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Anal. Chem.

2003, 75, 288-295

Microfabricated Device for DNA and RNA


Amplification by Continuous-Flow Polymerase
Chain Reaction and Reverse
Transcription-Polymerase Chain Reaction with
Cycle Number Selection
Pierre J. Obeid,† Theodore K. Christopoulos,*,†,‡ H. John Crabtree,§ and Christopher J. Backhouse|

Department of Chemistry, University of Patras, Patras, Greece GR-26500, Institute of Chemical Engineering and
High-Temperature Chemical Processes, P.O. Box 1414, Patras, Greece GR-26500, Micralyne Inc., 1911 94th Street,
Edmonton, Alberta, T6N 1E6, Canada, and Department of Electrical and Computer Engineering, University of Alberta,
Edmonton, Alberta, T6G 2V4, Canada

We have developed a high-throughput microfabricated, monolithic microdevice that integrates continuous-flow RT


reusable glass chip for the functional integration of reverse and PCR with cycle number selection.
transcription (RT) and polymerase chain reaction (PCR)
Nucleic acid analysis techniques have a major impact in diverse
in a continuous-flow mode. The chip allows for selection
areas, such as molecular diagnosis of disease and assessment of
of the number of amplification cycles. A single micro-
therapy, environmental testing, food technology, agriculture, and
channel network was etched that defines four distinct
forensic science.1 The polymerase chain reaction (PCR)2,3 entails
zones, one for RT and three for PCR (denaturation,
the in vitro exponential enzymatic amplification of specific DNA
annealing, extension). The zone temperatures were con- sequences to levels that are several orders of magnitude higher
trolled by placing the chip over four heating blocks. than the starting material. RNA amplification is accomplished by
Samples and reagents for RT and PCR were pumped synthesizing a copy of complementary DNA, using reverse
continuously through appropriate access holes. Outlet transcriptase, and then performing PCR. DNA or RNA amplifica-
channels were etched after cycles 20, 25, 30, 35, and tion has become an essential step of most procedures used for
40 for product collection. The surface-to-volume ratio for nucleic acid detection, quantification, and sequencing. The Human
the PCR channel is 57 mm-1 and the channel depth is Genome Project including projects on various model organisms
55 µm, both of which allow very rapid heat transfer. As a have provided a vast amount of sequence data and therefore
result, we were able to collect PCR product after 30 initiated a new era of nucleic acid-based tests. High throughput
amplification cycles in only 6 min. Products were col- and automation of DNA/RNA analysis techniques, however, is
lected in 0.2-mL tubes and analyzed by agarose gel required in order to exploit the accumulated genetic information.
electrophoresis and ethidium bromide staining. We stud- In recent years, significant advances have emerged in the area of
ied DNA and RNA amplification as a function of cycle miniaturization of analytical instrumentation and development of
number. The effect of the number of the initial DNA and micro total analysis systems (µTAS).4-6 Key benefits of miniatur-
RNA input molecules was studied in the range of 2.5 × ization include high throughput, low consumption of sample and
106-1.6 × 108 and 6.2 × 106-2 × 108, respectively. reagents, portability, and the prospect of integration of all steps
Successful amplification of a single-copy gene (β-globin) * To whom correspondence should be addressed: (tel) (+30) 2 610 997130;
(fax) (+30) 2 610 997118; (e-mail) tkc@chemistry.upatras.gr.
from human genomic DNA was carried out. Furthermore, † University of Patras.

PCR was performed on three samples of DNA of different ‡ Institute of Chemical Engineering and High-Temperature Chemical Pro-

cesses.
lengths (each of 2-µL reaction volume) flowing simulta- § Micralyne Inc.

neously in the chip, and the products were collected after | University of Alberta.

various numbers of cycles. Reverse transcription was also (1) Christopoulos, T. K. Anal. Chem. 1999, 71, 425R-438R.
(2) Mullis, K. B.; Faloona, F. A. Methods Enzymol. 1987, 155, 335-350.
carried out on four RNA samples (0.7-µL reaction volume) (3) Christopoulos, T. K. Polymerase Chain Reaction and Other Amplification
flowing simultaneously in the chip, followed by PCR Systems. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; Wiley:
Chichester, 2000; pp 5159-5173.
amplification. Finally, we have demonstrated the concept
(4) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74,
of manually pumped injection and transport of the reac- 2623-2636.
tion mixture in continuous-flow PCR for the rapid genera- (5) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74,
2637-2652.
tion of amplification products with minimal instrumenta- (6) Kopp, M. U.; Crabtree, H. J.; Manz, A. Curr. Opin. Chem. Biol. 1997, 1,
tion. To our knowledge, this is the first report of a 410-419.

