Integrated Genetic Analysis Microsystems

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Critical Reviews in Solid State and Materials Sciences, 30:207233, 2005

Copyright c Taylor and Francis Inc.

ISSN: 1040-8436 print

DOI: 10.1080/10408430500332149

Integrated Genetic Analysis Microsystems

E. T. Lagally

California Nanosystems Institute, University of CaliforniaSanta Barbara, Santa Barbara, CA, USA

H. T. SohCalifornia Nanosystems Institute and Department of Mechanical and Environmental Engineering,

University of CaliforniaSanta 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-lelismleading to increased assay throughput. In addition, when fully integrated, this technology

will enable portable systems for high-speed in situanalyses, 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 .........................................................................................................................................................210

A. PCR and PCR Microsystems ..........................................................................................................................210

1. PCR .................................................................................................................................................... 210

2. Microscale PCR ...................................................................................................................................212

3. Portable PCR Microsystems .............. ............... ............... ............... ............... ................ ............... ......... 212

4. Microscale PCR: Materials and Design Considerations ..................... ................ ............... ............... ......... 212

a. Substrate Material and Surface Chemistry .............. ............... ................ ............... ............... ......... 212

b. Heaters and Temperature Sensors .............. ............... ............... ............... ............... ............... ....... 214

c. Enclosed Chambers ...................................................................................................................214

5. Significance .........................................................................................................................................214

B. Capillary Electrophoresis and Microchannel CE .............. ............... ............... ................ ............... ............... .... 215

1. Capillary Electrophoresis Background ............... ............... ............... ............... ................ ............... ......... 2152. Microchannel CE .................................................................................................................................215

3. Entropic Trap Separations .............. ............... ................ ............... ............... ............... ............... ............ 216

4. Materials Issues ...................................................................................................................................217

a. Surface Chemistry .....................................................................................................................217

5. Significance .........................................................................................................................................218

E-mail: [email protected]

207


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

IV. INTEGRATION ...............................................................................................................................................218

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

1. Microvalves .........................................................................................................................................218

2. Micropumps ........................................................................................................................................218

B. Examples of Integrated Microsystems .............. ............... ............... ............... ............... ................ ............... .... 219

C. Integrated Optics ............. ............... ................ ............... ............... ............... ............... ............... ................ .... 221

V. MICROSYSTEMS FOR REAL-WORLD APPLICATIONS .............................................................................222

A. Epidemiology Applications of PCR-CE ......... ............... ................ ............... ............... ............... ............... ....... 222

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

B. Forensic Identification .......... ................ ............... ............... ............... ............... ............... ................ .............. 224

VI. FUTURE DIRECTIONS ..................................................................................................................................224

A. Analysis from Complex Sample Mixtures ..................... ................ ............... ............... ............... ............... ....... 224

1. Isolation of Cells .................................................................................................................................. 224

2. Isolation of Molecules .............. ............... ............... ................ ............... ............... ............... ............... .. 226

B. Advanced Detection Methodologies ................. ............... ............... ............... ............... ................ ............... .... 226

1. Optics-Free Detection ............... ............... ............... ................ ............... ............... ............... ............... .. 227

2. Reagentless Detection ............... ............... ............... ................ ............... ............... ............... ............... .. 227

C. Microsystems for Parallel Information Gathering ................ ............... ............... ............... ............... ............... .. 2271. Motivation ...........................................................................................................................................227

2. Interface Challenges .............. ................ ............... ............... ............... ............... ................ ............... .... 228

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

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

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

I. INTRODUCTION

The analysis of genetic material is one of the most important

facets of molecular biology, health sciences, and forensics. The

necessary technology has advanced tremendously, with some of

the most dramatic advances occurring within the past five to ten

years. Analyses that used to require large sample volumes and

needed hours can be performed in minutes in volumes as low

as hundreds of picoliters (1012 L). The fundamental paradigm

through 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 that

are capable of performing multiple steps of a conventional ge-

netic analysis. Such integration not only reduces the time scale

and volumes (and therefore the costs) of analyses, but also de-

creases or eliminates external contamination. Furthermore, themonolithic parallel integration of multiple devices within a chip

promises to increase the throughput as well as facilitating the

fabrication of disposable devices.

The genesis of integrated genetic analysis systems began with

the fabrication of microchannels capable of conducting liquids

from one point to another within a chip using processes simi-

lar to IC and solid-state MEMS technology. Subsequently, the

integration 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 as

methodologies to control surface chemistry using a variety of

materials. The field of integrated genetic analysis systems is in

an active phase of research and development, and the number

of publications in this field continues to grow at a rapid rate.

With the expanding availability of entire genomes of increas-

ing numbers of organisms,13 such microsystems will begin to

address systems-level connections between genes both within

and among organisms. This review highlights the advances at

each of the major developmental stages of the technology, with

the emphasis on the materials science and surface chemistry as-

pects. The conclusion will attempt to provide a look forward at

possible 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 genetic

analysis may be broken down as a sample preparation step fol-

lowed by a detection or analysis step. Figure 1 schematically

presents the major steps of a conventional analysis. The first

step is the isolation of target cells, which may be as simple as

centrifugation or as complex as separation of different cell types

using 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 cell

lysis (DNA is thewhite strands floating in themixture). The nucleic acids are usually purified using a variety of techniques, followed

by amplification. Amplified products are again purified before analysis using capillary electrophoresis or real-time detection. Certain

purification steps, marked with dashed lines, may be omitted depending on the assay.

such as fluorescence activated cell sorters (FACS). Cell isolation

is followed by cell culture, on which cells are grown on media

preferential to specific cell types. The next step is nucleic acid

extraction, in which the cells of interest are first lysed. This can

be 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 accomplished

through a series of chemical steps leading to the nucleic acids

suspended in an aqueous solution, while selectively removing

the 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-based

methods, 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 contains

a sequence of repeated adenine (A) residues at one end due to

modification inside the cell, can be hybridizedto complementary


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poly-thymine (T) oligonucleotides, which themselves have been

covalently bound to microspheres.6 Such affinity-based meth-

ods may also be used with DNA or other nucleic acids if

the sequence of the desired nucleic acid is known. The non-

covalent bonding approach is similar in its approach except

that the nucleic acids are non-specifically bound to the solid

phase such as glass microspheres or a silica membrane throughhydrogen bonding.7 Huang et al.8 have reviewed the ways

MEMS technology has been applied to sample purification and

preparation.

