integrated multiprocess microfluidic systems for automating analysis

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2010 15: 198Journal of Laboratory AutomationWeichun Yang and Adam T. Woolley

Integrated Multiprocess Microfluidic Systems for Automating Analysis

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Keywords:

microfluidics,

integrated

microdevices,

microchips,

capillary

electrophoresis,

biomarker analysis,

separation,

sample preparation

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198 JALA June

Integrated Multiprocess MicrofluidicSystems for Automating Analysis

*CoChe846

153

Cop

doi

201

Weichun Yang, and Adam T. Woolley*Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT

Microfluidic technologies have been applied

extensively in rapid sample analysis. Some current

challenges for standard microfluidic systems are relatively

high detection limits, and reduced resolving power and

peak capacity compared with conventional approaches.

The integration of multiple functions and components

onto a single platform can overcome these separation and

detection limitations of microfluidics. Multiplexed systems

can greatly increase peak capacity in multidimensional

separations and can increase sample throughput by

analyzing many samples simultaneously. On-chip sample

preparation, including labeling, preconcentration, cleanup,

and amplification, can all serve to speed up and automate

processes in integrated microfluidic systems. This article

summarizes advances in integrated multiprocess micro-

fluidic systems for automated analysis, their benefits, and

areas for needed improvement. ( JALA 2010;15:198–209)

INTRODUCTION

Microfluidic analysis systems have advanced rapidlysince the early 1990s,1,2 providing new capabilities forchemistry, biology, and medicine. For instance,microfluidic devices offer low sample and reagentconsumption3 (which is critical for expensive phar-maceutical characterization or trace samples), smalldead volume,4 fast mixing,5e7 rapid analysis speed,8

high throughput,9 and valveless flow control.10

rrespondence: Adam T. Woolley, Ph.D, Department ofmistry and Biochemistry, Brigham Young University, Provo, UT02; E-mail: [email protected]

5-5535/$36.00

yright �c 2010 by The Association for Laboratory Automation

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Consequently, these advantages of microfabricateddevices have been exploited widely in bioanalysis,and reviews cover areas such as protein separation,2,11

cell analysis,12e14 genomics,15,16 and biomarker as-says.17,18 Because the field of microfluidics has be-come so broad, our focus here is on integratedmicrofluidic methods in separation-based analysiswith strong automation potential.

To date, many microfluidic designs have beenmade, but they are generally tested with low-complexity samples. For actual biological specimens,which are mixtures with wide analyte concentrationranges, it remains a challenge to directly separate eventens of components on microdevices. The smallmicrochip platform size usually results in a short sep-aration length, limiting the resolving power and peakcapacity, which are critical for separating complexmixtures.19 For instance, the peak capacity of a poly-dimethylsiloxane (PDMS) microchip for micellarelectrokinetic chromatography (MEKC) was w12for protein separation.20 Importantly, to completelyisolate a 20-component mixture with 95% probabil-ity, the peak capacitymust bew800.21Clearly, resolv-ing power and peak capacity in microfluidic systemscould be improved. In addition, tiny sample volumes(usually in themicroliter range)22 are placed onmicro-devices, and often nanoliter or smaller volumes areinjected. Furthermore, microchips generally havea short optical detection path,23 such that the detec-tion limit is another aspect ofmicrofluidic devices thatcould be improved.

Fortunately, these separation and detection limi-tations can be overcome by integrating multiplefunctions and components at the chip scale.Methods for microfluidic device fabrication are gen-erally based on photolithographic processes, whichmake complex designs possible.24 Moreover,

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fabrication techniques have been developed to transfer thesecomplex designs into low-cost materials like plastics.25,26 Byintegrating sample preparation processes into a single micro-device, trace samples can be preconcentrated before analysis.Multidimensional separations on-chip can significantly im-prove the sample capacity. Importantly, because the samplesin many integrated microdevices are manipulated by volt-ages, these microfluidic systems can be readily automated.Compared with traditional methods, automated sample anal-ysis can be more economical, requiring less human interven-tion, and enabling increased sample throughput.27

Consequently, these advantages make integrated microdevi-ces especially attractive for automating the characterizationof complex mixtures.