288 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003 10.1021/ac0260239 CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/12/2002
of an analytical procedure into a single device. The manufacturing flow-through devices. However, a significant limitation of this
of miniaturized and integrated analytical systems is based on the device is that the number of cycles is fixed by the channel layout
technologies developed in the microelectronics industry for at 20 cycles. Therefore, only a limited control of the reaction
production of integrated circuits. A major part of the research conditions is possible through changes of the flow rate and zone
activity in this area is directed toward biochemical analysis.7-9 temperatures.
Microfabricated devices for cell separation,10,11 electrophoresis,12,13 Despite the wide spectrum of applications of RNA amplification
DNA amplification,14-22 and DNA sequencing23,24 have been in research and molecular diagnosis, there is limited work on the
developed. coupling of reverse transcription (RT) and PCR on a single
Chip-based PCR systems reported so far have been based on microdevice. In a previous report, RT was performed in a
two configurations. In one configuration, the reaction mixture is stationary reaction mixture (50 µL) placed in a polypropylene tube
stationarysenclosed in a reaction chamber that is typically liner that was inserted in a silicon thermal cycled cavity.17
micromachined in Si or glass. The chamber is then placed on a Following one round of PCR, the sample was manually transferred
conventional thermocycling instrument for heating and cooling.14-16 to another chamber for a second round of amplification (nested
Alternatively, heaters and temperature sensors are integrated into PCR).
the device.17 Noncontact heating by infrared radiation has also In this work we report a microfabricated device for continuous-
been employed for fast thermocycling of microstructures.18 flow DNA and RNA amplification, which offers the following
Further developments in this basic configuration aim at the distinct advantages: (a) functional integration of reverse transcrip-
analysis of amplification products. This has been accomplished tion and polymerase chain reaction (RT-PCR) onto a single
by integrating optical windows in the silicon chamber allowing monolithic chip for direct RNA amplification and (b) cycle number
for real-time fluorometric monitoring of the reaction.17 There are selection at 20, 25, 30, 35, and 40 cycles. We first study the effect
also several reports on the combination of PCR with capillary of cycle number on the amount of amplification product. The
electrophoresis (CE).19-21 influence of the input DNA and RNA molecules on the amplified
More recently,22 a second configuration of chip-based PCR was product is also studied. Moreover, the high-throughput capability
reported that involves a continuous-flow amplification system in of continuous-flow PCR and RT-PCR is demonstrated, for the first
which the reaction mixture, instead of being stationary in a time, by running simultaneously multiple DNA samples of different
thermocycling chamber, is pumped continuously through the chip sizes followed by product collection at different cycle numbers.
and flows repeatedly through zones of different temperatures to Similarly, multiple small-volume RNA samples are subjected to
ensure denaturation, annealing, and extension. The system allows RT-PCR in a continuous-flow format. The amplification of a single-
for very rapid heat transfer and thermal cycling of the minute copy gene from human genomic DNA isolated from whole blood
fluidic element due to both the high surface-to-volume ratio and is performed successfully. Finally, it is shown that continuous-
the short linear thermal diffusion distance in the microfluidic flow PCR can be performed rapidly in this chip by a simple hand-
channels. This design advantage is best achieved by maintaining driven sample and reagent transport without the use of pumps.
the zones at preset constant temperatures. The continuous-flow
system is a true microfluidic device and is more suitable for high-
EXPERIMENTAL SECTION
throughput analysis and coupling with upstream and downstream
Microfabrication. The microfabricated chip used throughout
(7) Kricka, L. J. Clin. Chim. Acta 2001, 307, 219-223.
