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Anal. Chem.

2005, 77, 3887-3894

Advances in Polymerase Chain Reaction on


Microfluidic Chips
Michael G. Roper,† Christopher J. Easley,† and James P. Landers*,†,‡
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, and Department of Pathology, University of
Virginia Health Science Center, Charlottesville, Virginia 22908

Review Contents on high-throughput microdevices as a means of reducing the


Background 3887 backlog of samples awaiting analysis in the United States (4).
Genome Era 3887 Advances in sample preparation, at least those akin to what
Microfluidic Heating Methods 3888 microfabricated devices have brought to separations (enhanced
Microchip PCR Background 3888 speed and throughput, decreased cost), are more difficult to find.
Scoring Method for Device Comparison 3888 Sample preparation involves three general processes that, when
PCR Reaction Volume 3889 carried out sequentially, reduce samples of diverse origin and
High-Speed PCR 3890 character to a form amenable to separation. These processes often
Future Strategies 3891 include a cell sorting step (to isolate the cells of interest from a
Potential Improvements 3891
mixture) but almost universally involve the extraction of DNA from
Real-Time PCR 3891
the sample matrix, followed by DNA amplification via the poly-
Whole Genome Amplification 3891
Conclusions 3892
merase chain reaction (PCR). The role of the PCR is obvious: to
Literature Cited 3892 create adequate copies of select DNA sequences so that these
can be detected by separation. The role of the purification step is
simple: to rid the sample of any species that could potentially
BACKGROUND interfere with a successful and robust amplification by the PCR
Genome Era. The mapping and sequencing of the human process. Pre-PCR steps serve the sole function of presenting a
genome represents a monumental accomplishment that will DNA-containing sample that is amenable to optimal amplification,
impact medicine and biology in ways that we are only beginning while post-PCR separation serves to display the product(s) yielded
to comprehend. The successful sequencing reported in 2001 (1) from the PCR process. From this perspective, one can easily argue
ushered in what has been dubbed the “postgenome era”. While that PCR is the keystone of genetic analysis.
this implies that the genome era is over, it is, in fact, just getting The PCR is an enzyme-mediated process where, in response
underway in so many ways. A map of the more than 3 billion bases to temperature cycling, a single DNA molecule can be rapidly
and their corresponding 30 000+ genes will not only be critical amplified into many billions of molecules. PCR was introduced in
in fleshing out the structure and function of these genes but will 1985 by Mullis and co-workers (5) where he described the
leverage the discovery of new genes and a newfound understand- transitioning of a PCR mixture (bacterial polymerase, dNTPs,
ing of the potential roles the gene products play in health and template DNA, and primers) through different temperatures that
disease. Genetic analytical methods have been, and will continue facilitated the denaturation of the template DNA (94 °C), the
to be, critical to such discovery, providing the means to dissect annealing of primers to complimentary sequences (e.g., 55 °C),
gene structure and gene function. These methods have almost and the extension of complimentary sequence from the primer
invariably involved two general processes: sample preparation and (72 °C). Repeated cycling through these temperatures resulted
separation/detection. in an exponential increase in the number of copies of a specific
Capillary electrophoresis (CE), a means for separating DNA DNA sequence (in size up to ∼1000 base pairs, bp) relative to
fragments in a manner that allows for decoding DNA sequence, the original number of DNA template copies. The ability to create
has supplanted the tedious, time-consuming methods associated millions to billions of copies from a single DNA template has
with sequencing slab gels. As a consequence of this, the role of revolutionized the field of molecular biology, supported by the
CE in accelerating the sequencing of the genome is well- fact that this pioneering work earned Mullis a portion of the 1993
recognized (2). More recently, there is a palpable interest in the Nobel Prize in Chemistry.
next paradigm shiftshigh-resolution separations on microfabri- Since its conception, the PCR has become one of the main
cated devices that provide comparable resolution to CE separa- laboratory tools for scientists in the life sciences. An effective PCR-
tions but with enhancements in speed, throughput capabilities, based amplification is now an indispensable part of genetic
and cost-effectiveness (3). One example is the current interest analysis, whether it involves molecular biological-, diagnostic-,
from the forensics arena to carry out short tandem repeat typing forensic-, or agricultural-related analysis. PCR is nearing its quarter
century anniversary and plays a key role in the evolution of newer
* To whom correspondence should be addressed. Phone: (434)-243-8658. and more powerful molecular biological techniques; and the
Fax: (434)-924-3048. E-mail: landers@virginia.edu.