Because the applications and principles of integrated mi-crodevices have been reviewed elsewhere,2,24 we focus this re-view on integrated microfluidic methods with high potentialfor automating analysis. Multiplexed separation and on-chipsample preparation will be emphasized in this work. We notethat on-chip sample preparation is a broad topic, encompass-ing cell analysis,14 sample purification,28 and other technolo-gies. Hence, to provide an in-depth discussion, we limit thescope of this review to the sample preparation areas of label-ing, preconcentration, and polymerase chain reaction (PCR)amplification.

MULTIPLEXED SEPARATION

Multidimensional Systems

Because the overall peak capacity of multidimensionalseparations is the product of the peak capacities of the indi-vidual, orthogonal one-dimensional methods,29,30 these sys-tems are of great interest for complex mixture analysis. Forexample, two-dimensional gel electrophoresis (2DE) is an es-tablished approach for high-resolution profiling of pro-teins,31 separating analytes according to isoelectric point inthe first dimension (isoelectric focusing [IEF]) and then bymass-to-charge ratio in the second dimension (polyacryl-amide gel electrophoresis [PAGE]). Despite its enormouslysuccessful application in biochemistry and clinical studies,32

the downsides of 2DE are also significant: extensive hands-on labor (gel preparation, staining, etc.) and slow separation(w1 day).33 To increase throughput and facilitate automa-tion, 2DE has been transferred into a microfluidic platform.For instance, MEKC coupled with capillary electrophoresis(CE) was demonstrated for peptide separation in 2000.34

However, this approach used different buffers for the two di-mensions, which in turn increased the complexity of deviceoperation. More recently, Herr et al.30 developed a microchipIEFeCE system that used the same buffer for both dimen-sions. Microchip IEFePAGE systems have now been auto-mated, providing a separation time of !2 h.35 Anotherapproach for 2DE involves using a gel for the first dimensionand a solution electrophoresis method for the second dimen-sion, as implemented by Osiri et al.36 with a capillary gel elec-trophoresiseMEKC system that was used to profile fetal calf

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serum proteins. Chen and Fan37 recently reviewed two-dimensional microchip separations, and the reader can referto this reference for additional information.

Future multidimensional electrophoresis microdevices willneed to further increase peak capacity through more effectivecoupling of separation dimensions. Improvements to devicefabrication and operation should enhance the interface be-tween the first and second separation techniques, potentiallyproviding additional information.

Parallel Separations

Although multidimensional separations can increase peakcapacity, usually only one sample is analyzed per run. Sam-ple throughput or capacity can be increased by performingseparations in parallel columns, in a manner similar to whatis done in slab gel electrophoresis. In a microchip format,forming parallel capillaries is readily achieved via photolith-ographic patterning. The first capillary array system for mi-crochip electrophoresis was demonstrated in 1997 with 12separation channels in parallel (Fig. 1A).38 In this design,channels terminate at one anode reservoir, whereas the otherend of each lane has a cross-injection design with three reser-voirs; this device was successfully used in human HLA-Hgenotyping. Mathies’ group further explored a 48-channeldesign with 96 sample reservoirs.42 The unique rectilinearlayout facilitated sample loading via a multichannel micropi-pettor. However, detection constraints made scaling to morechannels difficult; moreover, in this design, two samplesshared one separation channel, which increased the possibil-ity of cross-contamination.

Because of the limitations of the rectilinear format, a ra-dial microplate design with 96 channels was developed.43

The radial design used a rotary confocal fluorescence scan-ning system to probe each channel. Importantly, with an in-crease in wafer size, more lanes or longer separationchannels could be obtained. For instance, 384 lane microchipDNA analyzers were constructed in 8-in. (200-mm)-diametersubstrates (Fig. 1B).9 Separation length and resolution aresomewhat limited in the radial design; a 6-in. (150-mm)-diameter wafer only offers a 5.5-cm separation length,44

which is not ideal for DNA sequencing. For a fixed devicesize, the separation length can be increased by folding thechannel in a serpentine fashion. However, because the insidetrack of a turn will be shorter than the outside track, a ‘‘U’’-shaped turn will cause band dispersion, reducing resolution.Paegel et al.45 found that this dispersion can be significantlyreduced by narrowing the width of a channel in a turn,which extended the separation length to w16 cm on a 6-in.-diameter wafer, yielding an average sequencing read of430 bases.46 Recently, Kumagai et al.39 developed a micro-fabricated DNA sequencing device with 384 lanes in a fanshape (Fig. 1C). The microplate formed a part of a muchlarger automated apparatus that yielded a throughput of5� 106 bases per day per instrument.

