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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. Soh
California 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 the
microsystems 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 situ analyses, permitting a new standard in dis-
ciplines such as clinical chemistry, personalized medicine, forensics, biowarfare detection, and
epidemiology. 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 current
developments 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. Signicance ......................................................................................................................................... 214
B. Capillary Electrophoresis and Microchannel CE .............................................................................................. 215
1. Capillary Electrophoresis Background .................................................................................................... 215
2. Microchannel CE ................................................................................................................................. 215
3. Entropic Trap Separations ..................................................................................................................... 216
4. Materials Issues ................................................................................................................................... 217
a. Surface Chemistry ..................................................................................................................... 217
5. Signicance ......................................................................................................................................... 218

E-mail: lagally@engineering.ucsb.edu
207
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 Identication of Bacterial Pathogens ................................................................................. 224
B. Forensic Identication ................................................................................................................................... 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 ............................................................................................. 227
1. 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 ve 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 (10
12
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, the
monolithic 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
uid control structures such as valves and pumps, as well as
methodologies to control surface chemistry using a variety of
materials. The eld of integrated genetic analysis systems is in
an active phase of research and development, and the number
of publications in this eld 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 FINISH
Typically, samples must rst 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 rst
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 byspecializedinstruments
INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 209
FIG. 1. The steps of a typical genetic analysis. Nucleic acids (DNA, RNA) are rst extracted from biological cells following cell
lysis (DNAis the white strands oating in the mixture). The nucleic acids are usually puried using a variety of techniques, followed
by amplication. Amplied products are again puried before analysis using capillary electrophoresis or real-time detection. Certain
purication steps, marked with dashed lines, may be omitted depending on the assay.
such as uorescence activated cell sorters (FACS). Cell isolation
is followed by cell culture, on which cells are grown on media
preferential to specic cell types. The next step is nucleic acid
extraction, in which the cells of interest are rst 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, purication is often re-
quired. Historically, efcient purication 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 afnity-based methods and non-covalent bonding-based
methods, are also in use. In the afnity-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
modication inside the cell, can be hybridized to complementary
210 E. T. LAGALLY AND H. T. SOH
poly-thymine (T) oligonucleotides, which themselves have been
covalently bound to microspheres.
6
Such afnity-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-specically bound to the solid
phase such as glass microspheres or a silica membrane through
hydrogen bonding.
7
Huang et al.
8
have reviewed the ways
MEMS technology has been applied to sample purication and
preparation.
Following nucleic acid purication, the next major step is
sample amplication. 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, signicant 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 amounts
of 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, specic, and rela-
tively rapid, and is effectively implemented in microfabricated
devices.
After purifying the amplication products, the nal stage in a
genetic analysis is the labeling and detection of the genetic mate-
rial. Depending on the requirements, the analysis may be as sim-
ple as conrmation that nucleic acids of a certain sequence are
present, or it may be as detailed as the length and the sequence of
the amplication 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
eld. There are a variety of electrophoresis methods including
slab gel electrophoresis,
4
pulsed eld 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 amplication.
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 rst is microchip PCR for amplica-
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 performsuch amplications compared to conventional
methodologies.
III. DEVICES
A. PCR and PCR Microsystems
1. PCR
In genetic analysis, the most materials-critical step is the sam-
ple preparation, and the case of PCR amplication 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 amplied, (2) a set of
short oligonucleotide primers specic to known sequences on
the template strand, (3) a thermostable DNA polymerase (Taq, a
modied DNA polymerase isolated from the thermophilic bac-
teria Thermus aquaticus is most commonly used), and (4) indi-
vidual dinucleotide triphosphates (dNTPs) of adenine, thymine,
guanine, and cytosine. As depicted in Figure 2, the reaction pro-
ceeds in repeated cycles of three temperatures. The rst temper-
ature, from 94

C96

C, separates or denatures the two template


strands (Figure 2A); at the second temperature, typically 45
60

C, the primers hybridize to their complementary sequences


on the parent strand (Figure 2B); during the third temperature
step, usually at 72

C, 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 efciency therefore generates 2
n
daughter strands,
where n 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
=
_
n

i =1
(1 +
i
)
_
[DNA]
i
, [1]
where [DNA]
f
is the nal DNAconcentration, [DNA]
i
is the con-
centration at the i
th
cycle, and
i
is the efciency of the reaction at
the i
th
cycle. The efciency of the reaction is theoretically unity
for small values of i and decreases with increasing cycle number.
This phenomenon may be explained by the Michaelis-Menten
equation:
v =
v
max
[T]
[T] + K
M
, [2]
where v is the rate of product formation at any point in the
reaction, v
max
is the maximum rate of product formation, [T] is
the concentration of target (uncatalyzed primer and dNTPs), and
K
M
is 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 efciency of
PCR as
18

