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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, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single micro- channel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were con- trolled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and

40 for product collection. The surface-to-volume ratio for

the PCR channel is 57 mm -1 and the channel depth is

55 µm, both of which allow very rapid heat transfer. As a

result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were col- lected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We stud- ied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 × 10 6 -1.6 × 10 8 and 6.2 × 10 6 -2 × 10 8 , respectively. Successful amplification of a single-copy gene ( -globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-µL reaction volume) flowing simulta- neously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-µL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reac- tion mixture in continuous-flow PCR for the rapid genera- tion of amplification products with minimal instrumenta- tion. To our knowledge, this is the first report of a

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

monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.

Nucleic acid analysis techniques have a major impact in diverse areas, such as molecular diagnosis of disease and assessment of therapy, environmental testing, food technology, agriculture, and forensic science. 1 The polymerase chain reaction (PCR) 2,3 entails the in vitro exponential enzymatic amplification of specific DNA sequences to levels that are several orders of magnitude higher than the starting material. RNA amplification is accomplished by synthesizing a copy of complementary DNA, using reverse transcriptase, and then performing PCR. DNA or RNA amplifica- tion has become an essential step of most procedures used for nucleic acid detection, quantification, and sequencing. The Human Genome Project including projects on various model organisms have provided a vast amount of sequence data and therefore initiated a new era of nucleic acid-based tests. High throughput and automation of DNA/RNA analysis techniques, however, is required in order to exploit the accumulated genetic information. In recent years, significant advances have emerged in the area of miniaturization of analytical instrumentation and development of micro total analysis systems (µTAS). 4-6 Key benefits of miniatur- ization include high throughput, low consumption of sample and reagents, portability, and the prospect of integration of all steps

* To whom correspondence should be addressed: (tel) (+30) 2 610 997130; (fax) (+30) 2 610 997118; (e-mail) tkc@chemistry.upatras.gr. University of Patras. Institute of Chemical Engineering and High-Temperature Chemical Pro- cesses. § Micralyne Inc. | University of Alberta. (1) Christopoulos, T. K. Anal. Chem. 1999, 71, 425R-438R. (2) Mullis, K. B.; Faloona, F. A. Methods Enzymol. 1987, 155, 335-350. (3) Christopoulos, T. K. Polymerase Chain Reaction and Other Amplification Systems. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; Wiley:

Chichester, 2000; pp 5159-5173. (4) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74,

2623-2636.

(5) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74,

2637-2652.

(6) Kopp, M. U.; Crabtree, H. J.; Manz, A. Curr. Opin. Chem. Biol. 1997, 1,

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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 of miniaturized and integrated analytical systems is based on the technologies developed in the microelectronics industry for production of integrated circuits. A major part of the research activity in this area is directed toward biochemical analysis. 7-9 Microfabricated devices for cell separation, 10,11 electrophoresis, 12,13 DNA amplification, 14-22 and DNA sequencing 23,24 have been developed. Chip-based PCR systems reported so far have been based on two configurations. In one configuration, the reaction mixture is stationarysenclosed in a reaction chamber that is typically micromachined in Si or glass. The chamber is then placed on a conventional thermocycling instrument for heating and cooling. 14-16 Alternatively, heaters and temperature sensors are integrated into the device. 17 Noncontact heating by infrared radiation has also been employed for fast thermocycling of microstructures. 18 Further developments in this basic configuration aim at the analysis of amplification products. This has been accomplished by integrating optical windows in the silicon chamber allowing for real-time fluorometric monitoring of the reaction. 17 There are also several reports on the combination of PCR with capillary electrophoresis (CE). 19-21 More recently, 22 a second configuration of chip-based PCR was reported that involves a continuous-flow amplification system in which the reaction mixture, instead of being stationary in a thermocycling chamber, is pumped continuously through the chip and flows repeatedly through zones of different temperatures to ensure denaturation, annealing, and extension. The system allows for very rapid heat transfer and thermal cycling of the minute fluidic element due to both the high surface-to-volume ratio and the short linear thermal diffusion distance in the microfluidic channels. This design advantage is best achieved by maintaining the zones at preset constant temperatures. The continuous-flow system is a true microfluidic device and is more suitable for high- throughput analysis and coupling with upstream and downstream

(7) Kricka, L. J. Clin. Chim. Acta 2001, 307, 219-223. (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-

197.

(10) Cheng, J.; Sheldon, E. L.; Wu, L.; Uribe, A.; Gerrue, L. O.; Carrino, J.; Heller,

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,

P. Anal. Biochem. 1998, 257, 95-100.

(12) Effenhauser, C. S.; Paulus, A.; Manz, A. Widmer, M. M. Anal. Chem. 1994,

66, 2949-2953.

(13) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11348-

11352.

(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 Acids Res. 1996, 24, 375-379. (16) Cheng, J.; Schoffner, M. A.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 380-385. (17) Northrup, M. A.; Benett, B.; Hadley, D.; Landre, P.; Lehew, S.; Richards, J.; Stratton, P. Anal. Chem. 1998, 70, 918-922. (18) Giordano, B. C.; Ferrance, J.; Swedberg, S.; Huhmer, A. F. R.; Landers, J.