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Figure 1. Parallel separations in microdevices. (A) A 12-channel microdevice with linear fluorescence scan detection; chip size was50� 75 mm. (B) A 384-lane radial microdevice for rotary fluorescence scanning detection on a 200-mm-diameter wafer. Lanes arew60 mm wide and 30 mm deep, and the effective separation length is 8.0 cm. (C) A 384-lane fan-shaped microdevice. The plate dimen-sions are 47 cm� 25 cm� 1.4 mm, and the dimensions of each channel are 40 mm (depth)� 90 mm (top width). The upper inset showsoval sample holes connected directly to the separation channels. The lower inset shows access holes for an anode reservoir. (D) Deviceswith 16 or 36 parallel separation channels used with CCD detection. (a) Design of a microfluidic network containing 16 parallel separationchannels on a 76� 76-mm2 Borofloat glass substrate. (b) Design of a 36-channel network. (c) Photograph of a 36-channel chip. (d) Image ofthe detection area on a 36-channel chip. (E) A 16-channel device with an integrated contact conductivity sensor array detector. The lengthsof the injection channel and separation channel were 9 and 54 mm, respectively. Channel sizes were 60 mm (width)� 40 mm (depth). To-pographical layout of the lithographically printed Au conductivity sensor array (gray); the outlet end consisted of 16 Au electrodes (7.62 mmlong� 500 mm wide) serving as the conductivity sensors. Adapted and reproduced from Refs. 9, 38e41, with permission from ACS andWiley.

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CCD-based fluorescence detection is also being pursued asa simpler setup than the scanning confocal arrangement. Peiet al.40 developed a multichannel system for enzyme assaysusing a CCD detector (Fig. 1D). In this radial system, paral-lel separation channels were directed to a common waste res-ervoir via a channel, and the CCD captured light at thecenter to obtain fluorescence images of all the channels.Recently, Dishinger et al.47 used a similar system to monitorinsulin secretion using 15 parallel channels.

Because fluorescence detection requires complex equip-ment, making miniaturization a challenge, alternative ap-proaches to parallel lane detection are being pursued.

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Shadpour et al.41 developed a 16-channel device with an in-tegrated contact conductivity sensor array (Fig. 1E). In thissystem, a gold conductivity sensor array was first patternedon a polycarbonate film and then aligned with a substratehaving hot-embossed microchannels, thus interfacing eachseparation channel with a pair of Au electrodes. Separationswere carried out for amino acids, peptides, proteins, and ol-igonucleotides. However, the limit of detection (LOD) waspoorer than that for conductivity detection in a single chan-nel, and the LOD was considerably worse than that for fluo-rescence detection. Moreira et al.48 developed a multichannelsystem with a single electrode for amperometric detection,

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which worked when the channels were operated serially,rather than in parallel.

A major emphasis for future work in parallel separationsshould be enhancing and simplifying detection, because fab-rication capabilities have become quite advanced. Simpler al-ternatives to scanning confocal fluorescence detection such asCCD fluorescence detection and electrochemical methods areattractive targets.

ON-CHIP SAMPLE PREPARATION

Labeling

Because of small channel dimensions, a good detector isan essential part of microfluidic systems. Demonstrateddetection methods in microdevices include ultraviolet (UV)absorbance,49 laser-induced fluorescence (LIF),50 mass spec-trometry,51 electrochemistry,52 and chemiluminescence.53 Ofthese methods, LIF is the most popular because of its lowLOD.54 Although many proteins can be directly detectedvia native fluorescence from tryptophan, an uncommondeep-UV light source is needed for optimal excitation.55

Therefore, derivatization of proteins with a fluorophore istypically needed for LIF detection. Fluorescein isothiocya-nate (FITC) is widely used in off-chip labeling because ofits high quantum yield (w0.7) and water solubility.56 How-ever, because of the slow rate of reaction for FITC withamine groups (12e24 h at room temperature),56 off-chipFITC labeling limits throughput and prolongs the analysistime. Thus, integrating the labeling process into a microflui-dic system can automate and speed up analysis. Typically,the derivatization of sample on-chip can be performed eitherbefore (precolumn) or after (postcolumn) separation.