† University of Virginia. continuous morphing of molecular technology is, indeed, real.
‡ University of Virginia Health Science Center. Despite the power of PCR technology for routine diagnostics, there
10.1021/ac050756m CCC: $30.25 © 2005 American Chemical Society Analytical Chemistry, Vol. 77, No. 12, June 15, 2005 3887
Published on Web 05/19/2005
exists trepidation among some diagnostic laboratories to adopt involving PCR on microfluidic devices that are not mentioned in
new molecular methods. Some speculate that it is the ever- the text. We refer the readers to other recent reviews for a further
morphing nature of molecular biological techniques that precludes survey of the literature (3, 8-12).
adoption by some diagnostic laboratories, primarily because of Microchip PCR Background. It is noteworthy to summarize
concerns that the technology will rapidly become outdated (6). the initial developments in microchip PCR before outlining the
The conventional format of PCR involves the temperature recent literature. The first report of translating PCR to a chamber
cycling of the PCR components in thin-walled, plastic tubes in a in a microchip device was by Wilding et al. (12), who used a
temperature-controlled metal block between the desired temper- silicon/glass hybrid device that held 5-10 µL of reaction mixture
atures to effect the amplification of a specific DNA sequence. The and was heated using an external copper block heater. The same
amplified volumes are typically in the 2-50-µL range with cycling group then emphasized the need for surface passivation for
speeds (the time to traverse through one denature/anneal/extend microchip PCR, and they determined that silanization of the
temperature cycle) on the order of 20-40 cycles are typically microchamber surface provided the best results (13). Later, Burns
carried out. Thermal mass is the key to fast thermocycling, and and co-workers (14) created an integrated microchip including a
with conventional PCR instrumentation, this resides in the heating resistive heating region with temperature sensors for DNA
block used to control solution temperature. If we look to microchip amplification, a sample loading region, and a gel-based separation
technology to do for PCR what it has done for electrophoretic region; this work demonstrated the ability to integrate single-
separations, then the volume of PCR mixture thermocycled will strand displacement assay with other sample analysis steps on
be reduced by 1-2 orders of magnitude, and the speed with which one device. Kopp et al. (15) exploited the geometric freedom of
thermocycling can occur will be increased, not only because the microchip design by developing the first flow-through PCR
volumes involved are reduced but because thermal mass should microdevice. By simply using the microchip reservoir as the
be as well. reaction chamber, Waters and co-workers (16) performed cell
Microfluidic Heating Methods. Temperature cycling on lysis, PCR amplification, and electrophoretic sizing on a single
microfluidic devices can be performed either with contact heating microchip device by placing the entire chip into a commercial
methods or noncontact heating methods. Here, we define contact thermocycler. The first report of noncontact PCR on microchips
heating methods as having heaters fabricated within the microchip was by our group (7e) using a polyimide substrate and cycling
or in thermal contact with the outside the microchip; with both, through selective infrared heating and air cooling. Lagally et al.
the common denominator is that the thermal mass of the (18) developed a device with microfabricated resistance heaters,
microfluidic device is in contact with the heating element. Not resistance temperature sensors, and PCR chambers seamlessly
surprisingly, noncontact methods are defined as using a heating connected to electrophoretic separation channels, including valves
and hydrophobic vents. These publications have been seminal to
method that is remote from the microfluidic device and not in
the development of microchip PCR, and as such, a few were
physical contact with the PCR chamber. While our group has
included in Table 1 for the purpose of comparison along with
focused extensively on noncontact heating methods for PCR
others (7e, 15, 16, 19-30, 33, 36-42).
amplification to reduce the thermal mass of the components to
Scoring Method for Device Comparison. Throughout this
be cycled (7) and, hence, achieve faster thermocycling rates, the
review, we found it necessary to attempt a normalization of the
majority of papers published during 2003-2005 rely on contact
various microfluidic platforms for performing PCR to enable a
heating. For this reason, the rest of this review will devote its
better comparison between the designs. To perform this normal-
attention to contact heating methods.
ization, three major facets of PCR amplification were included.
