Microfluidic Immunoassays

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DOI: 10.1016/j.jala.2010.01.013

2010 15: 253Journal of Laboratory AutomationChun-Che Lin, Jung-Hao Wang, Hui-Wen Wu and Gwo-Bin Lee

Microfluidic Immunoassays

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Keywords:

microfluidics,

immunoassay,

immobilization,

lab-on-a-chip,

MEMS

Technology Review

Microfluidic Immunoassays

*CoEngRoa633

153

Cop

doi

Chun-Che Lin, Jung-Hao Wang, Hui-Wen Wu, and Gwo-Bin Lee*Department of Engineering Science, National Cheng Kung University, Tainan,

Taiwan, R.O.C.

Immunoassays have long been widely used in a variety of

applications, such as for medical diagnostics,

pharmaceutical analysis, environmental, food safety

testing, and for basic scientific investigations because of

its simplicity, sensitivity, and specificity. Microfluidic

systems, also well known as a ‘‘lab-on-a-chip’’ or

a ‘‘micro-total-analysis-system’’ have attracted a lot of

attention in the past two decades because of advantages

associated with miniaturization, integration, and

automation. A promising platform for the combination of

these two technologies, microfluidic immunoassays, has

been extensively explored in recent years. The aim of this

article is to review recent advancements in microfluidic

immunoassays. A brief introduction to immunoassays and

microfluidic devices will include a literature review,

followed by an in-depth discussion of essential techniques

in designing a microfluidic-based immunoassay from

different perspectives, including device substrates,

sample/reagent transportation, surface modification,

immobilization, and detection schemes. Finally, future

perspectives on microfluidic immunoassays will be

provided. These developments with microfluidic immu-

noassays may provide a promising tool for automatic,

sensitive, and selective measurements in practical

applications. ( JALA 2010;15:253–74)

rrespondence: Dr. Gwo-Bin Lee, Professor, Department ofineering Science, National Cheng Kung University, 1, Universityd, Tainan, Taiwan 701, R.O.C.;Phone: þ886.6.2757575, ext.47; Fax: þ886.62761687; E-mail: [email protected]

5-5535/$36.00

yright �c 2010 by The Association for Laboratory Automation

:10.1016/j.jala.2010.01.013

by Nan Hallock on Jajla.sagepub.comDownloaded from

INTRODUCTION

Immunoassays

Antibodies (Abs) are proteins produced in ani-mals and human bodies by immunological responsesto the presence of allochthonous substances calledantigens (Ags). They have a highly specific affinityfor these Ags in nature. Each Ab has a unique struc-ture recognized by a corresponding Ag in a lock-and-key mechanism. Immunoassays have a varietyof formats, all of which make use of the sensitivityand specificity of this AbeAg interaction, which al-lows for the quantification and monitoring of smallmolecules, such as drugs and metabolites,1 large pro-teins,2 nucleic acids,3 and even whole pathogens.4

They have been widely used in clinical analysis, foodsafety and environmental monitoring, and basic bio-technological investigations.

Immunoassays can be used to detect either Abs orAgs according to the needs of the experiments.Generally, immunoassays can be classified as eithera competitive or a noncompetitive format.5 In thecompetitive format as shown in Eqs. (1) and (2), theunlabeled Ags competes with labeled Ags (Ag*) fora limited number of Ab-binding sites. As the amountof unlabeled Ags in a sample increases, the amount oflabeled Ags bound to the Abs decreases, resulting ina decrease in the detection signal if the Ab-boundAgs (Ag*�Ab) are detected, or an increase in signal,if the labeled free Ags are detected.

nuary 30,

AgþAb4Ag�Ab ð1Þ

Ag � þAb4Ag � �Ab ð2Þ

Alternatively, in the noncompetitive format, asshown in Eq. (3), Ags in a sample conjugate withan excessive amount of labeled Abs (Ab*) to form

JALA June 2010 2532012

mailto:[email protected]


Technology Review

a complex that is strongly dependent on the number of Ags,resulting an increase in the detection signal as the Ags in thesample increases.

254

AgþAb � ðexcessÞ4Ag�Ab � þAb � ðexcessÞ ð3Þ
Immunoassays can also be divided into heterogeneous andhomogeneous formats.5 In a heterogeneous format, Abs orAgs are immobilized on a solid substrate where thecomplex forms. On the other hand, in a homogeneousformat, the conjugation takes place in the solution phase.Heterogeneous immunoassays take advantage of the highsurface area/volume ratio and result in good performancein sensitivity. However, inextricable steps are sometimesrequired for the immobilization of Abs or Ags on the solidsubstrate. Homogeneous immunoassays benefit from themultiplexing and fast electrophoretic separations. However,preconcentration steps are usually required because thesurface area of the substrate is not used for immobilizationof Abs or Ags. Both formats have been extensively studiedand can be easily implemented in microfluidic devices.This section only provides a brief introduction toimmunoassays. Detailed information about immunoassayscan be found in the literature.6e8
Microfluidics

Microfluidic systems fabricated by microelectromechani-cal systems (MEMS) technology are now usually referredto as ‘‘lab-on-a-chip (LOC),’’ ‘‘biochips,’’ or ‘‘micro-total-analysis-system.’’ They are often envisioned as miniaturizedversions of their large-scale counterparts. These miniaturizedsystems can carry out entire protocols traditionallyperformed in a laboratory. Sample pretreatment, sample/re-agent transport, mixing, reaction, separation, detection, andproduct collection can all be performed automatically ona single LOC system. Functional microfluidic devices, suchas micropumps, microvalves, microfilters, microreactors,and microseparators can be microfabricated and even inte-grated to perform a specific assay. The advantages of thesedeveloped LOC systems include less sample/reagentconsumption, a reduced risk of contamination, enhancedsensitivity, less unit cost, lower power consumption, anda higher reliability and functionality. More importantly, por-tability arising from their compact form is a key factor forpoint-of-care (POC) applications. Despite these advantages,there are still potential limitations, such as bubbles formationand dead volume in microfluidics that need to be addressed.The bubbles formed in microdevices disrupt the continuity ofthe liquids and may result in poor performance. This isparticularly serious in electrophoresis-based microdevicesbecause the bubbles disturb the applied electric field.9 Deadvolume is another key issue in microfluidics. In the micro-scale environments, dead volume may cause serious contam-inations and hence poor results.10 The works in LOC havegrown rapidly in the past two decades, and many review

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articles are available.11e15 In this article, only recent ad-vancements in microfluidic immunoassays are covered here.

Immunoassays in Microfluidic Systems

The process in most immunoassays includes a series ofwashing, mixing, and incubation steps, which are labor inten-sive and time consuming, which often takes several hours,sometimes even up to 2 days to perform one single assay.Most of the time required in a long immunoassay is mostlybecause of the long incubation time attributed to inefficientmass transport for the immunoagents to move from a solu-tion to the surface where the conjugation occurs becausethe immunoreaction itself is relatively rapid.16 Moreover,the immunoagents used in immunoassays are relativelyexpensive. The consumption of the immunoagents can begreatly reduced if the system is miniaturized. Therefore, thereis a demand to develop an automated and miniaturized plat-form for immunoassays. Such a platform must be capable ofsimplifying procedures, reducing the assay time and sample/reagent consumption and enhancing the reaction efficiency.The advantages of the microfluidic systems described previ-ously fulfill these important criteria for immunoassays.Therefore, extensive investigations using microfluidics forperforming immunoassays have been reported recently. Thefollowing sections introduce some representative originalarticles in microfluidic immunoassays published betweenthe years 2005 and 2009. It should provide readers a compre-hensive understanding of this promising technology.

SUBSTRATE MATERIALS FOR MICROFLUIDIC

IMMUNOASSAYS

Microfluidic devices for immunoassays can be fabricatedfrom a variety of materials. The most commonly used sub-strate materials are silicon, glass, and polymers. Each ofthese materials has its own advantages and limitations. Inthis section, a brief introduction to these materials is firstgiven and a comparison of material properties is listed inTable 1.