i
= 1
v
i
v
max
, [3]
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
rst cycle; each of the products and the original template may then participate further in the reaction during the next cycle.
where v
i
is the rate of product formation at the ith cycle. Be-
cause v
i
decreases with decreasing reactant concentration, the
efciency
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 the number of required
cycles.
PCR exhibits several notable advantages over competing
techniques, including exponential amplication, 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 uids
20
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 amplies
this cDNAexponentially to a detectable level. In addition, multi-
ple DNAtemplates may be simultaneously amplied in the same
reaction vessel using multiplex PCR. In cases where the melt-
ing temperatures of different primers within a multiplex reaction
prevent successful parallel amplication using a single anneal-
ing temperature, step-down PCR is used where a series of suc-
cessively lower annealing temperatures allow the hybridization
212 E. T. LAGALLY AND H. T. SOH
of widely varying primer sets to multiple templates.
24
Another
widely used PCR variant that combines amplication with u-
orescent detection is real-time PCR (rtPCR).
25,26
rtPCR is con-
ducted in one of two ways: in the rst method, an intercalating
uorescent dye present in the reaction mixture labels amplied
DNAas the reaction progresses.
25
In the second method, a dual-
labeled uorescence detection oligonucleotide probe comple-
mentary to the PCR product is included in the reaction mix-
ture and hybridizes to amplied product.
26
The probe has a
uorescent dye at one end and a uorescence quencher at the
other end, resulting in a non-uorescent probe in its native state.
Following hybridization to amplied DNA, however, the probe
is cleaved by the polymerase during extension in the next cy-
cle, separating the quencher from the uorophore and restoring
uorescence. The rtPCR method has been adapted for use in
microsystems.
2730
2. Microscale PCR
PCR can be easily miniaturized, and such reduction in scale
provides 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 rst
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 et al.
32
demonstrated a silicon PCRmicrochip. 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.
Chaudhari et al.
36
demonstrated thermal monitoring and mod-
eling for the optimization of PCR microchips. Daniel et al.
37
demonstrated successful PCR from a novel silicon microcham-
ber utilizingsmall volumes (1L) andthermal isolationfromthe
rest of the substrate using thin suspended silicon nitride lms.
Taylor et 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 amplication as a function of
time. In 1998, Kopp et al.
38
demonstrated a fundamentally dif-
ferent PCR architecture called continuous ow PCR (CPCR)
wherein the chemical amplication is achieved as reagent mix-
ture is made to pass through serpentine microuidic channels
with three isothermal zones for the denaturing, annealing, and
extension steps so that the chemical amplication occurs as a
function of spatial location (Figure 3). This continuous ampli-
cation 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
time of as little as 1.5 minutes, using a total volume of 8 Lusing
a channel with a cross-sectional area of 3600 m
2
. The initial
demonstration required very high starting template concentra-
tions (10
8
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.
Zhang et al.
41
presented nite-element models of CPCR for the
purposes of enhanced thermal design. Obeid et al.
27
presented
laser-induced uorescence detection of PCR products using an
intercalating dye introduced following amplication.
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 the
solution between three differently heated regions to achieve am-
plication. 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 amplication. 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 eld-portable PCR systems. Using the rtPCR
assay, Belgrader et al.
46
demonstrated silicon based PCR de-
vice, assembled with all the electronics for thermal actuation
and control, as wells as the optics for uorescence 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
amplication.
28
In addition, Higgins et al.
47
demonstrated a
handheldrtPCRmicrodevice. Pal andVenkataraman
48
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 signicant way. In early
work, it was discovered that silicon and silicon nitrides demon-
strate an interfering effect when conducting certain nucleic acid
amplication assays.
34
Theories surrounding these materials in-
teractions vary, but a large contingent of researchers maintains
the hypothesis that because the polymerase requires divalent
INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 213
FIG. 3. (A) Schematic of a chip for ow-through PCR. Three well-dened zones are kept at 95, 77, and 60 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 rst one includes a threefold increase in DNA melting time. Reprinted with permission from Reference 26. (Copyright
1998 AAAS.)
cations (preferably Mg
2+
) 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 PCRbe-
came evident in the necessity to prevent the nucleic acids from
non-specically adsorbing to the sidewalls of the reaction vessel.
In particular, glass, with its free silanol (SiOH) groups at the
surface, readily forms hydrogen bonds to nucleic acids, leading
to sample adsorption. It is well known that the surface to vol-
ume ratio increases as device sizes shrink. Thus in microdevices,
non-specically adsorbed molecules, which are unavailable to
214 E. T. LAGALLY AND H. T. SOH
the reaction, become a larger percentage of the total number
of molecules and signicantly limit the efciency of the reac-
tion. The use of non-specic 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 fromthe 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 modication 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 amplication. 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 modication 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 microuidic
devices. PDMS in particular has demonstrated signicant ver-
satility as a structural material. Duffy et al.
58
rst described a
soft lithography method for microfabrication through the cre-
ation of a master using a positive photoresist, followed by 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-
cial etch mask for the formation of structural features in other
applications.
62
Polyimide has many desirable characteristics due
to its ability to be easily spun on as a resist-like lm, and be-
cause its curing process can be integrated with wafer bonding
processes.
b. Heaters and Temperature Sensors. Thin metal lms 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 lms using sputtering or evaporation
processes, and can be etched using a variety of wet or dry etching
techniques. Subsequent bondingprocesses (see later) canrequire
temperatures above 650