Following nucleic acid purification, the next major step is

sample amplification. Although the molecules may be present

within the cell at concentrations detectable using conven-

tional detection techniques (pM to nM), the actual number of

molecules may be quite small, down to a single DNA strand

of interest. Thus upon lysis, significant dilution is typically an

unavoidable result (sub fM). At these low concentrations, the

number of molecules plays an increasingly important role as

stochastic effects begin to emerge. To increase the number of

target molecules, several methods for amplifying trace amountsof nucleic acids have been developed and subsequently applied

within a microfabricated format. The most common technique

is polymerase chain reaction (PCR).9 In this reaction, mul-

tiple cycles of three temperatures are used to generate new

copies 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 microfabricated

devices.

After purifying the amplification products, the final stage in a

genetic analysis is thelabelingand detectionof thegenetic mate-

rial. Depending on the requirements, the analysis may be as sim-

ple as confirmation that nucleic acids of a certain sequence are

present, or itmay be as detailed as the length and the sequence of

the amplification products. One of the most common techniques

for the detection and analysis is electrophoresis, in which nu-

cleic acids are separated by length under an applied electric

field. There are a variety of electrophoresis methods including

slab gel electrophoresis,4 pulsed field electrophoresis,10 and

capillary electrophoresis.11 In the conventional genetic analy-

sis protocol, the overall required time can be on the order of

hours; however, it is often on the scale of days if cell culture is

required.

In contrast, integrated genetic analysis microsystems have

demonstrated the capability to perform the same tasks in a

fraction of the time, and complete genetic analysis within

30 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 nucleic

acid amplification.17 To illustrate the evolution of microde-

vices for genetic analysis, this review will focus on two

of the major steps in the genetic analysis as a vehicle for

detailed 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 conventional

methodologies.

III. DEVICES

A. PCR and PCR Microsystems

1. PCR

In genetic analysis,the mostmaterials-critical step is the sam-

ple preparation, and the case of PCR amplification warrants a

detailed discussion. Since its initial description in 1985,9 PCR

has 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 of

short oligonucleotide primers specific to known sequences on

the template strand, (3) a thermostable DNA polymerase (Taq, a

modified DNA polymerase isolated from the thermophilic bac-

teria Thermus aquaticusis 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 94C96C, separates or denatures the two template

strands (Figure 2A); at the second temperature, typically 45

60C, the primers hybridize to their complementary sequences

on the parent strand (Figure 2B); during the third temperature

step, usually at 72C, the DNA polymerase forms new daughter

strands, extending the primer sequences by adding individual

dNTPs from solution (Figure 2C). Repetition of the sequence

at optimal efficiency therefore generates 2n daughter strands,

wheren is the number of cycles. The reaction can be described

in terms of the concentration of DNA molecules as a function

of the number of cycles completed:

[DNA]f=

ni=1

(1 + i )

[DNA]i , [1]

where [DNA]f is thefinalDNA concentration, [DNA]iisthe con-

centration at theith cycle, andiis theefficiencyof thereaction at

the ith cycle. The efficiency of the reaction is theoretically unity

forsmall values ofiand decreaseswith increasing cycle number.

This phenomenon may be explained by the Michaelis-Menten

equation:

v= vmax[T]

[T] + KM, [2]

where v is the rate of product formation at any point in the

reaction,vmaxis the maximum rate of product formation, [T] is

theconcentration of target (uncatalyzed primer and dNTPs), and

KMis the Michaelis-Menten rate constant in mol/L. Using this

equation, which describes the reaction rate as being hyperbolic

with reactant concentration, we may express the efficiency of

PCR as18

i = 1 vi

vmax, [3]


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FIG. 2. A schematic representation of the polymerase chain reaction (PCR). Template nucleic acids are cycled between three

temperatures, denaturation (A), annealing (B), and extension (C), respectively. The right hand side depicts the products after the

first 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, the

efficiency i will also decrease as the reaction progresses and

more primers and dNTPS are consumed. Careful control of tem-

peratures and initial reactant concentrations are necessary to

maximize reaction yield and to minimize thenumber of required

cycles.

PCR exhibits several notable advantages over competing

techniques, including exponential amplification, relatively few

reagents, and a simple reaction scheme consisting of three easily

attained temperatures. PCR technology has been commercial-

ized to the point that almost every lab using nucleic acids owns

a 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 generate

a cDNA complement to an RNA of interest, and then amplifies

this cDNA exponentially to a detectable level. In addition, multi-

pleDNA templatesmay be simultaneously amplified in thesame

reaction vessel using multiplex PCR. In cases where the melt-

ing temperatures of different primers within a multiplex reaction

prevent 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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of widely varying primer sets to multiple templates.24 Another

widely 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 intercalating

fluorescent dye present in the reaction mixture labels amplified

DNA 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 a

fluorescent dye at one end and a fluorescence quencher at the

other end, resulting in a non-fluorescent probe in its native state.

Following hybridization to amplified DNA, however, the probe

is cleaved by the polymerase during extension in the next cy-

cle, separating the quencher from the fluorophore and restoring

fluorescence. The rtPCR method has been adapted for use in

microsystems.2730

2. Microscale PCR

PCR can be easily miniaturized, and such reduction in scaleprovides several important advantages. First, the reduction in

volume allows faster temperature transitions, while simultane-

ously reducing reagent costs. In addition, microfabrication al-

lows further integration of other functionalities to enable highly

portable integrated genetic analysis microsystems. The first

demonstration of microchip PCR, by Northrup and cowork-

ers in 1993, used a Si microchamber and a microfabricated

resistive heating element.31 Subsequently, a large number of

groups have explored different strategies for miniaturization.

Wilding etal.32 demonstrated a silicon PCR microchip. Shoffner

et al.33 and Cheng et al.34 investigated the use of silicon-glass

microstructures. Poser et al.35 demonstrated a novel silicon

PCR microstructure and investigated optimal chamber volume

and geometry through thermal modeling and chamber arrays.

Chaudhariet 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 L) andthermalisolation from the

rest of the substrate using thin suspended silicon nitride films.

Tayloret al.30 discussed the fabrication of process control el-

ements within the microchip PCR. All such microfabrication

strategies mimic the conventional static PCR approach where

samples are placed in a reaction chamber, which then undergoes

thermal cycling to achieve desired amplification as a function of

time. In 1998, Koppet 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 channels

with three isothermal zones for the denaturing, annealing, and

extension steps so that the chemical amplification occurs as a

function of spatial location (Figure 3). This continuous ampli-

fication strategy is especially well suited for microsystems, as

it does not involve constraining a small volume without bubble

formation. In this work, 20 cycles of PCR were performed in a

timeof aslittle as1.5 minutes, using a total volumeof 8L using

a channel with a cross-sectional area of 3600 m2. The initial

demonstration required very high starting template concentra-

tions (108 DNA copies) and relatively large volumes; later work

has mitigated many of these initial problems. Shin et al. 39 fab-

ricated a CPCR microchip from PDMS that was passivated with

Parylene to avoid sample absorption into the PDMS substrate.Sun et al.40 fabricated a CPCR microsystem with transparent

indium tin oxide (ITO) heaters for easier optical observation.