In precolumn labeling, the dye and sample are generallymixed for a controlled time in a diffusion-based reaction cham-ber before separation. The first precolumn labeling in glass mi-crochips was demonstrated by Jacobson et al.,57 and similarresults have also been shown in polymeric microdevices.58,59

Yu et al.59 demonstrated a precolumn labeling system usingfluorogenic ‘‘chameleon’’ dyes. These labels offer fast reactiontimes, and the net charge onmolecules is unchanged after reac-tion. Recently,Mair et al.60 developed a periodic monolithmi-crofluidic system, which yielded a modest improvement in themixing efficiency, leading to a 22% greater fluorescence levelthan that in an open-channel design. Digital (droplet) micro-fluidics show promise for precolumn labeling; for instance,two droplets can be manipulated and mixed readily using thisapproach.7

Fluorescence tagging often results inmultiply labeled analy-tes having different separation properties that can result in sev-eral peaks for a single analyte.61 In addition, fluorescentlabeling can change the analyte charge or size, which can alsonegatively impact separation with precolumn tagging. Becauseof these limitations, postcolumn labeling is a desirable strategy.In this format, the fluorescent tag is added at the end of columnsuch that the numbers and positions of attached dyes have littletime to influence separation. For postcolumn labeling, the


reaction kineticsmust be fast, and themixing efficiency of sam-ple and dye streamsmust be thorough to reduce band broaden-ing. In the firstmicrofluidic systemwith postcolumn labeling,62

significant bandbroadening occurred and separation efficiencywas rather low. To improve efficiency, Fluri et al.63 found thatchannel widths should be narrow (w45 mm) to facilitate rapiddiffusion, and pH differences in the mixing solutions should beminimized. Liu et al.64 used a noncovalent label, NanoOrange,to bind to hydrophobic regions of proteins andprovide fluores-cence emission. Their results indicated that the labeling reac-tion rate was close to the diffusion limit, which resulted inlittle band broadening. Sieben and Backhouse65 developed de-vices that have two injection systems, one for the sample andtheother for the label.Byusingpinched injection,22a controlledplug of label could be loaded into the separation channel. Anadditional purge reservoir was integrated into the device toclean out the separation channel after labeling. A similarprotocol has also beenused for chemiluminescence detection.66

In this approach, the detection systemwas simplified comparedwith LIF, because no light source was needed.

A key area for future development in on-chip labeling willbe to improve detection limits to near what is achieved withoff-chip labeling. Another important future direction will beto implement on-chip labeling in parallel analysis systems.

Traditional Online Preconcentration Techniques

Sample concentration techniques in CE such as sweepingand stacking have been proven effective for pharmaceuticalspecies,67 herbicides,68 steroids,69 and peptides.70 Thesemethods have also been applied in microfluidic formats.For instance, Jung et al.71 developed a porous polymer plugin a microchannel to create a high-conductivity buffer zoneand enriched fluorescent analytes 1000-fold using field-amplified sample stacking. The same group developed CEmicrochips coupled with isotachophoresis, which could en-rich Alexa Fluor 488 nearly two million-fold,72 and underoptimized conditions, the detection limit of Alexa Fluor488 was w100 aM.73 However, it is important but difficultto find suitable leading and terminating electrolytes for isota-chophoresis. A review on stacking and sweeping in microchipsystems was recently published.74

Desirable future emphases in microchip use of traditionalcapillary preconcentration methods are apparent. First, di-rect comparisons of the performance of microchip versuscapillary methods should be done. Additionally, microchipexperiments should be carried out on real samples in complexmatrixes.

