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Critical Reviews in Solid State and Materials Sciences

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Integrated Genetic Analysis Microsystems

E. T. Lagally & H. T. Soh

To cite this article: E. T. Lagally & H. T. Soh (2005) Integrated Genetic AnalysisMicrosystems, Critical Reviews in Solid State and Materials Sciences, 30:4, 207-233, DOI:10.1080/10408430500332149

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Critical Reviews in Solid State and Materials Sciences, 30:207–233, 2005Copyright c© Taylor and Francis Inc.ISSN: 1040-8436 printDOI: 10.1080/10408430500332149

Integrated Genetic Analysis Microsystems

E. T. Lagally∗California Nanosystems Institute, University of California—Santa Barbara, Santa Barbara, CA, USA

H. T. SohCalifornia Nanosystems Institute and Department of Mechanical and Environmental Engineering,University of California—Santa Barbara, Santa Barbara, CA, USA

The advent of integrated microsystems for genetic analysis allows the acquisition of informa-tion at unprecedented length and time scales. The convergence of molecular biology, chemistry,physics, and materials science is required for their design and construction. The utility of themicrosystems originates from increased analysis speed, lower analysis cost, and higher paral-lelism leading to increased assay throughput. In addition, when fully integrated, this technologywill enable portable systems for high-speed in situ analyses, permitting a new standard in dis-ciplines such as clinical chemistry, personalized medicine, forensics, biowarfare detection, andepidemiology. This article presents an overview of the recent history of integrated genetic anal-ysis microsystems with an emphasis on materials aspects, and provides a perspective on currentdevelopments and future prospects.

Keywords microfabrication, genetics, integration, analysis, review

Table of Contents

I. INTRODUCTION ............................................................................................................................................ 208

II. GENETIC ANALYSIS FROM START TO FINISH .......................................................................................... 208

III. DEVICES ......................................................................................................................................................... 210A. PCR and PCR Microsystems .......................................................................................................................... 210

1. PCR .................................................................................................................................................... 2102. Microscale PCR ................................................................................................................................... 2123. Portable PCR Microsystems .................................................................................................................. 2124. Microscale PCR: Materials and Design Considerations ............................................................................ 212

a. Substrate Material and Surface Chemistry .................................................................................... 212b. Heaters and Temperature Sensors ................................................................................................ 214c. Enclosed Chambers ................................................................................................................... 214

5. Significance ......................................................................................................................................... 214B. Capillary Electrophoresis and Microchannel CE .............................................................................................. 215

1. Capillary Electrophoresis Background .................................................................................................... 2152. Microchannel CE ................................................................................................................................. 2153. Entropic Trap Separations ..................................................................................................................... 2164. Materials Issues ................................................................................................................................... 217

a. Surface Chemistry ..................................................................................................................... 2175. Significance ......................................................................................................................................... 218

∗E-mail: lagally@engineering.ucsb.edu

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208 E. T. LAGALLY AND H. T. SOH

IV. INTEGRATION ............................................................................................................................................... 218A. Fluid Manipulation: Materials and Fabrication ................................................................................................. 218

1. Microvalves ......................................................................................................................................... 2182. Micropumps ........................................................................................................................................ 218

B. Examples of Integrated Microsystems ............................................................................................................. 219C. Integrated Optics ........................................................................................................................................... 221

V. MICROSYSTEMS FOR REAL-WORLD APPLICATIONS ............................................................................. 222A. Epidemiology Applications of PCR-CE ........................................................................................................... 222

1. Detection and Identification of Bacterial Pathogens ................................................................................. 224B. Forensic Identification ................................................................................................................................... 224

VI. FUTURE DIRECTIONS .................................................................................................................................. 224A. Analysis from Complex Sample Mixtures ........................................................................................................ 224

1. Isolation of Cells .................................................................................................................................. 2242. Isolation of Molecules .......................................................................................................................... 226

B. Advanced Detection Methodologies ................................................................................................................ 2261. Optics-Free Detection ........................................................................................................................... 2272. Reagentless Detection ........................................................................................................................... 227

C. Microsystems for Parallel Information Gathering ............................................................................................. 2271. Motivation ........................................................................................................................................... 2272. Interface Challenges ............................................................................................................................. 228

VII. CONCLUSIONS .............................................................................................................................................. 229

ACKNOWLEDGMENTS ........................................................................................................................................... 229

REFERENCES .......................................................................................................................................................... 229

I. INTRODUCTIONThe analysis of genetic material is one of the most important

facets of molecular biology, health sciences, and forensics. Thenecessary technology has advanced tremendously, with some ofthe most dramatic advances occurring within the past five to tenyears. Analyses that used to require large sample volumes andneeded hours can be performed in minutes in volumes as lowas hundreds of picoliters (10−12 L). The fundamental paradigmthrough which these advances have been propagated is the ap-plication of microfabrication techniques combined with the uti-lization of novel materials to build integrated microsystems thatare capable of performing multiple steps of a conventional ge-netic analysis. Such integration not only reduces the time scaleand volumes (and therefore the costs) of analyses, but also de-creases or eliminates external contamination. Furthermore, themonolithic parallel integration of multiple devices within a chippromises to increase the throughput as well as facilitating thefabrication of disposable devices.

The genesis of integrated genetic analysis systems began withthe fabrication of microchannels capable of conducting liquidsfrom one point to another within a chip using processes simi-lar to IC and solid-state MEMS technology. Subsequently, theintegration of heaters, temperature sensors, and optical compo-nents emerged, followed by the development of active on-chip

fluid control structures such as valves and pumps, as well asmethodologies to control surface chemistry using a variety ofmaterials. The field of integrated genetic analysis systems is inan active phase of research and development, and the numberof publications in this field continues to grow at a rapid rate.With the expanding availability of entire genomes of increas-ing numbers of organisms,1−3 such microsystems will begin toaddress systems-level connections between genes both withinand among organisms. This review highlights the advances ateach of the major developmental stages of the technology, withthe emphasis on the materials science and surface chemistry as-pects. The conclusion will attempt to provide a look forward atpossible future challenges and areas of advancement.

II. GENETIC ANALYSIS FROM START TO FINISHTypically, samples must first undergo a series of steps to pre-

pare and purify the genetic material, thus the task of geneticanalysis may be broken down as a sample preparation step fol-lowed by a detection or analysis step. Figure 1 schematicallypresents the major steps of a conventional analysis. The firststep is the isolation of target cells, which may be as simple ascentrifugation or as complex as separation of different cell typesusing a variety of methods including chemical, mechanical, ul-trasonic, electrokinetic techniques, or by specialized instruments

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 209

FIG. 1. The steps of a typical genetic analysis. Nucleic acids (DNA, RNA) are first extracted from biological cells following celllysis (DNA is the white strands floating in the mixture). The nucleic acids are usually purified using a variety of techniques, followedby amplification. Amplified products are again purified before analysis using capillary electrophoresis or real-time detection. Certainpurification steps, marked with dashed lines, may be omitted depending on the assay.

such as fluorescence activated cell sorters (FACS). Cell isolationis followed by cell culture, on which cells are grown on mediapreferential to specific cell types. The next step is nucleic acidextraction, in which the cells of interest are first lysed. This canbe accomplished using a variety of methods including electrical(electroporation), thermal (boiling), or chemical (low salt caus-ing an osmotic imbalance, or immersion in a chaotropic salt,which disrupts membrane structure through disordering the wa-ter molecule structure) methods.

Following nucleic acid extraction, purification is often re-quired. Historically, efficient purification has been accomplishedthrough a series of chemical steps leading to the nucleic acids

suspended in an aqueous solution, while selectively removingthe membrane components and proteins in an organic phase(phenol and chloroform).4 The nucleic acids are then precipi-tated from the aqueous phase through the addition of ethanol.Other methods that do not require toxic organic reagents, in-cluding affinity-based methods and non-covalent bonding-basedmethods, are also in use. In the affinity-based approach, the nu-cleic acids are hybridized and trapped by complementary se-quences that are immobilized on a solid phase, and then selec-tively eluted.5 For instance, mRNA, which typically containsa sequence of repeated adenine (A) residues at one end due tomodification inside the cell, can be hybridized to complementary

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210 E. T. LAGALLY AND H. T. SOH

poly-thymine (T) oligonucleotides, which themselves have beencovalently bound to microspheres.6 Such affinity-based meth-ods may also be used with DNA or other nucleic acids ifthe sequence of the desired nucleic acid is known. The non-covalent bonding approach is similar in its approach exceptthat the nucleic acids are non-specifically bound to the solidphase such as glass microspheres or a silica membrane throughhydrogen bonding.7 Huang et al.8 have reviewed the waysMEMS technology has been applied to sample purification andpreparation.

Following nucleic acid purification, the next major step issample amplification. Although the molecules may be presentwithin the cell at concentrations detectable using conven-tional detection techniques (pM to nM), the actual number ofmolecules may be quite small, down to a single DNA strandof interest. Thus upon lysis, significant dilution is typically anunavoidable result (sub fM). At these low concentrations, thenumber of molecules plays an increasingly important role asstochastic effects begin to emerge. To increase the number oftarget molecules, several methods for amplifying trace amountsof nucleic acids have been developed and subsequently appliedwithin a microfabricated format. The most common techniqueis polymerase chain reaction (PCR).9 In this reaction, mul-tiple cycles of three temperatures are used to generate newcopies of nucleic acids with the same sequence, at an expo-nential rate. The PCR reaction is sensitive, specific, and rela-tively rapid, and is effectively implemented in microfabricateddevices.