Silicon

Silicon is used as a material for fabricating microfluidicdevices because of the well-established microfabrication pro-cesses developed by the microelectronic and the MEMS in-dustry and from extensively studied surface chemistry.Furthermore, it possesses good thermal conductivity and isresistant to high temperatures; therefore, is suitable for appli-cations requiring a relatively high operating temperature,such as for a polymerase chain reaction (PCR) and forbioreactions. The most popular fabrication method forsilicon-based microfluidic structures is wet etching,17 usuallyresulting in microstructures with low aspect ratio featuresthat may be a limitation in most applications. This issuemay be addressed by using dry etching,18 which is a relativelyexpensive microfabrication process. Moreover, silicon is notoptically transparent and is electrically conductive, thus

an Hallock on January 30, 2012


Table 1. Comparison of silicon, glass, and polymer as materials for microfluidic immunoassays

Silicon Glass Polymer

Development period Early Later Latest

Surface chemistry Well developed Well developed Well developed

Thermal conductivity Good Good Poor

Endurance to high temperature Good Good Poor

Structure aspect ratio Low (may be high if dry

etching has been adopted)

Low (may be high if dry

etching has been adopted)

High to low

Optically transparenta No Yes Yes

Electrical conductivity Yes No No

Substrate cost Expensive Less expensive Low cost

Fabrication cost Expensive Less expensive Low cost

Mass productionb Yes Yes Yes

aAlthough glass and polymers are optically transparent, they may adsorb light in the UV spectrum.bAlthough all the materials are capable of being mass produced, the fabrication processes for silicon and glass are relatively slow. For polymers, replication methods for PDMS are not as convenient asthose used for PC, PS, and PMMA in practical and commercial aspects.

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resulting in limitations for optical and electrochemical detec-tion and for electrokinetic transportation, which are key is-sues with microfluidic immunoassays. More importantly,silicon substrates are relatively expensive when comparedwith other materials, such as glass and polymers. Further-more, the fabrication process for silicon-based microfluidicdevices involving substrate cleaning, resist coating, photoli-thography, development, and wet/dry etching is relativelytime consuming and costly. These limitations hinder itspractical applications in commercial immunoassays.

Glass

In contrast to silicon, glass substrates are less expensive,optically transparent throughout the visible spectrum, andnot electrically conductive. These properties overcome someof the problems associated with silicon-based microfluidicdevices. Therefore, glass seems to be an ideal substrate forperforming microfluidic immunoassays. However, glass-based microfluidic devices are usually fabricated by a wetetching method,19 which still limits the etched microstruc-tures to low aspect ratio features. Again, some expensivedry-etching processes for glass can be applied to address thislimitation.20

Polymers

Polymers are alternative materials to silicon and glass inmicrofluidic immunoassays because of their relatively lowcost and simple fabrication process. Many polymers havebeen used in microfluidic applications, such as polymethylmethacrylate (PMMA),21 polycarbonate,22 polystyrene(PS),23 and polydimethylsiloxane (PDMS).24 Among thesepolymers, PDMS is one of the most commonly used mate-rials for microfluidic immunoassays in recent studies becauseof its desirable characteristics, such as flexibility, opticaltransparency (down to 230 nm), and biocompatibility.PDMS is a polymer composed of repeating OSi(CH3)2monomers, resulting in a hydrophobic surface, which is the

by Njla.sagepub.comDownloaded from

major limitation when used with immunoassays because ofthe nonspecific adsorption of proteins and other molecules,poor wettability with aqueous solutions, and the formationof bubbles.25 This disadvantage, however, can be easilyovercome by bulk/surface modification and well-developedfunctionalization techniques.26e31

Heterogeneous Substrate Materials

Recently, heterogeneous materials formed by using twodifferent types of materials for microfluidic devices have beenextensively explored. For example, PDMS/glass32 andsilicon/glass33 are commonly used for forming microfluidicdevices. These hybrid microfluidic devices provide advan-tages attributed to their component materials. For example,a microchip made of PDMS and glass can fulfill the require-ments of fast fabrication, low cost, flexibility, biocompatibil-ity, and good thermal conductivity.

SAMPLE/REAGENT TRANSPORT

Liquid transport plays an important role in microfluidic im-munoassays because the performance of the liquid drivingsystem can directly affect the results of the assays. Manystrategies for on-chip liquid transport in microfluidicimmunoassays have been reported in the literature, such aselectroosmotic,34,35 electrowetting,36e38 pneumatic,39e42 cen-trifugal,43e48 power-free,49 piezoelectric,50 and thermopneu-matic51 approaches. A comparison of these on-chip liquidtransport strategies is listed in Table 2. External power sour-ces, valve integration, and flow rates for each pumpingmethod are also listed.

Electroosmotic Pumping

Electroosmotic pumping based on electroosmotic flow(EOF)34 is a commonly used liquid transport strategy in mi-crofluidic systems because of the ease of automation and nomoving parts are required. EOF is the bulk flow resulting

JALA June 2010 255an Hallock on January 30, 2012


Table 2. Comparison of strategies for on-chip sample/reagent transportation in microfluidic immunoassays

External power source Valve integration Flow rate Refs.

Electroosmosis Electrical voltage Yes nL/s to mL/s 34,35

Electrowetting Electrical voltage Noa NAb 36e38

Micropump Compressed air pump Yes mL/min to mL/min 39e42

Centrifugal force Rotary motor Yes nL/s to submicroliter/s 43e48

Power-free Not necessary Yes nL/s 49

aAlthough on-chip valves are not integrated in the electrowetting-based transport method, liquids can be immediately stopped when the electric voltage is not applied.bThe flow rate for electrowetting is normally expressed as a flow velocity, and ranges from 0.01 to 0.1 m/s for a droplet with a radius less than 10�3 m.

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from the effect of the electric field on the solution doublelayer at the channel wall. Counterions (typically, cations)building up near the wall surface to maintain charge balanceform the double layer and create a potential difference veryclose to the wall surface, which is known as the zeta poten-tial. The zeta potential is determined by the surface chargeon the channel wall. Because this charge is strongly pH de-pendent, the magnitude of the EOF varies with pH. The zetapotential is also dependent on the ionic strength of thebuffer. Increased ionic strength results in double-layer com-pression, decreased zeta potential, and hence reduced EOF.As the electric field is applied, the cations forming the diffusedouble layer are attracted toward the cathode. Using thismechanism, the steps for flow switching, sequencing, andstopping can be easily achieved by controlling the appliedelectric field. For example, Gao et al.35 developed a microchipfor simultaneous detection of multiple microbial Ags basedon electroosmotic pumping. As shown in Figure 1, Ags werefirst adsorbed onto a PDMS-coated glass slide by using a mi-crofluidic network, followed by bonding to another PDMSslab-bearing H-shaped microchannels designed for deliveringsolutions of the primary Ab, the washing buffer, and the sec-ondary Ab. The sequential pumping of these solutions wasachieved by using a programmable high voltage sequencerequipped with eight independent outputs. Using this method,the determination for Escherichia coli (E. coli) lysate Ag hasbeen achieved with a detection limit of 3 mg/mL. Moreover,the simultaneous detection of E. coli and Helicobacter pylori(H. pylori) lysate Ags has also been achieved. This microflui-dic system represents the potential for the simultaneousdetection of multiple pathogenic infections.

Electrowetting Force

Electrowetting is a liquid transport strategy whereby liquiddroplets are manipulated in the presence of programmed volt-age sequences applied to an electrode array.36 It can be a prom-ising platform for performing immunoassays. For instance,combining electrowetting and magnetic microbead-based im-munoassays, Sista et al.37 demonstrated a microfluidic chipfor performing heterogeneous immunoassays. As shown inFigure 2, a sample droplet and a reagent droplet containingcapture Ab-conjugated magnetic beads, reporter-conjugatedAbs, and blocking proteins were dispensed on the surface ofthe chip. These two droplets were then merged, mixed, and

256 JALA June 2010 by Njla.sagepub.comDownloaded from

incubated by electrowetting-based transport, followed by ap-plying a magnetic field to capture the sandwiched microbeadcomplexes. Then, unbound components were split from themerged droplet, followed by the introduction of the washingbuffer. The splitting and washing steps were repeated severaltimes to thoroughly remove unwanted components. Finally,a reagent droplet was added for chemiluminescent detection.Using this electrowetting system, a sandwiched heterogeneousimmunoassay on human insulin and interleukin-6 (IL-6)has been successfully performed within 7 min. Thiselectrowetting-based microfluidic platform can be also suc-cessfully used for the identification of a cardiac marker, tropo-nin I, from whole blood samples,36 sample preparation forbacterial pathogens indicating infectious diseases,36 and theextraction of human genomic DNA.38 It is believed that theelectrowetting-based microfluidic immunoassay is adaptablefor POC applications because of its relatively low cost andsimple instrumentation.