The benefits of microfluidics have always been the ability to
First, we calculated the average experimental rate at which PCR
integrate multiple functionalities on one device, decrease analysis
was occurring from the amplicon size and reaction time. This term
time, and minimize dilution. Therefore, an ideal integrated genetic
therefore included not only the kinetic rate of nucleotide incor-
analysis system will accept a complex biological fluid (e.g., blood,
poration by Taq polymerase but also the effects of general reaction
serum, or semen), remove PCR inhibitors, amplify the DNA, and,
conditions such as passivation of the substrate. Second, the volume
finally, separate and detect the amplified products (amplicons) of
of the PCR chamber was used as a measure of the “cost” of
interest. Due to the multiple steps in this sequence, a short process
amplification. For instance, a small volume would reduce the
time for each step is ideal, which can be achieved in PCR via fast
overall cost of the system by allowing for lower amounts of
cycling times, which favors reduced volumes (and therefore
reagents to be used. Third, the limit of detection of the experiment
thermal mass) as mentioned previously. Therefore, this review is
was reflected in the score. This last parameter was included
divided into two sections focused on what we believe to be two of
because the reaction rate (as defined in the first facet) will be
the more significant parameters in chip PCR: the PCR reaction dependent on the copy number used. In this way, a device with
volume and the speed of amplification. a slow reaction rate could still receive a relatively high score if
Chemical Abstract Service was searched from 2003 to 2005 the limit of detection is good. To incorporate these parameters,
including the keywords “microfluidic AND amplification”, “mi- the formula for the score was calculated as follows:
crofluidic AND reaction”, “lab-on-a-chip AND amplification”, “lab-
bp
on-a-chip AND reaction”. This review is not meant to be a Score )
comprehensive summary of all literature during this time period, Vol × t × CN
only a cursory review and summary of notable achievements of
fast PCR and low-volume PCR during this era. Table 1 is a where bp is the number of base pairs of the amplified fragment,
summary of the literature cited in the text and also lists literature Vol is the volume of the PCR domain, t is time for amplification,
3888 Analytical Chemistry, Vol. 77, No. 12, June 15, 2005
Table 1. Abbreviated List of the PCR Performed within Microfluidic Devices

amplicon grade (bp


vol amplification LOD or amount length µL-1 s-1
ref (nL) time (s) template DNA of DNA used (bp) copy-1)
Low Volume
19 3 3600 1800-bp cDNA LOD ) 60 copies 294 0.45
20 29 5600 genomic Salmonella LOD ) 6 copies 559 0.57
21 40 >3400 genomic DNA LOD ) 0.4 copies 295
22 200 1200 intact E. coli cells LOD ) 2-3 cells 280 0.58
23 200 420 Topo PCR 2.1 vector 15 ng 127
3.5 × 109 copiesa
24 200 600 genomic DNA LOD ) 10 copies 150 0.12
25 240 1860 mouse DNA snrpn template n/ab
Fast PCR
26 total ) 2500 102 λ-phage DNA LOD ) 2 × 107 copies 500 9.8 × 10-8
at 10 mm/s
27 2000 360 1.02-kbp genomic DNA 2.5 × 106 copies 230
Others
15 240 1030c 106-bp M. tuberculosis DNA 9.6 pg, 8.8 × 107 copiesa 106
16 10 000 90 with 72.9 nL/s 1000-bp N. gonorrheae DNA 108 copies 176
7e 1700 240 λ-DNA 6.9 × 104 copies 500
28 500 600 λ-DNA 5 pg, 9.5 × 104 copiesa 500
29 15 000 1800 3.9-kbp genomic DNA 6 ng, 1.4 × 109 copiesa 295
30 119 000 4380 B. cereus DNA 1.6 µg, 2.9 × 108 copiesa 700
33 280 900 2686-bp M13/pUC19 LOD ) 1 copy 136 0.54
cloning vector
36 10 000 780 280-bp genomic DNA 1.56 × 105 copies 230
37 20 000 5400 intact E. coli cells 1000 E. coli in 1 mL blood 221
1 × 103 copiesa
38 15 000 5940 intact white blood cells 10000 cells, 2 × 105 copiesa 379
39 3500 3390 Vero E6 cells and nasal swabs n/a 438
40 3000 4680 genomic DNA 6 × 104-3 × 105 ng 350
2 × 107-9.8 × 107 copiesa
41 8000 4200 Topo PCR 2.1 vector LOD ∼500 copies 600
42 12 8280 λ-DNA 24 pg, 4.8 × 105 copiesa 199
a Indicates our calculation based on molecular weight of template DNA. b n/a, not available. c Single strand displacement assay.