this study was fabricated at Micralyne (Edmonton, AB, Canada)
(8) Sanders, G. H. W.; Manz, A. Trends Anal. Chem. 2000, 19, 364-378.
(9) Burke, D. T.; Burns, M. A.; Mastrangelo, C. Genome Res. 1997, 7, 189- and is depicted in Figure 1. This device is composed of two
197. Corning 0211 borosilicate glass plates (each 40 × 45 × 0.55 mm)
(10) Cheng, J.; Sheldon, E. L.; Wu, L.; Uribe, A.; Gerrue, L. O.; Carrino, J.; Heller,
that were fusion-bonded together to form a closed structure. A
M. J.; O’Connell, J. P. Nat. Biotechnol. 1998, 16, 541-546.
(11) Wilding, P.; Kricka, L. J.; Cheng, J.; Hvichia, G. E.; Schoffner, M. A.; Fortina, continuous channel network was etched into the bottom plate by
P. Anal. Biochem. 1998, 257, 95-100. standard photolithographic and wet chemical (HF) etching
(12) Effenhauser, C. S.; Paulus, A.; Manz, A. Widmer, M. M. Anal. Chem. 1994,
techniques. The channels were 194 µm wide (at the top) and 55
66, 2949-2953.
(13) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11348- µm deep for the reverse transcription section of the device, and
11352. 120 µm wide and 55 µm deep for the PCR section (Figure 1).
(14) Wilding, P.; Schoffner, M. A.; Kricka, L. Clin. Chem. 1994, 40, 1815-1818.
(15) Schoffner, M. A.; Cheng, J.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic
The cover plate contains access holes (400-µm diameter, drilled
Acids Res. 1996, 24, 375-379. ultrasonically) that were used for sample injection and product
(16) Cheng, J.; Schoffner, M. A.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic collection. These were aligned with the inlet and outlet channels
Acids Res. 1996, 24, 380-385.
(17) Northrup, M. A.; Benett, B.; Hadley, D.; Landre, P.; Lehew, S.; Richards, J.;
on the bottom plate. After cleaning, the cover and bottom plates
Stratton, P. Anal. Chem. 1998, 70, 918-922. were fusion bonded to form the sealed microfluidic device.
(18) Giordano, B. C.; Ferrance, J.; Swedberg, S.; Huhmer, A. F. R.; Landers, J. Both the RT and PCR sections contain two inlets each and
P. Anal. Biochem. 2001, 291, 124-132.
(19) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.;
are marked on the chip by RT1, RT2, PCR1, and PCR2. The PCR
Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. channel incorporates 40 identical cycles that pass over three
(20) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. temperature-controlled zones, to perform DNA denaturation,
S.; Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176.
(21) Khandurina, J.; McKnight, T. E.; Jacobson, S. C.; Waters, L. C.; Foote, R. primer annealing, and enzymatic extension steps of amplification
S.; Ramsey, J. M. Anal. Chem. 2000, 72, 2995-3000. at time ratios of 4:4:9, respectively. The channel allows for a 3-fold
(22) Kopp, M. U.; deMello, A. J.; Manz, A. Science 1998, 280, 1046-1048. extended denaturation step prior to entering the first cycle. Outlet
(23) Liu, S.; Shi, Y.; Ja, W. W.; Mathies, R. A. Anal. Chem. 1999, 71, 566-573.
(24) Backhouse, C.; Caamano, M.; Oaks, F.; Nordman, E.; Carrillo, A.; Johnson, channels were designed after cycles 20, 25, 30, 35, and 40 for
B.; Bay, S. Electrophoresis 2000, 21, 150-156. product collection. The capacity of the PCR channel (from cycles
Analytical Chemistry, Vol. 75, No. 2, January 15, 2003 289
Figure 1. (A) Schematic diagram of the microfabricated device for continuous-flow PCR and RT-PCR with cycle number selection. There are
two inlets for PCR (PCR1 and -2), two inlets for RT (RT1 and -2), and five outlets for product collection at 20, 25, 30, 35, and 40 cycles. The
chip is placed over four heating blocks, a-d, with appropriate temperatures for denaturation, extension, and annealing, respectively. I, intersection
between the RT and the PCR channels. (B) A simple holding apparatus for the chip. This configuration provides quick leak-proof connections
between capillaries and the access holes of the chip (inlets and outlets) by using silicone disks. It also facilitates the accurate positioning of the
chip over the heated blocks and eliminates the direct gluing of the capillaries to the access holes. (C) Diagram of the etched channels. For the
RT channel, the radial depth (D) is 55 µm and the width (W) is 84 µm. This yields a 194-µm channel width at the top. The PCR channel has a
radial depth of 55 µm and width of 10 µm. Hence, the channel width at the top is 120 µm.