Micropneumatic Pumping

Themicropneumatic pump is composed of a liquid flowmi-crochannel, flexible PDMS membranes, and air chambers topneumatically deflect the PDMS membranes in sequence viathe introduction of compressed air.39e42 As shown inFigure 3, the peristaltic effect driven by the time-phased deflec-tion of PDMSmembranes along themicrochannel can be usedto deliver liquid in themicrochannel. It has been demonstratedthat micropneumatic pumps, incorporated with microvalves,can be used to determine the presence of Abs specific to hepa-titis C virus and syphilis from serum samples.39 In this work,theAgwas first coated on the surface of a detection area. Then,serum samples, a washing buffer, a horseradish peroxidase(HRP)-labeled secondary Ab, a developing buffer, and a stop-ping buffer in individual reservoirs were sequentially trans-ported to the detection area by the micropneumatic pump,followed by optical detection via the measurement of absor-bance. Usually it takes at least three electromagnetic valvesto control the sequential deflection of the PDMS membranes.Alternatively, similar micropneumatic pumps have beenreported, in which different-sized air chambers were intercon-nected by air channels to transport liquids in microchannels.40

Because the interconnecting air channels allowed for thesequential actuation of the air chambers, this generated a peri-staltic-like activation of the PDMS membranes, only one



Figure 1. The fabrication process of the microchip for simulta-neous detection of multiple microbial antigens based on electroki-netic pumping. (AeB) Antigen immobilization and (C) bondingwith a PDMS slab-bearing H-shaped microchannels and formingthe reaction regions. Reprinted from Ref. 35 with permission fromSpringer ScienceþBusiness Media (Copyright 2009 Springer Scien-ceþBusiness Media).

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electromagnetic valve was required to perform the pumping.By using this micropneumatic pump, bead-based flowcytometry using immunoassays was performed in a micro-chip.41 The delivery of virus samples, a washing buffer,virus-specific-Ab-conjugated magnetic beads, dye-labeledAbs, and sheath flows for focusing the sample stream wereall transported by themicropneumatic pump.Using thismicroflow cytometer chip, the entire process, including sample/


reagent delivery, mixing, incubation, washing, detection, andthe collection of beadswas completedwithin 40 min. Recently,anothermicropneumatic pumpwas reported,whichwas incor-porated with a normally closed valve for generating highpumping rates and to prevent any backflow from the destina-tionwells after releasing the compressed air.42Using this liquidtransport method, it avoided contamination from the wastechambers, and this design is expected to be applicable formanyfuture microfluidic applications.

Centrifugal Force

The centrifugal force has been used for liquid transport incompact disc (CD)-based immunoassays in recent years, andsome review articles are available.43,44 Immunoassays ona CD are a promising platform to realize many POC applica-tions by embedding many analytical functions performed ina laboratory, such as sample/reagent transport, metering, di-lution, mixing, separation, and detection. All these functionscan be performed automatically in a disc format. It has beenexpected to be a powerful platform for medical and clinicaldiagnostics. It has great commercial potential because of sim-ple instrumentation, ease for economic mass production, andeasy adaptation to existing detection methods, particularlythose based on optical detection. Moreover, with the abilityto perform simultaneous and identical functions in parallellayouts, immunoassays on a CD are an ideal platform forhigh throughput screening of analytes. Figure 4 gives a sche-matic illustration of a CD-based sandwich enzyme-linked im-munosorbent assay (ELISA). The key concept is to overcomethe capillary force, which prevents the liquid in one chamberfrom moving out, by using the centrifugal force controlled bythe rotational speed of the CD. Furthermore, the increasingcentrifugal force from the center toward the edge of the CDis used for flow sequencing which replaces the stepwiseprocedures in a conventional ELISA. Not only can ELISAbe performed on this CD-based platform but also otherimmunoassays can be realized using a very similar approach.For more detailed information such as the operatingtheory and instrumentation, readers can refer to variousreview articles.43,44

This section will focus on some representative works onCD-based immunoassays published between the years 2005and 2009. The first centrifugal fluidic platform was developedin 196945 and has been extensively studied since then. Compa-nies, such as Gyros AB, Tecan, and Burstein Technologiesalso developed commercial products. Recently, Gyros ABdeveloped a CD-based immunoassay for the determinationof a-fetoprotein (AFP), IL-6, and carcinoembryonic antigen(CEA). The disc structure is an intricate web of microchannelsand reaction chambers packed with streptavidin-coated PSmicroparticles for conjugating with biotinylated Abs againstAFP, IL-6, and CEA. Alexa 647-labeled bovine serum albu-min (BSA) was used as a detection Ab. All the flow controlis through the rotation of the CD mounted on a workstationequipped with a detection system (laser-induced fluorescence,



Figure 2. Merging (A), incubation (B), immobilization (C), splitting (D), and washing (E) processes in an electrowetting-based immunoassay.Reprinted from Ref. 37 with permission from the Royal Society of Chemistry (RSC) (Copyright 2009 The Royal Society of Chemistry).

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LIF) and robotic arms for dispensing liquids into the inlets onthe CD. The experimental results showed that the detectionlimits for AFP, IL-6, and CEA were 0.15, 1.25, and1.31 pmol/L, respectively.46 The inter- and intra-assay coeffi-cients of variation were less than 10% and 20%, respectively.This disc also demonstrated an excellent analytical efficiency(104 assays in 50 min) when compared with the performanceof traditional 96-well ELISA plates (96 assays, a few hours).Moreover, the relationship between the fluorescent imagingprofiles of the column structure and the kinetic properties ofthe Ag and Ab was also investigated.

Although the disc developed byGyrosAB gives an excellentanalytical efficiency within acceptable variations, an expensiverobotic unit is inevitably required. For practical applications,a cheap and fully automated CD immunoassay withouta robotic system is required. To achieve this, valves capableof precisely controlling the flow sequences in an immunoassayare required. For instance, a disc with superhydrophobic capil-lary valves was reported to perform the sequential flow steps.47

A capillary valve is themost popular valving component in CDimmunoassays because it requires no moving parts or externalactuation. Unfortunately, proteins present in solution may ad-sorb onto conventional capillary valves to gradually makethem hydrophilic, leading to the failure of the valve. To solvethis problem, polyaniline nanofibers along with a noncrystal-line fluorine coating to create superhydrophobic nanostruc-tures as valves have been reported.47 Measurements showed


that the contact angle before and after protein blocking were170� and 165�, respectively, indicating that these valvesmaintain their superhydrophobic properties after treatmentwith protein solutions. This excellent result is because of the in-creased surface roughness and decreased surface energy attrib-uted to the coating of polyaniline nanofibers andnoncrystallinefluorine, respectively. Furthermore, to improve the detectionsensitivity of the assay, the detection surface was sequentiallytreated by an oxygen plasma, polyethyleneimine (PEI), tyrosi-nase, and protein A to increase its binding capacity based onorientation-controlled immobilization. This disc integratesfunctions, including pumping, valving, fluid splitting, washing,and mixing. Automation, without using a robotic unit, wasachieved because the primary Ab and blocking protein werepreimmobilized onto the detection area. The substrate, conju-gate, washing, secondaryAb, andAg solutionwere loaded intocorresponding wells for flow sequencing. Comparing witha conventional 96-well ELISA plate, this disc has advantagesthat included minimized sample/regent consumption, shorterassay time, and a broader dynamic range.

Another strategy to control the transport of liquid ina CD immunoassay is to use active valves. For example,a disc with a laser irradiated ferrowax microvalve (LIFM)that can be actively controlled to open and close by laserirradiation was reported.48 LIFMs are valves made of nano-composite materials in which nanosized iron oxide particlesare dispersed in paraffin wax. They are phase change-based



Figure 3. The sequential deflection of PDMS membranes in a micropneumatic pump.

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microvalves and can be fabricated as normally open, nor-mally closed, and reversible microvalves. Low-intensity laserlight can melt the paraffin wax with the embedded iron oxidenanoparticles, whereas laser light with a high intensity can-not melt the wax. Using this mechanism, the LIFMs canbe actively opened or closed by the irradiation of laser lightduring the assay process. This disc contains plasma separa-tion channels and chambers for a substrate solution, washingbuffer, stopping buffer, mixing, waste, and detection. Thesechambers are connected by microchannels where LIFMsare fabricated in a normally open or normally closed state.Figure 5 shows a schematic illustration of the disc designand the reaction steps involving in the active opening andclosing of the LIFMs. In this disc, valves 3, 6, 9, and 12are the normally open LIFMs where all other valves arethe normally closed LIFMs. Using these LIFMs togetherwith precoated PS microbeads, the concentrations of theantibody to the hepatitis B surface antigen (anti-HBs) andhepatitis B virus surface antigen (HBsAg) from whole blood


samples can be determined in an automated manner within30 min without using robotic arms, which is a much shortertime when compared with conventional ELISA plates (longerthan 90 and 120 min for anti-HBs and HBsAg, respectively).

