Anal. Chem. 2005, 77, 658-666
Electrokinetically Synchronized Polymerase Chain Reaction Microchip Fabricated in Polycarbonate Jifeng Chen,† Musundi Wabuyele,‡ Hengwu Chen,§ Donald Patterson,† Mateusz Hupert,† Hamed Shadpour,† Dimitris Nikitopoulos,| and Steven A. Soper*,†
Department of Chemistry and Center for BioModular Multi-Scale Systems, and Department of Mechanical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6101, and Chemistry Department, Zhejiang University, Hangzhou, PR China 310028
This paper presents a novel method for DNA thermal amplification using the polymerase chain reaction (PCR) in an electrokinetically driven synchronized continuous flow PCR (EDS-CF-PCR) configuration carried out in a microfabricated polycarbonate (PC) chip. The synchronized format allowed patterning a shorter length microchannel for the PCR compared to nonsynchronized continuous flow formats, permitting the use of smaller applied voltages when the flow is driven electrically and also allowed flexibility in selecting the cycle number without having to change the microchip architecture. A home-built temperature control system was developed to precisely configure three isothermal zones on the chip for denaturing (95 °C), annealing (55 °C), and extension (72 °C) within a single-loop channel. DNA templates were introduced into the PCR reactor, which was filled with the PCR cocktail, by electrokinetic injection. The PCR cocktail consisted of low salt concentrations (KCl) to reduce the current in the EDS-CF-PCR device during cycling. To control the EOF in the PC microchannel to minimize dilution effects as the DNA “plug” was shuttled through the temperature zones, Polybrene was used as a dynamic coating, which resulted in reversal of the EOF. The products generated from 15, 27, 35, and 40 EDS-CFPCR amplification cycles were collected and analyzed using microchip electrophoresis with LIF detection for fragment sizing. The results showed that the EDS-CF-PCR format produced results similar to that of a conventional block thermal cycler with leveling effects observed for amplicon generation after ∼25 cycles. To the best of our knowledge, this is the first report of electrokinetically driven synchronized PCR performed on chip. The polymerase chain reaction (PCR) is a powerful tool for creating large numbers of copies of specific DNA fragments for both sequencing and genotyping applications.1-3 PCR has the * To whom correspondence should be addressed. E-mail:
[email protected]. † Department of Chemistry and Center for BioModular Microsystems, and Department of Mechanical Engineering, Louisiana State University. ‡ Oak Ridge National Laboratory. § Zhejiang University. | Department of Mechanical Engineering, Louisiana State University. (1) White, T. J.; Arnheim, N.; Erlich, H. A. Trends Genet. 1989, 5, 185-189. (2) Mullis, K. B.; Faloona, F. A. Methods Enzymol. 1987, 155, 335-350.
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potential to rapidly amplify even a single DNA molecule into billions of identical molecules, making it attractive for the analysis of low-abundant targets even in heterogeneous samples. The common devices used for PCR consist of metal blocks that are continually thermal cycled between the various temperatures required for the amplification process. The advantage of these “block” thermal cyclers is that they can simultaneously amplify 96-384 samples placed in titer wells that are sandwiched between the thermal block and cover plate. The disadvantage associated with this format is the poor thermal management produced by the need for heating/cooling large thermal masses and the slow thermal equilibrium between the block and the fluid contained within the titer well. The result is limitations on the speed of the amplification due to slow thermal equilibration. In addition, block thermal cyclers set limits on reducing the volume of a PCR reaction to ∼0.5 µL due to the need for manually pipetting reagents into the wells. Recently, several microfluidic PCR devices have been introduced4-11 to overcome some of the shortcomings associated with benchtop thermal cyclers. They are based on one of two different operational configurations; chamber-type devices or continuous flow devices. For devices with chamber-type configurations, microfabricated silicon- or glass-based reaction chambers are used to carry out the PCR on a conventional or integrated thermal controlling unit.4-7 These formats allow ultrasmall volumes of sample and reagents to be amplified using short cycling times in a confined chamber. Chamber-type devices also provide ways to integrate the PCR with other downstream processing steps on a single monolithic chip.4,7 As an alternative, continuous flow PCR (CF-PCR) microfluidic devices have been reported,8-11 which consist of a single channel that is continuously looped through different temperature zones (3) Vosberg, H. P. Hum. Genet. 1989, 83, 1-15. (4) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162. (5) Zhao, Z.; Cui, Z.; Cui, D.; Xia, S. Sens. Actuators, A 2003, A108, 162-167. (6) Cheng, J.; Shoffner, M. A.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 380-385. (7) Khandurina, J.; McKnight, T. E.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 2995-3000. (8) Kopp, M. U.; De Mello, A. J.; Manz, A. Science (Washington, D. C.) 1998, 280, 1046-1048. (9) Park, N.; Kim, S.; Hahn, J. H. Anal. Chem. 2003, 75, 6029-6033. (10) Obeid, P. J.; Christopoulos, T. K.; Crabtree, H. J.; Backhouse, C. J. Anal. Chem. 2003, 75, 288-295. (11) Obeid, P. J.; Christopoulos, T. K. Anal. Chim. Acta 2003, 494, 1-9. 10.1021/ac048758e CCC: $30.25