1 to 40) is 15.3 µL. The RT channel has a capacity of 5 µL and is Chip Peripherals. Fluids were pumped through the chip by
also in contact with its own temperature-controlled zone. hydrostatic pressure. Two PC-controlled precision syringe pumps
Assembly of the Chip. To introduce samples through the chip (model 50300, Kloehn, Las Vegas, NV), each with a syringe
and collect amplification products, we have constructed a simple capacity of 25 µL, were used for sample delivery. A 2-µm in-line
holding apparatus that facilitates the connection between capillary microfilter (model M-532, Upchurch Scientific, Oak Harbor, WA)
tubes and the inlets and outlets of the device (Figure 1). A piece was connected between the chip and the pumps.
of clear Plexiglas (80 × 55 × 5 mm) was used, which contained Temperature control was achieved by placing the chip as-
0.5-mm drilled holes that matched exactly those of the micro- sembly on top of a suitable heating base constructed in-house by
device. Capillary tubes (3 cm long, 375-µm o.d., 100-µm i.d., using four copper blocks, 80 × 20 × 10 mm each. Inside each
Polymicro Technologies, Phoenix, AZ) were cut and glued with block, and along its length, we placed a heating cartridge (Omega,
epoxy to the Plexiglas holes such that 1 mm of their ends CSS-03365/110) and a temperature sensor (Omega, 1PT 100
protruded out from the holes. To provide leak-proof connections, kn2528). The block temperatures were controlled by using four
disks (2-4-mm diameter and 0.8 mm thick) were prepared from digital PID temperature controllers (Jumo iTron microprocessor
silicone glue and were perforated with the protruding ends of the controller, model 702040 from Enercorp, Islington, ON, Canada)
capillaries. For assembly, the capillary ends carrying the disks housed in a single unit containing a power supply and electric
were inserted carefully into the access holes of the device and circuits for the heating cartridges and sensors. The blocks were
the apparatus was held tight with two screws. The free ends of then positioned, with a 5-mm spacing between each other, on a
the capillaries, therefore, become the inlets and outlets for the nonthermally conducting base (85 × 85 × 10 mm) and their
channels. There are four inlet and five outlet capillaries. Cycle surfaces were evened out with respect to each other. This ensured
selection was accomplished by blocking the ends of all outlet that the chip made a good contact throughout, when it was placed
capillaries with Teflon caps except of the one corresponding to on top of the heating blocks. To minimize heat transfer between
the selected cycle number. Sample collection was accomplished the blocks, a small fan was placed in front of them, allowing air
by simply placing an inverted plastic 0.2-mL tube on top of the to pass through the spacing. Temperature control accuracy was
uncapped capillary. The caps used for blocking the free ends of within (0.1 °C.
unutilized capillaries were prepared by using a 5-mm-long Teflon Channel Surface Passivation. Although some work has been
tubing (∼380-µm i.d.). One end of the tubing was plugged with done to ascertain the steps necessary to prevent poisoning of the
epoxy glue while the open end was tightly fitted over the capillary. PCR by contaminants from the microfabrication process,25 we have
Prior to the first PCR of the day, the whole chip was filled with developed a more extensive method that effectively prevents
water. Thus, the unused (capped) outlet channels prior and after contamination in our devices. The walls of the microchannels were
the selected cycle number contain water. This ensures that the passivated by silanization as follows. The channels were cleaned
reaction mixture exits only from the uncapped capillary. No losses (25) Taylor, T. B.; Harvey, S. E.; Albin, M.; Lebak, L.; Ning, Y.; Mowat, I.;
of sample into the capped outlets occur. Scluerlein, T.; Principe, E. Biomed. Microdevices 1998, 1, 65-70.

