compensating for variations in

• The steady-state nature of this method makes the pH of the blood sample, which affectslong signal integration times possible. The ionized calcium readings.interface zone of the reagent and sample in the • T-Sensor manufacturing is not limited to silicondetection window is continuously renewed as micromachining. Several other methods havethe fluids flow past the detector, while the zone been demonstrated that yield low-cost mass-that is imaged remains visually the same. This manufacturable microstructures with similarcan provide for higher sensitivities than in sizes and geometries.comparable flow detection systems that rely onthe injection of sample plugs into a carrier A critical requirement for achieving a micro totalstream such as Flow Injection Analysis [64], as analytical system for the analysis of cells and their


constituent proteins is to integrate the lysis andfractionation steps on-chip [65] for combined celllysing and enzymatic detection in an LFDI devicethat combines an H-Filter and a T-Sensor. Anintegrated microfluidic system for the continuouslysis of bacterial cells and the extraction ofb-galactosidase with detection by a fluorogenic enzymeassay was demonstrated. Such a continuous flowdevice would be a valuable upstream component of Fig. 8. Schematic of flow injection analysis, FIA.further separations by isoelectric focusing or variouschromatographic techniques, as well as for analyticaltechniques such as immunoassays. Here, a microfl-uidic system integrating the lysis of bacterial cells would permit downstream analysis of cell contentsand the extraction of a large intracellular enzyme, [72]. More recently, pressure-driven flow was usedb-galactosidase, was demonstrated. in conjunction with a micromachined silicon/glass

device to transport and position mammalian embryos[71] for processing in preparation for further cell

4 .2. Flow injection analysismanipulations, such as cell fusion and/or assistedreproduction [74]. Mobilization of biological cells in

One of the earliest inspirations for Labs-on-a-Chipa microfluidic system by electroosmotic and/or

were the integrated Flow Injection Analysis circuitselectrophoretic pumping was demonstrated, includ-

(see Fig. 8) described by Ruzicka et al. [64]. Whileing steps for cell selection and subsequent reaction

fluid volumes and circuit dimensions were somewhat[72].

bigger than modern microfluidic circuits (mostlybecause of the size of active elements such asvalves), and the fact that the circuits were not

4 .3.1. Flow cytometrymicrofabricated but machined out of plastic blocks,

Flow cytometry is based on focusing of cells intothey still showed all the elements typical for mi-

a single line for individual analysis. Altendorf et al.crofluidic analysis systems. More recently, Ruzicka

demonstrate a microfluidic disposable capable ofet al. and others [66–68] have shown microfabri-

performing a three-part white blood cell differentialcated versions of FIA systems. Here, sample plugs

[7]. Another device integrates upstream chemicalare injected into a carrier stream and transported past

cell-treatment with downstream cytometric detectiona detector, thus allowing the rapid manipulation and

[61].sequential detection of a large number of samples in

PMMA and polycarbonate microfluidic devicesa single integrated analyzer.

with electroosmotic flow and a confocal epi-illumi-nation system were used to detect single labeled

4 .3. Cell manipulation and sensing DNA (double-stranded) molecules [75]. Excited by a7 micron Gaussian-profile laser beam, higher sam-

Both human cells and bacteria have been success- pling efficiency was obtained by narrowing thefully transported in microfluidic systems, using either sampling channel from 50 to 10 microns than bypressure-driven [69,70,60,71] or EOF-driven flow hydrodynamic focusing with sheath electrokin-[72]. Bacteria were concentrated using either ZE or etically-driven from side channels. Molecule sizingIEF [60]. As early as 1987, a silicone microfluidic using photon-burst detection was inconclusive due todevice was used to transport and fuse individual cells the Gaussian profile of the excitation beam.[73]. One group used EOF-driven in combination An electrophoretically-driven flow microcytometerwith a flow injection-type channel assembly to select using coincident light scattering and fluorescenceeither bacteria or mammalian cells and then bring the detection was developed [76]. Labeled Escherichiachosen cells in contact with a lysing agent, which coli were counted at 30 to 85 Hz.