and CN is the copy number at the limit of detection. As can be and pneumatic pumps makes integration facile. An added benefit
seen, bp/t represents the average experimental rate of the of this design was the reduction in the amount of manual pipetting
reaction, while the volume and copy number at the limit of needed to address an N × N matrix of reaction centers from 3N2
detection are also included. Thus, a high score will occur if a large to 2N + 1, thereby reducing the possibility of contamination and
amplicon is generated from a small-volume, high-speed, low-copy errors. Although this work set the lower limit of reaction volume,
number assay (an example of an “ideal” high score would be speed of analysis was not the major focus and thermocycling was
generation of a 200-bp amplicon from a single copy of template performed by placing the entire device on a conventional ther-
DNA in a 10-nL reactor in 90 ssall numbers that have been mocycler. Thus, even with a dramatically reduced PCR reaction
achieved at least individually in chip-based PCRswould provide volume, one that would be amenable to extremely fast thermocy-
a score of 222). We are aware that this is a simplistic formula; cling, 1 h was still required for amplification of a 294-bp fragment
however, it has helped us to provide a more clear comparison of the human β-actin cDNA fragment. With an LOD of 60 template
between these PCR chips (discussed in the text and shown in copies, the calculated score for this work was 0.45 bp µL-1 s-1
Table 1), and we hope that it will also help the readers. All scores
copy-1.
were based on parameters at the limit of detection, and if no limit
A 29-nL PCR reactor was demonstrated on a poly(cyclic olefin)
of detection was given, no score was assigned. Additionally, the
microfluidic chip that used paraffin wax valves to contain the PCR
scoring method assumes similar PCR reagent concentrations
mixture followed by electrophoretic separation of the amplified
(other than DNA). In the conclusion of this review, we address
product (20). Silver/graphite inks were printed onto the plastic
issues of “weighting” the various parameters in an applications-
substrate prior to bonding to be used as both the heaters and
dependent manner.
electrophoretic contacts, and a resistive temperature detector
(RTD) was used to sense the temperature during PCR amplifica-
PCR REACTION VOLUME
With microfabricated devices come the ability to react ex- tion. Limit of detection for this device was shown to be six copies
tremely small volumes of solutionsPCR is not excluded from this for both purified Escherichia coli and Salmonella DNA per 29-nL
quest. The smallest PCR reaction volume reported to-date comes reaction vessel. Heating rates attained were 12 °C/s, and cooling
from work in the Quake group (19). PCR in a volume of 3 nL rates were 2 °C/s. Although it was stated that PCR could be
reduces, by almost an order of magnitude, the volume required performed with shorter hold times, 80 min was still required for
by the second lowest report. This volume of solution is amenable amplification of DNA, thus not providing much advantage in speed.
to upstream and downstream components of a completely inte- With this favorable LOD and low reaction volume a very high
grated genetic analysis system, and their use of hydraulic valves score could have been obtained with more optimization of reaction
Analytical Chemistry, Vol. 77, No. 12, June 15, 2005 3889
time, but with the 80-min reaction time, the work registered a advantages of low-volume design (see Table 1), which is reflected
score of 0.57 bp µL-1 s-1 copy-1. by the fact that the scores are an order of magnitude below ideal.