After purifying the amplification products, the final stage in agenetic analysis is the labeling and detection of the genetic mate-rial. Depending on the requirements, the analysis may be as sim-ple as confirmation that nucleic acids of a certain sequence arepresent, or it may be as detailed as the length and the sequence ofthe amplification products. One of the most common techniquesfor the detection and analysis is electrophoresis, in which nu-cleic acids are separated by length under an applied electricfield. There are a variety of electrophoresis methods includingslab gel electrophoresis,4 pulsed field electrophoresis,10 andcapillary electrophoresis.11 In the conventional genetic analy-sis protocol, the overall required time can be on the order ofhours; however, it is often on the scale of days if cell culture isrequired.

In contrast, integrated genetic analysis microsystems havedemonstrated the capability to perform the same tasks in afraction of the time, and complete genetic analysis within30 minutes have been demonstrated.12 This capability is en-abled by the advent of microchannel capillary electrophoresis(CE),13,14 DNA hybridization arrays,15,16 and on-chip nucleicacid amplification.17 To illustrate the evolution of microde-vices for genetic analysis, this review will focus on twoof the major steps in the genetic analysis as a vehicle fordetailed discussion. The first is microchip PCR for amplifica-tion, and the second example is microchannel CE for separa-tion. Both devices contributed to dramatic increases in speed,

decreases in necessary volume, and reductions in the power re-quired to perform such amplifications compared to conventionalmethodologies.

III. DEVICESA. PCR and PCR Microsystems1. PCR

In genetic analysis, the most materials-critical step is the sam-ple preparation, and the case of PCR amplification warrants adetailed discussion. Since its initial description in 1985,9 PCRhas established itself as the foremost sample preparation tech-nology for nucleic acids. The reaction requires four major com-ponents: (1) the template DNA to be amplified, (2) a set ofshort oligonucleotide primers specific to known sequences onthe template strand, (3) a thermostable DNA polymerase (Taq, amodified DNA polymerase isolated from the thermophilic bac-teria Thermus aquaticus is most commonly used), and (4) indi-vidual dinucleotide triphosphates (dNTPs) of adenine, thymine,guanine, and cytosine. As depicted in Figure 2, the reaction pro-ceeds in repeated cycles of three temperatures. The first temper-ature, from 94◦C–96◦C, separates or denatures the two templatestrands (Figure 2A); at the second temperature, typically 45–60◦C, the primers hybridize to their complementary sequenceson the parent strand (Figure 2B); during the third temperaturestep, usually at 72◦C, the DNA polymerase forms new daughterstrands, extending the primer sequences by adding individualdNTPs from solution (Figure 2C). Repetition of the sequenceat optimal efficiency therefore generates 2n daughter strands,where n is the number of cycles. The reaction can be describedin terms of the concentration of DNA molecules as a functionof the number of cycles completed:

[DNA] f =[

n∏i=1

(1 + εi )

][DNA]i , [1]

where [DNA] f is the final DNA concentration, [DNA]i is the con-centration at the ith cycle, and εi is the efficiency of the reaction atthe ith cycle. The efficiency of the reaction is theoretically unityfor small values of i and decreases with increasing cycle number.This phenomenon may be explained by the Michaelis-Mentenequation:

v = vmax[T ]

[T ] + KM, [2]

where v is the rate of product formation at any point in thereaction, vmax is the maximum rate of product formation, [T ] isthe concentration of target (uncatalyzed primer and dNTPs), andKM is the Michaelis-Menten rate constant in mol/L. Using thisequation, which describes the reaction rate as being hyperbolicwith reactant concentration, we may express the efficiency ofPCR as18

εi = 1 − vi

vmax, [3]

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 211

FIG. 2. A schematic representation of the polymerase chain reaction (PCR). Template nucleic acids are cycled between threetemperatures, denaturation (A), annealing (B), and extension (C), respectively. The right hand side depicts the products after thefirst cycle; each of the products and the original template may then participate further in the reaction during the next cycle.

where vi is the rate of product formation at the ith cycle. Be-cause vi decreases with decreasing reactant concentration, theefficiency εi will also decrease as the reaction progresses andmore primers and dNTPS are consumed. Careful control of tem-peratures and initial reactant concentrations are necessary tomaximize reaction yield and to minimize the number of requiredcycles.

PCR exhibits several notable advantages over competingtechniques, including exponential amplification, relatively fewreagents, and a simple reaction scheme consisting of three easilyattained temperatures. PCR technology has been commercial-ized to the point that almost every lab using nucleic acids ownsa thermal cycler, and PCR has been successfully applied to such

diverse samples as polar ice,19 bodily fluids20 and tissues,21 un-treated wastewater,22 and soil.23

Several extremely useful variants of PCR have been devel-oped that enhance its utility and broadens the scope of its appli-cation. Reverse transcriptase PCR (RT-PCR) is used to generatea cDNA complement to an RNA of interest, and then amplifiesthis cDNA exponentially to a detectable level. In addition, multi-ple DNA templates may be simultaneously amplified in the samereaction vessel using multiplex PCR. In cases where the melt-ing temperatures of different primers within a multiplex reactionprevent successful parallel amplification using a single anneal-ing temperature, step-down PCR is used where a series of suc-cessively lower annealing temperatures allow the hybridization

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212 E. T. LAGALLY AND H. T. SOH

of widely varying primer sets to multiple templates.24 Anotherwidely used PCR variant that combines amplification with flu-orescent detection is real-time PCR (rtPCR).25,26 rtPCR is con-ducted in one of two ways: in the first method, an intercalatingfluorescent dye present in the reaction mixture labels amplifiedDNA as the reaction progresses.25 In the second method, a dual-labeled fluorescence detection oligonucleotide probe comple-mentary to the PCR product is included in the reaction mix-ture and hybridizes to amplified product.26 The probe has afluorescent dye at one end and a fluorescence quencher at theother end, resulting in a non-fluorescent probe in its native state.Following hybridization to amplified DNA, however, the probeis cleaved by the polymerase during extension in the next cy-cle, separating the quencher from the fluorophore and restoringfluorescence. The rtPCR method has been adapted for use inmicrosystems.27−30

2. Microscale PCRPCR can be easily miniaturized, and such reduction in scale

provides several important advantages. First, the reduction involume allows faster temperature transitions, while simultane-ously reducing reagent costs. In addition, microfabrication al-lows further integration of other functionalities to enable highlyportable integrated genetic analysis microsystems. The firstdemonstration of microchip PCR, by Northrup and cowork-ers in 1993, used a Si microchamber and a microfabricatedresistive heating element.31 Subsequently, a large number ofgroups have explored different strategies for miniaturization.Wilding et al.32 demonstrated a silicon PCR microchip. Shoffneret al.33 and Cheng et al.34 investigated the use of silicon-glassmicrostructures. Poser et al.35 demonstrated a novel siliconPCR microstructure and investigated optimal chamber volumeand geometry through thermal modeling and chamber arrays.Chaudhari et al.36 demonstrated thermal monitoring and mod-eling for the optimization of PCR microchips. Daniel et al.37

demonstrated successful PCR from a novel silicon microcham-ber utilizing small volumes (1 µL) and thermal isolation from therest of the substrate using thin suspended silicon nitride films.Taylor et al.30 discussed the fabrication of process control el-ements within the microchip PCR. All such microfabricationstrategies mimic the conventional static PCR approach wheresamples are placed in a reaction chamber, which then undergoesthermal cycling to achieve desired amplification as a function oftime. In 1998, Kopp et al.38 demonstrated a fundamentally dif-ferent PCR architecture called “continuous flow PCR” (CPCR)wherein the chemical amplification is achieved as reagent mix-ture is made to pass through serpentine microfluidic channelswith three isothermal zones for the denaturing, annealing, andextension steps so that the chemical amplification occurs as afunction of spatial location (Figure 3). This continuous ampli-fication strategy is especially well suited for microsystems, asit does not involve constraining a small volume without bubbleformation. In this work, 20 cycles of PCR were performed in a

time of as little as 1.5 minutes, using a total volume of 8 µL usinga channel with a cross-sectional area of 3600 µm2. The initialdemonstration required very high starting template concentra-tions (108 DNA copies) and relatively large volumes; later workhas mitigated many of these initial problems. Shin et al. 39 fab-ricated a CPCR microchip from PDMS that was passivated withParylene to avoid sample absorption into the PDMS substrate.Sun et al.40 fabricated a CPCR microsystem with transparentindium tin oxide (ITO) heaters for easier optical observation.Zhang et al.41 presented finite-element models of CPCR for thepurposes of enhanced thermal design. Obeid et al.27 presentedlaser-induced fluorescence detection of PCR products using anintercalating dye introduced following amplification.

Other researchers have investigated means of increasing thespeed of the PCR beyond reducing the volume and using resis-tive heating elements. Non-contact heating, in which the solu-tions within a microchamber or microchannel are heated usinginfrared radiation, provides very fast heating while eliminatingsubstrate heating.42 Liu et al.43 presented a novel rotary PCRmicrochip utilizing a series of PDMS microvalves to drive thesolution between three differently heated regions to achieve am-plification. Bu et al.44 presented a PCR system that used peri-staltic pumps to shuttle a drop linearly between three differentlyheated regions to achieve amplification. Heap et al.45 used anAC current to heat a PCR solution electrolytically for thermalcycling.

3. Portable PCR MicrosystemsAdvances in microfabricated devices have recently led to

the fabrication of field-portable PCR systems. Using the rtPCRassay, Belgrader et al.46 demonstrated silicon based PCR de-vice, assembled with all the electronics for thermal actuationand control, as wells as the optics for fluorescence detection,in a suitcase-sized instrument. The system was able to oper-ate on battery power, making it a truly portable system for anon-site genetic analysis. The same group later demonstrated aneven smaller notebook-sized, battery-operated system for PCRamplification.28 In addition, Higgins et al.47 demonstrated ahandheld rtPCR microdevice. Pal and Venkataraman48 presenteda portable PCR system based on inductive heating. These im-pressive microsystems are making their way into clinical andforensic investigations, and their roles are sure to increase withfurther advances in technology.