Power-Free Transport

Most of the liquid transport devices require externalpower sources to drive the liquids. For example, the electro-wetting method requires the application of a voltage, the mi-cropneumatic pump needs compressed air generated bya bulky pump to deflect the membranes, and the centrifugalforce-based transport method uses a rotary motor to spin theCD. Taking into consideration the demands of POC applica-tions, a power-free liquid transport method, such as thatused in commercial immunoassay strips, is also a promisingalternative. For example, Hosokawa et al.49 developeda power-free injection method for a microfluidic immunoas-say by air evacuation. As shown in Figure 6, a microchip



Figure 4. ELISA in a lab-on-a-CD. (A) The 4-array CD-ELISA design, and (B) a single assay. Chambers 2, 4, 6, and 8 are loaded withwashing buffer, whereas chambers W, D, 1, 3, 5, 7, 9 are designed for waste, detection, first antibody, blocking solution, sample, secondantibody, and substrate, respectively. As the CD rotates, the solutions in chambers 1 to 9 flow out sequentially because of the decreasingcentrifugal force and then complete the ELISA procedures. The most common valves are hydrophobic and capillary valves. Hydrophobicvalves stop the flow by the hydrophobic surfaces created in the microchannel, and capillary valves stop the flow by a capillary pressurebarrier at junctions where the channel diameter suddenly expands.

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composed of PDMS microchannels and a glass bottom platewas first degassed in a vacuum system to store the pumpingenergy, followed by covering them with a piece of adhesivetape. When this microchip was brought back to atmosphericconditions, air dissolved into the PDMS flows again throughthe walls of the microchannels and reservoirs. Once analiquot was pipetted at the inlet of the microchannel, thedissolution of air reduces air pressure in the microchannel-reservoir system, and hence pumps the solution into the mi-crochannel. Because the inlet of the microchannel is narrowand wettable, a capillary retention valve is naturally formedto prevent the microchannel from drying out. When a secondaliquot was pipetted at the inlet of the microchannel, thepumping effect occurred again. Using this method, sampleand reagents required for performing an immunoassay weresequentially injected into the microchannels for immobiliza-tion, blocking, washing, reaction, and detection. It has beenused to demonstrate the detection of rabbit IgG and humanC-reactive protein (CRP) with a detection limit of 0.21 and0.42 nM, respectively, which can be performed within20 min. Power-free liquid transportation is promising be-cause one of the ultimate goals for microfluidic immunoas-says is to perform these chips for POC applications outsidethe laboratory with minimum support equipment.

HETEROGENEOUS MICROFLUIDIC IMMUNOASSAYS

Immobilization is a key step in heterogeneous immunoassaysbecause it greatly influences the specificity and sensitivity.Briefly, the surface of these microfluidic chips has to betreated so that proteins can be immobilized for further detec-tion. In general, heterogeneous microfluidic immunoassayscan be classified as surface-based and bead-based


immobilization methods. In heterogeneous microfluidic im-munoassays, the Abs or Ags are usually immobilized onthe surfaces of the microchannels or microbeads. Immobili-zation on surfaces of microchannels requires additional stepsduring the microfabrication process and may suffer frompoor reproducibility and reliability. In contrast, microbead-based immobilization can be performed outside themicrochip and offers a significantly large surface for immobi-lization, and hence improved performance of theimmunoassays.52e54

Surface-Based Immobilization

Many surface-based immobilization strategies have beendeveloped for microfluidic immunoassays. Different substratematerials, such as PDMS,55e59 cycloolefin-copolymer,60

PS,61,62 silicon nitride (SN),63 and PMMA,64 have been re-ported for use in microfluidic devices. Among them, surfaceimmobilization on PDMS will be emphasized in this sectionbecause it is now the most popular material for fabricatingmicrofluidic devices. For example, a method called ‘‘macro-molecules to PDMS transfer’’ was reported to directly entrapmacromolecules for capturing target analytes during thePDMS polymerization step.55 In this work, rabbit IgG oranti-CRP solutions were spotted on a Teflon master by a pie-zoelectric arrayer, followed by pouring liquid PDMS on top.The PDMS membrane was then peeled off after curing, andthe molecules (rabbit IgG or anti-CRP) were thus transferredto the PDMS membrane. The rabbit IgG and anti-CRP-conjugated PDMS membranes were then used to capturerheumatoid factor (RF) and free CRP in human serum sam-ples, respectively, followed by conjugating with peroxidase-labeled detection Abs for chemiluminescence detection. Usingthis method, the sensitivities for CRP and RF measurements



Figure 5. (A) Schematic illustration of the disk design. (BeG) The reaction principle of the CD immunoassay to detect anti-HBs. The light-ning symbols indicate the laser irradiation on the LIFMs. Reprinted from Ref. 48 with permission from the Royal Society of Chemistry (RSC)(Copyright 2009 The Royal Society of Chemistry).

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were 12.5 mg/L and 5.3 IU/mL, respectively. Due to itssimplicity and efficiency for immobilization, this methodhas a great potential to be applied to fabricate PDMS-based microchip for immunoassays.

Alternatively, Hashimoto et al.56 developed an electro-chemical method to perform localized immobilization of pro-teins within the channels of a microchip. In this work, a layerof PDMS containing micropost structures was first attachedto the inner surface of an upper glass substrate by an oxygenplasma treatment before assembling with a bottom glass sub-strate where Pt electrodes were deposited. After assembly,a solution containing PEI and heparin was introduced toelectrostatically form a PEI/heparin multilayer on the wallof the channel. To obtain an area for protein immobilization,the channel was filled with a solution of potassium bromide,followed by applying an electrical pulse to the patternedelectrodes to locally produce Br2 and HBrO. The oxidationaction of HBrO then caused the PEI/heparin multilayer tolocally detach from the substrate, and the exposed area(PDMS) was thus ready for localized immobilization becauseheparin is resistant to the adhesion of many proteins. Using


this method, antiglutathione peroxidase (GPX) was locallyimmobilized to capture GPX, followed by conjugation withCy3-labeled anti-GPX for fluorescent detection. This break-through work provides a simple method for immobilizingproteins in predefined areas and is believed to be promisingfor surface immobilization.

Supported bilayer membranes (SBMs), such as phospho-lipid bilayer membranes are biomimetic membranes capableof providing long-term hydrophilic and protein-resistantproperties. Therefore, they have been reported for surfaceimmobilization. For example, Phillips and Cheng57 usedphosphatidylcholine membranes assembled on PDMS asa supported membrane for protein immobilization. In thiswork, a phosphatidylcholine membrane was first assembledon an oxygen plasma-treated PDMS surface, followed byvesicle fusion to form cell surface ligand GM1-integratedSBMs for capturing the cholera toxin (CT). Then, rabbitanti-CT and a dye-labeled goat antirabbit Ab were sequen-tially conjugated for fluorescent detection. Using thismethod, the entire assay can be completed within 25 minwith a detection limit of 8 fmol for the measurement of



Figure 6. Schematic illustration of a microchip based on power-free liquid transport. (A) The chip design, and (B) the pumpingmechanism. Reprinted from Ref. 49 with permission from theRoyal Society of Chemistry (RSC) (Copyright 2006 The Royal So-ciety of Chemistry).

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CT. The SBMs, however, may peel away from the solidsupport when exposed to air. Therefore, reinforced SBMs(r-SBMs) were developed to solve this problem and sensingof Staphylococcus enterotoxin B (SEB) was demonstrated.58

In this work, the supported bilayer was prepared by fusingvesicles containing biotin phosphoethanolamine and phos-phatidylcholine to the channels, followed by incubation witha streptavidin solution to form the r-SBMs. The biotinlyatedrabbit anti-SEB was then introduced to be immobilized onthe streptavidin-conjugated SBMs (r-SBMs) to captureSEB, followed by attaching sheep anti-SEB and a dye-labeled detection Ab for fluorescent detection. Using thismethod, a detection limit of 0.5 ng/mL was obtained. Moreimportantly, the r-SBMs-modified solid support is air stableand is expected to have the capability for long-term storagein a dehydrated format for disposable applications.