Matsubara et al. (21) performed PCR amplification in 40-nL This is certainly the case when thermocycling involves heating
reaction vessels. Forty nanoliters of a solution containing primers the entire device and not simply the PCR domain. This is
and a fluorescent probe were first loaded into the vessels and supported by the fact that the fastest thermocycling described
allowed to dry, and 200 µL of oil was applied to avoid evaporation. here involved chip-integrated heaters, where only the PCR domain
Then, 40 nL of PCR mixture containing human genomic DNA (and the enclosed PCR mixture) was heated (22-24). A drawback
minus primers was added under the oil to each well. The entire to integrating heaters and temperature sensors directly into the
chip was then placed on a conventional thermocycler (amplifica- device is the complicated and costly method of fabrication.
tion time was 1 h) and successful amplification determined by Although several of these reports did not have small-volume
detecting fluorescence from the probe using a charge-coupled PCR with high-speed amplification as the ultimate goal of the
device. A 0.4 DNA copy number was reported for their limit of project, these systems are much faster compared to conventional
detection, but it is important to note that only 2 out of 60 vessels thermocyclers. However, if a complete genetic analysis system
had detectable amounts of DNA at this concentration. The use of for time-sensitive diagnostics (e.g., biowarfare detection) is
oil as a cover for their system is a distinct disadvantage and raises needed, the PCR must be performed with greater amplification
questions as to the applicability for integrated genetic analysis speed.
systems. Due to the LOD discrepancy, a score was not reported
for this work. Again a similar theme is observed, where the volume HIGH-SPEED PCR
and LOD were particularly low, but the reaction time gave no To obtain high-speed PCR, reducing the thermal mass of the
improvement over conventional PCR. system can increase the heating rates during temperature cycling.
Other reports have described PCR with volumes of 200 nL or However, as noted above, small mass (or volume) does not
higher. Lagally et al. (22) have set the bar with a portable genetic necessarily translate to fast PCR thermocycling. The fastest PCR
system that uses an 20.3 cm × 25.4 cm × 30.5 cm box for housing during 2003spresent was performed with flow-through devices
all optics and electronics for a microdevice that performs PCR in which the PCR Mastermix and template DNA are pumped past
and electrophoretic separation with a 200-nL PCR chamber. Limit preheated domains. Thus, only thermal mass was the solution
of detection for their system was 2-3 E. coli cells with amplifica- and not the microdevice.
tion performed in ∼20 min. With intermediate values of volume The work performed by Hashimoto et al. (26) exemplifies the
and reaction time combined with a good LOD, this work scored fast speed attainable with PCR performed in microfluidic chips.
the highest among those reviewed at 0.58 bp µL-1 s-1 copy-1. They utilized a flow-through device consisting of discrete tem-
Lee’s group (23) developed a hybrid silicon/poly(methyl meth- perature zones produced by electrical resistance heaters. This
acrylate) chip with platinum thin-film heaters and RTD sensors work leveraged the original work of Manz and co-workers (16),
fabricated on the Si wafer. The authors reported successful PCR who performed 20 cycles of amplification in 90 s using a glass
amplification in 390 s although, due to the 200-nL volume in the microdevice. In the Hashimoto et al. work, amplification of a 500-
PCR chamber, amplified DNA could not be removed from the bp region of λ-phage DNA was performed in 1.7 min while a 997-
chip for analysis. Therefore, SYBR Green I dye was added to the bp fragment took 3.2 min, which the authors attributed to the
reaction chamber and increased fluorescence, compared against kinetic limit of nucleotide incorporation by Taq polymerase. Finite
fluorescence prior to PCR, was used as an indirect measure of element analysis revealed the steady-state temperature distribution
PCR success. Guttenberg et al. (24) used a surface acoustic wave and the temperature of the PCR reagents in each zone as a
pump to drive a 200-nL droplet of PCR mixture to two temperature function of velocity. It was found that if the flow was too fast
zones produced by thin-film heaters on a planar substrate. between the denaturation and renaturation step during amplifica-
Detection was accomplished by pumping the solution to a tion, cooling to the desired temperature was not attained with the
hybridization probe spotted on the same device. Amplification passive system used. The starting copy number of DNA will affect
times for this two-temperature PCR were as low as 10 min the speed at which PCR amplification can be performed, and with
beginning with 10 genomic DNA copies. This work registered a a linear velocity of 10 mm/s, the lowest DNA starting copy to
score of 0.12 bp µL-1 s-1 copy-1. Finally, a low-volume PCR provide amplified signal in 20 cycles was 1 × 107 copies. The
microdevice was developed by Burns and co-workers (25), with authors attempted lower concentrations at this velocity; however,
240-nL reaction chambers isolated from other domains within the amplified product was undetectable in their system. Even with
microchip through the use of phase-changing valves. Heaters and the extremely fast cycling, combining such an unfavorable LOD
temperature sensors were integrated within the device, and with the large volume used (2.5 µL) resulted in a score of 9.8 ×
amplification of mouse DNA snrpn template was achieved in 31 10-8 bp µL-1 s-1 copy-1.