C, and so it is important that the metals


exhibit minimal thermal effects, including expansion, oxidation,
and diffusion at these temperatures. Platinumin particular is well
suited for these applications, although gold has also been used.
12
Due to its linearity in temperature coefcient 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 PCRreac-
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 uid
ow. 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 bonding
65,66
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 (>500

C) prevents the use of most polymer lms and may


lead to oxidation and diffusion of metal lms used in these sys-
tems. Microsystems with polymer lms may 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 sufcient; they
include bonding of PDMS to glass, as well as the use of thin pho-
toresist lms 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 sufciently 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 has been used in the fabrication of PDMS microvalves
and peristaltic pumps for directing liquid ows in microchannel
environments.
43,51,7173
Bonding processes are difcult 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 ow, and because bonding steps are generally at the end
of a fabrication process, much work may be lost if successful
bonding of two substrates is not achieved. Second, bonding yield
is a non-linear function of lm thicknesses, temperature, time,
and pressure, making optimization of such processes difcult.
Thus more research into a mechanistic description of bonding
processes of heterogeneous substrates is needed.
5. Signicance
PCR microsystems demonstrate a number of interesting
characteristics. First, they can amplify miniscule volumes of
nucleic acids with comparable efciency to that of conven-
tional technologies at a fraction of the time, power, and re-
quired reagents. Such systems can be fabricated using relatively
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
amplication.
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 amplication.
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 molecules
based 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 nd 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 or
vertical slab of gel into which DNA was loaded. Applied volt-
age resulted in a length-dependent separation of DNA in which
smaller molecules traversed the gel faster than larger ones. How-
ever, these systems suffered from numerous problems, includ-
ing high temperatures due to the large currents (10100 mA)
applied, which resulted in high DNAdiffusivity and band broad-
ening and poor resolution due to the initial 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 eld 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
cm
2
/V*second) and E is the applied electric eld (V/cm). The
resolution of a CE separation is dened 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 =
t
2
t
1
1
2
(w
1
+w
2
)
=
L(
1

2
)
4
_

1
_
(
1
E
inj
t
inj
)
2
12
+
2DL

1
E
_
1/2
_
[5]
where L is the column length,
1
and
2
are the mobilities of the
two DNA fragments of interest, E
inj
is the applied electric eld
for injection, t
inj
is the injection time, E is the applied electric
eld for the separation, and Dis the average diffusion coefcient
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 the rst regime, the band 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 are difcult to engineer, it is imperative to minimize the 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 the injected plug 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 et al.
13
Later work by others extended these
approaches toward high-resolution and parallel operation. Wool-
ley and Mathies
14,78
demonstrated the rst DNAfragment sizing
and DNA sequencing separations on a glass microchannel CE
device in which DNAwas introduced electrokinetically through
an injection cross-channel and separated on a 5 cm-long, gel-
lled microchannel in only 120 seconds. The DNA was labeled
on-column using an intercalating uorescent dye and detected
with laser-induced confocal uorescence detection. Aschematic
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 ef-
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
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 DNAfragments (>1000 bp), CE is not
effective, because the difference in the mobilities of DNA frag-
ments decreases as the average length of the DNA increases.
Extremely long DNA fragments eventually enter the 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-eld 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
longDNAfragments ina microdevice. Hanet al.
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 for long DNAfragments where the mobility of DNA
fragments depends on the average size of the DNAin its random
coil conguration. Thus the mobility can be directly correlated
to the DNA length. The underlying equation governing the resi-
dence time of a DNAcoil in the entropic trap has been elucidated
as:
87
=
0
e
Fmax
k
B
T
, [6]
where
0
is a prefactor with a dependence on the length of the
random DNA coil in solution, and F
max
is the entropic energy
barrier requiredfor DNAtoescape the nanometer-sizedconstric-
tion. Because the entropic energy barrier is a function only of the
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 eld separates the DNA molecules by length in seconds,
as opposed to hours as is required by conventional techniques.
Cabodi et al.
88
also demonstrated a novel nanopillar array uti-
lizing an AC electric eld 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 DNAmolecules, 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,
sufciently shallow channels have not yet been demonstrated.
4. Materials Issues
CE typically employs electric eld strengths up to
300 V*cm
1
. In addition, because the detection of nucleic acids
commonly require uorescence 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 exibility, 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-specic
adsorption to device sidewalls. The solution to this problemwas
the use of the coating rst introduced by Hjert en, a version of
the silanization protocol also used to control DNA adsorption to
oxide surfaces during PCR.
50
The use of this coating also con-
tributed to a signicant 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 specically, the native surface
charge of the fused silica gives rise to a charged double layer and
subsequent bulk electroosmotic ow (EOF) in the presence of
an electric eld that transport the uid in an opposite direction
with respect to the electrophoretic movement of the molecules.
The bulk EOF velocity may be expressed in the following way:
v
EOF
=
_