P. Anal. Biochem. 2001, 291, 124-132.

(19) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.;

Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (20) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R.

S.; Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176.

(21) Khandurina, J.; McKnight, T. E.; Jacobson, S. C.; Waters, L. C.; Foote, R.

S.; Ramsey, J. M. Anal. Chem. 2000, 72, 2995-3000.

(22) Kopp, M. U.; deMello, A. J.; Manz, A. Science 1998, 280, 1046-1048. (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,

B.; Bay, S. Electrophoresis 2000, 21, 150-156.

flow-through devices. However, a significant limitation of this device is that the number of cycles is fixed by the channel layout at 20 cycles. Therefore, only a limited control of the reaction conditions is possible through changes of the flow rate and zone temperatures. Despite the wide spectrum of applications of RNA amplification in research and molecular diagnosis, there is limited work on the coupling of reverse transcription (RT) and PCR on a single microdevice. In a previous report, RT was performed in a stationary reaction mixture (50 µL) placed in a polypropylene tube liner that was inserted in a silicon thermal cycled cavity. 17 Following one round of PCR, the sample was manually transferred to another chamber for a second round of amplification (nested PCR). In this work we report a microfabricated device for continuous- flow DNA and RNA amplification, which offers the following distinct advantages: (a) functional integration of reverse transcrip- tion and polymerase chain reaction (RT-PCR) onto a single monolithic chip for direct RNA amplification and (b) cycle number selection at 20, 25, 30, 35, and 40 cycles. We first study the effect of cycle number on the amount of amplification product. The influence of the input DNA and RNA molecules on the amplified product is also studied. Moreover, the high-throughput capability of continuous-flow PCR and RT-PCR is demonstrated, for the first time, by running simultaneously multiple DNA samples of different sizes followed by product collection at different cycle numbers. Similarly, multiple small-volume RNA samples are subjected to RT-PCR in a continuous-flow format. The amplification of a single- copy gene from human genomic DNA isolated from whole blood is performed successfully. Finally, it is shown that continuous- flow PCR can be performed rapidly in this chip by a simple hand- driven sample and reagent transport without the use of pumps.

EXPERIMENTAL SECTION Microfabrication. The microfabricated chip used throughout this study was fabricated at Micralyne (Edmonton, AB, Canada) and is depicted in Figure 1. This device is composed of two Corning 0211 borosilicate glass plates (each 40 × 45 × 0.55 mm) that were fusion-bonded together to form a closed structure. A continuous channel network was etched into the bottom plate by standard photolithographic and wet chemical (HF) etching techniques. The channels were 194 µm wide (at the top) and 55 µm deep for the reverse transcription section of the device, and 120 µm wide and 55 µm deep for the PCR section (Figure 1). The cover plate contains access holes (400-µm diameter, drilled ultrasonically) that were used for sample injection and product collection. These were aligned with the inlet and outlet channels on the bottom plate. After cleaning, the cover and bottom plates were fusion bonded to form the sealed microfluidic device. Both the RT and PCR sections contain two inlets each and are marked on the chip by RT1, RT2, PCR1, and PCR2. The PCR channel incorporates 40 identical cycles that pass over three temperature-controlled zones, to perform DNA denaturation, primer annealing, and enzymatic extension steps of amplification at time ratios of 4:4:9, respectively. The channel allows for a 3-fold extended denaturation step prior to entering the first cycle. Outlet channels were designed after cycles 20, 25, 30, 35, and 40 for product collection. The capacity of the PCR channel (from cycles

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Figure 1. (A) Schematic diagram of the microfabricated device for continuous-flow PCR and RT-PCR with

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 also in contact with its own temperature-controlled zone. Assembly of the Chip. To introduce samples through the chip and collect amplification products, we have constructed a simple holding apparatus that facilitates the connection between capillary tubes and the inlets and outlets of the device (Figure 1). A piece of clear Plexiglas (80 × 55 × 5 mm) was used, which contained 0.5-mm drilled holes that matched exactly those of the micro- device. Capillary tubes (3 cm long, 375-µm o.d., 100-µm i.d., Polymicro Technologies, Phoenix, AZ) were cut and glued with epoxy to the Plexiglas holes such that 1 mm of their ends protruded out from the holes. To provide leak-proof connections, disks (2-4-mm diameter and 0.8 mm thick) were prepared from silicone glue and were perforated with the protruding ends of the capillaries. For assembly, the capillary ends carrying the disks were inserted carefully into the access holes of the device and the apparatus was held tight with two screws. The free ends of the capillaries, therefore, become the inlets and outlets for the channels. There are four inlet and five outlet capillaries. Cycle selection was accomplished by blocking the ends of all outlet capillaries with Teflon caps except of the one corresponding to the selected cycle number. Sample collection was accomplished by simply placing an inverted plastic 0.2-mL tube on top of the uncapped capillary. The caps used for blocking the free ends of unutilized capillaries were prepared by using a 5-mm-long Teflon tubing (380-µm i.d.). One end of the tubing was plugged with epoxy glue while the open end was tightly fitted over the capillary. Prior to the first PCR of the day, the whole chip was filled with water. Thus, the unused (capped) outlet channels prior and after the selected cycle number contain water. This ensures that the reaction mixture exits only from the uncapped capillary. No losses of sample into the capped outlets occur.