Zhanget al.41 presented finite-element models of CPCR for the

purposes of enhanced thermal design. Obeid et al.27 presented

laser-induced fluorescence detection of PCR products using an

intercalating dye introduced following amplification.

Other researchers have investigated means of increasing the

speed 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 using

infrared radiation, provides very fast heating while eliminating

substrate heating.42 Liu et al.43 presented a novel rotary PCR

microchip 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 differently

heated regions to achieve amplification. Heap et al.45 used an

AC current to heat a PCR solution electrolytically for thermal

cycling.

3. Portable PCR Microsystems

Advances in microfabricated devices have recently led to

the fabrication of field-portable PCR systems. Using the rtPCR

assay, 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 an

on-site genetic analysis. The same group later demonstrated an

even smaller notebook-sized, battery-operated system for PCR

amplification.28 In addition, Higgins et al.47 demonstrated a

handheldrtPCR microdevice. Paland Venkataraman48 presented

a portable PCR system based on inductive heating. These im-

pressive microsystems are making their way into clinical and

forensic investigations, and their roles are sure to increase with

further advances in technology.

4. Microscale PCR: Materials and Design Considerations

a. Substrate Material and Surface Chemistry. Choice of

substrate material affects the biochemical function of PCR

reagents within a microsystem in a significant way. In early

work, it was discovered that silicon and silicon nitrides demon-

strate an interfering effect when conducting certain nucleic acid

amplification assays.34 Theories surrounding these materials in-

teractions vary, but a large contingent of researchers maintains

the hypothesis that because the polymerase requires divalent


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FIG. 3. (A) Schematic of a chip forflow-through PCR. Three well-defined zones arekept at 95, 77, and60 by means of thermostated

copper blocks. The sample is hydrostatically pumped through a single channel etched into the glass chip. (B) Channel layout. The

device 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 or

semiconductor cations in solution could interfere with the proper

operation of the polymerase. Passivation of these materials with

oxides 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 adsorbingto the sidewalls of the reaction vessel.

In particular, glass, with its free silanol (SiOH) groups at thesurface, readily forms hydrogen bonds to nucleic acids, leading

to sample adsorption. It is well known that the surface to vol-

ume ratio increasesas device sizes shrink. Thus in microdevices,

non-specifically adsorbed molecules, which are unavailable to


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the reaction, become a larger percentage of the total number

of molecules and significantly limit the efficiency of the reac-

tion. The use of non-specific surface coatings are often used to

overcome such restrictions; inclusion of carrier molecules in

reaction mixtures that are designed to coat the chamber surfaces

are 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 to

prevent DNA adsorption have also been explored. For example,

Giordano et al.49 presented work on optimization of dynamic

polymer coatings for microscale DNA amplification. Most of

these coatings rely on the reaction of a bifunctional silane moe-

ity with the silanol groups on a fully deprotonated silicon oxide

surface, followed by chemical modification of the other end of

the silane molecule to present a hydrophilic surface that inhibit

hydrogen-bonding with DNA in solution.50

A series of polymers has recently become important for

genetic analysis microsystems. Some of these polymers, such

as poly(dimethylsiloxane) (PDMS), poly(methylmethacrylate)(PMMA), and poly(carbonate) (PC) are useful as substrate

materials. A variety of microfabrication strategies including

casting,51 laser ablation,52,53 hot embossing,54 or injection

molding.5557 have been developed for polymer microfluidic

devices. 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 masterusing a positivephotoresist, followedby casting

of the mold negative in PDMS. This technique has been used

widely 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 used

extensively as a substrate material, has been adapted for the

fabrication of microchannels.42 It has also been used as a sacri-

ficial etch mask for the formation of structural features in other

applications.62 Polyimidehas many desirable characteristics due

to its ability to be easily spun on as a resist-like film, and be-

cause its curing process can be integrated with wafer bonding

processes.

b. Heaters and Temperature Sensors. Thin metal films of

platinum, palladium, and to a lesser extent, gold are used to

form electrodes, heaters, and temperature sensors in integrated

genetic analysis microsystems, as they provide low chemical

reactivity, low resistivity, and high melting point. These metals

are easily deposited as thin films using sputtering or evaporation

processes, andcan be etchedusing a variety of wetor dryetching

techniques. Subsequent bonding processes (seelater) can require

temperatures above 650C, and so it is important that the metals

exhibit minimal thermal effects, including expansion, oxidation,

and diffusion at these temperatures. Platinumin particular is well

suitedfor 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 temperature

detectors (RTD).30,31,33,34,46 Indium tin oxide is an example of

a transparent conductor that can be used to fabricate electrodes

or heaters in applications requiring optical transparency.40,63

c. Enclosed Chambers. Initial microfabricated PCR reac-

tors consisted of etched wells into which reagents were loaded

and covered with mineral oil to prevent evaporation.31,64 The

availability of wafer bonding processes now allows fabrication

of 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 from

the 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 fuse

two or more glass substrates together. Such bonds have high

mechanical strength; however, the necessity of high tempera-

tures (>500C) prevents the use of most polymer films and may

lead to oxidation and diffusion of metal films used in these sys-

tems. Microsystems with polymer filmsmay undergo bonding in

similar ways, generally requiring the polymer to be raised above

its glass transition temperature in non-oxidizing environments.In limited cases, microsystems can be fabricated where low-

mechanical-strength, non-permanent bonds are sufficient; they

include bonding ofPDMSto glass, as wellas the use ofthinpho-

toresist films that have been cured between two substrates. The

bonding of PDMS to glass and silicon substrates has proven to

be useful and interesting. Current theories hold that the PDMS,

when exposed to air or oxygen plasmas, undergoes an oxidation

reaction at the surface, leading to diffusion of unaltered oxy-

gen groups from the bulk.69 This process is self-reversing on a

time scale of hours, depending on conditions. However, when

the 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 bonding

technique hasbeen used in the fabrication of PDMS microvalves

and peristaltic pumps for directing liquid flows in microchannel

environments.43,51,7173

Bonding processes are difficult to generalize, because they

depend on the substrate and other fabrication details, but certain

trends are evident across most bonding processes. First, bonding

processes may cause lower process yields than other steps in a

process flow, and because bonding steps are generally at the end

of a fabrication process, much work may be lost if successful

bonding of twosubstrates is notachieved. Second, bonding yield

is 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 bonding

processes of heterogeneous substrates is needed.

