92Over the past year there have been a number of recentadvances in the fields of miniaturized reaction and separationsystems, including the construction of fully integrated ‘lab-on-a-chip’ systems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, arenow used in several other chemical and biochemical assays.Miniaturized separation columns are currently employed foranalyzing a wide variety of samples including DNA, RNA,proteins and cells. Although significant advances have beenmade at the component level, the realization of an integratedanalysis system still remains at the early stages of development.AddressesDepartment of Chemical Engineering and Department of BiomedicalEngineering, 2300 Hayward, 3022 HH Dow, University of Michigan,Ann Arbor, MI 48109-2136, USA*e-mail: [email protected]†e-mail:[email protected]‡e-mail: [email protected]§e-mail: [email protected]#e-mail: [email protected] Opinion in Biotechnology 2001, 12:92–980958-1669/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.AbbreviationsCE capillary electrophoresisCL chemiluminescenceEC electrochemical detectionIntroductionPhotolithographic microfabrication is a mature technologydeveloped and optimized by the microprocessor industry.Semiconductor microfabrication serves as an excellent plat-form for developing miniature integrated analysis systemswith short analysis times, reduced sample-volume require-ments and cost efficiency. These microanalysis devices canbe classified into two broad categories based on the com-plexity of the fluidics involved: microarray-based (DNA orprotein immobilized on the chip) and microfluidic-based(DNA or proteins being transported, reacted and separatedon the chip) microdevices [1]. In this review, we focus onrecent developments in microfluidic systems for the analy-sis of DNA, proteins and other biomolecules. There has been a burst of activity in the analysis of biomol-ecules other than DNA and proteins using microdevices.Biosensors have been developed for the detection and analy-sis of physiologically relevant molecules, such as glucose,lactic acid and ascorbic acid [2•], and detection of environ-mental agents and herbicides, such as 2,4-diphenoxyaceticacid [3]. In the area of DNA analysis, reaction volumes rangefrom a few microliters [4•] to a few hundred nanoliters [5],and although reaction volumes can easily be reduced further,speculation persists on whether this is indeed a viable ven-ture when developing diagnostic devices for the detection ofinfectious agents. This caution is because of the fact thatsample volume statistically limits the number of availabletargets for the detection in a given assay [6].In this review, we discuss recent advances in various com-ponents of the microfluidic analysis system and also reportattempts at constructing integrated microanalysis systems.Microfabricated reaction systemsThe general trend in the area of microfabricated DNAanalysis is toward devices with multiple functions that per-form multiple reactions in series and/or parallel. The majoroperations now performed in DNA analysis devicesinclude cell lysis, sample concentration and enzymaticreactions such as reverse transcription, PCR, DNase diges-tion and terminal transferase labeling [4•,7] (Figure 1).Several groups also report combined PCR and elec-trophoretic analysis of reaction products [5,7,8]. SangerDNA sequencing was performed in a solid-phase nanore-actor directly coupled to capillary gel electrophoresis [9].With the current interest in performing PCR in amicrochip, the choice of substrate material has become animportant issue. The trade-off in materials reduces theneed for low-thermal conductivity for thermal isolation ina multiple reaction device versus the need for higher con-ductivity for effective heat removal for rapid cycling inPCR. Polycarbonate and glass devices have been reportedfor on-chip PCR in conjunction with other reactions [4•]and an electrophoresis module [5,7]. In addition to thermalisolation, a significant challenge in performing PCR inmicrofabricated devices is associated with controlling theevaporation of the reaction. This problem has beenaddressed through the use of diaphragm valves [4•].An important area in DNA analysis is the analysis of geneexpression. Gene expression analysis on the microscale hastraditionally been performed by hybridization in microarrays.A recent development on the conventional method of pas-sive, non-active hybridization is dynamic hybridization usingDNA probes pumped through target-bearing paramagneticbeads [10]. Traditional hybridization systems are not elec-tronically active; Radtkey et al. [11•] describe a method forthe discrimination of short tandem repeat (STR) allelesbased on active microarray hybridization. A rapid analysis ofSTR alleles using passive hybridization techniques (requiredin non-active microarrays) is currently very difficult. In the area of protein analysis in microchips, a device hasbeen demonstrated that performs enzymatic reactions,Microfabricated reaction and separation systemsMadhavi Krishnan*, Vijay Namasivayam†, Rongsheng Lin‡, Rohit Pal§andMark A Burns#Microfabricated reaction and separation systems Krishnan et al. 93electrophoretic separation of the reactants from the prod-ucts, and post-separation labeling of proteins and peptidesprior to detection [12]. In this work, the authors performedtryptic digestion of the insulin B chain and reduction of thedisulfide bridges of insulin on a microchip [12]. Anotherarea that appears to have benefited significantly fromincreasing interest in the development of microanalysisdevices for a wide variety of biomolecules has been thearea of immunoassays. Immunoassays typically requirevery high specificity and are time-consuming and expen-sive. Reports on chip-based immunoassays usually focuson separation of the free form of the antigen from the anti-gen–antibody complex by capillary electrophoresis (CE);there have been few reports on the antigen–antibody reac-tion performed on-chip [13]. In a recent report on amicrodevice-based immunosorbent assay, detection ofsecretory human
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