_
E. [7]
In this presentation, is the surface charge density, is the
dynamic viscosity of the solution, E is the applied electric eld,
and is the Debye-Huckel constant, dened as
= F
_
_
2

c
i
z
2
i

r
RT
_
, [8]
218 E. T. LAGALLY AND H. T. SOH
where c is the concentration of an individual ion in solution, z
is that ions valence state, F is the Faraday constant,
0

r
is the
dielectric constant of the solution, R is the Boltzmann constant,
and T is the temperature. Equation 8 reveals that the EOF
velocity depends on the pH of the solution, the temperature, and
the surface charge density of the capillary sidewall. In capillary
electrophoresis, the EOF manifests itself as a uid migration
direction opposite to that of DNA movement, which results
in distorted peak shapes and very low separation resolution.
Application of the Hjert en or similar covalent couplings
alleviates this EOF through eliminating the charge presented to
the solution with an acrylamide group.
5. Signicance
The evolution from slab gel electrophoresis to CGE to mi-
crochannel CE has facilitated dramatic improvements in res-
olution as a function of channel length. These improvements
have come through reductions in diffusive band broadening as
well as injection-dominated band broadening. The end result
is a monolithic system in which DNA and other nucleic acids
may be separated in seconds instead of hours, using volumes of
only hundreds of picoliters. Most importantly, the advent of the
cross injector design allows the integration of these microchan-
nel CE devices with upstream sample preparation and sample
purication.
IV. INTEGRATION
Following the demonstration of discrete devices on chip, ef-
forts began to combine these modules to form integrated mi-
crosystems. Such integration promises to harness the advantages
of batch fabrication through further reduction of volumes and
analysis times as well as elimination of manual transfers be-
tween analysis steps. In order to enable such integration, it was
necessary to develop efcient methodologies for microuidic
connections between the on-chip components. The microvalves
and micropumps continue to serve as the unifying microuidic
interconnection elements in current microsystems.
A. Fluid Manipulation: Materials and Fabrication
1. Microvalves
The ability to direct and manipulate uids of very small vol-
umes (pL to nL) over micrometer length scales is one of the
essential tasks in integrated analysis systems, and the advent of
robust microvalve technology has been seminal; it provided a
method to accurately dene analysis volumes and restrict the
contact between solutions in space and time. Furthermore, it
provided the ability to actively control uid movement, making
a single chamber useful for multiple steps of an analysis. Many
types of microvalves and uid control elements have been de-
veloped, but only a small subset of these share the advantages
of being robust, biocompatible, simple to fabricate, and allow-
ing massively parallel operation. The normally open PDMS
microvalve architecture developed by Unger et al.
51
has been
widely used. The device is comprised of two PDMS microchan-
nels, one of which ows over the other on a separate layer. The
owcontrol is achieved by the application of pneumatic pressure
through the top PDMS microchannel selectively collapsing or
opening the bottom microchannel that transports the uid. Be-
cause PDMS microchannels are relatively simple to fabricate,
this approach has been utilized to fabricate an extraordinarily
large array of zero-dead-volume valves on a single substrate.
73
Another noteworthy PDMS microvalve architecture has been
developed by Grover et al.,
70
which used a similar approach but
provided two advantages; the valves may be normally closed
instead of normally open, and the PDMS surface area contacting
the sample is minimized, which reduces non-specic adsorp-
tion. The PDMS membrane that performs the valve actuation is
formed over the entire substrate, and covers a series of drilled
valve vias. Upon the application of positive pneumatic pressure
from an external manifold, the PDMS membrane locally de-
ects forming an open uid path. Application of vacuum seals
the membrane against the vias, effectively blocking uid ow.
Both the normally closed and normally open valve designs have
seen further application in a series of integrated microsystems
for genetic analysis.
43,89
Besides the pneumatic actuation, other modes of microvalve
transduction have been explored including the phase change
valve. In this construction, liquid parafn wax is loaded into
a microsystem and solidied in a desired location, forming a
restriction to uid ow. Upon application of temperature, the
wax re-melts, allowing uid ow. Recent work by Pal et al.
90
has recently tested a variety of waxes for biocompatibility and
demonstrated pressure sealing up to 250 psi applied backpres-
sure. Another type of phase change valve was introduced by
Eddington et al.,
91
in which a pH-sensitive hydrogel is selec-
tively patterned on a vertical post situated within a microchan-
nel. Upon application of low pH, the polymer swells to ll the
microchannel, blocking uid ow. Such smart valve technol-
ogy is passive and requires no external actuation; however, it
relies on the chemical characteristics of the solution to deter-
mine its state. Further investigation of several types of hydrogels
and their optimized fabrication within microuidic systems have
been reported.
92
2. Micropumps
The trade-off between speed and power is the chief constraint
to the development of genetic analysis microsystems. Espe-
cially for eld-portable applications, low-power systems are of
paramount importance. It is in these same types of analysis sit-
uations, however, that one would like to conduct a fast analysis
(e.g., the detection of neonatal meningitis in a remote hospital).
Most current published work uses manually applied pressure or
a syringe pump to move uids through a device. However, man-
ually applied pressure is not quantitative and syringe pumps are
bulky, expensive, and not conducive to miniaturization. Elec-
trolytic pumping, in which a DC current is applied through two
electrodes immersed in a salt solution to generate gases, which
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 uid movement
within microfabricated systems. Some of these technologies also
allow for other desirable processes, such as mixing in laminar
ow conditions. Electroosmotic ow is the main motive force
used in these systems, in which a voltage is applied to a uid
surrounded by a charged substrate (usually untreated glass), giv-
ing rise to a zeta potential and bulk uid motion.
94,95
Although
these systems require high electric eld 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. Chen et al.
96
recently presented a rotary
PCRmicrosystemthat used electrokinetic forces to transport the
PCRsolution through three differently heated regions to achieve
amplication.
B. Examples of Integrated Microsystems
Concomitant with the development of microuidic manipula-
tion technologies, efforts began to integrate nucleic acid ampli-
cation technologies with microchannel CE for analysis of the
products. The rst 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-
plied within the microchamber was electrokinetically injected
directly into the glass CE microchannel for separation and uo-
rescence detection. This microsystemwas capable of amplifying
5 L of sample in a time of 15 minutes and the subsequent CE
separation took place in a 5 cm-long CE microchannel in a time
of 120 seconds. This work demonstrated correct product sizing
and good correlation between amplication 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. Waters et al.
64
demonstrated a series
of all-glass PCRCE systems that were capable of thermal cell
lysis, amplication of several targets and subsequent separation
ona single CEchannel. Inaddition, the same grouphas presented
a microdevice for enzymatic digestions of DNA followed by
microchannel 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 et al.
98
published a fully
integrated DNA analysis system (Figure 6) employing SDA, an
220 E. T. LAGALLY AND H. T. SOH
exponential and isothermal amplication reaction to conduct a
miniature slab gel separation under a small electric eld to sep-
arate the products. The microsystem also integrated sample ma-
nipulation in the formof selective hydrophobic coatings, as well
as photodetectors using a single-crystal Si photodiode. Although
a fully integrated functionality was not demonstrated, their sys-
tem was able to meter liquid volumes as small as a few hundred
nL, amplify the DNA present in these volumes, and separate
the resulting products with a resolution of 100 bp. The use of
SDA 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 rst to demonstrate
the potential for complete integration in a single chip.
The rst functional monolithic integration of PCRCE sys-
tem was achieved by Lagally et al.
74
This system was able to
amplify as few as ve 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-
derson et al.,
97,99
in which positive pressure was applied to the
PDMS microvalves to obtain efcient 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 PCRamplication, the DNAwas electrokinetically in-
jected into the gel-lled microchannel and labeled in situ using
an intercalating uorescent dye, thiazole orange. This microsys-
temdemonstrated 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 belowthe level of
approximately ve template molecules, PCR enters a stochastic
regime, in which the amplication yield for a series of reac-
tions of a certain average concentration will obey the Poisson
distribution:
P(x) =