5. Significance

PCR microsystems demonstrate a number of interesting

characteristics. First, they can amplify miniscule volumes of

nucleic 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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simple fabrication processes and integrated thermal control can

be easily accomplished. Although the reaction is sensitive to

temperature, some results have demonstrated that even highly

anisotropic temperature distributions can result in successful

amplification.74 Undisputedly, PCR is an important component

of an integrated genetic analysis microsystem.

B. Capillary Electrophoresis and Microchannel CE

The second example of microfabricated genetic analysis sys-

tems centers on the development of capillary electrophoresis

microchannel systems and their integration with the PCR mi-

crosystems discussed earlier. Such CE systems are frequently

used to separate DNA by length and function as the analysis

step following amplification.

1. Capillary Electrophoresis Background

Early DNA separation systems relied on the knowledge that

DNA has a net negative charge due to regular phosphate groups

in its backbone. However, electrophoresis separates moleculesbased on their charge-to-mass ratios, and application of voltage

to DNA in a free-zone separation (buffer only) cannot separate

based on DNA length because the number of phosphate groups

scales 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 have

an average size that is small enough (10 nm200 nm) to restrict

the 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 therefore

length) 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 which

smaller moleculestraversed the gelfaster than larger ones. How-

ever, these systems suffered from numerous problems, includ-

ing high temperatures due to the large currents (10100 mA)

applied, which resulted in high DNA diffusivity and band broad-

ening andpoor resolution dueto theinitial plug formation within

the gel (see later). Later work resulted in the development of gel

electrophoresis separations in drawn fused-silica capillaries, and

this technique became known as capillary gel electrophoresis

(CGE).75

In this technique, nucleic acids are separated by length

through a sieving matrix under an applied electric field within

a glass capillary (inner diameter 50200 m). The velocity of

DNA fragments in the capillary is described as a function of the

electrophoretic mobility

v= E [4]

where is a constant for particular length of DNA (units of

cm2/V*second) and Eis the applied electric field (V/cm). The

resolution of a CE separation is defined as the difference (in

elution time) of adjacent bands of DNA of constant length over

their average widths. Theoretically, the resolution may be ex-

pressed as:76

R= t2 t1

12

(w1 +w2)=

L(1 2)

41(1Einjtinj)

2

12 + 2DL

1E 1/2

[5]

whereL is thecolumn length, 1and 2are the mobilities of the

two DNA fragments of interest, Einjis the applied electric field

for injection,tinj is the injection time, E is the applied electric

field for the separation, andDis the average diffusion coefficient

of the DNA fragments. Depending on the operating regime, the

resolution depends on either the length of the channel or the

square root of the length. In thefirst regime, theband broadening

caused by the electrokinetic injection dominates, and as a result,

the resolution scales with length. In the second regime, the band

broadening is governed by diffusion resulting in a square-root

dependence of the resolution on length. As diffusion characteris-

tics aredifficult to engineer, it is imperative to minimizethe band

broadening caused by the electrokinetic injection in a microsys-tem. Microchannel CE is advantageous compared to standard

CE systems because microfabrication allows precise determina-

tion of the shape and size of theinjectedplug of genetic material,

thereby enabling short separation lengths and high-performance

separations.

2. Microchannel CE

The initial descriptions of microchannel CE were by Manz

et al.77 and Harrison etal.13 Later work by others extended these

approaches toward high-resolution and paralleloperation. Wool-

ley andMathies14,78 demonstrated the first DNA fragment sizing

and DNA sequencing separations on a glass microchannel CE

device in which DNA was introduced electrokinetically through

an injection cross-channel and separated on a 5 cm-long, gel-

filled microchannel in only 120 seconds. The DNA was labeled

on-column using an intercalating fluorescent dye and detected

with laser-induced confocal fluorescence detection. A schematic

diagram of the microchannel geometry and experimental set-up

is presented in Figure 4. The key feature of this device leading

to exceptional performance was an injection cross-channel de-

sign that intersects the main separation channel. This feature is

critical 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 of

parallel 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 challenges

beyond microfabrication. The operation of 96 CE channels re-

quired a nearly 100-fold increase in current, which led to a rapid

exhaustion of buffering capacity as protons were quickly de-

pleted. A recirculation system was necessary to replenish the

buffer during the course of a full sequencing run and the device

utilized folded hyperturns to achieve a separation length of


10/28


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 of

microchip CE include the work by Emrich et al.81 that used a

straight-channel design with a direct injection scheme to demon-

strate a 384-channel DNA fragment sizing separation. Medintz

et al.8285 demonstrated a number of clinically relevant DNA

separations using microchannel CE.

3. Entropic Trap Separations

For separation of long DNA fragments (>1000bp), CEis 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 biased

reptation regime, and they all move with equal velocities re-

gardless of their length. In applications where long fragments

need to be separated, pulsed-field gel electrophoresis has been

successful.10 Unfortunately, this method suffers from the same

disadvantages as other slab gel techniques, and many research

groups proceeded to develop alternate methods for separating

longDNA fragmentsin a microdevice.Han etal.86,87 have devel-

oped an elegant method, consisting of a series of nanochannels

etched into a Si substrate. In their construction, they exploited

the fact that shallow (10 nm) channels form an entropic en-ergy barrier forlong DNA fragmentswherethe mobility of DNA

fragments depends on the average size of the DNA in its random

coil configuration. Thus the mobility can be directly correlated

to the DNA length. The underlying equation governing the resi-

dence time of a DNA coil in theentropictrap hasbeen elucidated

as:87

= 0eFmax

kB T , [6]

where0 is a prefactor with a dependence on the length of the

random DNA coil in solution, and Fmaxis the entropic energy

barrier requiredfor DNAto escape the nanometer-sized constric-

tion. Because the entropic energy barrier is a function only of the


11/28


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 isolated

buffer moats that lie above the drilled cathode and waste ports.

(C) Expanded view of the injector. Each doublet features two

sample 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 m, a turn

channel width of 65 m, and a radius of curvature of 250 m.

Reprinted with permission from Reference 56.

channel height, the separation may be achieved on the basis of

0, 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.

Cabodiet 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 have

the advantage that they do not require a polymer sieving matrix.

However, for small DNA molecules, the separation performance

trails that of CE, because the entropic energy barrier depends on

the average coil size of the DNA. For small DNA molecules,

sufficiently shallow channels have not yet been demonstrated.