4 .3.2. Living cells on self-assembled layers endothelial cells to differentiate forming capillaryTechniques for immobilizing viable cells in pat- tube-like structures containing a central lumen.

terns on microchannel walls have been developed. Martinoia [91] established patterned populationsMiniaturized sensing techniques for detecting the of neurons on silicon microchips using hydraulically-

response of cells has been demonstrated. For exam- driven deposition of adhesion molecules. Patternedple, bacterial antibiotic sensitivity has been detected biological neural networks were formed with chickin cultured cells by microphysiometry based on a embryo spinal-cord neurons. Takayama [92] usedlight-addressable potentiometric sensor [77,78]. To laminar flow in capillaries to pattern both cellmonitor individual cells surface-creation techniques deposition and the media to which they are exposed.were developed to position cells accurately inside a They demonstrated patterning of the substrate withmicrofluidic device. Stable nanometer-scale struc- different proteins, patterning of adjacent areas withtures joined by non-covalent bonds were produced different types of cells, patterning of the chemicalsby molecular self-assembly [79]. Precisely en- delivered to the cells, and enzymatic reactions overgineered surfaces for self-assembly were obtained selected cells or portions of a cell.through materials science nanotechnology to obtain Microcontact printing and self-assembly have alsooptimized reactions with proteins and cells [80]. been used to create a pattern of lines as narrow as 5Condensation figures, the patterns resulting from microns of self-assembled streptavidin beads on thecondensation of vapor on an incompletely-wetted internal walls of a microchannel [93]. Single beadssurface, are a nondestructive method for assessing were precisely positioned and able to withstandthe heterogeneities of the surface [81]. forces applied by flowing water in the channel.

Microcontact printing was demonstrated to be aflexible method to position cells. Cells placed in 4 .4. Electrokinetic techniquesmicrometer-scale arrays by imprinting gold surfaceswith an elastomeric stamp [82] provide control over One can differentiate between molecules/particlescell growth and protein secretion. Gold structures on the basis of many properties, such as density orwith edge resolution of less than 100 nm were color, and use such differences to separate, identify,obtained by lithography of self-assembled mono- and concentrate these molecules/particles. One suchlayers (SAMs) of alkanethiolates by beams of meta- property, surface charge, forms the basis for manystable argon atoms [83]. Sub-micrometer patterns well-developed sample handling and analytical meth-have been produced on curved substrates [84] and ods, referred to as ‘‘electrokinetic’’ methods. Exam-other topologically-complex surfaces [85] by mi- ples of electrokinetic techniques include electropho-crocontact printing of SAMs. SAMs of alkanethiol- resis and isoelectric focusing (IEF). IEF and electro-ates along with microcontact printing and mi- phoresis are the two techniques that comprise two-cromolding in capillaries were used to prepare tissue dimensional (2-D) gel analysis, which has become aculture substrates in which both topology and molec- major tool in proteome analysis, (see Fig. 9). Aular structure of the interface were controlled related technique, dielectrophoresis, relies on the[86,87]. Patterned SAMs were also demonstrated on dielectric properties of the particle(s) under inves-optically transparent films of gold or silver on which tigation.endothelial cells were then attached and visualizedby inverted and/or fluorescence microscopy [88]. 4 .4.1. Electrophoresis

The dependence of cell function on cell shape was Electrophoresis is a ubiquitous technique in medi-shown by restricting endothelial cells to progressive- cal analyses, commonly used to fractionate andly smaller micropatterned islands generated by mi- analyze both DNA and proteins. In recent years itscrocontact printing of SAMs of alkanethiolates on implementation has moved to smaller and smallergold [89]. A size-related transition from growth to scales, from gels to capillaries to microchannels.apoptosis was revealed. A follow-on study [90] Electrophoresis is simply the movement of a chargedshowed that intermediate-size islands, too small for particle in an electric field and has been used for twocell growth and too large to cause apoptosis, caused related but distinct approaches. Particles can be


[96] with higher efficiency than in conventionalfused-silica capillaries and comparable to gel electro-phoresis methods. A microfluidic DNA-typing elec-trophoresis device was developed for an allelicprofiling assay of short tandem repeats that is 10 to100 times faster than capillary electrophoresis [97].Electrophoretic separation of neurotransmitters andDNA restriction fragment and polymerase chainreaction product-sizing were demonstrated in a mi-crofabricated glass-based system with integratedelectrochemical detection [98].