Another method for immobilization is to use dextran,which is a biocompatible polymer. The immobilization ofdextran on a microchip surface by introducing linkers forprotein immobilization is a less toxic and inexpensiveprocess. More importantly, the hydrophilicity and


immobilization efficiency can be enhanced because of theabundant oxidation-resultant aldehyde groups. For instance,Yu et al.59 used this dextran-based immobilization to simul-taneously detect three biomarkers. In this work, a solutioncontaining H2O and HCl was first introduced to oxidizethe surface of the PDMS channels, followed by the introduc-tion of a solution containing 3-aminopropyltriethoxylsilane(APTES) and ethanol to make the surface abundant withamine groups. Then, the partially oxidized dextran (with al-dehyde groups) solution was introduced to conjugate ontothe channel surface via the formation of shiff’s base betweenthe amines and aldehydes, followed by treating with NaIO4

to oxidize the dextrans again. The surface is now aldehydeabundant and is ready to react with the amine groups ofproteins. Capture Abs, such as anti-IL5, anti-HBsAg, andanti-rabbit IgG were immobilized in different channels tocapture IL5, HBsAg, and rabbit IgG, respectively, followedby introducing HRP-labeled detection Abs for colorimetricdetection. Using this method, a detection limit of 100 pg/mL was obtained for these three markers.

In addition to PDMS, dextran can be also used in a cyclo-olefin-copolymer substrate for protein immobilization. Forinstance, Jonsson et al.60 first treated a copolymer chip withan oxygen plasma and APTES to expose amine groups, fol-lowed by a sequentially treatment with partially oxidizeddextran and NaIO4 to make the channel surface aldehydeabundant. Then, capture Abs for CRP were immobilizedfor performing immunoassays. Using Alexa 647-labeleddetection Abs for fluorescent detection, a detection limit of2.6 ng/mL was obtained. These dextran-based immobiliza-tions are long-term stable, inexpensive, and capable of beingproduced in large quantities, resulting in the possibility forcommercialization.

Othermaterials, such as PS, SN, andPMMAhave also beenused as substrates for microfluidic immunoassays. For exam-ple, Darain et al.61,62 used different strategies to immobilizeproteins on PS substrates. In one work,61 the PS substratewas sequentially treated with ultraviolet (UV)/O3 (to introduceOH group), APTES (to introduceNH2 group), and glutaralde-hyde (to introduce CHO group), followed by conjugating withantimyoglobin for capturing myoglobin. In another work,62

the PS substrate was sequentially treated with sputtered gold(to introduce a Au surface), 16-mercapto-hexadeconic acid(to introduce COOH groups), and N-hydroxy succinimideester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (to introduce active NSH esters), followed byconjugatingwith antihorse IgG, for capturing horse IgG.Afterfluorescent detection, the detection limit for myoglobin andhorse IgG was obtained as 16 ng/mL and 0.71 mg/mL, respec-tively. These two PS-based immobilization strategies can beextended for the measurement of other important biomarkersfor diagnostic applications.

SN has been widely used in the semiconductor industrybecause of its excellent chemical, electrical, and opticalproperties. It can also be used as a substrate for microfluidicimmunoassays. For instance, He et al.63 used SN as the



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substrate for immunosensing of CRP in two formats. Thefirst format is a two-layer system in which anti-CRP was di-rectly patterned on a SN-coated silicon wafer as the firstlayer to capture dye-labeled CRP; the second format isa four-layer system in which biotinylated BSA was first ad-sorbed on a SN-coated silicon wafer, followed by sequentialconjugation with avidin, biotinylated anti-CRP, and dye-labeled CRP. The biotinylated BSA works as the firstadhesion layer and as a blocking material as well. Therefore,another blocking solution is not required in this format.Using 1.76 mM of CRP as the analyte, the fluorescence inten-sity obtained from the four-layer system is 3.2-fold strongerthan that obtained from a two-layer system.

PMMA has been used as a substrate for microfluidicdevices because of its relatively low cost and optical transpar-ency. For example, Wang et al.64 used PMMA microchan-nels filled with an Al2O3 solegel network to encapsulatecapture Abs for immunoassays. In this work, the innersurface of the PMMA microchannel was first treated witha copolymer to bear silanol groups, followed by introducinga solution containing anti-IgG and Al2O3 solegel to allowpolycondensation between the solegel and the silanolgroups. The anti-IgG was thus encapsulated in the networkfor capturing target IgG. After conjugating with alkalinephosphatase (ALP)-labeled secondary Abs, electrochemicaldetection was used with a detection limit of 1 pg/mL. Theexcellent sensitivity is attributed to the high surface area-to-volume ratio of the network. Table 3 summarizes theaforementioned surface-based immobilization strategies.The substrate selection, target analyte, immobilizationscheme, detection strategy, and detection limits have all beensummarized.

Bead-Based Immobilization

As mentioned previously, microbead-based immobiliza-tion offers a large surface area and hence may improve theperformance of the immunoassays. In the following sections,bead-based immobilization methods classified as either non-magnetic or magnetic microbeads in microfluidic immunoas-says will be reviewed and discussed.

Nonmagnetic Microbeads. The most commonly used non-magnetic microbeads in microfluidic heterogeneous immuno-assays are PS beads and glass beads because of theirextensively studied characteristics. For example, Ko et al.65

developed a microfluidic electroimmunosensing chip forsimultaneous determination of cancer biomarkers, includingAFP, CEA, and prostate-specific antigen by a combinationof Ab-conjugated PS beads (for immobilization), goldnanoparticles (for labeling), and silver enhancer (for signalamplification). In this work, PS beads were coated withstrepavidin for conjugation with biotinylated capture Abs.These capture Ab-conjugated beads were then introducedand trapped in the reaction area by PDMS micropillars,followed by introducing samples containing cancer markers,


gold nanoparticles-labeled detection Ab, and silver enhancer,respectively. Gold nanoparticles were used to catalyze thereduction of silver ions into metallic silver, which was thendeposited onto the gold nanoparticles and enlarged theparticles to 30e100 nm. Finally, an electrical bridge wasformed between the underlayered Pt electrodes and increasedthe electrical path, resulting in a decrease in resistance(Fig. 7). Compared with conventional immunoassays, theanalysis time was reduced from several hours to 55 min.

Alternatively, Lee et al.66 used glass beads to detect a bac-terial pathogen. In this work, glass beads were sequentiallytreated with 3-aminopropyl trimethoxysilane, glutaralde-hyde, and a capture Ab (anti-O157 Ab). These beads werethen packed in spatially isolated microchambers, followedby introducing L-lysine solution for blocking unreacted sur-faces. Samples containing E. coli O157:H7 expressing greenfluorescence protein were then introduced and followed byfluorescence detection.

Most of the previous works only used the outer surface ofthe beads for immobilization. To improve the sensitivity ofan immunoassay, Yang et al.67 used superporous agarosebeads (superpores with diameters ranging from 10 to 80 mm)as a solid support for performing microfluidic immunoassays.In this work, superporous agarose beads were sequentiallytreatedwith proteinAand anti-goat IgG to capture target goatIgG, followed by treatment with ALP-conjugated anti-goatIgG and 5-bromo-4-chloro-3-indolyphosphate /nitroblue ter-azolium for colorimetric detection. Using these superporousbeads as immobilization materials, a detection limit of100 pg/mL of goat IgG was obtained, which is 10 times moresensitive than conventional bead-based microfluidic immuno-assays.68 The improved sensitivity is mainly because of thelarge surface area attributed to the inner matrices of the super-porous beads.

Most nonmagnetic bead-based microfluidic immunoas-says require the fabrication of microstructures to retain thebeads for immunoreactions. Alternatively, Sivagnanamet al.69 used a brilliant strategy to retain the beads. In thiswork, positively charged APTES were patterned on a glasssubstrate, followed by the electrostatic assembly of nega-tively charged streptavidin-coated microbeads. These self-assembled streptavidin-coated microbeads were then usedto conjugate with a biotinylated capture Ab to capture thetarget Ags. Using this method, a target mouse IgG Ag wasdetected with a detection limit of 15 ng/mL and 250 pg/mLin a stop-flow and a continuous-flow mode, respectively. Thisapproach requires no microstructures to retain beads and ispromising for patterning complicated immobilization beadsfor multiplexed applications.