4. Materials Issues

CE typically employs electric field strengths up to

300 V*cm1. In addition, because the detection of nucleic acids

commonly 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 optical

transparency, glass is a preferred substrate, and the success of

microchannel CE may be attributed to the advances in materi-

als and surface chemistry developed from earlier work in drawn

fused-silica capillaries.

a. Surface Chemistry. As described earlier, nucleic acids

have a net negative charge because of the presence of phosphate

groups in their backbones. In addition, nucleic acids readily form

hydrogen bonds to glass, resulting in an undesired, non-specific

adsorption to device sidewalls. The solution to this problem was

the use of the coating first introduced by Hjerten, a version ofthe silanization protocol also used to control DNA adsorption to

oxide surfaces during PCR.50 The use of this coating also con-

tributed to a significant increase in the resolution capability of

electrokinetic separation. The resolution of DNA separations in

early constructions of electrophoresis systems using standard,

uncoated glass capillaries was poor due to electrokinetic effects

that exist at charged surfaces in contact with conductive solu-

tions under applied voltage. More specifically, the native surface

charge of the fused silica gives rise to a charged double layer and

subsequent bulk electroosmotic flow (EOF) in the presence of

an electric field that transport the fluid in an opposite direction

with respect to the electrophoretic movement of the molecules.

The bulk EOF velocity may be expressed in the following way:

vEOF=

E. [7]

In this presentation, is the surface charge density, is the

dynamic viscosity of the solution, E is the applied electric field,

and is the Debye-Huckel constant, defined as

= F

2

ciz2i

0r RT

, [8]


12/28


13/28


then generates pressures for pumping, has been investigated as

an 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 sought

to develop electrokinetic techniques for bulk fluid movement

within microfabricated systems.Someof these technologies also

allow for other desirable processes, such as mixing in laminarflow conditions. Electroosmotic flow is the main motive force

used in these systems, in which a voltage is applied to a fluid

surrounded by a charged substrate (usually untreated glass), giv-

ing rise to a zeta potential and bulk fluid motion. 94,95 Although

these systems require high electric field strengths (200 V/cm),they operate at currents of 100 A or less, resulting in lower

power than the technologies mentioned previously. Integrated

application of such electrokinetic transport on the microscale

has been demonstrated. Chenet al.96 recently presented a rotary

PCR microsystem that used electrokinetic forces to transport the

PCR solution through three differently heated regions to achieve

amplification.

B. Examples of Integrated Microsystems

Concomitant with the development of microfluidic manipula-

tion technologies, efforts began to integrate nucleic acid ampli-

fication technologies with microchannel CE for analysis of the

products. 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-processing

electronics, is an excitation light source placed above the electrophoresis channel. (Bottom) Optical micrograph of the device from

above. Wire bonds to the printed circuit board can be seen along the top edge of the device. Reprinted with permission from

Reference 79. (Copyright 1998 AAAS.)

was performed by Woolley et al.,17 which included a Si PCR

microchamber attached to a glass CE microchannel. DNA am-

plified within the microchamber was electrokinetically injected

directly into the glass CE microchannel for separation and fluo-

rescence detection. This microsystem was capable of amplifying

5L of sample in a time of 15 minutes and the subsequent CE

separation took place in a 5 cm-long CE microchannel in a timeof 120 seconds. This work demonstrated correct product sizing

and good correlation between amplification time and product

yield, which proved the feasibility of such microsystems to har-

ness the advantages of both miniaturized sample preparation and

analysis. Subsequently, Anderson et al.97 demonstrated a PCR

device integrated with hybridization array technology for DNA

and RNA analysis.97 Their technology utilized multiple lami-

nated polycarbonate sheets to form microchannels, the analysis

chamber, and microvalves. Waterset al.64 demonstrated a series

of all-glass PCRCE systems that were capable of thermal cell

lysis, amplification of several targets and subsequent separation

on a singleCE channel. Inaddition, thesamegroup haspresented

a microdevice for enzymatic digestions of DNA followed bymicrochannel CE. Unfortunately, these initial monolithic glass

systems required placing the entire device on a conventional

thermal cycling block, removing some of the advantage of con-

ducting microscale PCR. In 1998,Burns etal.98 published a fully

integrated DNA analysis system (Figure 6) employing SDA, an


14/28


exponential and isothermal amplification reaction to conduct a

miniature 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 well

as photodetectors usinga single-crystalSi photodiode. Although

a 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 separate

the resulting products with a resolution of100 bp. The use ofSDA instead of PCR provided the advantage of eliminating the

need for thermal cycling but compromises in analysis speed was

necessary. Nevertheless, this work was the first to demonstrate

the potential for complete integration in a single chip.

The first functional monolithic integration of PCRCE sys-

tem was achieved by Lagally et al.74 This system was able to

amplify as few as five copies of a double-stranded DNA tem-

plate in a time of 10 minutes. The products were separated on

a 5 cm-long CE microchannel in 120 seconds. Critical to the

success of this microsystem was containment and isolation of

the sample within the 280-nL chamber during thermal cycling.The strategy employed was one adapted from the work of An-

dersonet al.,97,99 in which positive pressure was applied to the

PDMS microvalves to obtain efficient sample containment. The

original work had presented microvalves with dead volumes in

the microliter range; however, for the purposes of a 280 nL PCR

chamber, 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 labeledin situusing

an intercalating fluorescent dye, thiazole orange. This microsys-

tem demonstrated an excellent linear correlation, as expected for

the linear regime of PCR, and moreover, the extrapolation indi-

cated a molecular limit of detection of only two DNA template

molecules. This is an important result because below the level of

approximately five template molecules, PCR enters a stochastic

regime, in which the amplification yield for a series of reac-

tions of a certain average concentration will obey the Poisson

distribution:

P(x) = xex

x![9]

where is the mean of the distribution and x is the number

of template molecules. To test the ability of such microsystems

to amplify single DNA template molecules, an internal control

template was added to separate the effects of the statistical am-

plification from the possibility of a failed reaction. The ensuing

multiplex reactionutilized two setsof primers and two templates,

one stochastic template present at approximately one molecule

within the PCR chamber and the other outside the stochastic

regime at approximately five molecules in the chamber. Figure

7 presents the results of 60 separate amplifications. The data

are fit to the presumptive Poisson distribution and provide a

good fit (KomologorovSmirnov 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 peak

area ratiosfroma seriesof 60 multiplex PCRamplifications from

stochastic single-molecule template (136 bp product) and con-

trol template (231 bp product). Distinct clusters are suggestive

of amplification from single DNA template molecules. (B) Fit

of histogram in (A) to expected Poisson distribution. The mean

of the fitted distribution is = 0.9 0.1 molecules, demon-strating successful amplification from single DNA template

molecules.