Microfluidic CE has been used to analyze manyFig. 9. Electrophoresis and isoelectric focusing.

classes of molecules beyond DNA. The affinitycoefficient of a monoclonal antibody was measured

fractionated on the basis of electrophoretic mobility, using a microfluidic device to perform affinity CEwhich is the ratio of the velocity of a particle in an [99]. Detection of phenols by a glass micromachinedelectric field to the strength of that field. Alternately, capillary electrophoresis system using indirect fluo-electrophoresis can be combined with a sieving rescence was compared with a conventional capillarymatrix, most commonly a gel, to fractionate particles electrophoresis system using direct ultraviolet de-on the basis of size if the particles have been treated tection [100]. Two phenols were separated in twoto insure a uniform electrophoretic mobility. orders-of-magnitude less time, but with only one-

One of the most common methods of analyzing third the precision. A protein biological-threat agentproteins and DNA is capillary electrophoresis (CE), detection system that combines laser-induced fluores-in which a homogenous solution is loaded into a high cence with a microfluidic electrophoresis device wasaspect-ratio channel. A field is applied length-wise developed and tested with the simulant, ovalbuminacross the channel, inducing charged particles to [101]. The achieved concentration detection limitmigrate towards the electrode of opposite sign. Once was comparable to detection of 900 out of 4560the fractionation has been completed, the channel injected molecules. A microfluidic device in PDMScontents can either be analyzed in situ or pumped to that employs a microavalanche photodiode was useda downstream detector. Some of the possible advan- to detect the separation of a mixture of proteins andtages of a microfluidic implementation of CE over small molecules by capillary electrophoresis [102].traditional CE have been summarized by Manz et al. The excitation light was brought to the microchanneland include higher efficiency, faster response time, by optical fiber and the photodetector, operated inand reduced reagent consumption [94]. These advan- Geiger mode, was close enough to the microchanneltages are particularly important when working with to make transfer optics unnecessary. A microfluidicclinical samples, which are typically complex and system using capillary electrophoresis laser-inducedlimited in both total volume and concentration [95]. fluorescence was developed for assessment of cellMicrofluidic devices offer the possibility of inte- viability [103]. It was demonstrated with two bac-grating sample preconditioning and analysis of small teria and yeast in single and mixed samples.volumes. Advances in 1997 in DNA and protein sepa-

Microfluidic CE systems using either pressure- rations, cell manipulations, immunoassays and poly-driven flow or EOF have been successfully demon- merase chain reaction using electrophoretic sepa-strated. One of the first groups to demonstrate on- ration on microchips as part of a miniaturized totalchip microfluidic capillary electrophoresis used a analysis system were reviewed by Kopp et al. [104].glass device with EOF-driven flow [94,20]. Electro- Sample handling and separation in microchip-basedphoresis on a planar glass microchip with laser- electrophoresis was reviewed with emphasis oninduced fluorescence detection was used to produce applications in human serum protein analysis, im-chromatographic separations of biological samples munoassay, and DNA studies [95]. In 1999, Regnier


[105] examined the role of microchip-based sepa- and simpler handling [112]. An electrode-definedration systems in the evolution of chemical analyses sample compaction and injection technique achievedin biology, medical research, and health care. The sample compaction without migration into the gel.emphasis was on protein purification, peptide frac- To develop a better way to introduce samples fromtionation and sequencing, amino acid analysis, and the macro environment, a pressure-driven sample-DNA sequencing. Microfabricated electrophoretic introduction channel for a manifold of electrokineticdevices were able to perform rapid genotyping flow channels was designed and tested [113]. Leak-assays and sequenced single-strand DNA in less than age flow was less than 1% of the electroosmotic flow15 min [106]. In 2000, Baldwin [107] reviewed three rate and the quantitative results obtained from elec-years of advances in capillary electrophoresis de- trophoretic separations in the electrokinetic channelstection systems with a focus on the non-electro- were unaffected by the pressure-driven channel.chemist audience. He includes devices based both onamperometry and on potentiometry and discusses the4 .4.2. Isoelectric focusingevolution toward microfabricated lab-on-a-chip anal- Isoelectric focusing (IEF) is similar to electro-ysis. In 2001, Krishnan [108] reviewed miniaturized phoresis in that both techniques rely on the move-reaction and separation systems including the micro- ment of a charged particle in an electric field.scale separation columns used to analyze DNA, However, IEF selects on the basis of isoelectric pointRNA, proteins, and cells. (pI), which is defined as the pH at which a particle

