integrated genetic analysis microsystems
TRANSCRIPT
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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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210 E. T. LAGALLY AND H. T. SOH
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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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 211
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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212 E. T. LAGALLY AND H. T. SOH
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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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 213
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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214 E. T. LAGALLY AND H. T. SOH
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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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 215
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
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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 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
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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 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]
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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 219
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
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220 E. T. LAGALLY AND H. T. SOH
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
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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-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
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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 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
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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. 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
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224 E. T. LAGALLY AND H. T. SOH
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
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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 225
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.
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226 E. T. LAGALLY AND H. T. SOH
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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INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 227
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