min as determined by off-chip separation. Another flow-through PCR device capable of either PCR or
Small-volume PCR is advantageous in reducing the cost of reverse transcriptase PCR (RT-PCR) developed by Obeid et al.
reagents used for the amplification (especially costly Taq poly- (27) uses four heating blocks, three for PCR amplification and
merase) but should also allow for more rapid thermocycling and, the fourth for reverse transcription. The device contained outlet
as a result, faster temperature transitions and more efficient PCR reservoirs after 20, 25, 30, 35, or 40 cycles to allow the user to
amplification. However, as described here, the use of small-volume collect samples at different cycle numbers. With 107 starting copies
PCR chambers does not necessarily coincide with reduced of DNA in 10 µL of reaction buffer, 30 cycles of amplification could
amplification times if the mode of heating does not exploit the be performed in 6 min using a flow rate of 21 nL/s. RT-PCR was
3890 Analytical Chemistry, Vol. 77, No. 12, June 15, 2005
performed in starting volumes as low as 700 nL with the lower than 300 bp. They also note that these convection-based amplifica-
limit of starting copy number tested at 6.25 × 106. The ability to tion devices may be useful on centrifugal microfluidic platforms.
perform multiple PCR or RT-PCR amplifications was also shown. The majority of papers presented in this review have relied
Our laboratory has pioneered the use of infrared-mediated on physical heaters. A completely orthogonal approach was
heating for performing PCR amplification (7). While the IR described by Verpoorte and co-workers (31), who take advantage
radiation impinges only on the solution in the PCR chamber, the of exothermic and endothermic chemical reactions on a microf-
surrounding glass will heat due to transfer of the energy and luidic chip to heat and cool microchannels. Evaporation of acetone
partial absorption of the IR light by the glass. By completely from a channel produced a cooling effect down to -5 °C, while
etching glass away near the PCR zone, IR-mediated PCR (25 on-chip dilution of concentrated sulfuric acid provided heating to
cycles) has been performed in 5 min, one of the fastest cycling 76 °C. While not demonstrated as functional for PCR amplification,
times using a non-flow-through system. this clever approach for controlling temperature is simple and does
These reports represent several of the faster analyses reported not require macrocomponents or multiple fabrication steps for
within the past 2 years. Unfortunately, both flow-through devices construction of the microfluidic chip.
used a large amount of template DNA. Though scores were not Finally, an interesting approach to expediting genetic analysis
assigned to most of these, a similar problem with time and volume may be to avoid PCR altogether. This can be achieved with
optimization was observed among the fast cycling reports, in which detection that has single-molecule sensitivity. Park et al. (32) used
reaction volumes were frequently in the microliter range. The true a microfluidic chip to mix fluorescently labeled sex-determining
value of high-speed PCR amplification will be realized when low region Y gene with 65-70-nm-diameter silver particles. Confocal
starting copy numbers can be amplified in a short time in low- surface-enhanced Raman spectroscopy was used to monitor the
volume chambers. oligonucleotide, and a 10 pM limit of detection was reported.
Although significant technical hurdles exist, it may be feasible to
use this type of system to detect hybridization of a labeled probe
FUTURE STRATEGIES with gene sequences on template DNA, thereby, circumventing
While the previous sections have shown small volume and fast the need for PCR.