There are been many technological advances since has no net charge. To perform IEF, a pH gradientthe first microfluidic CE device. Thermoresponsive must be constructed between the two electrodespolymer matrices that change reversibly from high- generating the electric field, oriented such that theviscosity solutions at 258C to low-viscosity colloidal acidic region is proximal to the anode and the basicdispersions at elevated temperatures were demon- region is proximal to the cathode.strated in electrophoresis microchannels [109]. Ma- Isoelectric focusing (IEF) of sample biologicaltrix loading flows were accelerated by three orders- analytes, bovine hemoglobin and bovine serum al-of-magnitude while still retaining the optimal se- bumin, was performed in microchannels to illustratequencing performance of highly entangled solutions the potential of ‘‘microfluidic transverse IEF’’ forof high molar mass polymers. A microfabricated use in continuous concentration and separation sys-glass chip that integrates separation, staining, virtual tems [59]. An optically-based pH-gradient detectiondestaining, and detection to perform a protein-sizing method and a one-dimensional model were de-assay was demonstrated [110]. It used a novel veloped to interpret the physics of the system [114].electrophoretic dilution step to bring the sample The pH gradients were formed by electrochemicalcomplexes below the critical micelle concentration to decomposition of water at 2.5 V and opticallyreduce background and increase peak amplitude by quantified by acid–base indicators. Zone electro-one order-of-magnitude. Synchronized cyclic capil- phoresis and IEF were used in a continuous-flowlary electrophoresis was demonstrated in which a microfluidic device to concentrate bacterial solutionssample is separated during many cycles in a closed [60] and protein (bovine serum albumin in a singleloop separation channel using a polygon geometry of ampholyte buffer) [115].3 to 5 sides [111]. Optimizations included reductionof losses and dispersion at the intersections by 4 .4.3. Dielectrophoresisdesigning voltage connections that are more shallow Unlike electrophoresis and IEF, dielectrophoresisthan the separation channel, and reduction of disper- (DEP) works on both charged and uncharged par-sion in the turns by narrowing the channels at the ticles. In DEP, a non-uniform electric field is appliedcorners. A microfabricated separation system using to the solution under investigation. This field resultsphotodefinable polyacrylamide gels as a sieving in a polarization of both the particles suspended inmedium for DNA electrophoresis instead of non- solution and the solution itself. If the particle is morecross-linked polymer solutions was shown to offer polarizable than the surrounding medium, the in-shorter curing times, locally-controlled gel interface, duced dipole aligns with the applied field and the


particle moves towards regions of higher field typically using sodium dodecyl sulphate poly-strength; this effect is described as ‘‘positive DEP’’. acrylamide gel electrophoresis (SDS–PAGE) tech-If the particle is less polarizable than the surrounding niques [122]. In 2001, Figeys [123] surveyed themedium, the induced dipole aligns against the ap- recent developments in microfluidic and array tech-plied field and the particle moves to regions of lower nologies that were being applied to proteomics. Leefield strength (‘‘negative DEP’’) [116]. Since the [124] surveyed the tools available for proteomedirection of movement is relative to the strength, not analysis including 2D protein electrophoresis, massthe direction, of the field, DEP can be performed spectrometry, spotted-array-based methods, and mi-with either DC or AC fields. crofluidic devices. He pointed out that additional

Dielectrophoretic forces generated by microelec- technology was required to be able to fully integratetrodes on the bottom surface of microchannel were proteomic information with information obtainedable to differentially levitate different types of cells about DNA sequence, mRNA profiles and metabolitein a dielectrophoretic field-flow-fractionation device concentrations into effective models of biological[117]. Stem cells were displaced higher than breast systems. Jain [125] reported on advances in mi-cancer cells producing a separation efficiency of crofluidic devices for genomics, drug discovery and99%. Human breast cancer cells were separated from molecular diagnostics with a focus on biochip andnormal blood cells by dielectrophoretic /gravitational microarray technology. In 2002, Mouradian [126]field-flow fractionation by exploiting the difference reported on advances in applications of microfluidicin dielectric and density properties between the cell chips in proteomics including protein sizing, two-populations [118]. dimensional separation, and integration of trypsin

Linear traveling wave dielectrophoresis was dem- digestion, separation, and injection into a massonstrated in a microchip with latex beads and rabbit spectrometer within a single device.heart cells [119]. The dependence of latex beadmotion on applied potential and frequency, suspend- 4 .6. Sample preconditioninging medium conductivity, bead size, and surfacecharacteristics was studied. Their surface conduct- A key component of successful assays is properance was determined from the dielectrophoretic sample preparation and handling upstream of thecrossover frequency. assay itself. Very often the target analyte is initially

An analytic solution was derived to study the present in low concentration and in solution witheffects of electrode spacing and electrode width on numerous other constituents that can impair assaythe gradient in electric field intensity produced by a function. Sample preconditioning, by removing thesetwo-dimensional array of parallel electrodes [120]. interferent constituents and concentrating the ana-The intensity gradient was found to be dependent on lytes of interest, can significantly improve the opera-electrode width squared and increase exponentially tion of the downstream assay. The macroscopicwhen the electrode spacing is less than two widths. techniques for sample preparation such as precipi-

tation, specialized membranes, and centrifuges are4 .5. Proteomics not directly amenable to microfluidics.