achieved using the integrated PCRCE paradigm, and was the

first such demonstration on a microdevice.100 Lagally et al.12

also produced a PCRCE microdevice with integrated heaters

and temperature sensors, which yielded temperature transitions

of 20C s1. Figure 8 presents a schematic drawing of the tem-

perature control elements used in this work. The microheaters

were fabricated from Ti and Pt thin films and were located

on the reverse of the glass device. The heaters had very low

resistance (812 ) and possessed electroplated gold leads in


15/28


FIG. 8. Perspective diagram of the relative orientation of three microfabricated elements used in a fully integrated PCR-CE

microsystem. The heater is located on the bottom side of the device, the RTD is located between two bonded glass wafers forming

the enclosed chambers and channels of the device. Adapted from Reference 5.

order to localize theheatingunderthe PCRchamber andto lever-

age the second-order dependence of Joule heating on the current

(P = I2R). The temperature sensors were of the four-wire re-sistance temperature detector (RTD) form, used to minimize the

impact of self-heating effects on the sensing system. The highly

linear temperature coefficient of resistance of Pt enabled sen-

sors with extremely high fidelity. This new system was used to

conduct a sex determination assay from human genomic DNA,described later.

C. Integrated Optics

Fluorescence provides the invaluable capability of multi-

color detection with exquisite sensitivity; however, it typically

requires bulky optical sources and detectors that pose signifi-

cant challenges in integration. For example, the PCRCE sys-

tems described in the previous section required laser diodes and

conventional PMTs for conducting confocal fluorescence de-

tection, which limited the system size and prevented avenues of

further miniaturization. In order to address this bottleneck, many

groups are investigating the fabrication of fluorescence detection

optics directly onto the integrated microsystem that may allow

precise positioning of the optical detection hardware in rela-

tion to the analyte, removing the necessity for time-consuming

alignment procedures. Roulet et al.101 fabricated arrays of mi-

crolenses and thin-film metal apertures on a glass microdevice

for fluorescence detection. The detection was conducted off-

chip using either a CCD camera or a photomultiplier tube, and

demonstrated a limit of detection of 3 nM for a common fluores-

cent dye. Chabinycet al.102 presented an avalanche photodiode

coupled to a PDMS microdevice using a fiber optic cable. Na-

masivayamet al.103 have investigated the use of Si photodiodes

for on-chip fluorescence detection and have fabricated these

within integrated genetic analysis systems. It is important to

note that the choice of substrates plays an important role in the

integration of optoelectronic components. For example, the use

of 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 elegant

integration of VCSEL/photodiodes,104 however the requirement

for high-temperature processing may eliminate the possibility to

use polymer-based materials.

Kameiet al.105 presented a novel microfabricated photode-

tector in the form of a hydrogenated amorphous silicon a-Si:H

photodiode (Figure 9). The photodiodes are fabricated from

successive layers of doped amorphous silicon and the fabrica-

tion process occurs below 300C in a plasma-enhanced chem-

ical vapor deposition (PECVD) system, allowing the use of

glass 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 optimized

for the detection wavelength. Recently, this photodiode was

used to detect the results of a PCR-based assay to distin-

guish pathogenic strains ofStaphylococcus aureus bacteria.106

In a different approach, Kwon and Lee107 fabricated an en-

tire scanning confocal fluorescence detection apparatus on a

microdevice, including microlenses, scanning hardware, pin-

holes, and pupils. This impressive system demonstrates the


16/28


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 from

Reference 86. (Copyright 2003 American Chemical Society.)

feasibility of creating entire integrated optical detection sys-

tems on the microscale, harnessing the power of fluorescence

imaging while conserving the advantages of miniaturization and

portability.

V. MICROSYSTEMS FOR REAL-WORLD APPLICATIONS

Integrated genetic analysis microsystems, and particularly

the PCR-CE systems described previously, demonstrate several

advantages that make them applicable to several key areas of

modern genetic analysis. Their small size, fast operation, lower

operating powers, and autonomous operation allow them to be

used in remote environments and by untrained or minimallytrained operators. Batch fabrication allows the devices to be dis-

posable, enabling assays requiring sampling from bodily fluids

or pathogenic samples. Following the development of the first

integrated PCR-CE microsystems, researchers began to apply

these systems to real-world problems of clinical and forensic

utility.

Lagallyet al.89 demonstrated the construction and testing of

the first field-portable, fully integrated PCRCE microsystem.

This system is based on the integrated PCRCE systems de-

scribed earlier. In this case, the microsystem contains a single

PCR 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 dead

volumes as low as 8 nL and are actuated with small pressures

and vacuums. Pt electrodes were also fabricated within the de-

vice, allowing application of a high voltage without the need

for external electrodes. The microsystem is the size of a micro-

scope slide, and is placed into a portable analysis instrument

that contains all the necessary electronics, optics, and control

hardware for conducting a genetic analysis. The analysis instru-

ment contains a miniature confocal fluorescence set-up, includ-

inga laser diode,filters, anda photomultipliertube forcollecting

fluorescence data. Figure 10 presents a picture of the portable

analysis 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 human

sex determination.

A. Epidemiology Applications of PCR-CE

Epidemiology plays a central role in food safety, infectious

disease research, and anti-bioterrorism efforts. Of particular

concern is the detection and identificationof 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.112116 Detection and identification

of bacterial pathogens presents unique challenges that genetic

analysis microsystems, and PCR-CE microsystems in particular,

are well poised to confront. First, such pathogens can be present

in very small quantities and in very small concentrations. For

instance,E. coli O157:H7 is a major food pathogen causing as

many as 20,000 infections a year in the United States alone,

and has been the causative pathogen in food-borne outbreaks

in the United States.113 Importantly, the minimum infectious


17/28


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. Adapted

from Reference 68.

dose of this organism is as low as 50 cells, depending on the

route of introduction.114 Second, because pathogens are closely

related 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 human

intestine, so differentiation of these organisms from pathogenic

O157:H7 strains must use unique genetic markers or known

immunological differences. Finally, pathogens vary widely in

their routes of infections, and so genetic analysis microsystems

must be able to adapt to multiple types of sample preparation

technologies.