4. Microscale PCR: Materials and Design Considerationsa. Substrate Material and Surface Chemistry. Choice of

substrate material affects the biochemical function of PCRreagents within a microsystem in a significant way. In earlywork, it was discovered that silicon and silicon nitrides demon-strate an interfering effect when conducting certain nucleic acidamplification assays.34 Theories surrounding these materials in-teractions vary, but a large contingent of researchers maintainsthe hypothesis that because the polymerase requires divalent

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 213

FIG. 3. (A) Schematic of a chip for flow-through PCR. Three well-defined zones are kept at 95, 77, and 60 by means of thermostatedcopper blocks. The sample is hydrostatically pumped through a single channel etched into the glass chip. (B) Channel layout. Thedevice has three inlets on the left side of the device and one outlet to the right. The whole chip incorporates 20 identical cycles,except the first one includes a threefold increase in DNA melting time. Reprinted with permission from Reference 26. (Copyright1998 AAAS.)

cations (preferably Mg2+) to function correctly, other metal orsemiconductor cations in solution could interfere with the properoperation of the polymerase. Passivation of these materials withoxides has resulted in removal of such inhibition.34

Another major materials consideration of microchip PCR be-came evident in the necessity to prevent the nucleic acids from

non-specifically adsorbing to the sidewalls of the reaction vessel.In particular, glass, with its free silanol (−SiOH) groups at thesurface, readily forms hydrogen bonds to nucleic acids, leadingto sample adsorption. It is well known that the surface to vol-ume ratio increases as device sizes shrink. Thus in microdevices,non-specifically adsorbed molecules, which are unavailable to

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214 E. T. LAGALLY AND H. T. SOH

the reaction, become a larger percentage of the total numberof molecules and significantly limit the efficiency of the reac-tion. The use of non-specific surface coatings are often used toovercome such restrictions; inclusion of “carrier” molecules inreaction mixtures that are designed to coat the chamber surfacesare effective at shielding the analyte of interest from the surface.The addition of bovine serum albumin (BSA) or large concen-trations of inert carrier DNA have been used for this purpose.Strategies for covalent modification of the chamber sidewalls toprevent DNA adsorption have also been explored. For example,Giordano et al.49 presented work on optimization of dynamicpolymer coatings for microscale DNA amplification. Most ofthese coatings rely on the reaction of a bifunctional silane moe-ity with the silanol groups on a fully deprotonated silicon oxidesurface, followed by chemical modification of the other end ofthe silane molecule to present a hydrophilic surface that inhibithydrogen-bonding with DNA in solution.50

A series of polymers has recently become important forgenetic analysis microsystems. Some of these polymers, suchas poly(dimethylsiloxane) (PDMS), poly(methylmethacrylate)(PMMA), and poly(carbonate) (PC) are useful as substratematerials. A variety of microfabrication strategies includingcasting,51 laser ablation,52,53 hot embossing,54 or injectionmolding.55−57 have been developed for polymer microfluidicdevices. PDMS in particular has demonstrated significant ver-satility as a structural material. Duffy et al.58 first described a“soft lithography” method for microfabrication through the cre-ation of a master using a positive photoresist, followed by castingof the mold negative in PDMS. This technique has been usedwidely in many areas of bioscience, including surface pattern-ing of biological materials,59 fabrication of microchannels,60

and targeted cell adhesion.61 Polyimide (PI), although not usedextensively as a substrate material, has been adapted for thefabrication of microchannels.42 It has also been used as a sacri-ficial etch mask for the formation of structural features in otherapplications.62 Polyimide has many desirable characteristics dueto its ability to be easily spun on as a resist-like film, and be-cause its curing process can be integrated with wafer bondingprocesses.

b. Heaters and Temperature Sensors. Thin metal films ofplatinum, palladium, and to a lesser extent, gold are used toform electrodes, heaters, and temperature sensors in integratedgenetic analysis microsystems, as they provide low chemicalreactivity, low resistivity, and high melting point. These metalsare easily deposited as thin films using sputtering or evaporationprocesses, and can be etched using a variety of wet or dry etchingtechniques. Subsequent bonding processes (see later) can requiretemperatures above 650◦C, and so it is important that the metalsexhibit minimal thermal effects, including expansion, oxidation,and diffusion at these temperatures. Platinum in particular is wellsuited for these applications, although gold has also been used.12

Due to its linearity in temperature coefficient of resistance, plat-inum is especially suitable for its use as resistive temperaturedetectors (RTD).30,31,33,34,46 Indium tin oxide is an example of

a transparent conductor that can be used to fabricate electrodesor heaters in applications requiring optical transparency.40,63

c. Enclosed Chambers. Initial microfabricated PCR reac-tors consisted of etched wells into which reagents were loadedand covered with mineral oil to prevent evaporation.31,64 Theavailability of wafer bonding processes now allows fabricationof fully enclosed structures that are capable of channeling fluidflow. There are multiple bonding strategies and typically the pro-cess needs to be tailored for a particular application. The bond-ing techniques used in early systems were taken directly fromthe semiconductor industry, including Si-Si direct bonding65,66

and anodic bonding of silicon to thin oxide layers.67,68

High-temperature compression bonding may be used to fusetwo or more glass substrates together. Such bonds have highmechanical strength; however, the necessity of high tempera-tures (>500◦C) prevents the use of most polymer films and maylead to oxidation and diffusion of metal films used in these sys-tems. Microsystems with polymer films may undergo bonding insimilar ways, generally requiring the polymer to be raised aboveits glass transition temperature in non-oxidizing environments.In limited cases, microsystems can be fabricated where low-mechanical-strength, non-permanent bonds are sufficient; theyinclude bonding of PDMS to glass, as well as the use of thin pho-toresist films that have been cured between two substrates. Thebonding of PDMS to glass and silicon substrates has proven tobe useful and interesting. Current theories hold that the PDMS,when exposed to air or oxygen plasmas, undergoes an oxidationreaction at the surface, leading to diffusion of unaltered oxy-gen groups from the bulk.69 This process is self-reversing on atime scale of hours, depending on conditions. However, whenthe polymer is sufficiently cleaned and activated, for example,through a UV-ozone cleaner, the bond formation becomes irre-versible, resulting in a high mechanical strength.70 This bondingtechnique has been used in the fabrication of PDMS microvalvesand peristaltic pumps for directing liquid flows in microchannelenvironments.43,51,71−73

Bonding processes are difficult to generalize, because theydepend on the substrate and other fabrication details, but certaintrends are evident across most bonding processes. First, bondingprocesses may cause lower process yields than other steps in aprocess flow, and because bonding steps are generally at the endof a fabrication process, much work may be lost if successfulbonding of two substrates is not achieved. Second, bonding yieldis a non-linear function of film thicknesses, temperature, time,and pressure, making optimization of such processes difficult.Thus more research into a mechanistic description of bondingprocesses of heterogeneous substrates is needed.

5. SignificancePCR microsystems demonstrate a number of interesting

characteristics. First, they can amplify miniscule volumes ofnucleic acids with comparable efficiency to that of conven-tional technologies at a fraction of the time, power, and re-quired reagents. Such systems can be fabricated using relatively

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 215

simple fabrication processes and integrated thermal control canbe easily accomplished. Although the reaction is sensitive totemperature, some results have demonstrated that even highlyanisotropic temperature distributions can result in successfulamplification.74 Undisputedly, PCR is an important componentof an integrated genetic analysis microsystem.

B. Capillary Electrophoresis and Microchannel CEThe second example of microfabricated genetic analysis sys-

tems centers on the development of capillary electrophoresismicrochannel systems and their integration with the PCR mi-crosystems discussed earlier. Such CE systems are frequentlyused to separate DNA by length and function as the analysisstep following amplification.

1. Capillary Electrophoresis BackgroundEarly DNA separation systems relied on the knowledge that

DNA has a net negative charge due to regular phosphate groupsin its backbone. However, electrophoresis separates moleculesbased on their charge-to-mass ratios, and application of voltageto DNA in a free-zone separation (buffer only) cannot separatebased on DNA length because the number of phosphate groupsscales directly with the mass of the DNA, resulting in a con-stant charge to mass ratio. As a result, a sieving matrix (gel)was added in the path of the DNA. The pores of the gel havean average size that is small enough (10 nm–200 nm) to restrictthe straight-line movement of different length DNA molecules.Larger molecules, with their larger radii of hydration, must en-counter more pores to find pores those big enough to traverse,resulting in a mobility that is hydration radius (and thereforelength) dependent.