Other techniques, such as those based on elec-Biologically-based research has moved increasing- trokinesis, offer viable microfluidic solutions to the

ly towards a ‘‘holistic’’ approach, leading to the question of sample preconditioning. The successfuldevelopment of genomics and proteomics [121]. concentration of proteins and vegetative bacteria hasAlthough genomics can provide significant infor- been demonstrated using free-flow continuous IEF inmation, protein expression is necessary to understand a laminate device driven with pressure driven flowthe function and regulation of an organism. The [59,60]. A microchip-based sample purification tech-current standard for proteomics is the 2D gel, in nique was demonstrated by removing primers from awhich a complex solution of proteins is first fraction- sample of amplified DNA [127], leaving only prod-ated on the basis of isoelectric point, via IEF, and uct DNA. A microchamber for electrophoretic ac-then fractionated on the basis of molecular weight, cumulation of charged biomolecules was designed


for use as a preconditioning step in the analysis of diffusive mass transfer of cephradine and other lowDNA and protein samples [128]. Concentration of molecular weight compound is taking place (seeDNA oligomers and streptavidin in aqueous solu- Fig. 12). The red blood cells and other blood par-tions was increased up to 200 times at the center of ticles remain in the sample stream and are removedthe chamber. into the waste loop WL. Under optimized conditions,

Simple differences in the diffusion speed of a large portion of high molecular weight compoundsmolecules with different molecular weights have also (i.e. proteins) also remains in the original sample andbeen utilized for sample preconditioning. Jandik et ends up in waste.al. [15] describe microfluidics-based sample prepara-tion prior to high-pressure liquid chromatography 4 .7. Immunoassays(HPLC). The H-Filter� makes possible a diffusionaltransfer of an analyte from a sample stream into a Using the LFDI technology discussed earlier, astream of a ‘‘receiver’’ fluid (see Fig. 10). The rapid microfluidic diffusion immunoassay has beenauthors discuss the extraction of an antibiotic cep- developed (see Fig. 13) and shown to detect sub-hradine from blood to demonstrate the utility of the nanomolar concentrations of phenytoin, a typicalnew device. The new method completely avoids the small drug molecule used to treat epilepsy, in lessuse of centrifuges that is otherwise typical for most than 1 min [6]. The assay was performed on dilutedcurrent methodologies for the preparation of blood whole blood; no removal of blood cells was neces-samples prior to HPLC analysis. sary. Results compared well to those obtained with a

Fig. 11 illustrates the position of liquids at the end commercially available macroscopic assay. A com-of a sample preparation. The dark segment represents petitive binding reaction is established in which aan unprocessed aliquot of the whole blood. As known concentration of labeled analyte is added torepresented in the enlarged portion of Fig. 11, a the unknown sample, which is then injected into the

device and allowed to flow adjacent to a streamcontaining a known concentration of antibody to theanalyte, permitted interdiffusion between the twostreams. Once bound to antibody, the rate of diffu-sion of the analyte drops sharply, resulting in anaccumulation of antibody–antigen complex at theinterface between the two streams. The degree ofaccumulation, optically tracked with the labeledanalyte, is modulated by the concentration of un-known analyte, which competes for binding siteswith the labeled analyte.

A glass-microchip system for competitive im-munoassay of serum theophylline was developed[129] that includes a mixing of sample with tracerstep and a mixing/ reaction with antibody step, bothcontrolled by electroosmotic pumping, followed byan electrophoretic separation step. A microfluidicchip based on Borofloat glass performed electro-phoretic separations of many samples over several

Fig. 10. H-Filter Schematic. Two streams are introduced into a months to measure affinity constants of monoclonalcommon channel, where the form a laminar fluid diffusion antibodies to bovine serum albumin [99].interface (LFDI). Depending on their diffusion coefficient, par- Biotinylated lipopolysaccharide (LPS) was im-ticles start to diffuse across the LFDI, with the smaller particles

mobilized on streptavidin-coated sensor chips todiffusing more rapidly. Under optimized conditions, the majoritystudy the binding affinities of different LPS-bindingof a population of small particles can be removed from a sample

also containing larger particles. proteins and peptides [130].


Fig. 11. Fluid positions at the end of a sample preparation in an H-Filter card.

analysis, would be performed on the same device.Several groups have recently been moving towardssuch integrated systems. Voltammetric analysis withon-chip chemical reactions and fluid manipulationswere demonstrated on a microfluidic device [131].Manipulation of the electroosmotic flow enablesstopped-flow and reversed-flow operations allowingextended residence times in the detector compart-ment.