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to carry out DNA denaturing, annealing, and extension. The PCR cocktail and DNA template are pumped through the channel with the cycle number typically determined by the number of loops through each isothermal zone required for the PCR. CF-PCR formats have some particularly attractive advantages including better thermal management, since each temperature required for the PCR is at equilibrium prior to target amplification, and therefore, only small volumes of fluid need to be heated/cooled at each step significantly reducing amplification time, it can be operated in a segmented flow format for high-throughput applications, and it does not require valves, simplifying the fabrication process.12 CF-PCR chips have recently been shown to produce 500-bp amplicons (20 thermal cycles) from λ-DNA in as little as 1.5 (7.5 s/cycle) and 3.5 min (9.5 s/cycle) for a 1000-bp amplicon,13 a direct result of the reduced thermal equilibration time required with the CF-PCR format. To date, CF-PCR amplification has been conducted with hydrodynamic flow in a long channel with the number of thermal cycles fixed by the length of the channel. Disadvantages associated with this format includes the high back pressure induced by the long and narrow channel, which may cause leakage between adjacent channels; the hyperbolic-shaped flow profile, which may lead to serious dispersion of a sample band injected into a continuous flowing PCR cocktail; the fixed number of PCR cycles; and because PCR is frequently followed by capillary electrophoretic (CE) analysis of the DNA fragment produced by PCR, coupling pressure-driven systems with an electrokinetically driven CE system is not straightforward. Electrokinetically driven systems adapted to a CF-PCR format could potentially alleviate some limitations associated with pressure-driven formats, such as minimization of sample dispersion due to the plug flow geometry, ease of flow coupling to microchip electrophoresis platforms, elimination of the need for “off chip” pumps, and negligible pressure drops induced by movement of fluid. However, to shuttle a DNA sample electrophoretically (µep ) -3.75 × 10-4 cm2/V‚s)14 through a CF-PCR channel of 1.5-m length at a linear velocity of 0.1 cm/s (20 cycles, total cycling time 1500 s, 25 min) would require an applied voltage of 37.5 KV (E ) 250 V/cm). This voltage may result in transverse fields of several hundred thousand volts per centimeter between adjacent tracks in a spiral or serpentine configuration. This “leaky” field may induce electrical arcs between adjacent tracks providing a field-free region (i.e., no potential drop). Recently, researchers have shown that extended length separation columns can be produced within a small footprint by using a synchronized electrokinetic pumping format.15-17 In this technique, electrode reservoirs are placed at each corner of a single loop patterned on a substrate photolithographically. Once the sample plug is injected into the loop, a field is applied at two opposite corners of the loop. The section of the loop in which the field is (12) Chiou, J.; Matsudaira, P.; Sonin, A.; Ehrlich, D. Anal. Chem. 2001, 73, 20182021. (13) Hashimoto, M.; Chen, P.-C.; Mitchell, M. W.; Nikitopoulos, D. E.; Soper, S. A.; Murphy, M. C. Lab Chip. In press. (14) Stellwagen, N. C.; Gelfi, C.; Righetti, P. G. Biopolymers 1997, 42, 687-703. (15) Burggraf, N.; Manz, A.; Verpoorte, E.; Effenhauser, C. S.; Widmer, H. M.; Derooij, N. F. Sens. Actuators, B 1994, 20, 103-110. (16) Manz, A.; Bousse, L.; Chow, A.; Metha, T. B.; Kopf-Sill, A.; Parce, J. W. Fresenius J. Anal. Chem. 2001, 371, 195-201. (17) Zhao, J. G.; Hooker, T.; Jorgenson, J. W. J. Microcolumn Sep. 1999, 11, 431-437.
applied is synchronized with the plug position to allow cycling the sample through the single-loop channel. After securing the appropriate number of cycles through the loop, the sample plug can be easily ejected. Using this synchronized cyclic format for electrophoresis, high-efficiency separations have been generated on a channel that is only a few centimeters in length. In this study, we demonstrate the use of a microfabricated polymer chip for DNA amplification using electrokinetically driven synchronized pumping for a CF-PCR configuration (EDS-CF-PCR). The microchannel pattern for EDS-CF-PCR was fabricated on a polycarbonate (PC) substrate by micromachining to fabricate a mold insert and, then, replication of parts in PC using hot embossing. A temperature control system assembled in-house provided precise isothermal temperature zones for denaturing, annealing, and extending of the target DNA. The specific 500base pair target was amplified on chip and collected after 15, 27, 35, and 40 cycles. Due to the small volume of the PCR mix, polymer microchip electrophoresis with laser-induced fluorescence (LIF) was used for detection of PCR fragments for recognition and sizing. The effect of cycle number on the amount of PCR products obtained using EDS-CF-PCR was in agreement with DNA amplification obtained by conventional PCR. EXPERIMENTAL SECTION Microchip Design and Fabrication. The microchip layout (see Figure 1a) was designed using AutoCAD (Autodesk Inc., San Rafael, CA). The channel size for the PCR reactor was 100 µm in width, 70 µm in depth, and 7.9 cm long, producing a total volume for the reactor of 0.55 µL. Access channels, which were positioned at each corner of the reactor, possessed the same dimensions as the channels in the reactor. Reservoirs 1-4 (see Figure 1a) were designed to accommodate electrodes to apply voltages between points 1-3, and 2-4 for moving the DNA plug through the three temperature zones in a synchronized fashion. Reservoir 5 and 6 were used for electrokinetic sample loading. The autocad geometry was transferred to a brass plate by a micromilling machine (Kern Micro-und Feinwerktechnik GmbH & Co. KG, Murnau, Germany). The fabricated molding die is depicted in Figure 1b. The fluidic channels were hot embossed from the brass master in polycarbonate (MSC Industrial Supply, Melville, NY). The hot embossing system consisted of a PHI Precision Press model TS21-H-C (4A)-5 (City of Industry, CA). A vacuum chamber was installed into this press to remove air (pressure
