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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.
3892 Analytical Chemistry, Vol. 77, No. 12, June 15, 2005
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