We implement microfluidic technology to miniaturize a thermal cycling system for amplifying DNA fragments. By using a microfluidic thermal heat exchanger to cool a Peltier junction, we have demonstrated rapid heating and cooling of small volumes of solution. We use a miniature K-type thermocouple to provide a means for in situ sensing of the temperature inside the microrefrigeration system. By combining the thermocouple, two power supplies controlled by a relay system, and computer automation, we reproduce the function of a commercial polymerase chain reaction thermal cycler and demonstrate amplification of a DNA sample of about 1000 base pairs

Gomez, Alvaro

Gomez, Frank A.

Maltezos, George

Scherer, Axel

Zhong, Jiang

English

Caltech Authors - Main

Microfluidic polymerase chain reaction
George Maltezos,1,a Alvaro Gomez,2 Jiang Zhong,3 Frank A. Gomez,2 and Axel Scherer1
1Department of Electrical Engineering, California Institute of Technology, 1200 East California
Boulevard, MS 200-36 Pasadena, California 91125, USA
2Department of Chemistry and Biochemistry, California State University, Los Angeles,
5151 State University Drive, Los Angeles, California 90032-8202, USA
3Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA
Received 4 July 2008; accepted 12 November 2008; published online 16 December 2008
We implement microfluidic technology to miniaturize a thermal cycling system for amplifying DNA
fragments. By using a microfluidic thermal heat exchanger to cool a Peltier junction, we have
demonstrated rapid heating and cooling of small volumes of solution. We use a miniature K-type
thermocouple to provide a means for in situ sensing of the temperature inside the microrefrigeration
system. By combining the thermocouple, two power supplies controlled by a relay system, and
computer automation, we reproduce the function of a commercial polymerase chain reaction thermal
cycler and demonstrate amplification of a DNA sample of about 1000 base pairs. © 2008 American
Institute of Physics. DOI: 10.1063/1.3046789
The polymerase chain reaction PCR has emerged as
one of the most sensitive analysis tools available to research-
ers in molecular diagnostics.1 DNA genotyping, virus identi-
fication, and forensic applications are a few of the applica-
tions that rely on PCR amplification and analysis.1–3 Clearly,
thermal cycling PCR is one of the leading techniques for the
amplification of DNA fragments.1 It is a rapid and straight-
forward method for generating many copies of DNA mol-
ecules. PCR amplification typically consists of thermally cy-
cling DNA fragments through many heating denaturation,
cooling annealing, and extension temperatures of target
DNA mixed with a primer and a polymerase involved in the
catalysis of DNA.
To obtain accurate amplification of the desired DNA
chains, it is necessary to carefully control the three tempera-
tures and to ensure good temperature uniformity in the ther-
mal cycling process. Traditionally, resistive heating or forced
air heating has been used to control the temperature. More
recently, Peltier junctions have been the refrigeration and
heating system of choice for PCR applications.4 This choice
is a result of the relative simplicity of integrating electrically
controlled Peltier junctions within electronic feedback loops
using thermocouple measurement data.4
Herein, we compare two alternatives of combining ther-
mocouple device with thermovoltaic solid-state devices a
Peltier junction to form a miniaturized PCR cycling system
within a microfluidic and glass capillary system with rapid
ramp rates. One of the advantages of developing such a mi-
crofluidic cooling system is that liquid cooling can be used
for effective heat sinking of the Peltier junctions. The other
advantage of integrating PCR cyclers within microfluidic
systems is that rather complex sample preparation can be
performed microfluidically and the transfer of amplicon from
the fluidic chip into a capillary-based thermal cycler can be
avoided. However, more conventional glass capillary tubes
offer the great advantage of well-defined surface chemistries
and the avoidance of evaporation through porous elastomer
channels—a major problem with previous polydimethylsi-
loxane PDMS approaches.5
Two Peltier junction systems were constructed: the first
was based on a multilayer replication molded PDMS micro-
fluidic chip developed for the purpose of integrating fluidic
sample delivery, where both the junction and the thermo-
couple are assembled to run a DNA sample using the PCR
technique described above.6,7 Therefore, the process of incor-
porating different elements such as the junction and the ther-
mocouple into the chip can be thought as a “lab-on-a-chip”
device that can both miniaturize and reduce costs when deal-
ing with the nowadays-expensive techniques of PCR. An al-
ternative system, in which a commercially available glass
microcapillary tube Polymicro Technologies TSG320450
with a 320 m inner diameter is inserted into a copper heat
exchanger, was also constructed. PCR amplification was
demonstrated on large DNA molecules, and the ramping
speed of the system was characterized. The microfluidic de-
vice was able to perform very fast cycling that consisted of a
rise in temperature from 22 to 95 °C and then holding at
95 °C for 900 s, followed by a 35-cycle PCR amplification
reaction.3 The control thermocouple was calibrated to actual
sample temperatures by performing a dummy run with a
aAuthor to whom correspondence should be addressed. Tel.: 626-395-4691.
FAX: 626-577-8442. Electronic mail: maltezos@caltech.edu.