Bead-based immunoassays have also been used in flow cy-tometry since the 1980s,70 with an improved sensitivity, spec-ificity, and dynamic range.71,72 Recently, Holmes et al.53

developed a bead-based immunoassay performed on a micro-chip-based flow cytometer. In this work, commercial glycidylmethacrylate microbeads were sequentially treated with suc-cinic anhydride, protein A, and human IgG or rabbit IgG to



Table 3. Summary of surface immobilization strategies in microfluidic immunoassays

Ref. Substrate Target analyte Immobilization schemeDetection strategy

(detection limit)

55 PDMS 1) RF

2) CRP

PDMS/RF; CRP (direct entrapment) Chemiluminescent,

1) 5.3 IU/mL

2) 12.5 mg/L

56 PDMS GPX PDMS/(PEIþ heparin)/anti-GPX Fluorescent, 5 ng/mL

57 PDMS CT PDMS/SBMs/GM1 Fluorescent, 8 fmol

58 PDMS SEB PDMS/rSBMs/biotinlyated rabbit anti-SEB Fluorescent,0.5 ng/mL

59 PDMS 1) IL5

2) HBsAg

3) rabbit IgG,

PDMS/Dextran/anti-IL5; anti-HBsAg;

anti-rabbit IgG

Colorimetric,

1) 100 pg/mL

2) 100 pg/mL

3) 100 pg/mL

60 Cycloolefin-copolymer CRP cycloolefin-copolymer/dextran/anti-CRP Fluorescent, 2.6 ng/mL

61 PS Myoglobin PS/UVþO3/APTES/glutaraldehyde/anti-myoglobin Fluorescent, 16 ng/mL

62 PS Horse IgG PS/Au/16-MHA/NHS esterþEDC/anti-horse IgG Fluorescent, 0.71 mg/mL

63 SN CRP 1) SN/anti-CRP

2) SN/biotinylated BSA/avidin/

biotinylated anti-CRP

Fluorescent, less than 1.76 mM

64 PMMA IgG PMMS/Al2O3 solegel/anti-IgG (encapsulate) Electrochemical, 1 pg/mL

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form human IgG or rabbit IgG-conjugated microbeads, re-spectively. These microbeads were then reacted with samplescontaining Cy3-labeled anti-human IgG and Cy5-labeledanti-rabbit IgG to form the complexes, followed by analysisin a micro flow cytometer. The micro flow cytometer usednegative dielectrophoresis to focus the microbeads into a par-ticle stream, and the impedance and fluorescence signals weremeasured for indicating particle size and for obtaining scatterplots, respectively. The results were comparable to those ob-tained by a commercial flow cytometer. Although the datafrom measurements of particle sizes were not used (accuratesizing of beads with diameters ranging from 2 to 10 mm), ithas a great potential for bead encoding. Combining the beadencoding with fluorescence detection in a micro flow cytom-eter is believed to be a promising platform for multiplexeddiagnosis.

Magnetic Beads. Magnetic beads are popular for a fullyautomated detection system because they have a large surfacearea, are easy to manipulate by a magnetic field, and allow easyseparation of target analytes from the reaction mixture. Incontrast to nonmagnetic beads that usually require microstruc-tures to retain thebeads,magneticbeads canbe easily retainedatdesired areas by the application of a magnetic field.73e75 Forexample, Lien et al.76 developed an integrated microfluidicsystem for virus detection by using Ab-conjugated magneticbeads to capture target viruses. In this work, the target viruses(dengue virus serotype 2 and enterovirus 71) were firstcaptured by the Ab-conjugated magnetic beads for enrichmentunder a magnetic field. Then, the virus RNA was extractedand transcripted to complementary deoxyribonucleic acid,followed by an amplification process using a micro-RT-PCRmodule. The required components for the entire assay, such as


micropumps/microvalves for liquid delivery, microcoils formagnetic field generation, and microheaters/microsensors fortemperature control were all integrated on a single chip. Usingthis highly integrated microdevice, viruses were successfullypurified, collected, and detected in an automatic fashion witha detection limit of 102 pfu/mL. Compared with large-scaleequipment, this integrated microsystem can automaticallyperform mixing, incubation, purification, transportation, andnucleic acid amplification of a virus, making it a flexibleplatform for future diagnostic applications. Magnetic bead-based microfluidic chip for the separation and detection ofbacteria was also reported. For example, Qiua et al.77 useda U-type microchip in which anti-Staphylococcus aureus(S. aureus) and anti-Salmonella-modified magnetic beads weremagnetically immobilized to specifically capture S. aureus andSalmonella, followed by fluorescent detection throughfluorescein isothiocyanate-labeled anti-S. aureus and anti-Salmonella, respectively.

There are a lot of studies on magnetic bead-basedmicrofluidic immunoassays. Readers can refer to severalarticles for different applications, such as the determinationof AFP,78 cholera toxin subunit B,79 and multiplexedimmunoassays.80

HOMOGENEOUS MICROFLUIDIC IMMUNOASSAYS

Unlike heterogeneous immunoassays performed in microflui-dic devices, a concentration step is usually required inmicrochip-based homogeneous immunoassays because thesurface of the microchannel is not used for immobilization.Homogeneous microfluidic immunoassays are mostlyperformed in an electrophoresis-based microchip format,and many concentration strategies, such as the use of



Figure 7. Schematic illustration of the detection method based on immunogold silver staining. Reprinted from Ref. 65 with permissionfrom Wiley InterScience (Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA).

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a preconcentration membrane or isotachophoresis (ITP) tostack the target analytes have been used for improving thesensitivity of detection. For instance, Meagher et al.81 devel-oped an electrophoretic immunoassay-based microchipincorporated with a photopolymerized membrane for pre-concentration and polyacrylamide gel-filled microchannelsfor sample/reagent loading and Ab/complex separation. Asshown in Figure 8, protein biotoxins (sample) were loadedthrough a larger pore-size polyacrylamide gel to upstreamof the membrane, followed by trapping for preconcentrationunder the continuous application of an electric field. The ca-pability of the membrane to perform preconcentration arisesfrom its porous structure that allows the transport of bufferions but excludes the proteins. Then, dye-labeled Abs wereloaded and concentrated in the same area. After mixingand conjugating, unbound dye-labeled Abs and complexeswere separated in the separation channel in which a smallerpore-size polyacrylamide gel was filled on switching thedirection of the electric field. Using this method, the entireimmunoassay can be performed within 20 min with a minimalsample volume of less than 10 mL and a detection limit of lessthan 10 pM for SEB determination. This microchip was alsoused for the determination of collagen-cleaving enzyme ma-trix metalloproteinase-8 for rapid saliva-based clinical diag-nostics.82 Although the aforementioned microchip performsvery well for preconcentration and analysis of target analy-tes, a microchannel for the separation of unbound labeledAbs and the complexes was required. In another work,


Reichmuth et al.83 fabricated a membrane with proper pores(about 10 nm) in the microchannel. Due to these largerpores, this membrane allows the transport of both bufferions and unbound Abs but excludes the viral particles (about80 nm). Using this membrane-based concentration and filtra-tion method, rapid detection of swine influenza virus wassuccessfully performed within 6 min with a detection limitof 610 TruckeeeCarson Irrigation District (TCID) 50/mLwithout an electrophoretic separation.

ITP84 is a commonly used preconcentration method incapillary electrophoresis and microchip electrophoresis. Forinstance, Kawabata et al.85 developed an ‘‘electrokinetic an-alyte transport assay’’ microchip in which ITP was per-formed for on-chip concentration, mixing, and reaction,followed by electrophoretic separation and detection in thedownstream channel. As shown in Figure 9, an electropho-retic microchip was designed for the formation of plugs ofthe tailing buffer, the first Ab, the sample, the secondaryAb, and the leading buffer in the channel by the applicationof vacuum pressure at the waste wells. On the application ofan electric field between reservoirs 1 and 6, the first Ab wasconcentrated by ITP and migrated into the sample plug toform the first Abesample complex. This complex then mi-grated into a dye labeled-secondary Ab plug to form thesandwich complex (first Abesample-labeled secondary Ab).This sandwich complex was then separated from the othercomponents by using a microchip gel electrophoresisthrough the application of an electric field between reservoirs



Figure 8. Schematic illustration of the electroosmotic microchipincorporated with a preconcentration membrane and a separationchannel. S¼ sample, SW¼ sample waste, Ab¼ antibody,B¼ buffer (not used here), LB¼ loading buffer, RB¼ runningbuffer, RW¼ running waste. Reprinted from Ref. 81 with permis-sion from the Royal Society of Chemistry (RSC) (Copyright 2008The Royal Society of Chemistry).

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5 and 6. Using this ITP-integrated electrophoretic microflui-dic immunoassay chip, AFP was successfully detected within136 s with a detection limit of 5 pM. Similarly, Kagebayashiet al.86 also used a microchip together with a desktop instru-ment containing components functionalized for chip load-ing/transportation/storage, pipetting, vacuum applications,conductivity monitoring, electric field generation, sampleand reagent storage, and LIF detection to determine totalAFP from serum samples. Good correlation with a commer-cially available reference method (LiBASys) for 68 patientserum samples was obtained. In summary, these on-chipelectrophoretic immunoassays using membranes orITP-based concentration and gel separation have a great po-tential to provide a rapid, sensitive, portable, andcost-effective method for both clinical and researchapplications.