Early gel electrophoresis systems consisted of a horizontal orvertical slab of gel into which DNA was loaded. Applied volt-age resulted in a length-dependent separation of DNA in whichsmaller molecules traversed the gel faster than larger ones. How-ever, these systems suffered from numerous problems, includ-ing high temperatures due to the large currents (10–100 mA)applied, which resulted in high DNA diffusivity and band broad-ening and poor resolution due to the initial plug formation withinthe gel (see later). Later work resulted in the development of gelelectrophoresis separations in drawn fused-silica capillaries, andthis technique became known as capillary gel electrophoresis(CGE).75

In this technique, nucleic acids are separated by lengththrough a sieving matrix under an applied electric field withina glass capillary (inner diameter 50–200 µm). The velocity ofDNA fragments in the capillary is described as a function of theelectrophoretic mobility

v = µE [4]

where µ is a constant for particular length of DNA (units ofcm2/V*second) and E is the applied electric field (V/cm). Theresolution of a CE separation is defined as the difference (in

elution time) of adjacent bands of DNA of constant length overtheir average widths. Theoretically, the resolution may be ex-pressed as:76

R = t2 − t112 (w1 + w2)

= L(µ1 − µ2)

4(µ1

[ (µ1 Einjtinj)2

12 + 2DLµ1 E

]1/2) [5]

where L is the column length, µ1 and µ2 are the mobilities of thetwo DNA fragments of interest, Einj is the applied electric fieldfor injection, tinj is the injection time, E is the applied electricfield for the separation, and Dis the average diffusion coefficientof the DNA fragments. Depending on the operating regime, theresolution depends on either the length of the channel or thesquare root of the length. In the first regime, the band broadeningcaused by the electrokinetic injection dominates, and as a result,the resolution scales with length. In the second regime, the bandbroadening is governed by diffusion resulting in a square-rootdependence of the resolution on length. As diffusion characteris-tics are difficult to engineer, it is imperative to minimize the bandbroadening caused by the electrokinetic injection in a microsys-tem. Microchannel CE is advantageous compared to standardCE systems because microfabrication allows precise determina-tion of the shape and size of the injected plug of genetic material,thereby enabling short separation lengths and high-performanceseparations.

2. Microchannel CEThe initial descriptions of microchannel CE were by Manz

et al.77 and Harrison et al.13 Later work by others extended theseapproaches toward high-resolution and parallel operation. Wool-ley and Mathies14,78 demonstrated the first DNA fragment sizingand DNA sequencing separations on a glass microchannel CEdevice in which DNA was introduced electrokinetically throughan injection cross-channel and separated on a 5 cm-long, gel-filled microchannel in only 120 seconds. The DNA was labeledon-column using an intercalating fluorescent dye and detectedwith laser-induced confocal fluorescence detection. A schematicdiagram of the microchannel geometry and experimental set-upis presented in Figure 4. The key feature of this device leadingto exceptional performance was an injection cross-channel de-sign that intersects the main separation channel. This feature iscritical in controlling the plug volume and shape, thereby min-imizing band-broadening effects from injection, allowing effi-cient separation over short times and channel lengths.

Paegel et al.79 later extended the work to 96 channels ofparallel DNA sequencing, with 500 bp of DNA electrophoret-ically separated in under 30 min. (Figure 5). The practical im-plementation of this system revealed other technical challengesbeyond microfabrication. The operation of 96 CE channels re-quired a nearly 100-fold increase in current, which led to a rapidexhaustion of buffering capacity as protons were quickly de-pleted. A recirculation system was necessary to replenish thebuffer during the course of a full sequencing run and the deviceutilized folded ‘hyperturns’ to achieve a separation length of

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216 E. T. LAGALLY AND H. T. SOH

FIG. 4. Top: Schematic drawing of a CE microchannel showing the four arms and reservoirs (cathode, anode, waste, and sample).Bottom: An exploded view of the injection cross channel region, with diagram of injection plug formation during the inject (left)and run (right) phases.

15.9 cm on a 150-mm diameter substrate.80 Other examples ofmicrochip CE include the work by Emrich et al.81 that used astraight-channel design with a direct injection scheme to demon-strate a 384-channel DNA fragment sizing separation. Medintzet al.82−85 demonstrated a number of clinically relevant DNAseparations using microchannel CE.

3. Entropic Trap SeparationsFor separation of long DNA fragments (>1000 bp), CE is not

effective, because the difference in the mobilities of DNA frag-ments decreases as the average length of the DNA increases.Extremely long DNA fragments eventually enter the “biasedreptation” regime, and they all move with equal velocities re-gardless of their length. In applications where long fragmentsneed to be separated, pulsed-field gel electrophoresis has beensuccessful.10 Unfortunately, this method suffers from the samedisadvantages as other slab gel techniques, and many research

groups proceeded to develop alternate methods for separatinglong DNA fragments in a microdevice. Han et al.86,87 have devel-oped an elegant method, consisting of a series of nanochannelsetched into a Si substrate. In their construction, they exploitedthe fact that shallow (∼10 nm) channels form an entropic en-ergy barrier for long DNA fragments where the mobility of DNAfragments depends on the average size of the DNA in its randomcoil configuration. Thus the mobility can be directly correlatedto the DNA length. The underlying equation governing the resi-dence time of a DNA coil in the entropic trap has been elucidatedas:87

τ = τ0e�Fmax

kB T , [6]

where τ0 is a prefactor with a dependence on the length of therandom DNA coil in solution, and � Fmax is the entropic energybarrier required for DNA to escape the nanometer-sized constric-tion. Because the entropic energy barrier is a function only of the

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 217

FIG. 5. 96-channel microchannel CE device for DNA sequenc-ing. (A) Overall layout of the 96-lane DNA sequencing mi-crochannel plate (MCP). (B) Vertical cut-away of the MCP.The concentric PMMA rings formed two electrically isolatedbuffer moats that lie above the drilled cathode and waste ports.(C) Expanded view of the injector. Each doublet features twosample reservoirs and common cathode and waste reservoirs.(D) Expanded view of the hyperturn region. The turns are sym-metrically tapered with a tapering length of 100 µm, a turnchannel width of 65 µm, and a radius of curvature of 250 µm.Reprinted with permission from Reference 56.

channel height, the separation may be achieved on the basis ofτ0, which varies proportionally with length. Using this system,the DC field separates the DNA molecules by length in seconds,as opposed to hours as is required by conventional techniques.Cabodi et al.88 also demonstrated a novel nanopillar array uti-lizing an AC electric field to cause entropically based differen-tial relaxations of long DNA molecules, leading to separation.Such nanopillar arrays and nanochannel device geometries havethe advantage that they do not require a polymer sieving matrix.However, for small DNA molecules, the separation performancetrails that of CE, because the entropic energy barrier depends onthe average coil size of the DNA. For small DNA molecules,sufficiently shallow channels have not yet been demonstrated.

4. Materials IssuesCE typically employs electric field strengths up to

300 V*cm−1. In addition, because the detection of nucleic acidscommonly require fluorescence at optical wavelengths (400–700 nm), it is necessary for the substrate material to be trans-parent at these wavelengths. Silicon, with its exceptional fabri-cation flexibility, was initially considered for use in microfabri-cated CE technology. However, due to the limitations in opticaltransparency, glass is a preferred substrate, and the success ofmicrochannel CE may be attributed to the advances in materi-als and surface chemistry developed from earlier work in drawnfused-silica capillaries.

a. Surface Chemistry. As described earlier, nucleic acidshave a net negative charge because of the presence of phosphategroups in their backbones. In addition, nucleic acids readily formhydrogen bonds to glass, resulting in an undesired, non-specificadsorption to device sidewalls. The solution to this problem wasthe use of the coating first introduced by Hjerten, a version ofthe silanization protocol also used to control DNA adsorption tooxide surfaces during PCR.50 The use of this coating also con-tributed to a significant increase in the resolution capability ofelectrokinetic separation. The resolution of DNA separations inearly constructions of electrophoresis systems using standard,uncoated glass capillaries was poor due to electrokinetic effectsthat exist at charged surfaces in contact with conductive solu-tions under applied voltage. More specifically, the native surfacecharge of the fused silica gives rise to a charged double layer andsubsequent bulk electroosmotic flow (EOF) in the presence ofan electric field that transport the fluid in an opposite directionwith respect to the electrophoretic movement of the molecules.The bulk EOF velocity may be expressed in the following way:

vEOF =(

σ

κη

)E . [7]

In this presentation, σ is the surface charge density, η is thedynamic viscosity of the solution, E is the applied electric field,and κ is the Debye-Huckel constant, defined as

κ = F

√[2

∑ci z2

i

∈0∈r RT

], [8]

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218 E. T. LAGALLY AND H. T. SOH

where c is the concentration of an individual ion in solution, zis that ion’s valence state, F is the Faraday constant, ∈0∈r is thedielectric constant of the solution, R is the Boltzmann constant,and T is the temperature. Equation 8 reveals that the EOFvelocity depends on the pH of the solution, the temperature, andthe surface charge density of the capillary sidewall. In capillaryelectrophoresis, the EOF manifests itself as a fluid migrationdirection opposite to that of DNA movement, which resultsin distorted peak shapes and very low separation resolution.Application of the Hjerten or similar covalent couplingsalleviates this EOF through eliminating the charge presented tothe solution with an acrylamide group.

5. SignificanceThe evolution from slab gel electrophoresis to CGE to mi-

crochannel CE has facilitated dramatic improvements in res-olution as a function of channel length. These improvementshave come through reductions in diffusive band broadening aswell as injection-dominated band broadening. The end resultis a monolithic system in which DNA and other nucleic acidsmay be separated in seconds instead of hours, using volumes ofonly hundreds of picoliters. Most importantly, the advent of thecross injector design allows the integration of these microchan-nel CE devices with upstream sample preparation and samplepurification.

IV. INTEGRATIONFollowing the demonstration of discrete devices on chip, ef-

forts began to combine these modules to form integrated mi-crosystems. Such integration promises to harness the advantagesof batch fabrication through further reduction of volumes andanalysis times as well as elimination of manual transfers be-tween analysis steps. In order to enable such integration, it wasnecessary to develop efficient methodologies for microfluidicconnections between the on-chip components. The microvalvesand micropumps continue to serve as the unifying microfluidicinterconnection elements in current microsystems.