A complete microfluidic system of DNA sizingand quantitation was developed, including: dispos-able glass microchannels, reagents, and software andinstrumentation for electrophoretic separations andFig. 12. Mean cephradine concentration as extracted by the H-fluorescence detection of double-stranded DNAFilter from whole blood without preconditioning.[132]. Analysis of polymerase chain reaction prod-ucts, sizing of plasmid digests, and detection of point

4 .8. Integrated multi-step systems mutations were demonstrated.A 330 picoliter chromatographic bed was fabri-

Ideally, all aspects of a medical assay, from cated in a glass microchannel by trapping 1.5–4.0sample injection to sample preconditioning and micron octadecylsilane-coated silica beads between


Fig. 13. Diffusion Immuno-Assay (DIA) schematic and experimental results from a DIA for Phenitoin [6].

two weirs [133]. EOF was used to repeatedly ex- Screening one compound against multiple enzymechange the beads through a bead-introduction chan- targets achieved identical performance to individ-nel and to sequentially introduce a non-polar analyte ually-performed assays.solution followed by acetonitrile to achieve a con- Coupling two different pre-column enzymaticcentration increase of up to 500 times. reactions of the same substrate followed by electro-

A microfluidic device that combines capillary phoretic separation of the reaction products gener-electrophoresis with an integrated planar radio-fre- ated two electrophoretic peaks for a single analyte,quency detector coil used for nuclear magnetic glucose [137] (and the simultaneous measurement ofresonance (NMR) spectroscopy was developed glucose and ethanol [138]). The peak current ratio is[134]. Separations were demonstrated, but high useful for confirming the peak identity, estimatingconcentrations were required to obtain satisfactory the peak purity, addressing co-migrating interfer-NMR spectra. ences, and deviations from linearity. A similar

Electrokinetic pumping was used in a microfluidic microdevice that adds a post-column reaction dem-216chip with biospecific layer immobilized on the onstrated a detection limit of 2.53 10 g/ml for a

reaction chamber walls [135] (protein A with mouse IgG model analyte [139].fluorescently labeled rabbit IgG as sample). Con- An quartz electrophoretic microchip was demon-centration gains of 30 of antibody from dilute strated that can be directly coupled to pressure-solution onto solid phase were shown for concen- driven flow analyzers with no electrical potentialtrations down to 50 nM. applied because the pressure-filled sampling channel

Multiplexed enzyme assays were demonstrated in of the microchip is kept floating [140]. Dye wasa microchip capillary electrophoretic device [136]. injected into the sample stream in continuous or


discrete mode, then separated and detected on-chip and low-resistance channels, to create automatedby laser-induced fluorescence. protocols with fixed incubation times and fluidic flow

A microfluidic immunoassay device that integrates rates.six independent mixing, reaction, and separation Microfluidic modules were assembled to combinemanifolds and two optical alignment channels for the electroosmotic pumping of solvent gradient, a re-scanned fluorescence-detection system was de- verse-phase microcolumn, and an electrospray ioni-veloped [141]. It offers direct immunoassays of six zation ion trap mass spectrometer for analysis ofsamples simultaneously or, even more interesting, peptide mixtures from proteolytic digestion of pro-simultaneous calibration and analysis. teins [144]. Filters in the reservoirs and exit of the

A microfabricated glass electrochemiluminescence microfluidic system and a mount to connect it to thedetector for micellar electrokinetic chromatographic electrospray mass spectrometer were developedseparation was demonstrated using the legs of a [145]. Chemical noise observed by the mass spec-‘‘U’’-shaped floating platinum electrode placed ac- trometer was greatly reduced. A polymeric microfl-ross the separation channel as the working and uidic chip made of Zeonor 1020 provided directcounter electrodes [142]. The high voltage at the electrospray mass spectrometric detection of polardetector did not interfere with the electrochemistry. small molecules separated by capillary electropho-

resis [146]. No surface treatment of the polymer was4 .9. Sample preparation for mass spectrometry required. Emitters for electrospray have also been

microfabricated in polydimethylsiloxane [147–149].Mass spectroscopy is a very sensitive and versatile Another integrated monolithic device combined an

detection technique that is now being applied more electrospray interface to a mass spectrometer with aand more in the biological sciences, especially in capillary electrophoresis channel, an injector and ahigh-throughput genomics and proteomics. However, protein digestion bed with immobilized trypsin onthe technique requires that samples are provided in a 40–60 micron beads [150]. Digestion, separation andreproducible and ionizable format. In electrospray identification of melittin, cytochromec and bovineand Matrix-Assisted Laser Desorption Ionization serum albumin were demonstrated. Coupled capillaryTime-of-Flight (MALDI-TOF) mass spectroscopy electrophoresis and mass spectrometric techniquesapplications, microfluidic devices have been used in have been used to measure the values of the pK ofa

sample preparation circuits to remove interacting the amino groups of the aminoglycoside antibioticparticles and ions from the sample prior to ioniza- amikacin and of its acetylated derivatives [151].tion. Two microfluidic devices were reported by Jiang