290 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003


thoroughly with filtered double-distilled water to remove the °C. At the end of this period, the RT heating block temperature
presence of chromium and other contaminants due to the micro- was raised to 95 °C for 5 min to inactivate the reverse tran-
fabrication process. The water was then removed by washing with scriptase. Subsequently, both the RT mixture and the PCR
acetone and the channel dried by applying vacuum. The channel reagents were pumped continuously and simultaneously with a
was filled with a filtered (0.45-µm filter) 5% dichlorodimethylsilane ratio of volumetric flow rates set at 1:9, respectively. Therefore, a
(DDMS) solution in chloroform. The chip along with a small 10-µL total volume of RT-PCR contains 1 µL of the RT product.
beaker containing 1 mL of the DDMS solution was placed in a The RT-PCR product was collected at the desired cycle number.
desiccator. Vacuum was applied until the solution in the beaker It should be noted that, after mixing of the two solutions, the final
started boiling. The vacuum inlet was then shut, and the desiccator concentrations of the PCR components are as above. In the second
was left for 1 h. During this period, the DDMS solution in the RT-PCR protocol (protocol B), the RT mixture was pumped
chip and beaker evaporates completely and the vapors form a thin continuously into the RT channel at a flow rate of 2 nL/s. Under
film on the walls of the channels. The chip was removed and these conditions, 30 min is required for the mixture to reach the
washed twice with 25 µL of chloroform, twice with 25 µL of filtered intersection with the PCR channel. At this point, the pump
acetone, and twice with 25 µL of filtered water. Washings were delivering the PCR reagents is activated at 18 nL/s to carry out
performed by applying vacuum. the amplification. Between samples, the RT channel and the PCR
DNA and RNA Templates. The DNA template was a 0.23- channel are washed once with a volume of water eaqual to the
kbp fragment synthesized by amplifying the prostate-specific sample volume.
antigen (PSA) mRNA as described elsewhere.26 The RNA template Analysis of Amplification Products. PCR and RT-PCR
was a 1-kb fragment synthesized by in vitro transcription of PSA products were analyzed by agarose gel (2%) electrophoresis and
cDNA. The primers used for continuous-flow PCR and RT-PCR ethidium bromide staining. DNA markers were from a φX174
of these templates are as in ref 26. The upstream and downstream HaeIII digest with fragment sizes of 1353, 1078, 872, 603, 310,
primers used for PCR amplification of the human β-globin gene 281, 271, 234, 194, 118, and 72 bp (MBI, Fermentas). The products
from genomic DNA correspond to sequences 62 253-62 272 were quantitated by densitometry of pictures taken with a Kodak
(exon 1) and 62 468-62 487 (exon 2) of the gene and generate a DC120 digital camera while the gels were under UV illumination.
0.23-kbp product. The plasmid used as a template was a 4-kbp The markers were used as standards for construction of calibration
GFP control vector, and the primers were the T7 promoter and curve.
terminator primers (plasmid from kit Catalog No. 3186148; primers
from kit Catalog No. 3186237; Roche Applied Sci., Mannheim, RESULTS AND DISCUSSION
Germany). This generates a 1.02-kbp PCR product. The 4 × 4.5 cm RT-PCR chip (Figure 1) enables the functional
Continuous-Flow PCR. The PCR mixture contained 10 integration of reverse transcription and polymerase chain reaction
mmol/L Tris-HCl, pH 9.0, 2.5 mmol/L MgCl2, 50 mmol/L KCl, 1 using a single continuous channel 343.2 cm long. The lengths of
mL/L Triton X-100, 1 mL/L Tween 20, 0.2 g/L bovine serum channel segments corresponding to RT and PCR (for 40 cycles)
albumin (BSA; New England BioLabs, MA), 2.8 µmol/L poly- are 54.4 and 288.8 cm, respectively, covering most of the chip. In
(vinylpyrrolidone) (MW 44 000), 0.2 mmol/L each of the dNTPs the design process, the needs for compact device design,
(MBI, Fermentas, Lithuania), 0.5 µmol/L each of the upstream minimum linear thermal diffusion distance (e.g., minimum etch
and downstream primers, 0.5 unit/µL thermostable DNA poly- depth) for a given channel volume, and higher cycle count and
merase (HT Biotechnology, Cambridge, England), and DNA channel volumes were balanced with microfabrication yield
template. The heating block temperatures were set at 95 °C for challenges associated with densely packed channel meanders and
denaturation, 58 °C for primer annealing, and 72 °C for extension. having only one device per substrate if the design was not
Between samples, the PCR channel was washed with a volume sufficiently compact. Microfabrication of this chip required that
of water equal to the sample volume. particular care be paid to reduce surface damage to the glass
Continuous-Flow RT-PCR. The reverse transcription mixture during subsequent processing. Careful handling minimized the
contained 50 mmol/L Tris-HCl, pH 8.3, 8 mmol/L MgCl2, 30 occurrence of significant channel defects upon etching. Microchips
mmol/L KCl, 10 mmol/L dithiothreitol, 0.2 g/L BSA, 2.8 µmol/L with smaller active areas are considerably less affected by
poly(vinylpyrrolidone), 0.5 µmol/L downstream primer, 0.5 mmol/L occasional scratches or other surface damage. The 4 × 4.5 cm,
each of the dNTP, 2 units/µL human placental ribonuclease 40-cycle design used afforded relatively shallow channels (55 µm),
inhibitor (HT Biotechnology), 10 units/µL reverse transcriptase sufficient volume (5.0 and 15.5 µL for the RT and PCR sections,
(M-MuLV from Finzyme), and RNA template. The RNA template respectively), yet well-spaced channels and four devices per 4-in.
and the primer were first heated at 70 °C for 5 min, to reduce substrate to enhance yield. The surface-to-volume ratio for the
any secondary structures of the RNA, and then cooled on ice. PCR channel is 57 mm-1 as opposed to a surface-to-volume ratio
Afterward the rest of the components were added and the mixture on the order of 1 mm-1 and a diffusion distance of 1-2 mm for a
was introduced into the RT inlet of the chip. typical 25-µL macroscale PCR. The length and the volume for each
Two protocols were used for performing RT-PCR. In one PCR cycle are 7.2 cm and 0.38 µL, respectively. Reaction mixtures
protocol (protocol A), the RT reaction mixture was pumped into for RT and PCR are introduced into the chip through appropriate
the RT zone of the chip at a fast flow rate until it reached the access holes, and the amplification products are collected from
channel intersection with the PCR reagents. At this point, the flow various outlets, thus allowing for cycle number selection. For
was stopped and the RT was allowed to incubate for 30 min at 42 example, at a flow rate of 21 nL/s, the observed cycling time is
13 s/cycle. Also, at this flow rate, product collection starts at 5, 6,
(26) Verhaegen, M.; Christopoulos, T. K. Anal. Chem. 1998, 70, 4120-4125. 7, 8, and 9 min for cycles 20, 25, 30, 35, and 40, respectively. The
Analytical Chemistry, Vol. 75, No. 2, January 15, 2003 291
Figure 2. (Left panel) Effect of the cycle number on the concentration of amplification product in continuous-flow PCR with on-chip cycle
selection. The input DNA was 107 molecules. (Right panel) Study of the concentration of the amplification product as a function of the input DNA
molecules. All products were collected at 30 cycles. The 0.23-kbp PSA DNA template was used. M, DNA markers. N, negative (no DNA template).