x
e
x
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-
plication from the possibility of a failed reaction. The ensuing
multiplexreactionutilizedtwosets of primers andtwotemplates,
one stochastic template present at approximately one molecule
within the PCR chamber and the other outside the stochastic
regime at approximately ve molecules in the chamber. Figure
7 presents the results of 60 separate amplications. The data
are t to the presumptive Poisson distribution and provide a
good t (KomologorovSmirnov statistic = 0.88) with a mean
number of stochastic template molecules of 0.9 0.1. This re-
sult veried that single-molecule DNA amplication had been
FIG. 7. (A) Histogram showing clustering of normalized peak
area ratios froma series of 60multiplexPCRamplications from
stochastic single-molecule template (136 bp product) and con-
trol template (231 bp product). Distinct clusters are suggestive
of amplication from single DNA template molecules. (B) Fit
of histogram in (A) to expected Poisson distribution. The mean
of the tted distribution is = 0.9 0.1 molecules, demon-
strating successful amplication from single DNA template
molecules.
achieved using the integrated PCRCE paradigm, and was the
rst 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 20

C s
1
. 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 lms and were located
on the reverse of the glass device. The heaters had very low
resistance (812 ) and possessed electroplated gold leads in
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 the heating under the PCRchamber and to lever-
age the second-order dependence of Joule heating on the current
(P = I
2
R). The temperature sensors were of the four-wire re-
sistance temperature detector (RTD) form, used to minimize the
impact of self-heating effects on the sensing system. The highly
linear temperature coefcient of resistance of Pt enabled sen-
sors with extremely high delity. 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 signi-
cant challenges in integration. For example, the PCRCE sys-
tems described in the previous section required laser diodes and
conventional PMTs for conducting confocal uorescence 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 uorescence 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-lm metal apertures on a glass microdevice
for uorescence detection. The detection was conducted off-
chip using either a CCD camera or a photomultiplier tube, and
demonstrated a limit of detection of 3 nMfor a common uores-
cent dye. Chabinyc et al.
102
presented an avalanche photodiode
coupled to a PDMS microdevice using a ber optic cable. Na-
masivayam et al.
103
have investigated the use of Si photodiodes
for on-chip uorescence 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.
Kamei et 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 300