A conventional pathogen detection and differentiation exper-

iment involves culturing from a clinical sample onto a specific

set of media depending on which organism is suspected. Such

media will generally screen to the species level, enabling fur-

ther analysis using pulsed-field gel electrophoresis techniques

following PCR amplification of known toxicity genes. PCRCE

has been shown to be a versatile technique for the detection and

identification of bacterial pathogens. Kohet al.117 demonstrated

a lab-based microdevice with integrated valves, PCR and CE us-

ing multiplex PCR to detect different strains ofEscherichia coli

O157:H7. In their work, a glass microdevice containing a PCR


18/28


chamber directly connected to a CE microchannel was used

to generate and subsequently separate the PCR products. On-

chip thermal lysis ofE. coliO157:H7 organisms was achieved,

negating the need for further upstream sample preparation. The

fluorescently labeled PCR products were detected using con-

focal laser-induced fluorescence. Such a system points the way

toward remote analyses on water supplies, for example, to detectfecal contamination or for food testing. For many applications,

portable PCRCE microsystems would provide a robust, quanti-

tative analysis method for detection of infectious disease. A key

attribute of this system is the ability to confirm product sizes

and glean important genetic information about the analyte in a

timely manner at any location.

1. Detection and Identification of Bacterial Pathogens

The 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 ofE. coli

and antibiotic-resistant Staphylococcusaureus, a pathogen caus-

ing local and systemic infections. In the first series of experi-

ments, a triplex PCR was used to detect and differentiate two

pathogenic strains of E. coli from a laboratory strain. E. coli

K12, O55:H7, and O157:H7 were successfully differentiated

in 30 minutes, and the resulting serial dilution demonstrated a

limit of detection of only two cells (Figure 11A). Thermal lysis

of the bacteria was achieved within the PCR chamber, elimi-

nating further upstream sample preparation. In a second set of

experiments, E. coli O157:H7 was successfully detected from

within 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 small

fraction of the total population of any species of interest (Fig-ure 11B). The third series of experiments successfully differen-

tiated Gram positive antibiotic-resistantStaphylococcus aureus

from antibiotic-sensitive cells of the same species. Such detec-

tion of antibiotic resistance in bacteria, andS. aureusin particu-

lar, is of ever-growing importance as antibiotic resistance in this

species is spreading both through nosocomial and community-

acquired infections.111 Due to its small size, fast operation, and

low limits of detection, such integrated, portable microsystems

may become a critical tool in infectious disease detection.

B. Forensic Identification

PCRCE systems mayalso be employed for forensic identifi-

cation where only a small amount of sample is available. Using

the laboratory-based system described earlier, human sex de-

termination was demonstrated. In this assay, human genomic

DNA 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 primers

hybridized to a section of the Y chromosome, and produced a

200 bp product. Observation of thenumber andthe lengths of the

resulting PCR products then allowed a determination of the gen-

derof theindividualfrom whom theDNA hadbeen isolated.The

resulting electropherograms demonstrated clear discrimination

of DNA isolated from males and females, respectively.12 The

mass of DNA used in these experiments was 10 ng, the upper

bound typically encountered in real-world forensic investiga-

tions; however, the signal-to-noise ratio of the fluorescent PCR

products was sufficiently high for a reduction to 1 ng or lessof starting material to be theoretically achievable. Such systems

can therefore be applied to real-world situations, in which the

availability of the starting material is usually limiting, and such

forensic applications are therefore also within the purview of

field-portable PCR-CE microsystems.

VI. FUTURE DIRECTIONS

The 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, the

routine use of such microsystems in a general set of situations in

both developed and developing countries requires microsystemsthat are more robust, simple for untrained operators to use, and

low power. A series of emerging technologies are discussed that

may serve to advance the field of microsystems for wider access

and utility.

A. Analysis from Complex Sample Mixtures

Many sample mixtures are complex and heterogeneous, and

contain inhibitory components that prevent the success of an

assay. One of the most troublesome challenges in genetic anal-

ysis in real-world situations is the simplification of the sample

mixture 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 these

samples (particularly in the case of pathogen analysis) can be

exceedingly low (110 cells/mL). Therefore, the development

of 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 discussed

here.

1. Isolation of Cells

The initial isolation and purification of cells from com-

plex sample mixtures is an important step prior to these ge-

netic analyses. Traditionally, centrifugation, immunomagnetic

separation,119 and use of sophisticated equipment such as

FACS120 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 neutral

particles in a non-uniform electric field arising from differences

in dielectric properties between the particles and the suspend-

ing fluid. The time-averaged force on a homogeneous sphere of


19/28


FIG. 11. (A) A series of amplifications and separations from different strains ofEscherichia coli cells conducted using the portable

PCR-CE microsystem. Top frame:E. coliK12, a non-pathogenic lab strain; middle frame:E. coliO55:H7, a pathogenic strain that

does not express Shiga-like toxin; bottom frame, E. coli O157:H7, a pathogenic strain expressing Shiga-like toxin. White peaks

are co-injected DNA ladder peaks, black peaks represent PCR products (280 bp: 16S species-specific marker; 625 bp: fliCgene

encoding H7 flagellar antigen; 348 bp:sltIgene encoding Shiga-like toxin). (B) Histogram showing relative product peak areas for

PCR product peak areas for sltIproduct (black) and 16S product (gray) for a series of serial dilutions ofE. coliO157:H7 cells into

non-pathogenicE. coli K12 cells. The fliCproduct is still visible to 0.1% pathogenic cells, indicating pathogenicity is detectable

to the level of 1 cell in 1000 using the portable PCR-CE microsystem. Adapted from Reference 68.


20/28


radiusrp can be approximated as:

FDEP= 2m r3pRe(K)Erms

2 [10]

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

defined as:

K=

p m

p + 2m [11]

where p is the complex permittivity of the particle and m is

the complex permittivity of the medium. Trapping of certain

cell types may be achieved by specifically attracting them to

electrodes (positive K) while repelling others (negative K).121

In the case of bacteria, and E. coli in particular, the cross-over

frequency is reported to be a stronger function of medium per-

mittivity than frequency. For media with conductivities smaller

than the measured conductivity of the cell ( 44 mS/m),K is positive for frequencies smaller than about 1 MHz.122,123