respective times for completion of the collection of a 10-µL product The reproducibility of continuous-flow PCR was assessed by
are 13, 14, 15, 16, and 17 min. running four identical mixtures containing 107 DNA template
Sample introduction into the chip as well as product collection molecules. The products were collected at 30 cycles, analyzed by
is accomplished through capillaries connected to the access holes. agarose gel electrophoresis, and quantified densitometrically. The
Epoxy glue is most commonly used to connect microfluidic calculated CV was 5.7% (n ) 4).
devices with capillary tubes. However, we have found it to be both To investigate the capability of the chip for high-throughput
time-consuming and often unsuccessful as the epoxy seeps around PCR, three samples, containing DNA templates of different sizes,
the capillary into the access holes, clogging the channel, and were run simultaneously on the chip by continuous-flow and the
making the chip unusable. Even if a successful connection is made products were collected at different cycle numbers. Sample 1
with epoxy glue, it is still troublesome to remove the glued contained 107 molecules of a 0.23-kbp DNA template (PSA), and
capillaries for cleaning the chip. The simple apparatus described the amplification product was collected at 25 cycles. Sample 2
in the Experimental Section and shown in Figure 1b ensures both contained 109 molecules of a 4-kbp supercoiled plasmid, and the
an easy connection (within minutes) of the capillaries to the access product was collected at 30 cycles. Sample 3 also contained 107
holes and convenient disassembly of the chip for cleaning. molecules of the 0.23-kbp template (PSA) and was collected at 35
The effect of the number of PCR cycles on the amount of cycles. The PCR volume was only 2 µL. The samples along with
amplification product was studied by using 107 molecules of DNA their appropriate primers were introduced at 5 nL/s from the
template (0.23 kbp of PSA DNA) in 10 µL of reaction mixture. PCR1 inlet. The rest of the PCR components were introduced at
The products were collected at 20, 25, 30, 35, and 40 cycles, and 5 nL/s from the PCR2 inlet (total flow rate of 10 nL/s). A 2-µL
the flow rate was set at 21 nL/s. A negative control, containing water plug was used as a wash between samples. A small air
no DNA template, was also subjected to PCR for 40 cycles to bubble (0.4 µL) was introduced between the sample and the wash.
ensure the absence of contamination. An electropherogram of the It was observed that the length of the bubble remained constant
products and a plot of product concentration as a function of the as the solutions were flowing through the chip. When a wash was
number of cycles are presented in Figure 2. In all cases, a single encountered, the pump delivering the PCR mix was stopped, and
band was observed at the expected size (0.23 kbp). Initially the the flow rate of the other pump was increased accordingly in order
amplification is exponential, but at high cycle numbers the plateau to maintain the 10 nL/s flow rate. After the wash was complete,
phase of PCR was reached. the flow rate was decreased back to 5 nL/s and the PCR mix pump
The dependence of the amount of amplification product on the was activated for the next sample. The whole process from
input DNA template molecules was studied in the range of 2.5 × injection of samples to the collection of products after amplification
106-160 × 106 molecules (10-µL reaction) at a constant number took 35 min to be completed. The products (2 µL) were subjected
of 30 cycles and a flow rate of 21 nL/s. The results are presented to agarose gel electrophoresis and the results are presented in
in Figure 2. We observe a linear relationship between the Figure 3. The three samples are amplified independently, without
concentration of accumulated product and the number of input any evidence of cross-contamination. Therefore, the chip offers
DNA molecules. Nonspecific amplification products due either to the advantage of multiple sample processing.
mispriming or to primer dimers were not observed, even at a high The amplification of a single-copy gene (β-globin) from human
number of template molecules. genomic DNA was also tested by using continuous-flow PCR on
292 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
Figure 3. (a) Schematic diagram of the PCR channel of the chip containing reaction mixtures (2 µL) from three samples (black segments) run
simultaneously. Intervening washes (2 µL) are shown as gray segments. A small air bubble (0.4 µL) is introduced between sample and wash.
The sample (with its primers) and the wash are introduced through PCR inlet 1. PCR reagents (enzyme, dNTPs, buffer) are introduced through
PCR inlet 2. (b) Agarose gel (2%) electrophoresis of the amplification products (2 µL) from three continuous-flow PCRs run simultaneously. M,
DNA markers. S1, sample 1 contains 107 DNA molecules (PSA DNA, 0.23 kbp) and the product is collected at 25 cycles. S2, sample 2 contains
109 supercoliled plasmid DNA molecules and the product is collected at 30 cycles. S3, sample 3 contains 107 DNA molecules (PSA DNA, 0.23
kbp) and the product is collected at 35 cycles. (c) Agarose gel (2%) electrophoresis of the amplification product from PCR of 2 (lane 1) and 4
µL (lane 2) of the β-globin gene from human genomic DNA isolated from whole blood. M, DNA markers. N, negative (no DNA template).