C 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 of Staphylococcus aureus bacteria.
106
In a different approach, Kwon and Lee
107
fabricated an en-
tire scanning confocal uorescence detection apparatus on a
microdevice, including microlenses, scanning hardware, pin-
holes, and pupils. This impressive system demonstrates the
222 E. T. LAGALLY AND H. T. SOH
FIG. 9. (A) Schematic cross-sectional view of the hybrid in-
tegrated a-Si:H uorescence detector with a microuidic elec-
trophoresis device. (B) Optical micrograph of the top view of
the 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 uorescence
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 minimally
trained operators. Batch fabrication allows the devices to be dis-
posable, enabling assays requiring sampling from bodily uids
or pathogenic samples. Following the development of the rst
integrated PCR-CE microsystems, researchers began to apply
these systems to real-world problems of clinical and forensic
utility.
Lagally et al.
89
demonstrated the construction and testing of
the rst eld-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
PCRchamber 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 the
top 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 uorescence set-up, includ-
ing a laser diode, lters, and a photomultiplier tube for collecting
uorescence data. Figure 10 presents a picture of the portable
analysis instrument. This section reviews two major areas of cur-
rent application of such eld-portable PCR-CE microsystems
detection and identication 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
concernis the detectionandidenticationof 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 identication
of bacterial pathogens presents unique challenges that genetic
analysis microsystems, and PCR-CEmicrosystems 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
INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 223
FIG. 10. Photograph of the rst 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, and


pneumatics 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 amplication 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.
Aconventional pathogen detection and differentiation exper-
iment involves culturing from a clinical sample onto a specic
set of media depending on which organism is suspected. Such
media will generally screen to the species level, enabling fur-
ther analysis using pulsed-eld gel electrophoresis techniques
following PCRamplication of known toxicity genes. PCRCE
has been shown to be a versatile technique for the detection and
identication of bacterial pathogens. Koh et al.
117
demonstrated
a lab-based microdevice with integrated valves, PCRand CEus-
ing multiplex PCR to detect different strains of Escherichia coli
O157:H7. In their work, a glass microdevice containing a PCR
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 of E. coli O157:H7 organisms was achieved,
negating the need for further upstream sample preparation. The
uorescently labeled PCR products were detected using con-
focal laser-induced uorescence. Such a system points the way
toward remote analyses on water supplies, for example, to detect
fecal contamination or for food testing. For many applications,
portable PCRCEmicrosystems would provide a robust, quanti-
tative analysis method for detection of infectious disease. A key
attribute of this system is the ability to conrm product sizes
and glean important genetic information about the analyte in a
timely manner at any location.
1. Detection and Identication of Bacterial Pathogens
The eld-portable PCR-CE microsystem described by La-
gally and coworkers
89
has been used to detect and identify mul-
tiple bacterial pathogens, including pathogenic strains of E. coli
and antibiotic-resistant Staphylococcus aureus, a pathogen caus-
ing local and systemic infections. In the rst 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-resistant Staphylococcus aureus
from antibiotic-sensitive cells of the same species. Such detec-
tion of antibiotic resistance in bacteria, and S. aureus in particu-
lar, is of ever-growing importance as antibiotic resistance in 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 Identication
PCRCEsystems may also be employed for forensic identi-
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 rst set of primers was specic 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 the number and the lengths of the
resulting PCRproducts then allowed a determination of the gen-
der of the individual fromwhomthe DNAhad been 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 uorescent PCR
products was sufciently high for a reduction to 1 ng or less
of 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
eld-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 microsystems
that 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 eld 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 simplication 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 purication of ge-
netic material from complex sample backgrounds is impera-
tive. There are two major regimes of sample purication, iso-
lation of cells and isolation of molecules, which are discussed
here.
1. Isolation of Cells
The initial isolation and purication 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
FACS
120
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 eld arising from differences
in dielectric properties between the particles and the suspend-
ing uid. The time-averaged force on a homogeneous sphere of
INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 225
FIG. 11. (A) Aseries of amplications and separations fromdifferent strains of Escherichia coli cells conducted using the portable
PCR-CE microsystem. Top frame: E. coli K12, a non-pathogenic lab strain; middle frame: E. coli O55: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-specic marker; 625 bp: iC gene
encoding H7 agellar antigen; 348 bp: sltI gene encoding Shiga-like toxin). (B) Histogram showing relative product peak areas for
PCR product peak areas for sltI product (black) and 16S product (gray) for a series of serial dilutions of E. coli O157:H7 cells into
non-pathogenic E. coli K12 cells. The iC product 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.
226 E. T. LAGALLY AND H. T. SOH
radius r
p
can be approximated as:

F
DEP
= 2
m
r
3
p
Re(K)E
rms
2
[10]
Here Re(K) is the real part of K, the Clausius-Mosotti factor,
dened as:
K =

p
+2

m
[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 specically 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 positive K are better dened, and typically lie in the
range between 10 and 90 kHz.
121
Therefore, by setting the DEP
frequency below the cross-over frequency of non-bacterial cells
and operating in a mediumwith sufciently lowpermittivity, 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-Encinas et al. demonstrated DEP con-
centration of bacteria using a series of insulating posts in an
electric eld, 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 ows 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, dening 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-specic fashion to the rRNA from E. coli cells. The
system is monitored using a confocal uorescence 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, purication of molecules from a sample mix-
ture is required before genetic analysis may proceed. For in-
stance, the specic amplication of RNA and its differentiation
fromDNArequires the rejection of DNAfromthe sample, which
can act as a contaminant. Detection of RNA yields information
that DNA cannot, namely the set of genes that are transcribed
within a cell at a given point in time under a dened set of
conditions. To this end, several groups have worked to develop
microsystems for the selective isolation and purication of RNA
from complex samples backgrounds. Jiang and Harrison
135
pre-
sented a microdevice using microbeads with poly-T oligonu-
cleotides immobilized on them that were selectively placed
withinanetchedmicrochannel toanmRNAcapture bed. Follow-
ing transcription from the DNA, mRNA is modied 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 efciency of 26%. The same group later used
magnetic microparticles coated with a monoclonal antibody to
capture T cells fromhuman blood at a capture efciency of 37%
using a series of parallel microchannels.
136
Post-amplication purication 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-amplication purication for DNA sequencing is ethanol
precipitation followed by resuspension in a suitable buffer
for CE separation. The microfabrication of such mid-stream
purication steps has proven difcult, but has been demon-
strated. Such systems utilize sequence-specic 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 modied to
contain a sequence-specic capture probe attached to the poly-
mer backbone. DNA sequencing samples were electrophoreti-
cally driven through this purication 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 (67