A. Fluid Manipulation: Materials and Fabrication1. Microvalves

The ability to direct and manipulate fluids of very small vol-umes (pL to nL) over micrometer length scales is one of theessential tasks in integrated analysis systems, and the advent ofrobust microvalve technology has been seminal; it provided amethod to accurately define analysis volumes and restrict thecontact between solutions in space and time. Furthermore, itprovided the ability to actively control fluid movement, makinga single chamber useful for multiple steps of an analysis. Manytypes of microvalves and fluid control elements have been de-veloped, but only a small subset of these share the advantagesof being robust, biocompatible, simple to fabricate, and allow-ing massively parallel operation. The “normally open” PDMSmicrovalve architecture developed by Unger et al.51 has been

widely used. The device is comprised of two PDMS microchan-nels, one of which flows over the other on a separate layer. Theflow control is achieved by the application of pneumatic pressurethrough the top PDMS microchannel selectively collapsing oropening the bottom microchannel that transports the fluid. Be-cause PDMS microchannels are relatively simple to fabricate,this approach has been utilized to fabricate an extraordinarilylarge array of zero-dead-volume valves on a single substrate.73

Another noteworthy PDMS microvalve architecture has beendeveloped by Grover et al.,70 which used a similar approach butprovided two advantages; the valves may be “normally closed”instead of normally open, and the PDMS surface area contactingthe sample is minimized, which reduces non-specific adsorp-tion. The PDMS membrane that performs the valve actuation isformed over the entire substrate, and covers a series of drilledvalve vias. Upon the application of positive pneumatic pressurefrom an external manifold, the PDMS membrane locally de-flects forming an open fluid path. Application of vacuum sealsthe membrane against the vias, effectively blocking fluid flow.Both the normally closed and normally open valve designs haveseen further application in a series of integrated microsystemsfor genetic analysis.43,89

Besides the pneumatic actuation, other modes of microvalvetransduction have been explored including the phase changevalve. In this construction, liquid paraffin wax is loaded intoa microsystem and solidified in a desired location, forming arestriction to fluid flow. Upon application of temperature, thewax re-melts, allowing fluid flow. Recent work by Pal et al.90

has recently tested a variety of waxes for biocompatibility anddemonstrated pressure sealing up to 250 psi applied backpres-sure. Another type of phase change valve was introduced byEddington et al.,91 in which a pH-sensitive hydrogel is selec-tively patterned on a vertical post situated within a microchan-nel. Upon application of low pH, the polymer swells to fill themicrochannel, blocking fluid flow. Such “smart valve” technol-ogy is passive and requires no external actuation; however, itrelies on the chemical characteristics of the solution to deter-mine its state. Further investigation of several types of hydrogelsand their optimized fabrication within microfluidic systems havebeen reported.92

2. MicropumpsThe trade-off between speed and power is the chief constraint

to the development of genetic analysis microsystems. Espe-cially for field-portable applications, low-power systems are ofparamount importance. It is in these same types of analysis sit-uations, however, that one would like to conduct a fast analysis(e.g., the detection of neonatal meningitis in a remote hospital).Most current published work uses manually applied pressure ora syringe pump to move fluids through a device. However, man-ually applied pressure is not quantitative and syringe pumps arebulky, expensive, and not conducive to miniaturization. Elec-trolytic pumping, in which a DC current is applied through twoelectrodes immersed in a salt solution to generate gases, which

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 219

then generates pressures for pumping, has been investigated asan alternative.93 Such electrochemical designs require compar-atively high currents (e.g., tens of mA for pumping of mL vol-umes), however. For these reasons, some groups have soughtto develop electrokinetic techniques for bulk fluid movementwithin microfabricated systems. Some of these technologies alsoallow for other desirable processes, such as mixing in laminarflow conditions. Electroosmotic flow is the main motive forceused in these systems, in which a voltage is applied to a fluidsurrounded by a charged substrate (usually untreated glass), giv-ing rise to a zeta potential and bulk fluid motion.94,95 Althoughthese systems require high electric field strengths (∼200 V/cm),they operate at currents of 100 µA or less, resulting in lowerpower than the technologies mentioned previously. Integratedapplication of such electrokinetic transport on the microscalehas been demonstrated. Chen et al.96 recently presented a rotaryPCR microsystem that used electrokinetic forces to transport thePCR solution through three differently heated regions to achieveamplification.

B. Examples of Integrated MicrosystemsConcomitant with the development of microfluidic manipula-

tion technologies, efforts began to integrate nucleic acid ampli-fication technologies with microchannel CE for analysis of theproducts. The first demonstration of an integrated microsystem

FIG. 6. Fully integrated nanoliter DNA analysis device. (Top) Schematic of integrated device with two liquid samples and elec-trophoresis gel present. The only electronic component not fabricated on the silicon substrate, except for control and data-processingelectronics, is an excitation light source placed above the electrophoresis channel. (Bottom) Optical micrograph of the device fromabove. Wire bonds to the printed circuit board can be seen along the top edge of the device. Reprinted with permission fromReference 79. (Copyright 1998 AAAS.)

was performed by Woolley et al.,17 which included a Si PCRmicrochamber attached to a glass CE microchannel. DNA am-plified within the microchamber was electrokinetically injecteddirectly into the glass CE microchannel for separation and fluo-rescence detection. This microsystem was capable of amplifying5 µL of sample in a time of 15 minutes and the subsequent CEseparation took place in a 5 cm-long CE microchannel in a timeof 120 seconds. This work demonstrated correct product sizingand good correlation between amplification time and productyield, which proved the feasibility of such microsystems to har-ness the advantages of both miniaturized sample preparation andanalysis. Subsequently, Anderson et al.97 demonstrated a PCRdevice integrated with hybridization array technology for DNAand RNA analysis.97 Their technology utilized multiple lami-nated polycarbonate sheets to form microchannels, the analysischamber, and microvalves. Waters et al.64 demonstrated a seriesof all-glass PCR–CE systems that were capable of thermal celllysis, amplification of several targets and subsequent separationon a single CE channel. In addition, the same group has presenteda microdevice for enzymatic digestions of DNA followed bymicrochannel CE. Unfortunately, these initial monolithic glasssystems required placing the entire device on a conventionalthermal cycling block, removing some of the advantage of con-ducting microscale PCR. In 1998, Burns et al.98 published a fullyintegrated DNA analysis system (Figure 6) employing SDA, an

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220 E. T. LAGALLY AND H. T. SOH

exponential and isothermal amplification reaction to conduct aminiature slab gel separation under a small electric field to sep-arate the products. The microsystem also integrated sample ma-nipulation in the form of selective hydrophobic coatings, as wellas photodetectors using a single-crystal Si photodiode. Althougha fully integrated functionality was not demonstrated, their sys-tem was able to meter liquid volumes as small as a few hundrednL, amplify the DNA present in these volumes, and separatethe resulting products with a resolution of ∼100 bp. The use ofSDA instead of PCR provided the advantage of eliminating theneed for thermal cycling but compromises in analysis speed wasnecessary. Nevertheless, this work was the first to demonstratethe potential for complete integration in a single chip.

The first functional monolithic integration of PCR–CE sys-tem was achieved by Lagally et al.74 This system was able toamplify as few as five copies of a double-stranded DNA tem-plate in a time of 10 minutes. The products were separated ona 5 cm-long CE microchannel in 120 seconds. Critical to thesuccess of this microsystem was containment and isolation ofthe sample within the 280-nL chamber during thermal cycling.The strategy employed was one adapted from the work of An-derson et al.,97,99 in which positive pressure was applied to thePDMS microvalves to obtain efficient sample containment. Theoriginal work had presented microvalves with dead volumes inthe microliter range; however, for the purposes of a 280 nL PCRchamber, valves with dead volumes of 50 nL were constructed.After the PCR amplification, the DNA was electrokinetically in-jected into the gel-filled microchannel and labeled in situ usingan intercalating fluorescent dye, thiazole orange. This microsys-tem demonstrated an excellent linear correlation, as expected forthe linear regime of PCR, and moreover, the extrapolation indi-cated a molecular limit of detection of only two DNA templatemolecules. This is an important result because below the level ofapproximately five template molecules, PCR enters a stochasticregime, in which the amplification yield for a series of reac-tions of a certain average concentration will obey the Poissondistribution:

P(x) = λx e−x

x![9]

where λ is the mean of the distribution and x is the numberof template molecules. To test the ability of such microsystemsto amplify single DNA template molecules, an internal controltemplate was added to separate the effects of the statistical am-plification from the possibility of a failed reaction. The ensuingmultiplex reaction utilized two sets of primers and two templates,one stochastic template present at approximately one moleculewithin the PCR chamber and the other outside the stochasticregime at approximately five molecules in the chamber. Figure7 presents the results of 60 separate amplifications. The dataare fit to the presumptive Poisson distribution and provide agood fit (Komologorov–Smirnov statistic = 0.88) with a meannumber of stochastic template molecules of 0.9 ± 0.1. This re-sult verified that single-molecule DNA amplification had been

FIG. 7. (A) Histogram showing clustering of normalized peakarea ratios from a series of 60 multiplex PCR amplifications fromstochastic single-molecule template (136 bp product) and con-trol template (231 bp product). Distinct clusters are suggestiveof amplification from single DNA template molecules. (B) Fitof histogram in (A) to expected Poisson distribution. The meanof the fitted distribution is λ = 0.9± 0.1 molecules, demon-strating successful amplification from single DNA templatemolecules.

achieved using the integrated PCR–CE paradigm, and was thefirst such demonstration on a microdevice.100 Lagally et al.12

also produced a PCR–CE microdevice with integrated heatersand temperature sensors, which yielded temperature transitionsof 20◦C s−1. Figure 8 presents a schematic drawing of the tem-perature control elements used in this work. The microheaterswere fabricated from Ti and Pt thin films and were locatedon the reverse of the glass device. The heaters had very lowresistance (8–12 �) and possessed electroplated gold leads in

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 221

FIG. 8. Perspective diagram of the relative orientation of three microfabricated elements used in a fully integrated PCR-CEmicrosystem. The heater is located on the bottom side of the device, the RTD is located between two bonded glass wafers formingthe enclosed chambers and channels of the device. Adapted from Reference 5.

order to localize the heating under the PCR chamber and to lever-age the second-order dependence of Joule heating on the current(P = I 2 R). The temperature sensors were of the four-wire re-sistance temperature detector (RTD) form, used to minimize theimpact of self-heating effects on the sensing system. The highlylinear temperature coefficient of resistance of Pt enabled sen-sors with extremely high fidelity. This new system was used toconduct a sex determination assay from human genomic DNA,described later.