FIG. 1. Color online A graph of temperature vs time showing the typical
temperature control temperature we can achieve. The actual temperature
control in the tube is better as the glass tube behaves as a low pass filter. We
calibrate the control temperature to a thermocouple in the liquid during a
dummy run.
APPLIED PHYSICS LETTERS 93, 243901 2008
0003-6951/2008/9324/243901/3/$23.00 © 2008 American Institute of Physics93, 243901-1
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thermocouple placed in the reaction chamber, which was
filled with PCR mastermix Fig. 1. A calibration between
the control temperature and the sample temperature was then
performed. We noticed little need for recalibration of the
system once this procedure was carried out. The schedule for
this experiment is summarized as follows: 50 °C for 1200 s
and 95 °C for 900 s; PCR 35 cycle: 94 °C for 15 s, 55 °C
for 20 s, 72 °C for 40 s.
A solution with 1000 base pair DNA molecules was run
through 35 cycles, and the amplified product was passed
through a gel using the gel electrophoresis technique to de-
termine whether or not the PCR run amplified the appropri-
ate DNA sample. A silica capillary tube was prepared with
the amplicon, and mineral oil was used to avoid evaporation
of the sample at the high denaturation temperature. When
single or double Peltier junctions were used on the copper
heat sink holding the capillary tube, 20 °C /s ramp rates
were measured, whereas the introduction of four parallel
Peltier junctions resulted in ramp rates of approximately
100 °C /s, measured in the amplicon mixture. Figure 2
shows a time-temperature plot in which heating rates of
above 100 °C /s and cooling rates of 90 °C /s were mea-
sured. In this system, the direction of current flow was re-
versed for heating and cooling of the primer/sample mixture.
The 0.32 mm capillary enabled the amplification of 0.4 l
DNA+primer samples. A 1000 base pair beta-actin DNA
mixture, as well as a DNA positive control, and a DNA lad-
der were tested in parallel by using agarose gel electrophore-
sis after the DNA sample was amplified. Figure 3 shows the
gel electrophoresis results indicating that the amplified prod-
uct did consist of 1000 base pair molecules.3 Furthermore,
once the DNA sample was cycled and gel electrophoresis
tests were completed, the same sample was used to obtain a
melting curve to determine the melting temperature of the
amplified DNA mixture. Figure 4 shows the resulting melt-
ing curve, indicating a very sharp melting temperature at
approximately 85 °C, indicative again of the amplification
of rather large DNA fragments. It is of interest to note that no
primer-dimer signals were detected from this melting curve,
indicating a uniform temperature within the amplification
system.8
We have described a simple and small PCR-on-a-chip
system to amplify DNA fragments. Recent work on PCR
technologies has focused on reducing the size of PCR sys-
tems. For on-chip PCR amplification, the amplicon is often
pumped over constant temperature heaters located in the path
of the fluidic channels. However, three important problems
arise when such microfluidic flow-controlled PCR systems
are to be used: 1 the flow rate of DNA molecules within a
channel depends on the distance between the molecule and
the side walls of the channel, 2 the PDMS commonly em-
ployed is porous in air and steam, and water evaporation at
high temperatures requires careful designs to avoid fluid loss
during the PCR amplification, and 3 the surface-to-volume
ratios of such fluidic systems are very large and not well
understood. The system described herein is based on cham-
bers that can be rapidly heated and cooled rather than fluid
flow based thermal cycling systems and on the use of Peltier
junctions. We show that modern Peltier junctions can be used
for rapid thermal control with heating and cooling rates of
100 °C /s—with temperature control accuracies of 0.1 °C.
The authors gratefully acknowledge financial support for
this research by grants from the National Science Foundation
Grant Nos. CHE-0515363 and DMR-0351848.
FIG. 3. Color online a Electrophoresis gel of the amplified product 2 l
PCR products were loaded into each lane. Lane 1: beta-actin amplified with
Roche Lightcycler. Lane 2: negative control with template. Lane 3: beta-
actin amplified with Caltech device. and b melting curve of the amplified
product on the Roche Lightcycler.
FIG. 4. Color online Maximum heating and cooling rates of a 400 nl
sample in our microfluidic thermal cycler.
FIG. 2. Color online An optical micrograph of our microfluidic PCR sys-
tem. The glass capillary tube is heated and cooled by the P and N Peltier
junctions attached to the copper jacket. Also visible to the right is the control
thermocouple.
243901-2 Maltezos et al. Appl. Phys. Lett. 93, 243901 2008
Downloaded 19 Dec 2008 to 131.215.225.137. Redistribution subject to AIP license or copyright; see http://apl.aip.org/apl/copyright.jsp
1K. B. Mullis, F. Ferre, and R. A. Gibbs, The Polymerase Chain Reaction
Birkhäuser, Boston, 1994.
2M. J. Adler, C. Coronel, E. Shelton, J. E. Seegmiller, and N. N. Dewji,
Proc. Natl. Acad. Sci. U.S.A. 88, 16 1991.
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243901-3 Maltezos et al. Appl. Phys. Lett. 93, 243901 2008
Downloaded 19 Dec 2008 to 131.215.225.137. Redistribution subject to AIP license or copyright; see http://apl.aip.org/apl/copyright.jsp


Microfluidic polymerase chain reaction

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Microfluidic polymerase chain reaction

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