DETECTION IN MICROFLUIDIC IMMUNOASSAYS

Generally speaking, in an immunoassay, one of the immuno-agents must be conjugated with specific labeling molecules tofacilitate the detection of the analytes. The most commondetection techniques used in microfluidic immunoassays arefluorescent41,46,47,49,53,56e58,60e63,65,69,77,81e83,86 and lumines-cencent37,55 detections because of their excellent sensitivity.Electrochemical detection is also popular because of its capa-bility for miniaturization and simple instrumentation.64,65

Readers can refer to the recommended references for moreinformation about these detection methods. In the followingsections, recent works in microfluidic immunoassays usingdifferent detection strategies, except those previouslyaforementioned detection schemes, will be reviewed and dis-cussed. We mainly focus on label-free detection methods, in-cluding surface plasmon resonance (SPR), a thermal lensmicroscope (TLM), quartz crystal microbalance (QCM),


and wavelength-interrogated optical sensor (WIOS) schemes.Detection methods based on the labeling of gold nanopar-ticles (colorimetric detection) and magnetic microbeads(magnetoelectric detection) are also introduced. Thesemethods are classified as optical (SPR, TLM, WIOS, colori-metric detection) and nonoptical (QCM, magnetoelectronicdetection) detections in the following sections.

Optical Detection

Surface Plasmon Resonance. SPR is a powerful detectionstrategy for immunoassays because of its capability to performreal-time sensing of interactions between Ab and Ag withoutthe use of labeling molecules. Information, such as theconcentration of analytes, kinetics, and affinity can bereadily obtained. Recently, Lee et al.87 developeda microfluidic system integrated with 2-D SPR phaseimaging systems for microarray immunoassays (Fig. 10). Inthis work, the microfluidic device was composed of anarrayed sample/reagent delivering module for well-controlled and high throughput fluidic transportation anda microtemperature control module for maintaining thetemperature field within the arrayed detection area forreducing the thermal noise during SPR sensing. The fluidictransportation module was composed of arrayedmicrochannels, micropumps, microvalves, and flow sensors,whereas the temperature control module was composed ofmicromachine-based temperature heaters and sensorsfabricated on a soda-lime glass substrate. Using this system,the interaction of anti-rabbit IgG and IgG was successfullydetected and a detection limit of 1� 10�4 mg/mL for IgGwas obtained. Microfluidic devices based on SPR sensing forthe determination of disease markers88 and environmentaltoxins89 have also been reported. Furthermore, portable,compact, and low-cost SPR instruments, such as the Spreeta2000 developed by Texas Instruments Inc. are nowcommercially available. It is believed that combininga microfluidic immunoassay platform with these cheap andminiaturized SPR instruments provides a promising tool forpractical biochemical sensing applications.

Thermal Lens Microscope. Fluorescence detection is themost commonly used detection strategy in bioanalysis be-cause of its high sensitivity. However, the protocol for label-ing fluorophores is tedious and laborious. Alternatively,TLM is highly suitable for the quantization of an ultra-lowtrace amount of analytes in small-volume samples,especially for nonfluorescent samples.90 In TLM, thecoaxial beams of the excitation and the probe beam arefocused into the sample stream. The sample molecules areexcited by the irradiation of the excitation beam, followedby a decay back to a ground state through the releasing ofheat and hence causing localized temperature increases insamples. The refractive index of the localized medium thenchanges because of the temperature increase and producesan effect to form a lens in the medium, which is named as



Figure 9. Schematic illustration of the ITP-integrated microfluidic immunoassay chip. W¼waste, 1¼ tailing buffer, 2¼ first antibody(DNA coupled anti-AFP WA1 antibody), 3¼ sample (AFP), 4¼ second antibody (Hilyte-labeled anti-AFP WA2 antibody), 5¼ handoff well,6¼ leading buffer. Reprinted from Ref. 85 with permission from Wiley InterScience (Copyright 2008 Wiley-VCH Verlag GmbH & Co.KGaA).

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a ‘‘thermal lens.’’ This thermal lens then causes thedivergence of the probe beam. Because the size of thethermal lens linearly depends on the number of excitedsample molecules, quantitative analysis can be performedby measuring this probe beam divergence. For example,Henares et al.91 developed a multiple micro ELISA systemto simultaneously determine human, goat, and chickenIgGs by combining a capillary-assembled microchip andTLM. In this work, plugs of square capillaries possessingvalving and immunoreaction functions were inserted intoa PDMS microchannel network (Fig. 11). The valving andimmunoreaction functions were performed by insertedcapillaries immobilized by a thermally responsive polymerand anti-IgGs, respectively. Using this multiple microELISA system, multiple determinations to a precision of0.1 ng/mL for IgGs (human, goat, and chicken) can beperformed within 30 min. Although only the determinationof IgGs was performed in this work, it is expected that thissystem can be applied to many other immunoapplicationsbecause of the flexible design of the multiple micro ELISAchip, which allows for the insertion of any required Ab-immobilized capillaries. Moreover, a miniaturized TLMwas developed by making use of laser diode modules andfiber-based optics.92 By combining this multiple micro


ELISA chip with a miniaturized TLM, it is believed that itwill have practical use in the near future.

Wavelength Interrogated Optical Sensing. WIOS is a recentlydeveloped, label-free detection method.93 As shown inFigure 12, a wavelength-tunable light source (a collimatedlight beam from a vertical cavity surface emitting laser) wasused to excite waveguide modes in a dielectric waveguide byan input grating coupler. Then, the light is transmitted toa photodetector through an output grating coupler if anyresonance occurs. Because the wavelength is continuouslyscanned, the output signal represents a resonance peak fromthe grating coupler in which the wavelength positionindicates the amount of the analytes adsorbed. This methodhas been used for microfluidic immunoassays. For instance,Suarez et al.94 developed a WIOS-based microfluidic chip forthe simultaneous determination of residual antibiotics inraw milk. In this work, the microfluidic chip was composedof a PMMA reservoir plate, a PMMA holder, and a UV-curable hyperbranched polymer sensing chamber, wherespecific haptens for antibiotics, including fulfonamides,fluorequinolones, and neutravidin were coated. The wholeimmunoassay procedure including the introduction ofa sample solution (raw milk), washing buffer, signal



Figure 10. (A) Schematic illustration of a microfluidic system integrated with a 2-D SPR phase imaging system. (B) Schematic illustration ofthe microfluidic device comprising of a fluidic transport module and a temperature control module. Reprinted from Ref. 87 with permissionfrom Elsevier (Copyright 2007 Elsevier).

Figure 11. A multiple micro ELISA system composed of valvingand immunoreaction capillaries and a PDMS microchannel net-work. Reprinted from Ref. 91 with permission from Elsevier(Copyright 2007 Elsevier).

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amplification buffer (secondary Ab), and detection can beperformed within 10 min with an accuracy of 95%. Incontrast to techniques requiring a laboratory environment,such as high-performance liquid chromatographyeUV/visible spectroscopy,95 high-performance liquidchromatography-mass spectrometry,96 ELISA,97 andparallel affinity sensor array,98 this WIOS-based microchip isa promising platform for daily, fast, and automateddeterminations of antibiotics outside the laboratory byintegrating micropumps and microvalves onto the microchip.

Colorimetric Detection. Although microfluidic immunoas-says are a potential platform for POC applications, there isstill a need for an easy-to-use, portable, user-friendly, andcheap detection strategy to make it acceptable for practicalor even personal use. Colorimetric detection may fulfillsuch requirements.99 Recently, Lin et al.100 developeda colorimetric detection strategy by using a nanoparticle-labeled Ab to realize the detection of H. pylori- and E. coliO157:H7-specific Ags based on the light-scatteringproperties of gold nanoparticles. In this work, lysate Agsof H. pylori were adsorbed onto the surfaces of themicrochannels, followed by sequentially introducing biotin-conjugated rabbit polyclonal anti-H. pylori Ab and goldnanoparticles functionalized with goat anti-biotin Ab. Forthe detection of an E. coli O157:H7-specific Ag, similarsteps were performed by replacing the lysate Ags of the H.pylori and biotin-conjugated rabbit polyclonal anti-H.pylori Ab with lysate Ags of E. coli O157:H7 andbiotin-conjugated goat polyclonal anti-E. coli O157:H7 Ab,respectively. With the light-scattering properties of goldnanoparticles, the color differences quantified by softwarecustom developed for this study increases with theincreased concentrations of H. pylori- and E. coli O157:H7-specific Ags. The detection limits for both Ags are 10 ng,which is comparable to those performed by conventionaldot blot assays. Another work by Lu et al.101 used a goldnanoparticles-enhanced silver staining technique to detect


human IgG (hIgG). In this work, the microfluidic devicewas composed of a PS bottom layer and PDMSmicrochannels. The PS plate was first immobilized withgoat anti-human IgG (g-hIgG) for capturing hIgG inanalytes. After the introduction and incubation of ananalyte solution, gold nanoparticle-labeled g-hIgG wasintroduced to form the complex. Then, to facilitate thevisualization of nanoparticle labels, a signal amplificationmethod was performed in which silver ions were reducedby hydroquinone to silver metal at the surfaces of the goldnanoparticles. Finally, a cell phone with an embeddedcamera was used to snap photos of the microchannelsfollowed by analysis of these photos in a personal



Figure 12. The sensing principle of a WIOS. Reprinted from Ref. 94 with permission from the Royal Society of Chemistry (RSC) (Copy-right 2009 The Royal Society of Chemistry).