For mammalian cells, however, the cross-over frequencies from

negative to positiveKare better defined, and typically lie in the

range between 10 and 90 kHz.121 Therefore, by setting the DEPfrequency below the cross-over frequency of non-bacterial cells

and operating in a medium with sufficiently low permittivity, se-

lective capture of bacteria is possible while rejecting larger cells

in the sample. Gascoyne and coworkers have presented a series

of microdevices for the separation and characterization of mul-

tiple cell types using DEP, including separation of cancer cells

from normal cells and separation of multiple types of immune

cells from one another.121,124128

Much recent work has applied DEP to integrated genetic

analysis systems; in particular, Cheng et al.129 fabricated an

integrated microsystem for the selection and concentration of

cells on microelectrodes, and the subsequent chemical inter-

rogation of these cells using the electrodes. Grodzinski and

coworkers presented a microfabricated system for cell concen-

tration and genetic sample preparation from complex sample

backgrounds.130 Manaresi et al. fabricated a CMOS chip for

manipulation and concentration of cells on a 320 320 elec-trode array.131 Lapizco-Encinaset al. demonstrated DEP con-

centration of bacteria using a series of insulating posts in an

electric field, and used this system to differentiate live from

dead bacteria.132,133 Recently, Lagally et al. have described a

microsystem for the concentration and detection of genetic ma-

terial from bacterial pathogens.134 Their system flows a sample

mixture through a polyimide microchannel and utilizes positive

dielectrophoresis (DEP) to trap any bacterial cells present in

the sample on a set of interdigitated microelectrodes. Following

trapping, a set of PDMS microvalves is closed around the micro-

electrodes, defining a 100 nL chamber that greatly concentrates

the target cells. A cell lysis buffer containing an optical molecu-

lar beacon is then introduced. The molecular beacon hybridizes

in a species-specific fashion to the rRNA from E. colicells. The

system is monitored using a confocal fluorescence microscope,

and the limit of detection is 25 cells. Importantly, cells can be

detected in 20 minutes, allowing rapid detection of bacteria.

2. Isolation of Molecules

In 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 differentiation

from DNA requires therejection of DNA from thesample,which

can act as a contaminant. Detection of RNA yields information

that DNA cannot, namely the set of genes that are transcribedwithin a cell at a given point in time under a defined set of

conditions. To this end, several groups have worked to develop

microsystems for the selective isolation and purification of RNA

from complex samples backgrounds. Jiang and Harrison135 pre-

sented a microdevice using microbeads with poly-T oligonu-

cleotides immobilized on them that were selectively placed

within an etched microchannelto an mRNA capture bed.Follow-

ing transcription from the DNA, mRNA is modified to contain

a poly-A tail, which hybridizes to the poly-T oligonucleotides

present on the microspheres. Their results showed that capture

of mRNA from total RNA was possible down to a minimum of

2.8 ng at a capture efficiency of 26%. The same group later used

magnetic 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 in

genetic analysis, particularly for DNA sequencing. DNA se-

quencing, due to the single base-pair resolution required, ne-

cessitates a high purity cycle sequencing sample. Conventional

post-amplification purification for DNA sequencing is ethanol

precipitation followed by resuspension in a suitable buffer

for CE separation. The microfabrication of such mid-stream

purification steps has proven difficult, but has been demon-

strated. Such systems utilize sequence-specific capture probes

immobilized within a certain section of a microsystem. Paegel

et al.137 described a three-dimensional monolithic capture gel,

consisting of linear polyacrylamide that had been modified to

contain 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 (the

authors used the known sequence directly 3 to the primers

to design the capture probe) were immobilized within the gel.

Application of higher temperatures (67C) released the bound

fragments, and these were electrophoretically injected onto and

separated using microchannel CE. The reduced system could

purify 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 possibility

of sequencing large genomes at greatly reduced cost.138

B. Advanced Detection Methodologies

Another importantarea of futureresearchwill be theelimina-

tion of the power-hungry and cost-intensive components from

integrated genetic analysis microsystems in order to enhance

their field-portability, disposability, and to reduce their cost of

production.


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1. Optics-Free Detection

Use of fluorescence has typically dominated genetic analy-

sis due to its extremely low (picomolar) detection limits. For

applications in remote environments with minimally trained op-

erators (e.g., thegenetic detectionof HIVin Africa), such optical

components may prove to be impractical due to size, cost, and

fragility. Alternative detection strategies are needed that sup-plantopticaldetection whilemaintainingmany of its advantages.

Electrochemical detection, relying on the transfer of electrons to

generate oxidative and reductive currents, is one such technique.

Although electrochemical detection has typically exhibited lim-

its of detection only in the nanomolarmicromolar range, the

use of PCR to amplify DNA can be used to generate sufficient

product such that electrochemical means may also be used for

detection. The first work to demonstrate the integration of mi-

crochannel CE with electrochemical detection was presented by

Woolley et al.139 In this work, PCR-amplified DNA was sepa-

rated on a CE microchannel and detected using electrochemical

electrodes placed past the end of the separation channel. Later

work 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 supported

electrochemical detector for use in integrated CE microsystems.

The sheath flow carries the DNA analyte from the gel separation

region into a free-solution detection region, electrically isolating

the electrochemical detector from the high electric fields inher-

ent to CE. Other recent advances in electrochemical detection,

such as differential measurements and use of electrochemical

intercalators141 and Ag-coated Au nanoparticles,142,143 have de-

creased the limits of detection of electrochemical means even

further 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 Lee

et al.144 In this work, gold electrodes within the PCR chamber

used 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 of

detecting as few as ten molecules of starting DNA template in

an 8 L PCR chamber. Such a system will inherently encounter

difficulty in differentiating similar DNA sequences, such as are

generated in forensic investigations through the amplification of

short tandem repeats (STRs); however, such systems avoid the

need 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 amenable

to mass fabrication. Toward this end, Liu et al. 145 published a

completely integrated genetic analysis system fabricated from

plastic substrates that incorporates cell concentration using mag-

netic bead capture, convective mixing, lysis, PCR amplification,

and electrochemical detection usinga sandwich assay. Their sys-

tem was capable of detecting 106 E. coli cells in 1 mL of whole

blood, and was also used to determine the presence of the human

HFE-C gene directly from human blood. Although the limits of

detection of this system were not investigated, the use of PCR

promises to provide high sensitivity and specificity over a wide

variety of targets. Such highly integrated systems may represent

the future of integrated microtechnologies for genetic analysis.

There are several outstanding limitations to be addressed; one of

themmay be cost, in thatthese assays require expensive reagents,

including gold or silver nanoparticles, magnetic microspheres,andPCR reagents. Workcontinues to develop a low-cost version

of such systems for wide accessibility in clinical and forensic

diagnostics.

2. Reagentless Detection

In many practical cases, the availability and storage of bio-

chemical reagents becomes a limiting constraint. The need for

refrigeration, storage, and handling infrastructure makes assays

that require such reagents impossible in many of the areas that

may need such systems the most. The ideal genetic detection

meth

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Integrated Genetic Analysis Microsystems