the chip. Genomic DNA was isolated from 200 µL of whole blood 106 molecules. Products were collected at 30 cycles. The results
using the QIAamp DNA blood mini kit (Qiagen, Germany). are presented in Figure 4. The amount of product is linearly
Aliquots of 2 and 4 µL were used for PCR in a total reaction volume related to the input RNA molecules. However, a plateau is reached
of 10 µL and a flow rate of 10 nL/s. Products were collected at 40 at a high number of RNA molecules because of saturation of
cycles and subjected to agarose gel electrophoresis (Figure 3c). reverse transcription and PCR.
A single band at the expected size (0.23 kbp) was observed. We performed various experiments to demonstrate high-
RNA amplification by RT-PCR is most often performed as a throughput RT-PCR on the chip. Four RT reaction mixtures, 0.7
two-step reaction. First, cDNA is synthesized by reverse transcrip- µL each, were pumped in a continuous-flow manner (protocol B)
tion. Then, the cDNA is mixed with the PCR reagents for into the RT channel at a flow rate of 2.6 nL/s. A 0.7-µL water plug
amplification. The difficulty in integrating the two processes on a was used as a wash between samples. Upon reaching the
single chip arises from the fact that the components of the RT intersection of RT and PCR channels, the solution was mixed with
mixture interfere with PCR. The Mg2+ concentration of the RT the PCR reagents that were flowing at 21 nL/s. When a water
mixture is 4 times higher than the optimum for PCR. Also, the plug was encountered, the PCR reagent pump was stopped,
reverse transcriptase enzyme inhibits PCR27 and must be inacti- allowing the RT pump to introduce the wash. Once the next RT
vated at high temperature (95 °C) prior to amplification. As a sample reached the intersection, the PCR reagent pump was
consequence, the RT mixture should constitute about 10% of the reactivated. The products (6.3 µL) were collected after 30 PCR
total PCR volume. This was overcome by using two pumps cycles and then were subjected to agarose electrophoresis. The
simultaneously, for RT and PCR, with flow rates at a ratio of 1:9. results are shown in Figure 5. Samples 1 and 3 contain 4.2 × 107
We studied the influence of the cycle number on the concen- RNA molecules and samples 2 and 4 contained 2.8 × 106 RNA
tration of amplification product in flow-through RT-PCR (protocol molecules. We observe (Figure 5b) clear characteristic bands for
A) by using a reverse transcription mixture containing 108 RNA each sample.
template molecules (1 kb)/µL. The total volume of RT-PCR was It is well known that a major problem of PCR and RT-PCR is
10 µL and contained 1 µL of the RT product (cDNA). A negative that of false positives arising from the contamination of the
control (with no RNA template) was also included. Figure 4 reaction mixture from previously amplified sequences. In continu-
presents an electropherogram of the amplified products and a ous-flow PCR and RT-PCR, the same reaction vessel (chip) is used
graph of the product concentration as a function of the number for amplification of various samples containing a wide range of
of cycles. The product increases exponentially until the plateau DNA and RNA molecules. As a consequence, we were concerned
phase of PCR is reached. A single band at the expected size was that the chip might become a source of contamination, i.e., that
observed in all cases. Nonspecific amplification products were not amplification products might remain in the chip and be carried
observed. over to the next sample. To examine this, we included a negative
The effect of input RNA template molecules on the amount of control after a series of samples with high DNA/RNA template
RT-PCR product was studied in the range of 6.25 × 106-200 × numbers. In all cases, no contamination was observed. Therefore,
(27) Chandler, D. P.; Wagnon, C. A.; Bolton, H. Appl. Environ. Microbiol. 1998, washing the chip with an equal volume of water after each sample
64, 669-677. is sufficient to avoid carryover. Generally, contamination in PCR
Analytical Chemistry, Vol. 75, No. 2, January 15, 2003 293
Figure 4. (Left panel) Effect of the cycle number on the concentration of amplification product in RT-PCR. The input RNA (1 kb) was 108
molecules. The RT mixture was mixed with the PCR mixture at a flow rate ratio of 1:9. (Right panel:) Study of the concentration of the amplification
product as a function of the input RNA molecules. All products were collected at 30 cycles. M, DNA markers. N, negative (no RNA template in
the RT mixture).

Figure 5. (a) Schematic diagram of the RT channel of the chip containing reverse transcription mixtures (0.7 µL) from four samples (black
segments) run simultaneously. Intervening washes (0.7 µL) are shown as gray segments. A small air bubble is introduced between sample and
wash. Upon reaching the intersection of RT and PCR channels (indicated by an asterisk), the RT solution is mixed with the PCR reagents. (b)
Agarose gel (2%) electrophoresis of the amplification products (6.3 µL) from four continuous-flow RT-PCRs run simultaneously. M, DNA markers.
S1, S3, samples 1 and 3 containing 4.2 × 107 RNA molecules. S2, S4, samples 2 and 4 containing 2.8 × 106 RNA molecules. The products were
collected at 30 cycles. (c) Hand-driven continuous-flow PCR. Electropherogram including DNA markers (M), a negative control (N), and the
product (10 µL) of a PCR mixture containing 108 input DNA molecules. (d) Organic material clogging a channel of the chip after prolonged
usage. (e) Burnt organic material after heating the chip at 300 °C. (f) Cleaned channels after treating the chip with concentrated HNO3. Pictures
in (d-f) were taken by using the Zeiss Axioscope 2 equipped with a Sony video camera model SSC-DC58AP.