C. Integrated OpticsFluorescence provides the invaluable capability of multi-

color detection with exquisite sensitivity; however, it typicallyrequires bulky optical sources and detectors that pose signifi-cant challenges in integration. For example, the PCR–CE sys-tems described in the previous section required laser diodes andconventional PMTs for conducting confocal fluorescence de-tection, which limited the system size and prevented avenues offurther miniaturization. In order to address this bottleneck, manygroups are investigating the fabrication of fluorescence detectionoptics directly onto the integrated microsystem that may allowprecise positioning of the optical detection hardware in rela-tion to the analyte, removing the necessity for time-consumingalignment procedures. Roulet et al.101 fabricated arrays of mi-crolenses and thin-film metal apertures on a glass microdevicefor fluorescence detection. The detection was conducted off-chip using either a CCD camera or a photomultiplier tube, anddemonstrated a limit of detection of 3 nM for a common fluores-cent dye. Chabinyc et al.102 presented an avalanche photodiode

coupled to a PDMS microdevice using a fiber optic cable. Na-masivayam et al.103 have investigated the use of Si photodiodesfor on-chip fluorescence detection and have fabricated thesewithin integrated genetic analysis systems. It is important tonote that the choice of substrates plays an important role in theintegration of optoelectronic components. For example, the useof Si substrates that facilitate the fabrication of PIN photodi-odes may limit capabilities in other areas of the microsystem,such as the application of high voltages for DNA separation.The use of III-V compound semiconductors can enable elegantintegration of VCSEL/photodiodes,104 however the requirementfor high-temperature processing may eliminate the possibility touse polymer-based materials.

Kamei et al.105 presented a novel microfabricated photode-tector in the form of a hydrogenated amorphous silicon a-Si:Hphotodiode (Figure 9). The photodiodes are fabricated fromsuccessive layers of doped amorphous silicon and the fabrica-tion process occurs below 300◦C in a plasma-enhanced chem-ical vapor deposition (PECVD) system, allowing the use ofglass or some plastic substrates. The photodiode was fabri-cated on a glass substrate as a detector for the microchan-nel CE separation, with spectral sensitivity that was optimizedfor the detection wavelength. Recently, this photodiode wasused to detect the results of a PCR-based assay to distin-guish pathogenic strains of Staphylococcus aureus bacteria.106

In a different approach, Kwon and Lee107 fabricated an en-tire scanning confocal fluorescence detection apparatus on amicrodevice, including microlenses, scanning hardware, pin-holes, and pupils. This impressive system demonstrates the

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222 E. T. LAGALLY AND H. T. SOH

FIG. 9. (A) Schematic cross-sectional view of the hybrid in-tegrated a-Si:H fluorescence detector with a microfluidic elec-trophoresis device. (B) Optical micrograph of the top view ofthe annular a-Si:H photodiode. Reprinted with permission fromReference 86. (Copyright 2003 American Chemical Society.)

feasibility of creating entire integrated optical detection sys-tems on the microscale, harnessing the power of fluorescenceimaging while conserving the advantages of miniaturization andportability.

V. MICROSYSTEMS FOR REAL-WORLD APPLICATIONSIntegrated genetic analysis microsystems, and particularly

the PCR-CE systems described previously, demonstrate severaladvantages that make them applicable to several key areas ofmodern genetic analysis. Their small size, fast operation, loweroperating powers, and autonomous operation allow them to beused in remote environments and by untrained or minimallytrained operators. Batch fabrication allows the devices to be dis-posable, enabling assays requiring sampling from bodily fluidsor pathogenic samples. Following the development of the firstintegrated PCR-CE microsystems, researchers began to applythese systems to “real-world” problems of clinical and forensicutility.

Lagally et al.89 demonstrated the construction and testing ofthe first field-portable, fully integrated PCR–CE microsystem.This system is based on the integrated PCR–CE systems de-scribed earlier. In this case, the microsystem contains a singlePCR chamber directly connected to a CE separation microchan-nel with hyperturns to increase its length. In contrast to pre-vious work, novel PDMS microvalves were assembled on thetop surface of the system.70 These microvalves simplify fabri-cation over the latex microvalves used previously, exhibit deadvolumes as low as 8 nL and are actuated with small pressuresand vacuums. Pt electrodes were also fabricated within the de-vice, allowing application of a high voltage without the needfor external electrodes. The microsystem is the size of a micro-scope slide, and is placed into a portable analysis instrumentthat contains all the necessary electronics, optics, and controlhardware for conducting a genetic analysis. The analysis instru-ment contains a miniature confocal fluorescence set-up, includ-ing a laser diode, filters, and a photomultiplier tube for collectingfluorescence data. Figure 10 presents a picture of the portableanalysis instrument. This section reviews two major areas of cur-rent application of such field-portable PCR-CE microsystems—detection and identification of bacterial pathogens and humansex determination.

A. Epidemiology Applications of PCR-CEEpidemiology plays a central role in food safety, infectious

disease research, and anti-bioterrorism efforts. Of particularconcern is the detection and identification of bacterial pathogens.Such pathogens are a ubiquitous part of the human environment,and are responsible for a large number of infectious diseases,including tuberculosis,108,109 wound infections,110,111 and nu-merous food-borne diseases.112−116 Detection and identificationof bacterial pathogens presents unique challenges that geneticanalysis microsystems, and PCR-CE microsystems in particular,are well poised to confront. First, such pathogens can be presentin very small quantities and in very small concentrations. Forinstance, E. coli O157:H7 is a major food pathogen causing asmany as 20,000 infections a year in the United States alone,and has been the causative pathogen in food-borne outbreaksin the United States.113 Importantly, the minimum infectious

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 223

FIG. 10. Photograph of the first portable PCR-CE analysis instrument with exploded schematic of portable PCR-CE microsystem.The portable analysis instrument measures 8′′ × 10′′ × 12′′ and includes all necessary electronics, optics, laser excitation, andpneumatics to control the microdevice. The microdevice contains a single PCR-CE system, including microfabricated heater,temperature sensor, and PCR chamber directly connected to a CE microchannel for analysis of the amplification products. Adaptedfrom Reference 68.

dose of this organism is as low as 50 cells, depending on theroute of introduction.114 Second, because pathogens are closelyrelated to non-pathogenic strains of the same species, differen-tiation of pathogens from commensal non-pathogens is a chal-lenge. Non-pathogenic E. coli is normally found in the humanintestine, so differentiation of these organisms from pathogenicO157:H7 strains must use unique genetic markers or knownimmunological differences. Finally, pathogens vary widely intheir routes of infections, and so genetic analysis microsystemsmust be able to adapt to multiple types of sample preparationtechnologies.

A conventional pathogen detection and differentiation exper-iment involves culturing from a clinical sample onto a specificset of media depending on which organism is suspected. Suchmedia will generally screen to the species level, enabling fur-ther analysis using pulsed-field gel electrophoresis techniquesfollowing PCR amplification of known toxicity genes. PCR–CEhas been shown to be a versatile technique for the detection andidentification of bacterial pathogens. Koh et al.117 demonstrateda lab-based microdevice with integrated valves, PCR and CE us-ing multiplex PCR to detect different strains of Escherichia coliO157:H7. In their work, a glass microdevice containing a PCR

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224 E. T. LAGALLY AND H. T. SOH

chamber directly connected to a CE microchannel was usedto generate and subsequently separate the PCR products. On-chip thermal lysis of E. coli O157:H7 organisms was achieved,negating the need for further upstream sample preparation. Thefluorescently labeled PCR products were detected using con-focal laser-induced fluorescence. Such a system points the waytoward remote analyses on water supplies, for example, to detectfecal contamination or for food testing. For many applications,portable PCR–CE microsystems would provide a robust, quanti-tative analysis method for detection of infectious disease. A keyattribute of this system is the ability to confirm product sizesand glean important genetic information about the analyte in atimely manner at any location.

1. Detection and Identification of Bacterial PathogensThe field-portable PCR-CE microsystem described by La-

gally and coworkers89 has been used to detect and identify mul-tiple bacterial pathogens, including pathogenic strains of E. coliand antibiotic-resistant Staphylococcus aureus, a pathogen caus-ing local and systemic infections. In the first series of experi-ments, a triplex PCR was used to detect and differentiate twopathogenic strains of E. coli from a laboratory strain. E. coliK12, O55:H7, and O157:H7 were successfully differentiatedin 30 minutes, and the resulting serial dilution demonstrated alimit of detection of only two cells (Figure 11A). Thermal lysisof the bacteria was achieved within the PCR chamber, elimi-nating further upstream sample preparation. In a second set ofexperiments, E. coli O157:H7 was successfully detected fromwithin a large background concentration of commensal K12 or-ganisms, demonstrating the utility of the device in epidemiolog-ical settings where pathogenic organisms may only be a smallfraction of the total population of any species of interest (Fig-ure 11B). The third series of experiments successfully differen-tiated Gram positive antibiotic-resistant Staphylococcus aureusfrom antibiotic-sensitive cells of the same species. Such detec-tion of antibiotic resistance in bacteria, and S. aureus in particu-lar, is of ever-growing importance as antibiotic resistance in thisspecies is spreading both through nosocomial and community-acquired infections.111 Due to its small size, fast operation, andlow limits of detection, such integrated, portable microsystemsmay become a critical tool in infectious disease detection.