Solid-Phase Extraction

Solid-phase extraction (SPE) is a widely used method forsample preparation. It can be fully automated with commer-cial systems like SPE-DEX (Horizon Technology, Salem,NH), OSP2 (Merck, Darmstadt, Germany), and MicroLabSPE (Hamilton, Reno, NV).75 In SPE, sample is retained ona solid medium, allowing the matrix to be rinsed away and



Figure 2. Retaining packed beads in microchannels. (A) Two-weirdesign, showing weir heights in relation to channel depth and particlesize. Electroosmotic flow is driven by walls and by free silanol groupson particles. Solvent flow direction is indicated for a preconcentrationstep. (B) Two-side etching/alignment protocol. Two prefabricatedplates were aligned and thermally bonded. On the right is a drawingof the cross section of a packed chip with its dimensions. (C) Photo-polymerized frits retaining 3-mm-diameter beads. (D) The keystoneeffect. A suspension of particles is flowed toward the taper by vac-uum. At the taper, the density of the particles increases, and thesefirst particles act as ‘‘keystones,’’ blocking the others and allowingthe packed segment to grow. Adapted and reproduced fromRefs. 82e85, with permission from ACS and Wiley.

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the retainedmaterial to be eluted for analysis.76 The promise ofsample enrichment and cleanup by SPE has led researchers toapply this approach inmicrodevices. An SPE column has beenfabricated by coatingmicrochip walls with silanes, and 80-foldpreconcentration of coumarin C460 was observed;77 however,because of the limited surface area, the loading capacity of thisapproach was relatively low.

Silica bead78 and polymer monolith79,80 SPE columns withhigh surface area and greater loading capacity have been in-tegrated into microchips. Because silica beads are commer-cially available and their properties are well characterized,microchip SPE columns made by packed beads are attrac-tive. However, it is necessary to localize these particles in tar-geted regions of microchips using physical barriers. Forexample, a solegel structure was fabricated to retain silicabeads, and this system was tested in on-chip DNA purifica-tion.81 In an alternate format, a two-weir design that createda cavity to trap beads was explored (Fig. 2A).82 Two photo-masks were used in device fabrication, one to pattern thetops of the weirs for etching and the other to pattern thechannels for etching to a different depth. In this manner,a 1-mm gap was formed to prevent beads from passing outfrom the SPE bed.78 Zhong et al.83 developed a two-sideetching and alignment protocol to construct weirs in a differ-ent manner (Fig. 2B). A top plate containing weirs and a bot-tom plate having the connection channels were aligned, anda 4-mm gap was created by sealing the plates together. In-stead of a microfabricated weir structure, a physical barriercan be prepared on-chip with a photopolymerized frit(Fig. 2C).84 In a different approach, beads can be packedthrough a tapered geometry by the keystone effect(Fig. 2D).85 The channel that contained the beads taperedfrom a 70- to a 16-mm width. When beads flowed throughthe channel, the density of the particles increased in the tapersuch that they aggregated without a physical barrier.

Packed-bead columns have disadvantages in terms of pack-ing procedures and frit fabrication,which complicatemicrode-vice preparation. On the other hand, monoliths are anattractive alternative to packed particles because of low backpressure and high surface area.86 Thermally polymerizedmonolith materials have been successfully applied as SPE col-umns.87 In 2001, Yu et al.79 photopolymerized amonolith col-umn in a microfluidic system and performed SPE. Enrichmentof peptides and proteins up to 1000-fold was achieved on thiscolumn. More importantly, because of low back pressure,the linear flow rate in these monoliths could reach 10 mL/min, which far exceeded flow in packed microchip columns.

Monolith columns have also been applied for DNA en-richment in complex mixtures like blood. However, nonspe-cific binding hindered elution of nucleic acids and decreasedsample loading capacity because of competitive adsorption;the presence of proteins lowered the monolith extraction ef-ficiency from w80% to !40%.88 Therefore, Wen et al.89 de-veloped a two-stage microchip SPE system. Before monolithcolumn extraction, a C18 reversed-phase column was used toremove proteins in the sample. Although the procedure was


more complex, whole-blood DNA extraction capabilitieswere significantly improved. For a 10-mL whole-blood sam-ple, w70% of the protein was removed by the C18 column,affording more interaction between DNA and the monolithicmaterial. This two-stage system enriched DNA w20-fold inthe reversed-phase portion, and the overall DNA extractionefficiency was w70%.