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computer with image processing and analysis software.Using this method, hIgG concentrations as low as 1 ng/mLcan be determined. This detection scheme offersadvantages, including low cost, portability, and ease of useand is promising for POC applications.

Nonoptical Detection

Quartz Crystal Microbalance. The QCM is a mass-sensitivedetection method for measuring the resonance frequencyshift attributed to mass changes on a surface and works wellwithout using any labeling molecules.102 Therefore, it issuitable for sensing the binding between Ags and Abs inmicrofluidic immunoassays. Recently, Michalzik et al.103

demonstrated a miniaturized system for the determination ofprotein A by integrating a QCM sensor and a PDMS-basedmicrofluidic cell. First, the quartz material was chemicallyetched to form a suitable geometry such that the masssensitivity can be enhanced, followed by the deposition ofgold electrodes. For immunosensing, a self-assembledmonolayer (SAM) was fabricated on top of the goldelectrodes, followed by immobilizing protein A, BSA, and


anti-protein A, sequentially. BSA was used to block thenonoccupied surface on the sensing area. Using this method,measurements of protein A with a concentration of 1 mg/mLcan be successfully performed. The reusability of thedeveloped system by regenerating the sensing surface withsodium hydroxide has also been demonstrated. Moreover,with the protein A layer, it is possible to immobilize thecorresponding Ab layer on the surface for sensing specificAgs. This system, therefore, provides advantages includingonline measurement, label-free sensing, and reusability,making it potentially useful for microfluidic immunoassays.

Magnetoelectronic Detection. In most immunoassays, nano-scale labeling molecules, such as fluorophores, chromophores,luminescent species, nanoparticles, enzymes, or electrochemi-cally active species are commonly used to conjugate with targetanalytes for detection because of their comparable sizes. Re-cently, Mulvaney et. al.104 reported that using micrometerscale labels, such as magnetic microbeads offers significantadvantages and demonstrated a platform combiningmicrofluidics and magnetoelectronics to determine theconcentration of ricin A chain (RCA) in samples. In this



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work, a standardmicroscope slidewas treated in a series of stepsto bear a NeutrAvidin layer followed by spotting biotinylatedgoat anti-ricin onto it. Samples containing RCA were thenintroduced for incubation, followed by introducing rabbitanti-RCA for incubation. Then, sheep anti-rabbit Ab-coatedmagnetic microbeads were introduced. Finally, a controlledlaminar flow generating a drag force on each bead that is lessthan the rupture strength of the immunocomplex but greaterthan that required to remove nonspecifically boundmicrobeads was applied at the capture surface. Counting ofmagnetoelectronic beads was finally performed by usinga magnetic sensor by measuring the resistance response. Thisplatform was also applied to the determination of femtomolarDNA concentrations without performing PCR amplification.This work demonstrated a platform capable of simplifyingassay procedures but providing excellent sensitivity andspecificity for the determination of DNA and proteins incomplex matrices. With a combination of portablemagnetoelectronic sensors, this platform is expected to bea simple but reliable method for microfluidic immunoassays.

CONCLUSIONS AND FUTURE PERSPECTIVES

Microfluidic immunoassays may play an important role inthe areas of clinical diagnostics, pharmaceutical analysis, en-vironmental and food safety surveillance, and basic scientificinvestigations because of the capabilities for miniaturization,integration, and automation. However, to achieve this objec-tive, integrating knowledge from fields, such as materialcharacterization, fabrication methods, liquid transportation,surface modification, immobilization, and detection schemesneed to be optimized and integrated. Therefore, critical issuesin the following fields are important and should be addressedproperly.

Mass Production

For commercial and disposability considerations, poly-meric substrates are the dominant candidates in this field.Although PDMS has been widely used for fabricating proto-types of microfluidic devices for immunoassays, the fabrica-tion process is still limited to a replication method involvingseveral hours of curing time. Materials with similar proper-ties and capabilities for mass production by techniques, suchas injection molding and embossing are in great demand forpractical applications.

Multiplicity

An important feature of microfluidic immunoassays isthe ability to perform a multiplexed assay in a single chip.Recently, Zhao et al.105 and Sun et al.106 developed a suspen-sion array for a multiplexed immunoassay using silica colloi-dal crystal beads (SCCBs) exhibiting different reflectionspectra as carriers. After the reaction between a samplesolution containing fluorescence dye-labeled anti-hIgG andanti-mouse IgG with a SCCBs suspension containing humanIgG or mouse IgG-conjugated SCCBs, a multiplexed


immunoassay can be successfully performed by a color-encoded method. By combining microfluidic devices withSCCBs-based multiplex immunoassays, it is believed to bea potential platform for clinical applications. The multi-plexed assay will continue to be the dominant method forcommercialization of these microfluidic immunoassays.

Surface Modification and Immobilization

Nonspecific adsorption or binding to molecules ratherthan analytes is a key concern in immunoassays because itmay greatly impair the sensitivity and selectivity. This isa challenging issue in microfluidic immunoassays. Fortu-nately, surface chemistry for the functional modification ofcommonly used materials including device substrates and mi-crobeads/microspheres has been well studied and developedand is expected to provide a solid foundation in microfluidicimmunoassays. However, surface modification and immobi-lization of these new materials still requires tremendousresearch efforts.

Purification and Concentration

Due to the complexity and the low trace of analytes inbiosamples, purification and concentration steps are oftenrequired in microfluidic immunoassays. Currently, micro-or nanoparticles and magnetic beads have been widely usedfor improving the sensitivity by performing a purificationprocess. With a high surface area and ease of manipulation,these beads are expected to provide a promising method forone-step purification and concentration by combing thesetwo techniques together (e.g., beads-on-beads configura-tion)107 in microfluidic immunoassays.

Detection

Detection systems for microfluidic immunoassays are usu-ally bulky and expensive, in contrast to other microcompo-nents, such as micropumps, microvalves, micromixers, andmicroheaters. Although some works on integrated detectionsystem108 have been developed, the cost, sensitivity, and fabri-cation processes still hinder their practical applications. There-fore, developing and integrating miniaturized, compact,portable, and inexpensive detection systemswith an acceptablesensitivity onto microfluidic devices are still in great demand.

Integration, Packaging, and Cost Down Issues

The ultimate goal for practical commercialization is to de-velop fully integrated, well packaged, disposable, and cheapmicrofluidic systems for immunoassays. Although many mi-crofluidic devices have been demonstrated, most of them areoperated by well-trained personnel in the laboratories andoff-chip peripheral units, such as power sources, controllers,and detection systems are used. Moreover, the device cost isstill relatively high because they are not massively produced.These factors greatly hinder their commercial applications.Fortunately, some efforts are made to address these problemsin recent years. For example, a small-sized blood analyzer



Technology Review

comprising a disc loader, a disc positioning, and rotationcontroller, a laser position controller, an optical detectionunit, a temperature controller, and a user interface controllerwas developed by Lee et al.48 Similarly, a prototype incorpo-rating electronics, optics, and chip architecture for highthroughput toxin diagnostics was developed by Meagheret al.81 A desktop instrument containing the LIF detectionsystem, mechanisms for chip loading and transport, electro-phoresis power supply, a means for monitoring the bufferconductivity, chip storage cassettes, and refrigerated reagentstorage compartment was developed by Kagebayashi et al.86

It is envisioned that an integrated low-cost microfluidicimmunoassays with a multiplexed detection function can berealized in the near future.

Storage of Reagents

Although many portable microfluidic devices have beenwell developed and applied in immunoassays in recent years,the long-term storage of bioreagents in portable microfluidicdevices is rarely discussed. This is one important issue in por-table devices because most bioreagents are not durable underroom temperature and some of them require special environ-ments for storage. Therefore, further efforts are required toaddress these demands.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the financial support provided by the

National Science Council in Taiwan (NSC 96-2628-E-006-238-MY3).

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

financial interests in this manuscript.

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Microfluidic Immunoassays

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