A new company, Gyros, AB, is producing com- [152]: one to detect aflatoxin via electrospray ioniza-mercially available integrated microfluidic systems tion mass spectrometry, and a second to detectfor MALDI, including sample volume measurement phenobarbital antibody and barbiturates. The micro-and sample preparation (desalting, elution, and crys- scale of these devices increased detection sensitivitytallization) [143]. These devices are fabricated onto significantly by analyte preconcentration during thecompact disks, in which processing steps are located affinity purification step, limited analyte dilution insequentially along a radius from the outer edge of the the microdialysis junction, minimized sample loss,disk towards the center. Using this configuration, and allowed the use of nanoscale sample flow rates.copies of a many-step process can be arrayed A method and microfluidic device to isolate aradially, allowing for parallel processing of multiple single cell by integrating a laser trap and dielec-samples. In these devices, fluid flow is driven either trophoresis was reported [153]. The target microbeby capillary action or centrifugal force. A second was trapped at the focal point of the laser and excesscompany, Gamera (now part of Tecan), also uses microbes were moved away electrokinetically beforerotational motion of the entire device to mediate fluid the target was transported to and taken out from thetransport and mixing. Their product, the LabCDTM, extraction port.relies on a program of rotational velocity variation, A microfluidic module for nanoelectrosprayin combination with chip design that includes high- ionization-tandem mass spectrometry was fabricated


in poly(methylmethacrylate) and tested [154]. Protein strated from whole blood in silicon-glass microchipsidentification from small samples was comparable to [160].commercially available nanospray capillary tips. A moderate density array method using DNA

A microfluidic device for protein identification chips arrays was developed for diagnostic sequenc-was developed that combines a membrane reactor for ing of PCR products to reduce their cost andprotein digestion with electrospray ionization mass complexity in use [161]. Antibiotic resistant clinicalspectrometry and an intermediate step of transient isolates were visually detected within 1 h after PCRcapillary isotachophoresis /capillary zone electropho- amplification. Another application is the DNA se-resis for further concentration of the peptide mixture, quence-based identification of toxic medicinal plantsif needed [155]. A nanogram of protein sample is used in Traditional Chinese medicine [162]. In 2001,sufficient and can be analyzed in minutes. Petrik [163] reviews the use of microarray devices

originally developed for genomic projects for mass4 .10. Polymerase chain reaction screening of blood donations for hepatitis C virus.

An integrated system for PCR analysis was at-The polymerase chain reaction (PCR) is a sensi- tained by combining dual Peltier thermoelectric

tive method for DNA analysis [156,157]. A target elements with electrophoretic sizing and detection onDNA sample is incubated with primers targeted a microchip [164]. Using a DNA concentrationtowards a specific DNA sequence or set of sequences injection scheme enabled detection of PCR productsand with all the materials needed to make new DNA within as few as ten thermal cycles.strands. This mixture is cycled through a series of A microfluidic cartridge was developed to preparechanges of a given experimental parameter, typically spores for PCR analysis by sonication, addition oftemperature, that controls the degree to which the PCR reagent to the disrupted spores, and insertion ofDNA is double- or singly-stranded. After each cycle the mixture in a PCR tube [165]. The processing andthe number of copies of the target DNA sequence is detection of the spore DNA was completed within 20doubled, thus allowing significant amplification of min.the initial DNA sequence concentration. An integrated microfluidic device was demon-

PCR was one of the first diagnostic techniques to strated that combines stochastic PCR amplification ofbe successfully implemented in a microfluidic for- a single DNA template molecule followed by capil-mat. The rate at which PCR can be performed is lary electrophoretic analysis of the products [166]. Atypically limited by instrumentation [158]. Microfl- histogram of the normalized peak areas from repeti-uidic implementation of PCR decreases cycle time tive PCR analyses revealed quantization due todue to improved control of solution temperature. The single viable template molecule copies in the reactor.high surface-area-to-volume ratio of microfluidic A microfluidic chip for detecting RNA amplifieddevices facilitates rapid heat transfer and smaller by nucleic-acid-sequence-based amplificationsample volumes have less heat capacity, allowing (NASBA) was developed [167]. Samples of cryptos-rapid temperature change. poridium parvum were detected and clearly dis-