In the previous applications described, microchip mono-lith SPE enriched analytes based on general interactions like



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hydrophobic absorption. To improve the selectivity, affinityelements can be immobilized on a monolith. Glycoproteinswere retained on a monolith with immobilized agglutininand then eluted in several fractions because of different affin-ities.90 Yang et al.91 prepared anti-FITC-modified samplepretreatment monoliths in microfluidic devices. FITC-labeled amino acids were enriched 20-fold and purified froma mixture containing a contaminant protein. However, theextraction and separation were performed on separate de-vices, which hindered automation. To circumvent this, Sunet al.92 coupled anti-FITC affinity monoliths with electro-phoretic analysis on a single device. Sample loading, rinsing,elution, and separation were all performed in an automatedmanner by controlling the potentials applied to various reser-voirs. FITC-tagged species were selectively retained by theimmunoaffinity column and separated from other contami-nants. The retained proteins were then eluted from themonolith with 200 mM acetic acid.

In addition to monoliths, other affinity columns havealso been integrated into a microfluidic format. For in-stance, microchannel surfaces were coated with silane andthen protein A via physisorption.93 Rabbit immunoglobulinG was concentrated and detected at 50-nM levels. However,this approach, as well as the other monolith work discussedpreviously, was only tested for capturing target analytes inbuffer solutions instead of complex matrixes like tissue orblood. Recently, Yang et al.94 developed an integrated mi-crofluidic system to perform quantitative determination ofalpha-fetoprotein (AFP) in human serum using both themethod of standard addition and a calibration curve(Fig. 3). The microdevices were made of poly(methyl meth-acrylate) (PMMA), and a photo-defined polymer wasformed on the microchannel surface, which allowed anti-body immobilization. All assay steps, including affinity ex-traction, elution, separation, and quantification, wereperformed on-chip in an automated manner via changingthe applied potentials in the reservoirs. AFP concentrationsin human serum measured in these microdevices using bothcalibration curve and standard addition methods comparedfavorably with those determined using a commercial assaykit. Phillips and Wellner95,96 have used immunoaffinity CEto measure biomarkers and neuropeptides in human biop-sies. The analytes were captured by a replaceable immu-noaffinity disk having attached antibodies. After removingnontarget materials, the captured analyte was labeled insitu, released, and then separated by microchip CE. The sys-tem was semiautomated, and the separation step was com-pleted within 5 min.

Nonelectrically driven immunoassays can also be per-formed in microchip devices. For instance, Kong et al.97

formed elastomeric microvalves in three-layer microchips tocontrol flow, although the valves were actuated by a vacuumpump and a compressor. Using this system, clenbuterol wasdetermined in pig urine samples in 30 min. Fan et al.98

designed a PDMS-on-glass microsystem to perform proteinassays on blood samples. Plasma was separated from


whole blood on chip, and selected proteins were detectedby antigeneantibody interaction.

The variety of column materials used thus far (commercialsilica particles, monoliths, etc.) indicates that consensus isstill lacking as to the optimal column properties. Future ef-forts should focus on determining which type of columnworks best for a given analysis. In addition, further work isneeded to streamline and simplify column fabrication.

Membrane Filtration

Another common method for sample preconcentrationand cleanup is membrane filtration, which uses the size differ-ence between analytes and buffer ions. Larger molecules can-not pass through a porous layer in a semipermeable hollowfiber,99 membrane,100 or joint,101 whereas smaller speciesare allowed to transit. In one design, a porous membranewas sandwiched between two PDMS pieces to createa three-dimensional microfluidic channel structure.102 Thissystem achieved 300-fold concentration of fluorescein inw40 min. The fluorescein was concentrated outsidea 10-nm pore membrane (with openings larger than themolecular size of fluorescein), because the negatively chargeddiffuse layer on the interior of the membrane repelled anions.Song et al.103 used a laser to pattern a nanoporous mem-brane at the junction of a cross-channel. This device couldconcentrate proteins more than 100-fold in 2 min, and the de-gree of concentration was limited only by analyte solubility.Similarly, an anionic polyacrylamide gel preconcentrator waslaser photopolymerized in one arm of a cross-channel ina PMMA microdevice.104 The negatively charged sulfonategroups in the gel repelled negatively charged proteins, en-abling concentration of proteins up to 100,000-fold. Footeet al.100 used a silicate membrane deposited between two ad-jacent microchannels, and a w600-fold signal increase forproteins was achieved. Kim and Han105 developed a simpleprotocol to fabricate a nanoporous membrane in microdevi-ces. They used razor blades to form a gap in microchannelsin a PDMS substrate; Nafion 117 was then filled into thegap and a portion of the microchannels via capillary forces.In this protocol, preconcentration was achieved in largechannels with dimensions up to 0.1� 1 mm.