B. Forensic IdentificationPCR–CE systems may also be employed for forensic identifi-

cation where only a small amount of sample is available. Usingthe laboratory-based system described earlier, human sex de-termination was demonstrated. In this assay, human genomicDNA with two sets of primers were mixed in a PCR cocktail,where the first set of primers was specific to the X chromo-some and generated a 157 bp product. The second set of primershybridized to a section of the Y chromosome, and produced a200 bp product. Observation of the number and the lengths of theresulting PCR products then allowed a determination of the gen-

der of the individual from whom the DNA had been isolated. Theresulting electropherograms demonstrated clear discriminationof DNA isolated from males and females, respectively.12 Themass of DNA used in these experiments was 10 ng, the upperbound typically encountered in real-world forensic investiga-tions; however, the signal-to-noise ratio of the fluorescent PCRproducts was sufficiently high for a reduction to 1 ng or lessof starting material to be theoretically achievable. Such systemscan therefore be applied to real-world situations, in which theavailability of the starting material is usually limiting, and suchforensic applications are therefore also within the purview offield-portable PCR-CE microsystems.

VI. FUTURE DIRECTIONSThe progress of integrated microsystem for genetic analysis

to this point has been rapid, with many critical assays being de-veloped and many useful microsystems emerging. However, theroutine use of such microsystems in a general set of situations inboth developed and developing countries requires microsystemsthat are more robust, simple for untrained operators to use, andlow power. A series of emerging technologies are discussed thatmay serve to advance the field of microsystems for wider accessand utility.

A. Analysis from Complex Sample MixturesMany sample mixtures are complex and heterogeneous, and

contain inhibitory components that prevent the success of anassay. One of the most troublesome challenges in genetic anal-ysis in real-world situations is the simplification of the samplemixture so that the genetic material is easily analyzed. For ex-ample, blood samples contain heme, which disrupts PCR,118

whereas urine contains urea, which acts as a DNA denaturant.4

In addition, the concentration of the genetic material in thesesamples (particularly in the case of pathogen analysis) can beexceedingly low (1–10 cells/mL). Therefore, the developmentof technologies for the concentration and purification of ge-netic material from complex sample backgrounds is impera-tive. There are two major regimes of sample purification, iso-lation of cells and isolation of molecules, which are discussedhere.

1. Isolation of CellsThe initial isolation and purification of cells from com-

plex sample mixtures is an important step prior to these ge-netic analyses. Traditionally, centrifugation, immunomagneticseparation,119 and use of sophisticated equipment such asFACS120 have been utilized in a laboratory setting. One tech-nology that is well suited to microsystems is dielectrophoresis(DEP). Dielectrophoresis (DEP) is a force on charge neutralparticles in a non-uniform electric field arising from differencesin dielectric properties between the particles and the suspend-ing fluid. The time-averaged force on a homogeneous sphere of

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 225

FIG. 11. (A) A series of amplifications and separations from different strains of Escherichia coli cells conducted using the portablePCR-CE microsystem. Top frame: E. coli K12, a non-pathogenic lab strain; middle frame: E. coli O55:H7, a pathogenic strain thatdoes not express Shiga-like toxin; bottom frame, E. coli O157:H7, a pathogenic strain expressing Shiga-like toxin. White peaksare co-injected DNA ladder peaks, black peaks represent PCR products (280 bp: 16S species-specific marker; 625 bp: fliC geneencoding H7 flagellar antigen; 348 bp: sltI gene encoding Shiga-like toxin). (B) Histogram showing relative product peak areas forPCR product peak areas for sltI product (black) and 16S product (gray) for a series of serial dilutions of E. coli O157:H7 cells intonon-pathogenic E. coli K12 cells. The fliC product is still visible to 0.1% pathogenic cells, indicating pathogenicity is detectableto the level of 1 cell in 1000 using the portable PCR-CE microsystem. Adapted from Reference 68.

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226 E. T. LAGALLY AND H. T. SOH

radius rp can be approximated as:

�FDEP = 2πεmr3pRe(K )∇Erms

2 [10]

Here Re(K ) is the real part of K , the Clausius-Mosotti factor,defined as:

K = ε∗p − ε∗

m

ε∗p + 2ε∗

m

[11]

where ε∗p is the complex permittivity of the particle and ε∗

m isthe complex permittivity of the medium. Trapping of certaincell types may be achieved by specifically attracting them toelectrodes (positive K ) while repelling others (negative K ).121

In the case of bacteria, and E. coli in particular, the cross-overfrequency is reported to be a stronger function of medium per-mittivity than frequency. For media with conductivities smallerthan the measured conductivity of the cell (σ ≈ 44 mS/m),K is positive for frequencies smaller than about 1 MHz.122,123

For mammalian cells, however, the cross-over frequencies fromnegative to positive K are better defined, and typically lie in therange between 10 and 90 kHz.121 Therefore, by setting the DEPfrequency below the cross-over frequency of non-bacterial cellsand operating in a medium with sufficiently low permittivity, se-lective capture of bacteria is possible while rejecting larger cellsin the sample. Gascoyne and coworkers have presented a seriesof microdevices for the separation and characterization of mul-tiple cell types using DEP, including separation of cancer cellsfrom normal cells and separation of multiple types of immunecells from one another.121,124−128

Much recent work has applied DEP to integrated geneticanalysis systems; in particular, Cheng et al.129 fabricated anintegrated microsystem for the selection and concentration ofcells on microelectrodes, and the subsequent chemical inter-rogation of these cells using the electrodes. Grodzinski andcoworkers presented a microfabricated system for cell concen-tration and genetic sample preparation from complex samplebackgrounds.130 Manaresi et al. fabricated a CMOS chip formanipulation and concentration of cells on a 320 × 320 elec-trode array.131 Lapizco-Encinas et al. demonstrated DEP con-centration of bacteria using a series of insulating posts in anelectric field, and used this system to differentiate live fromdead bacteria.132,133 Recently, Lagally et al. have described amicrosystem for the concentration and detection of genetic ma-terial from bacterial pathogens.134 Their system flows a samplemixture through a polyimide microchannel and utilizes positivedielectrophoresis (DEP) to trap any bacterial cells present inthe sample on a set of interdigitated microelectrodes. Followingtrapping, a set of PDMS microvalves is closed around the micro-electrodes, defining a 100 nL chamber that greatly concentratesthe target cells. A cell lysis buffer containing an optical molecu-lar beacon is then introduced. The molecular beacon hybridizesin a species-specific fashion to the rRNA from E. coli cells. Thesystem is monitored using a confocal fluorescence microscope,and the limit of detection is 25 cells. Importantly, cells can bedetected in 20 minutes, allowing rapid detection of bacteria.

2. Isolation of MoleculesIn other cases, purification of molecules from a sample mix-

ture is required before genetic analysis may proceed. For in-stance, the specific amplification of RNA and its differentiationfrom DNA requires the rejection of DNA from the sample, whichcan act as a contaminant. Detection of RNA yields informationthat DNA cannot, namely the set of genes that are transcribedwithin a cell at a given point in time under a defined set ofconditions. To this end, several groups have worked to developmicrosystems for the selective isolation and purification of RNAfrom complex samples backgrounds. Jiang and Harrison135 pre-sented a microdevice using microbeads with poly-T oligonu-cleotides immobilized on them that were selectively placedwithin an etched microchannel to an mRNA capture bed. Follow-ing transcription from the DNA, mRNA is modified to containa poly-A tail, which hybridizes to the poly-T oligonucleotidespresent on the microspheres. Their results showed that captureof mRNA from total RNA was possible down to a minimum of2.8 ng at a capture efficiency of 26%. The same group later usedmagnetic microparticles coated with a monoclonal antibody tocapture T cells from human blood at a capture efficiency of 37%using a series of parallel microchannels.136

Post-amplification purification is also often necessary ingenetic analysis, particularly for DNA sequencing. DNA se-quencing, due to the single base-pair resolution required, ne-cessitates a high purity cycle sequencing sample. Conventionalpost-amplification purification for DNA sequencing is ethanolprecipitation followed by resuspension in a suitable bufferfor CE separation. The microfabrication of such mid-streampurification steps has proven difficult, but has been demon-strated. Such systems utilize sequence-specific capture probesimmobilized within a certain section of a microsystem. Paegelet al.137 described a three-dimensional monolithic capture gel,consisting of linear polyacrylamide that had been modified tocontain a sequence-specific capture probe attached to the poly-mer backbone. DNA sequencing samples were electrophoreti-cally driven through this purification gel, and any DNA frag-ments containing the complement to the capture probe (theauthors used the known sequence directly 3’ to the primersto design the capture probe) were immobilized within the gel.Application of higher temperatures (67◦C) released the boundfragments, and these were electrophoretically injected onto andseparated using microchannel CE. The reduced system couldpurify and sequence samples within 30 minutes, a 10-fold re-duction in time using a 100-fold reduced volume compared toconventional samples. Such systems may lead to the possibilityof sequencing large genomes at greatly reduced cost.138

B. Advanced Detection MethodologiesAnother important area of future research will be the elimina-

tion of the power-hungry and cost-intensive components fromintegrated genetic analysis microsystems in order to enhancetheir field-portability, disposability, and to reduce their cost ofproduction.