depends on the amount of accumulated product. A more sensitive initial silanization. However, channel clogging is observed after
detection system requires less amplification product, thereby two weeks of continuous daily usage. The clogs always occur at
decreasing the possibility of contamination and enabling the use the high-temperature zone (95 °C) and are attributed to the
of lower ranges of starting DNA/RNA copies (or lower number accumulation of denatured protein. The blockage most frequently
of cycles). occurs during either the extended denaturation step or during
An advantage of the flow-through chip for PCR and RT-PCR is the first few PCR cycles. Perhaps the initial rapid rise of
that it can be reused. No special treatment is required after the temperature in these regions contributes to the deposition to the
294 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
channel wall. Figure 5d depicts a clogged channel wall obstructing oil was added over the PCR mixture to prevent evaporation during
the flow. To eliminate the problem, we have developed a simple cycling. Following RT-PCR, an aliquot of the mixture was
procedure for removing the clogs effectively. The chip is placed transferred manually to another tube for a second PCR (nested
on a ceramic base into a furnace (Thermolyne, type 48000), and PCR). On the contrary, in the present work, continuous-flow RT
the temperature is raised slowly (within 1 h) to 300 °C. This and PCR are truly integrated for the first time on a single
temperature is maintained for 1 h, and the furnace is turned off. monolithic device. No intervention is required between the RT
The chip is removed when ambient temperature is reached. This and PCR steps due to automated introduction and mixing of
process burns the organic material inside the channel. A clogged samples and reagents. Considerably faster cycling times are
chip, after baking, is shown in Figure 5e. The heating process achieved. For instance, at a typical flow rate of 21 nL/s, the cycling
does not damage the chip. Subsequently, 65% HNO3 is aspirated time is 13 s/cycle compared to 118 (“normal” protocol) and 70
into the channel by applying negative pressure to remove the s/cycle (“fast” protocol) reported in ref 17. No mineral oil is
burnt organic material. The acid-filled chip is left overnight at room required for PCR. Moreover, we have demonstrated that RT can
temperature and then washed thoroughly with chloroform. Figure be performed in final reaction volumes as low as 0.7 µL. Several
5f shows a chip section after the cleaning process. The clean chip RNA samples can be introduced into the chip in a continuous-
was silanized before use. flow mode for high-throughput analysis. Additionally, RT may
Finally, we investigated performing PCR in the flow-through precede PCR, or PCR can be chosen alone and to different
microdevice by a simple hand-driven injection and transport of numbers of cycles. This is a significant advantage because it allows
the reaction mixture, without using any pumps. The reaction control of the amount of amplification product depending on the
mixture (10 µL) was aspirated into a 0.4-mm (i.d.) Teflon tubing abundance of starting RNA or DNA copies in the sample. It was
connected to a 1-mL syringe containing water. PCR was then demonstrated that multiple samples containing DNA sequences
performed by applying moderate hand pressure to the syringe of different sizes can be run simultaneously on the chip and the
and collecting the amplification products at the desired cycle. products can be collected at different cycle numbers. Because it
Figure 5c presents an electropherogram of the products from two, is a continuous-flow system, the sample volume can be varied in
hand-driven, continuous-flow PCRs including a negative control order to optimize amplification.
and a positive containing 108 DNA template molecules. Products Throughout this work, the amplification products were ana-
were collected at 30 cycles. The reactions were completed in 6 lyzed by electrophoresis and ethidium bromide staining. It is
min (including injection and collection of 10 µL). This is 2.5 times conceivable that the chip may be coupled in-line with other
faster than injection and collection by pump-driven PCR (21 nL/ separation and detection systems such as capillary electrophoresis
s) for 30 cycles, which requires a total time of 15 min. Despite with laser-induced fluorescence detection.
the possible inconsistency of the pressure in the hand-driven flow,
a clear band is observed at the expected size. We have thus ACKNOWLEDGMENT
This work was supported by a grant to T.K.C. from the Institute
demonstrated a simple and robust system for rapid PCR, and we
of Chemical Engineering and High Temperature Chemical Pro-
envisage future implementations that are based on a flow-through
cesses (ICE/HT), Patras, Greece, and the General Secreteriat of
chip or a cartridge and that are operated by hand-driven injection
Research and Technology, Greece. We gratefully acknowledge
and transport of the reaction mixture without pumps. This system
the support of the Natural Sciences and Engineering Research
may be particularly useful for the rapid generation of amplified
Council of Canada to C.J.B.. We thank Martin Kopp and Andreas
DNA in the molecular biology research laboratory. Furthermore,
Manz for helpful discussions. We also acknowledge the financial
it could be used in field testing, especially for detection of the
support of Micralyne Inc.
presence of a specific target sequence.
In a previous report, RT and PCR were performed on a
microdevice.17 The RNA sample and the reagents, in a final volume Received for review August 5, 2002. Accepted November
of 50 µL, were placed in a polypropylene tube liner that was 11, 2002.
inserted into a thermally cycled silicon cavity. A layer of mineral AC0260239

Analytical Chemistry, Vol. 75, No. 2, January 15, 2003 295