Semipermeable membranes can also be integrated withother microchip functionalities. Herr et al.106 fabricated a sizeexclusion membrane at the injection junction of a microde-vice, allowing antibody enrichment at the membrane surface.Sample loaded on the membrane was captured through anti-geneantibody interaction, and enriched species were elutedinto a separation channel for electrophoretic immunoassay.This system measured a biomarker for periodontal diseasein saliva in !10 min with comparable results to conventionalmethods. A similar design has been developed into a portablediagnostic format for rapid detection of biological toxins.107

A membrane can also be used for SPE. Lion et al.108 inte-grated a poly(vinylidene difluoride) membrane to desaltand concentrate samples before analyzing with mass



Figure 3. Integrated affinity columnemicrochip CE devices for biomarker quantitation. (A) Layout of an integrated AFP analysis microde-vice. (B) Photograph of a microfluidic device with integrated anti-AFP affinity column. Scale bar is 1 cm. (C) Microchip CE of Alexa Fluor 488-labeled human serum and of AFP standard solutions after affinity column extraction. (D) Microchip CE of Alexa Fluor 488-labeled humanserum after standard addition and affinity column extraction. (E) Calibration curve generated from peak heights in (C), with the unknownsample data point indicated with a star. (F) Standard addition plot of concentration of standard added versus peak height generated from peakheights in (D). Adapted and reproduced from Ref. 94, with permission from ACS.

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spectrometry. Kim and Gale109 sandwiched an aluminum ox-ide membrane between PDMS pieces; when a blood samplepassed through the membrane, DNA was selectively enrichedand then eluted with buffer. The extraction time was!10 min, whereas the recovery was w40 ng of DNA per mi-croliter of blood. A membrane has also been applied to en-rich nonvolatile analytes by evaporation to reduce theamount of liquid phase.110 The membrane was located atthe interface between a gas and a liquid channel; samplewas introduced into the liquid channel, and water evaporatedinto the gas channel through which nitrogen was flowing.

Presently, membranes are formed through sandwichingbetween channels or photopolymerization. Although thesemethods work acceptably for prototyping efforts, morestraightforward fabrication techniques will be needed in thefuture. Moreover, the relatively slow rate of enrichment inmembrane-based systems is an issue that could be addressedwith high-speed membrane-based filtration devices.

Figure 4. Integrated PCR microdevices. (A) Continuous-flowPCR driven by the magnetic force. PCR reaction mixture, pushedby a ferrofluid plug, flows continuously through three temperaturezones in the circular channel. (B) PCR array device layout for DNAmethylation analysis. Nine functional units are arranged in a circularpattern. A total of 108 reactions can be done with nine sample in-jections, which significantly improves the throughput. (C) Func-tional unit layout. The sample inlet has a 0.64 mm diameter,and reaction chambers at the periphery have a 4 mm diameter.Primers specific for tumor suppressor promoters are depositedonto the bottom surface of designated reaction chambers by pi-petting. Adapted and reproduced from Refs. 116 and 118, withpermission from Springer and RSC.

PCR Amplification for DNA Analysis

PCR is an exponential amplification technique for DNAdiagnostics. The method relies on thermally cycling samplesin different temperature zones as follows: denaturationto single-stranded DNA, annealing primers to the single-stranded DNA template, and polymerase extension of theannealed duplex DNA. Miniaturization of PCR allows rapidthermal cycling, small sample quantities, and potential to in-tegrate with other microfluidic methods. Since the first reportof integrated PCReCE microdevices,111 considerable prog-ress has been made in the coupling of PCR with other tech-nologies (CE, immunoassay, cell isolation, DNA arrays, etc.)on microdevices. Because Chen et al.112 reviewed integratedPCR microfluidic technologies in 2007, we focus here onmore recent breakthroughs in the field.