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 227

1. Optics-Free DetectionUse of fluorescence has typically dominated genetic analy-

sis due to its extremely low (picomolar) detection limits. Forapplications in remote environments with minimally trained op-erators (e.g., the genetic detection of HIV in Africa), such opticalcomponents may prove to be impractical due to size, cost, andfragility. Alternative detection strategies are needed that sup-plant optical detection while maintaining many of its advantages.Electrochemical detection, relying on the transfer of electrons togenerate oxidative and reductive currents, is one such technique.Although electrochemical detection has typically exhibited lim-its of detection only in the nanomolar–micromolar range, theuse of PCR to amplify DNA can be used to generate sufficientproduct such that electrochemical means may also be used fordetection. The first work to demonstrate the integration of mi-crochannel CE with electrochemical detection was presented byWoolley et al.139 In this work, PCR-amplified DNA was sepa-rated on a CE microchannel and detected using electrochemicalelectrodes placed past the end of the separation channel. Laterwork has refined the technique, using electrical isolation tech-niques and different electrode geometries to improve the signal-to-noise ratio. Ertl et al.140 described a sheath-flow supportedelectrochemical detector for use in integrated CE microsystems.The sheath flow carries the DNA analyte from the gel separationregion into a free-solution detection region, electrically isolatingthe electrochemical detector from the high electric fields inher-ent to CE. Other recent advances in electrochemical detection,such as differential measurements and use of electrochemicalintercalators141 and Ag-coated Au nanoparticles,142,143 have de-creased the limits of detection of electrochemical means evenfurther and enabled the detection of hybridization events, allow-ing electrochemical sequence-specific detection.

The first demonstration of a microscale PCR chamber in-tegrated with electrochemical detection was published by Leeet al.144 In this work, gold electrodes within the PCR chamberused immobilized DNA probes and either electrochemical inter-calators or Ag-coated Au nanoparticles to detect the concentra-tion of the PCR product of interest. The system was capable ofdetecting as few as ten molecules of starting DNA template inan 8 µL PCR chamber. Such a system will inherently encounterdifficulty in differentiating similar DNA sequences, such as aregenerated in forensic investigations through the amplification ofshort tandem repeats (STRs); however, such systems avoid theneed for confocal optics and laser excitation, making them eas-ily portable. In addition, the fabrication of electrodes is low-costand can be accomplished on plastic or glass substrates amenableto mass fabrication. Toward this end, Liu et al. 145 published acompletely integrated genetic analysis system fabricated fromplastic substrates that incorporates cell concentration using mag-netic bead capture, convective mixing, lysis, PCR amplification,and electrochemical detection using a sandwich assay. Their sys-tem was capable of detecting 106 E. coli cells in 1 mL of wholeblood, and was also used to determine the presence of the humanHFE-C gene directly from human blood. Although the limits of

detection of this system were not investigated, the use of PCRpromises to provide high sensitivity and specificity over a widevariety of targets. Such highly integrated systems may representthe future of integrated microtechnologies for genetic analysis.There are several outstanding limitations to be addressed; one ofthem may be cost, in that these assays require expensive reagents,including gold or silver nanoparticles, magnetic microspheres,and PCR reagents. Work continues to develop a low-cost versionof such systems for wide accessibility in clinical and forensicdiagnostics.

2. Reagentless DetectionIn many practical cases, the availability and storage of bio-

chemical reagents becomes a limiting constraint. The need forrefrigeration, storage, and handling infrastructure makes assaysthat require such reagents impossible in many of the areas thatmay need such systems the most. The ideal genetic detectionmethod would be reagentless (requiring only the sample foranalysis), field-portable, low-power, reusable, and able to bestored for long times. Fan and colleagues146 have developed asystem that meets most of these requirements. Figure 12 presentsa schematic representation of such a system. Their system con-sists of a single strand of DNA attached at one end to an electro-chemical working electrode using standard Au-thiol chemistry.The other end of the probe DNA contains an electrochemicallyactive reporter molecule. The sequence of the DNA is chosento be self-complementary, such that the probe in the absenceof target forms a stem-loop structure. The electrochemical la-bel undergoes oxidation or reduction upon the application of apotential at the working electrode and transfers electrons to theworking electrode. Upon introduction of complementary targetDNA, however, the probe DNA hybridizes to the target and be-comes more rigid, moving the electrochemical label away fromthe surface. Because electron transfer is an exponential func-tion of distance, the electrochemical current in the presence oftarget is a small fraction of that in the stem–loop state, pro-viding a signal-off sensor. Xiao and coworkers have recentlyextended this work to protein detection through the use of ap-tamers, single-stranded DNA oligonucleotides that demonstratehigh affinity and high specificity for a certain target.147 Usingthe E-DNA architecture, an aptamer was bound to a gold elec-trode and contained an electrochemical label at the free end.Binding of thrombin, a protein involved in the human blood-clotting cascade, changed the aptamer structure, which in turnaffected the electrochemical current used for detection. The useof electrochemical reporters and efficient exploitation of ligand-induced folding of probe molecules may propel the field towardreagentless, portable, low-power, and disposable devices.

C. Microsystems for Parallel Information Gathering1. Motivation

Genetic analysis of samples from complex mixtures presentsan analytical challenge not only in the methods of analysis, but

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228 E. T. LAGALLY AND H. T. SOH

FIG. 12. The E-DNA system. A stem–loop oligonucleotide possessing terminal thiol and a ferrocene group is immobilized at agold electrode through self-assembly. In the absence of target, the stem–loop structure holds the ferrocene tag into close proximitywith the electrode surface, thus ensuring rapid electron transfer and efficient redox of the ferrocene label. On hybridization withthe target sequence, a large change in redox currents is observed, presumably because the ferrocene label is separated from theelectrode surface. Reprinted with permission from Reference 108.

also in the interpretation of the results. Most methods of ge-netic analysis are limited in the types of information that theycan present to the user. PCR is an excellent example of suchlimitations. Although fast, exponential, and highly specific, PCRexhibits a major limitation: one has to know what one is lookingfor. That is, the design of the PCR product requires appropri-ate primer sequences, and these primer sequences must be cho-sen from known sequence information a priori. All organismshave genomes that require regular random changes to their se-quences in order to adapt to new environments and to evolve.Thus, primer sequences that are chosen from currently availablegenome sequence data may become obsolete, and perhaps moreimportantly, the genome sequence may be unknown. In this as-pect, genetic analysis of bacteria presents an especially largechallenge, because such organisms are capable of very fast re-production (doubling times as fast as 30 minutes), and thereforeintrinsically higher rates of genetic variation.

There are several approaches to confront this analytical chal-lenge. The most adaptable solution in the long term is likely to bethe integration of multiple information streams about a particularsample, such that genetic analysis results can be crosscheckedagainst other information types (immunological, physical, elec-trical, etc.) to verify the results. One of the future areas of workin genetic analysis microsystems will therefore be integration toacquire multiple types of information about a particular sample.Such systems have already begun to emerge in the literature; thework by Lagally et al. involving DEP concentration followed bygenetic detection134 exhibits the advantage of combining dielec-trophoretic information about the bacteria with genetic data. Forexample, if bacteria are successfully captured but genetic anal-

ysis provides a negative result, at least the user is potentiallyaware of the presence of such bacteria, which would not be thecase with genetic analysis alone.

Genetic analysis integrated with other information types pro-vides advantages beyond analysis redundancy—it will allowthe user to rapidly characterize new or unknown samples morequickly. For instance, if a single system is capable of simul-taneous analysis of the immunological, genetic, electrical, andbiochemical characteristics of the bacteria in a mixed sample,this has the potential to reduce the time needed for deduction oftreatment methods. In addition, correlation of multiple parame-ters may be used to discover new relationships between differenttypes of organisms or components of a sample. Recent advancesin epigenetics, the self-regulation of phenotype without geneticchange,148 and relationships between the genome, proteome,149

and glycome150 of an organism call for integrated analysis sys-tems capable of aiding these fields with faster analysis, lowerdetection limits, higher sensitivity, and higher specificity.

2. Interface ChallengesThe development of such parallel-processing integrated

microsystems brings with it challenges in the “chip-to-world”interface. In analogy to the semiconductor industry, microfluidictechnologies have reached a stage where hardware such aszero-insertion force (ZIF) sockets have become a necessity. Asthe number of parallel analyses on a single microdevice grows,so do the number of distinct connections needed to externalhardware, which may be microfluidic, pneumatic, or electrical.Although some of the functionality may certainly be fabricated

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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 229

on the device itself in the future, some connections will have toremain off-device, and the number of connections will increasewith the functionality of the device. Fluidic connectionsare of particular concern, mostly because the interface mustremain rigid, yet sufficiently flexible for the device to bemoved. In addition, application of these systems to complexsample mixtures will increase the constraints on any fluidicconnection to prevent clogging, adsorption, or other detrimentalinteractions. Several other groups have proposed solutions tothese problems,151,152 but a common general solution to suchproblems has not emerged, making this area one of primaryfocus for future work.

VII. CONCLUSIONSThis review has presented a series of technologies for mi-

croscale and nanoscale analysis of genetic material. The ad-vances in such technology from the first examples in the early1990s to the hundreds of examples at present represents expo-nential growth in the field. Examples of advances in microfab-ricated “modules” representing individual components, as wellas efforts to combine these modules to form integrated systemsthat surpass the performance of their conventional predecessors,have been discussed. The design, fabrication, and testing of in-tegrated microsystems that collect multiple types of informationabout a sample have just begun to emerge, and will propel thefield to new levels of functionality and utility. The powerfulconvergence of materials science, chemistry, and biology withmicrosystems technology promise to advance the field to createsystems capable of unprecedented performance, utility, and ac-cessibility. It is hoped that the benefits from such technologieswill improve the lives of many.

ACKNOWLEDGMENTS

The authors thank Professors M. A. Burns, A. Manz, R. A.Mathies, and K. W. Plaxco for their permission to reprint figuresfrom their publications here.

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