temperature cycling for the PCR, several other noteworthy reports
have focused on unconventional strategies for amplification of
DNA via the PCR or for unconventional means of heating for the POTENTIAL IMPROVEMENTS
PCR. Many of these methodologies, while still conceptual in Real-Time PCR. Real-time PCR (or quantitative PCR, qPCR)
nature, have the potential to impact PCR in the future. has the advantage that the PCR amplification process can be
Soper and co-workers (28) have demonstrated an electroki- monitored as it occurs, presenting the opportunity to obtain
netically controlled flow-through PCR microdevice fabricated in quantitative PCR informationsthis is not possible with standard
a polycarbonate substrate. Two key features of their method PCR. There has been scant work reported on qPCR in the
included the use of a positive polyelectrolyte multilayer, Polybrene, literature, which is surprising given the availability of commercial
to coat the channel walls for reversed electroosmotic flow (EOF) qPCR kits. In addition, the optical setup used for detection is
and a low ionic strength PCR buffer to minimize Joule heating. simple considering a typical laser-induced fluorescence setup (that
By using EOF to control flow, cycle numbers could be altered most microfluidic labs use) is all that is needed. Chip-based qPCR
while one-channel design was maintained. Adequate amounts of would simplify the chip in many ways because the separation step
amplified product from a 500-nL PCR reaction could be attained could be eliminated. Moreover, the ability to obtain quantitative
after 15 cycles (600 s) beginning with 10 ng/µL λ-phage DNA. information lends itself perfectly to diagnostic applications where
One benefit of an electrokinetically controlled PCR flow-through the number of copies of starting template have clinical relevance
device is valveless integration of up- and downstream DNA (e.g., viral titer).
analysis. However, one must be aware that there is a possibility Whole Genome Amplification. While single-copy PCR has
of the anionic DNA binding to the cationic Polybrene-coated walls. been demonstrated on-chip (33), having access to a large amount
Ugaz and co-workers (29) have built upon previous work with of genomic DNA would be advantageous with samples where the
Rayleigh-Bénard convection systems for PCR thermocycling. By number of starting template copies is low. Whole genome
placing an array of 35 µL polycarbonate wells on an aluminum amplification (WGA) provides such a method (34) and is used in
heating block set at 95 °C and covering the wells with an many molecular biological assays associated with the analysis of
aluminum heating block at 61 °C, convection drives the solution human disease, where limited sample/tissue is available. For
through the various temperature zones, thereby, thermocycling. example, when a particular focus of interest consists of only a
To provide more uniform residence times in the temperature few hundred cells, the amount of extractable DNA may be
zones, closed-loop devices were developed that were in contact insufficient for genetic analysis. Comprehensive WGA is a chal-
with heaters and provided the same type of convective thermocy- lenge because it requires accurate replication of more than 3
cling. Amplification of a 191-bp fragment was observed in the 35- billion bases without the loss or mutation of any particular loci/
µL wells in 15 min, and a 295-bp fragment was amplified in 30 alleles. However, unlike the PCR (where specific sequences are
min using the 15-µL closed-loop system. Chen et al. (30) have targeted), WGA must represent the entire genome with minimal
also demonstrated successful amplification of both a 305- and 700- amplification bias. Methods for WGA have been developed that
bp DNA fragment using a closed-loop system. The authors point minimize this bias and allow, through conventional means, the
out that most reported closed-loop systems amplify fragments less generation of as much as microgram quantities of DNA (35). Since
Analytical Chemistry, Vol. 77, No. 12, June 15, 2005 3891
both thermal and isothermal WGA methods have been developed, Chrisopher J. Easley is a graduate student completing his doctoral
degree in analytical chemistry at the University of Virginia. He received
it is not unreasonable to anticipate that this can be accomplished his baccalaureate degree in chemistry from Mississippi State University
on-chip with the benefit of fast thermocycling and little dilution. in Starkville, MS, in 2002. His current reasearch involves rapid
noncontact microchip PCR integrated with electrophorectic separation for
genetic diagnoses of diseases or pathogen detection.