Liu et al.113 developed a portable microsystem for forensicgenetic analysis. All the electronics for temperature control,microfluidic manipulation, and CE separation, as well as op-tics for four-color LIF detection, were integrated ina 12� 10� 4-in.3 box. Oral swabs and human bone extractswere successfully analyzed by this system in less than 1.5 h.To improve on the efficiency of cross-injectors, an inline in-jection system114 with DNA-based affinity capture was devel-oped. Immobilized oligonucleotides in a polyacrylamide gelprovided more efficient injection of DNA fragments whilecleaning up sequencing samples. Subsequently, a photo-defined, cross-linked polyacrylamide gel affinity matrix wasintegrated with PCReCE microdevices.115 This affinity sys-tem increased the injection efficiency to nearly 100% andminimized band broadening. The devices were evaluated indetecting diluted Escherichia coli O157 in a high backgroundof E. coli K12.

Continuous-flow PCR offers another format for integrat-ing PCR into microdevices. In this approach, instead of heat-ing and cooling a sample in a fixed location, three regions ona device are maintained at constant temperature, and the


sample is passed repeatedly through these zones to thermallycycle. Recently, Sun et al.116,117 developed systems in whichPCR solution was driven magnetically around a loop ineither one channel or multiple channels. As shown inFigure 4A, the system had three temperature zones, a magnet,and a ferrofluid (a stable suspension of magnetic particles inoil). With the rotation of the magnet, the ferrofluid plugmoved accordingly and cycled the PCR mixture around thecircular channel. This system was applied in amplifying ge-netically modified soy and maize samples in less than 13 min.

Methylation-specific PCR is used to analyze DNA meth-ylation, which is associated with tumorigenesis. Zhanget al.118 developed a droplet-in-oil microfluidic system forhigh-throughput DNA methylation detection. The designlayout, which allowed 108 reactions to be performed in



Original Report

parallel, is shown in Figure 4B,C. The droplet-in-oil protocolreduced contamination and prevented evaporation duringthermal cycling.

Integrated PCR methods have become rather advancedsuch that many important areas for future work will be inthe application of these high-performance systems to real-world problems. Large-scale implementation of integratedmicrochip PCR is envisioned in fields such as food safetytesting and forensics.

SUMMARY

The advancement of microfluidic technologies has providedfaster and smaller analysis systems. However, the detectionlimit and peak capacity of many microdevices are not idealfor trace analyses in complex mixtures. Integrating multiplesample preparation functions into microfluidic formats canimprove throughput, simplify pretreatment, and, most im-portantly, automate processes.

Multidimensional microdevices can significantly increasespeed in complex sample analysis; however, the inability toanalyze highly complex mixtures remains a limitation forthese systems. Array platforms can analyze either one sam-ple in parallel or many samples simultaneously; to date,384 parallel lanes have been integrated into a microdevice.Online labeling can eliminate sample preparation steps andincrease analysis speed while automating processes. Multipledye attachment in precolumn labeling can result in bandbroadening; furthermore, choosing an appropriate dye iscritical for postcolumn labeling. To preconcentrate sampleor remove impurities before analysis, SPE and membrane fil-tration have been integrated into microdevices. Samples canbe concentrated by two to four orders of magnitude underoptimized conditions. For on-chip SPE, packed beads andmonolith columns are widely used as stationary phases.Packed particles in microchannels require a suitable retain-ing structure. Monoliths are attractive because of low backpressure and high surface area. Integrated affinity extractioncan effectively capture target components from a complexmatrix and therefore is desirable for biological sample anal-ysis. Semipermeable membranes can enrich samples up to100,000-fold, although further work is needed in developingstraightforward fabrication methods. PCR can be integratedinto portable microsystems for various genetic analyses, andprogress continues in this area.

Multifunctional microdevices have allowed researchers toanalyze biological samples in an automated manner. Impor-tantly, integrated microchips have provided improvements tosample throughput, peak capacity, and LOD. With contin-ued development, integrated microfluidic devices will bemore robust and fully automated for high-throughput com-plex sample analysis in the near future.

ACKNOWLEDGMENTS

We thank Elisabeth Pound and Chad Rogers for assistance with technical

editing. This work was supported by a Presidential Early Career Award


for Scientists and Engineers through the National Institutes of Health

(R01 EB006124).

Competing Interests Statement: The authors certify that they have no relevant

financial interests in this manuscript.

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