One of the first groups to perform a successful tinguishable from controls without separating themicrofluidic PCR combined the output with CE so amplified RNA from the NASBA mixture.that the output of the PCR reaction could be ana- An automated microfluidic system for nanoliterlyzed [159]. More recently, a micromachined chemi- DNA analysis directly from cheek cells was demon-cal amplifier, operated with continuous flow, demonstrated, including all the steps needed for DNAstrated a 20-cycle polymerase chain reaction (PCR) analysis: injection, mixing, lysis, PCR, separation,amplification of a 176-base pair fragment [158]. In sizing, and detection [168]. The possibility of furtherthis device the sample moves through regions of miniaturization of the system was established.various temperature, rather than changing the tem- An integrated microfluidic chip-based system wasperature of a stationary sample. PCR amplification of developed for quality control testing of a recombi-genomic DNA targets from white blood cells cap- nant, adenoviral, gene therapy product [169]. Thetured behind weirs in the flow chamber was demon- viral identity test sized and quantified the DNA


fragments and required 100-fold less sample than the erties. Mathematical modeling, once correlated toagarose gel method. experimental results, is useful for quantitative assess-

Polyimide microchips with a 1.7 ml chamber ment of local behavior as well as for device designcontaining a thermocouple were used to amplify a optimization. The fluid dynamics of nanoscale flow500-base-pair fragment of lambda-phage DNA dem- and mass transport have been studied by Ciofaloonstrating infrared-mediated temperature control [175]. Computational models, especially for particle-[170]. Adequate amounts of PCR product were fluid problems, are available for a variety of lengthobtained in 15 cycles, an amplification time of 240 s. scales.

Many analyses focus on various aspects of electro-4 .11. Novel concepts and applications kinetic flows. Mammen [176] points out that plots of

absorbance versus 1/ time could replace the tradition-A fluidic analogue of the field-effect transistor was al plots of absorbance versus time, since electro-

demonstrated with electro-osmotic flow inside a phoretic mobility is linearly proportional to 1/ timemicrofluidic channel controlled by an electric field and thus the spacing between peaks correspondsperpendicular to the fluid flow [171]. Two of these linearly to differences in mobility. Locally-asymmet-analogues combined with a channel junction were ric electric geometries were proposed by Ajdar [177]able to generate opposite flows inside a single flow for pumping liquid in microchannels and drivingchannel driven by electro-osmosis demonstrating the drops on surfaces. Fluid velocities were estimated byability to control and switch flows in a microfluidic a simple electroosmotic model.network. Electrokinetic injections on microfluidic devices

A microfabricated capillary electrophoretic device were studied by computer simulations to identifythat performs chiral separations of fluorescein iso- operating parameters that provide optimal perform-thiocyanate-labeled amino acids was developed for ance of the virtual valving [178]. In addition to studythe extraterrestrial search for life [172]. The device of pinched injection, gated injection was simulatedobtained enantiomeric ratios of amino acids from the and compared to experimental data to achieve leak-Murchison meteorite that agree with those obtained free valving during sample load. A variety of sampleby HPLC. loading profiles and dispensing schemes for pinched

Aqueous liquid droplets were encapsulated in a injection were studied experimentally [179]. Thehydrophobic powder creating ‘‘liquid marbles’’ that axial extent of the sample plug decreased as thewere easily moved by gravitational, electrical, and electric field strengths in the sample and samplemagnetic fields [173]. Quick displacement of the waste channels were raised relative to the analysisdroplets without leakage was demonstrated. channel.

A fluid-temperature measurement technique using Electroosmotic flow in microchannels with pat-a temperature-dependent fluorescent dye was demon- terned surface charge was investigated experimental-strated by measuring temperature distributions due to ly and theoretically assuming thin double-layers andJoule heating in electrokinetically-pumped microfl- low surface potential [180]. Surface charge variationuidic circuits [174]. Temperatures from 25 to 958C perpendicular to the driving electric field generatedwere measured with thermal, spatial, and temporal multidirectional flow along the direction of the field;resolutions of 0.038, 1 micron, and 33 ms, respective- charge variation parallel to the field generated re-ly. circulating cellular flow.

The effects of fluid inertia and pressure on thevelocity and vorticity field of electroosmotic flows

5 . Advances in modeling and analysis have been analyzed [181], assuming an inner flowregion dominated by viscous and electrostatic forces

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