C) 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 to
conventional samples. Such systems may lead to the possibility
of sequencing large genomes at greatly reduced cost.
138
B. Advanced Detection Methodologies
Another important area of future research will be the elimina-
tion of the power-hungry and cost-intensive components from
integrated genetic analysis microsystems in order to enhance
their eld-portability, disposability, and to reduce their cost of
production.
INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 227
1. Optics-Free Detection
Use of uorescence 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., the genetic detection of HIVin Africa), such optical
components may prove to be impractical due to size, cost, and
fragility. Alternative detection strategies are needed that sup-
plant optical detectionwhile maintainingmanyof 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 sufcient
product such that electrochemical means may also be used for
detection. The rst work to demonstrate the integration of mi-
crochannel CE with electrochemical detection was presented by
Woolley et al.
139
In this work, PCR-amplied DNA was sepa-
rated on a CE microchannel and detected using electrochemical
electrodes placed past the end of the separation channel. Later
work has rened 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-ow supported
electrochemical detector for use in integrated CE microsystems.
The sheath owcarries the DNAanalyte fromthe gel separation
region into a free-solution detection region, electrically isolating
the electrochemical detector from the high electric elds inher-
ent to CE. Other recent advances in electrochemical detection,
such as differential measurements and use of electrochemical
intercalators
141
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-specic detection.
The rst 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 DNAprobes 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
difculty in differentiating similar DNA sequences, such as are
generated in forensic investigations through the amplication 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-cost
and 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, PCRamplication,
and electrochemical detection using a sandwich assay. Their sys-
tem was capable of detecting 10
6
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 specicity 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
themmaybe cost, inthat these assays require expensive reagents,
including gold or silver nanoparticles, magnetic microspheres,
and PCRreagents. Work continues 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
method would be reagentless (requiring only the sample for
analysis), eld-portable, low-power, reusable, and able to be
stored for long times. Fan and colleagues
146
have developed a
systemthat meets most of these requirements. Figure 12 presents
a schematic representation of such a system. Their system con-
sists of a single strand of DNA attached at one end to an electro-
chemical working electrode using standard Au-thiol chemistry.
The other end of the probe DNA contains an electrochemically
active reporter molecule. The sequence of the DNA is chosen
to be self-complementary, such that the probe in the absence
of target forms a stem-loop structure. The electrochemical la-
bel undergoes oxidation or reduction upon the application of a
potential at the working electrode and transfers electrons to the
working electrode. Upon introduction of complementary target
DNA, however, the probe DNA hybridizes to the target and be-
comes more rigid, moving the electrochemical label away from
the surface. Because electron transfer is an exponential func-
tion of distance, the electrochemical current in the presence of
target is a small fraction of that in the stemloop state, pro-
viding a signal-off sensor. Xiao and coworkers have recently
extended this work to protein detection through the use of ap-
tamers, single-stranded DNA oligonucleotides that demonstrate
high afnity and high specicity for a certain target.
147
Using
the E-DNA architecture, an aptamer was bound to a gold elec-
trode and contained an electrochemical label at the free end.
Binding of thrombin, a protein involved in the human blood-
clotting cascade, changed the aptamer structure, which in turn
affected the electrochemical current used for detection. The use
of electrochemical reporters and efcient exploitation of ligand-
induced folding of probe molecules may propel the eld toward
reagentless, portable, low-power, and disposable devices.
C. Microsystems for Parallel Information Gathering
1. Motivation
Genetic analysis of samples from complex mixtures presents
an analytical challenge not only in the methods of analysis, but
228 E. T. LAGALLY AND H. T. SOH
FIG. 12. The E-DNA system. A stemloop oligonucleotide possessing terminal thiol and a ferrocene group is immobilized at a
gold electrode through self-assembly. In the absence of target, the stemloop structure holds the ferrocene tag into close proximity
with the electrode surface, thus ensuring rapid electron transfer and efcient redox of the ferrocene label. On hybridization with
the target sequence, a large change in redox currents is observed, presumably because the ferrocene label is separated from the
electrode surface. Reprinted with permission from Reference 108.
also in the interpretation of the results. Most methods of ge-
netic analysis are limited in the types of information that they
can present to the user. PCR is an excellent example of such
limitations. Although fast, exponential, and highly specic, PCR
exhibits a major limitation: one has to know what one is looking
for. That is, the design of the PCR product requires appropri-
ate primer sequences, and these primer sequences must be cho-
sen from known sequence information a priori. All organisms
have genomes that require regular random changes to their se-
quences in order to adapt to new environments and to evolve.
Thus, primer sequences that are chosen from currently available
genome sequence data may become obsolete, and perhaps more
importantly, the genome sequence may be unknown. In this as-
pect, genetic analysis of bacteria presents an especially large
challenge, because such organisms are capable of very fast re-
production (doubling times as fast as 30 minutes), and therefore
intrinsically higher rates of genetic variation.
There are several approaches to confront this analytical chal-
lenge. The most adaptable solution in the long termis likely to be
the integration of multiple information streams about a particular
sample, such that genetic analysis results can be crosschecked
against other information types (immunological, physical, elec-
trical, etc.) to verify the results. One of the future areas of work
in genetic analysis microsystems will therefore be integration to
acquire multiple types of information about a particular sample.
Such systems have already begun to emerge in the literature; the
work by Lagally et al. involving DEP concentration followed by
genetic detection
134
exhibits the advantage of combining dielec-
trophoretic information about the bacteria with genetic data. For
example, if bacteria are successfully captured but genetic anal-
ysis provides a negative result, at least the user is potentially
aware of the presence of such bacteria, which would not be the
case with genetic analysis alone.
Genetic analysis integrated with other information types pro-
vides advantages beyond analysis redundancyit will allow
the user to rapidly characterize new or unknown samples more
quickly. For instance, if a single system is capable of simul-
taneous analysis of the immunological, genetic, electrical, and
biochemical characteristics of the bacteria in a mixed sample,
this has the potential to reduce the time needed for deduction of
treatment methods. In addition, correlation of multiple parame-
ters may be used to discover newrelationships between different
types of organisms or components of a sample. Recent advances
in epigenetics, the self-regulation of phenotype without genetic
change,
148
and relationships between the genome, proteome,
149
and glycome
150
of an organism call for integrated analysis sys-
tems capable of aiding these elds with faster analysis, lower
detection limits, higher sensitivity, and higher specicity.
2. Interface Challenges
The development of such parallel-processing integrated
microsystems brings with it challenges in the chip-to-world
interface. In analogy to the semiconductor industry, microuidic
technologies have reached a stage where hardware such as
zero-insertion force (ZIF) sockets have become a necessity. As
the number of parallel analyses on a single microdevice grows,
so do the number of distinct connections needed to external
hardware, which may be microuidic, pneumatic, or electrical.
Although some of the functionality may certainly be fabricated
INTEGRATED GENETIC ANALYSIS MICROSYSTEMS 229
on the device itself in the future, some connections will have to
remain off-device, and the number of connections will increase
with the functionality of the device. Fluidic connections
are of particular concern, mostly because the interface must
remain rigid, yet sufciently exible for the device to be
moved. In addition, application of these systems to complex
sample mixtures will increase the constraints on any uidic
connection to prevent clogging, adsorption, or other detrimental
interactions. Several other groups have proposed solutions to
these problems,
151,152
but a common general solution to such
problems has not emerged, making this area one of primary
focus for future work.
VII. CONCLUSIONS
This review has presented a series of technologies for mi-
croscale and nanoscale analysis of genetic material. The ad-
vances in such technology from the rst examples in the early
1990s to the hundreds of examples at present represents expo-
nential growth in the eld. Examples of advances in microfab-
ricated modules representing individual components, as well
as efforts to combine these modules to form integrated systems
that surpass the performance of their conventional predecessors,
have been discussed. The design, fabrication, and testing of in-
tegrated microsystems that collect multiple types of information
about a sample have just begun to emerge, and will propel the
eld to new levels of functionality and utility. The powerful
convergence of materials science, chemistry, and biology with
microsystems technology promise to advance the eld to create
systems capable of unprecedented performance, utility, and ac-
cessibility. It is hoped that the benets from such technologies
will improve the lives of many.
ACKNOWLEDGMENTS
The authors thank Professors M. A. Burns, A. Manz, R. A.
Mathies, and K. W. Plaxco for their permission to reprint gures
from their publications here.
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