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http://jla.sagepub.com/content/15/3/253The online version of this article can be found at:

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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What is This?

- Jun 1, 2010Version of Record >>

by Nan Hallock on January 30, 2012jla.sagepub.comDownloaded from

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Keywords:

microfluidics,

immunoassay,

immobilization,

lab-on-a-chip,

MEMS

Technology ReviewMicrofluidic Immunoassays*CoEngRoa633

153

Cop

doiChun-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 ofapplications, 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;15:25374)rrespondence: Dr. Gwo-Bin Lee, Professor, Department ofineering Science, National Cheng Kung University, 1, Universityd, Tainan, Taiwan 701, R.O.C.;Phone: 886.6.2757575, ext.47; Fax: 886.62761687; E-mail: gwobin@mail.ncku.edu.tw5-5535/$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 INTRODUCTIONImmunoassays

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, AgAb4AgAb 1

Ag Ab4Ag Ab 2Alternatively, in the noncompetitive format, asshown in Eq. (3), Ags in a sample conjugate withan excessive amount of labeled Abs (Ab*) to formJALA June 2010 2532012

mailto:gwobin@mail.ncku.edu.twhttp://jla.sagepub.com/

Technology Reviewa complex that is strongly dependent on the number of Ags,resulting an increase in the detection signal as the Ags in thesample increases.254AgAb excess4AgAb Ab excess 3

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.6e8Microfluidics

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 reviewJALA June 2010 by Njla.sagepub.comDownloaded from 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 MICROFLUIDICIMMUNOASSAYS

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