CONCLUSIONS James P. Landers is a Professor of Chemistry at the University of


Virginia and Associate Professor of Pathology at the University of Virginia
Although PCR is a mature technique, its adaptation to the Health Sciences Center. He received his B.S. degree in biochemistry (minor
in biomedicine) at the University of Guelph (Canada in 1984 and his
microchip platform is an evolving technology. It is clear that the Ph.D. in biochemistry from the Department of Chemistry and Biochemistry
analytical challenges associated with doing efficient, fast, low- at the same university in 1988. After a postdoctoral fellowship at the
Banting Institute at the University of Toronto’s School of Medicine, he
volume PCR in a microfluidic chip have, by no means, been served as a Canadian Medical Research Council (MRC) Fellow at the
completely addressed and solved (see Table 1). The evolution Mayo Clinic under Thomas Spelsberg. Following his MRC Fellowship,
he was appointed as the Director of the Clinical Capillary Electrophoresis
involves trying to test and optimize a number of parameters that Facility in Mayo Clinic’s Department of Laboratory Medicine and
Pathology. His research interests include the development of analytical
include substrate (one that provides the best characteristics for microchip technology for use in human disease diagnostics. This not only
both PCR and fabrication), design of the PCR chamber, means of involves method development for electrophoretic separations on microchips
focused on expediting molecular diagnosis but also includes the develop-
temperature cycling, and whether the device is stand-alone or ment of microchip-based sample processing methodologies necessary to
integrated with other on-chip functions. This evolution is apparent create a totally integrated microchip system fir clinical diagnostics.
in the recent literature covered here. Ironically, some of the fastest
PCR reactions reported used the largest starting template copy
LITERATURE CITED
number and the largest volumes, while several of the lowest
(1) Venter, J. C.; Adams, M. D.; Myers, E. W.; Li, P. W.; Mural, R. J.;
volume PCR chambers thermocycled the slowest. While it may Sutton, G. G.; Smith, H. O.; Yandell, M.; Evans, C. A.; Holt, R. A.;
appear that a random logic dominates the evolution of chip-based Gocayne, J. D.; Amanatides, P.; Ballew, R. M.; Huson, D. H.;
Wortman, J. R.; Zhang, Q.; Kodira, C. D.; Zheng, X. H.; Chen, L.;
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number used depend largely on the type of heating utilized and Miklos, G. L.; Nelson, C.; Broder, S.; Clark, A. G.; Nadeau, J.;
McKusick, V. A.; Zinder, N.; Levine, A. J.; Roberts, R. J.; Simon,
the chip substrate. Other parameters, such a flow (for flow-through M.; Slayman, C.; Hunkapiller, M.; Bolanos, R.; Delcher, A.; Dew,
I.; Fasulo, D.; Flanigan, M.; Florea, L.; Halpern, A.; Hannenhalli,
systems) and temperature sensing, factor in as well. Consequently, S.; Kravitz, S.; Levy, S.; Mobarry, C.; Reinert, K.; Remington, K.;
the morphing that seems apparent will continue until there is Abu-Threideh, J.; Beasley, E.; Biddick, K.; Bonazzi, V.; Brandon,
R.; Cargill, M.; Chandramouliswaran, I.; Charlab, R.; Chaturvedi,
convergence to a few functional systems that, in all likelihood, K.; Deng, Z.; Di Francesco, V.; Dunn, P.; Eilbeck, K.; Evangelista,
will be detemined by the application addressed. Several functional C.; Gabrielian, A. E.; Gan, W.; Ge, W.; Gong, F.; Gu, Z.; Guan, P.;
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G. V.; Milshina, N.; Moore, H. M.; Naik, A. K.; Narayan, V. A.;
the need of the application. The choice of heating method will be Neelam, B.; Nusskern, D.; Rusch, D. B.; Salzberg, S.; Shao, W.;
determined by the speed deemed acceptable for the thermocy- Shue, B.; Sun, J.; Wang, Z.; Wang, A.; Wang, X.; Wang, J.; Wei,
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Cravchik, A.; Woodage, T.; Ali, F.; An, H.; Awe, A.; Baldwin, D.;
dependencies on the application can be better reflected in the PCR Baden, H.; Barnstead, M.; Barrow, I.; Beeson, K.; Busam, D.;
Carver, A.; Center, A.; Cheng, M. L.; Curry, L.; Danaher, S.;
score as well. A more valuable approach to scoring each device Davenport, L.; Desilets, R.; Dietz, S.; Dodson, K.; Doup, L.;
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Rodriguez, R.; Rogers, Y. H.; Romblad, D.; Ruhfel, B.; Scott, R.;
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Narechania, A.; Diemer, K.; Muruganujan, A.; Guo, N.; Sato, S.;
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