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Chip Peripherals. Fluids were pumped through the chip by hydrostatic pressure. Two PC-controlled precision syringe pumps (model 50300, Kloehn, Las Vegas, NV), each with a syringe capacity of 25 µL, were used for sample delivery. A 2-µm in-line microfilter (model M-532, Upchurch Scientific, Oak Harbor, WA) was connected between the chip and the pumps. Temperature control was achieved by placing the chip as- sembly on top of a suitable heating base constructed in-house by using four copper blocks, 80 × 20 × 10 mm each. Inside each block, and along its length, we placed a heating cartridge (Omega, CSS-03365/110) and a temperature sensor (Omega, 1PT 100 kn2528). The block temperatures were controlled by using four digital PID temperature controllers (Jumo iTron microprocessor controller, model 702040 from Enercorp, Islington, ON, Canada) housed in a single unit containing a power supply and electric circuits for the heating cartridges and sensors. The blocks were then positioned, with a 5-mm spacing between each other, on a nonthermally conducting base (85 × 85 × 10 mm) and their surfaces were evened out with respect to each other. This ensured that the chip made a good contact throughout, when it was placed on top of the heating blocks. To minimize heat transfer between the blocks, a small fan was placed in front of them, allowing air to pass through the spacing. Temperature control accuracy was within (0.1 C. Channel Surface Passivation. Although some work has been done to ascertain the steps necessary to prevent poisoning of the PCR by contaminants from the microfabrication process, 25 we have developed a more extensive method that effectively prevents contamination in our devices. The walls of the microchannels were passivated by silanization as follows. The channels were cleaned

(25) Taylor, T. B.; Harvey, S. E.; Albin, M.; Lebak, L.; Ning, Y.; Mowat, I.; Scluerlein, T.; Principe, E. Biomed. Microdevices 1998, 1, 65-70.

thoroughly with filtered double-distilled water to remove the presence of chromium and other contaminants due to the micro- fabrication process. The water was then removed by washing with acetone and the channel dried by applying vacuum. The channel was filled with a filtered (0.45-µm filter) 5% dichlorodimethylsilane (DDMS) solution in chloroform. The chip along with a small beaker containing 1 mL of the DDMS solution was placed in a desiccator. Vacuum was applied until the solution in the beaker started boiling. The vacuum inlet was then shut, and the desiccator was left for 1 h. During this period, the DDMS solution in the chip and beaker evaporates completely and the vapors form a thin film on the walls of the channels. The chip was removed and washed twice with 25 µL of chloroform, twice with 25 µL of filtered acetone, and twice with 25 µL of filtered water. Washings were performed by applying vacuum. DNA and RNA Templates. The DNA template was a 0.23- kbp fragment synthesized by amplifying the prostate-specific antigen (PSA) mRNA as described elsewhere. 26 The RNA template was a 1-kb fragment synthesized by in vitro transcription of PSA cDNA. The primers used for continuous-flow PCR and RT-PCR of these templates are as in ref 26. The upstream and downstream primers used for PCR amplification of the human -globin gene from genomic DNA correspond to sequences 62 253-62 272 (exon 1) and 62 468-62 487 (exon 2) of the gene and generate a 0.23-kbp product. The plasmid used as a template was a 4-kbp GFP control vector, and the primers were the T7 promoter and terminator primers (plasmid from kit Catalog No. 3186148; primers from kit Catalog No. 3186237; Roche Applied Sci., Mannheim, Germany). This generates a 1.02-kbp PCR product. Continuous-Flow PCR. The PCR mixture contained 10 mmol/L Tris-HCl, pH 9.0, 2.5 mmol/L MgCl 2 , 50 mmol/L KCl, 1 mL/L Triton X-100, 1 mL/L Tween 20, 0.2 g/L bovine serum albumin (BSA; New England BioLabs, MA), 2.8 µmol/L poly- (vinylpyrrolidone) (MW 44 000), 0.2 mmol/L each of the dNTPs (MBI, Fermentas, Lithuania), 0.5 µmol/L each of the upstream and downstream primers, 0.5 unit/µL thermostable DNA poly- merase (HT Biotechnology, Cambridge, England), and DNA template. The heating block temperatures were set at 95 C for denaturation, 58 C for primer annealing, and 72 C for extension. Between samples, the PCR channel was washed with a volume of water equal to the sample volume. Continuous-Flow RT-PCR. The reverse transcription mixture contained 50 mmol/L Tris-HCl, pH 8.3, 8 mmol/L MgCl 2 , 30 mmol/L KCl, 10 mmol/L dithiothreitol, 0.2 g/L BSA, 2.8 µmol/L poly(vinylpyrrolidone), 0.5 µmol/L downstream primer, 0.5 mmol/L each of the dNTP, 2 units/µL human placental ribonuclease inhibitor (HT Biotechnology), 10 units/µL reverse transcriptase (M-MuLV from Finzyme), and RNA template. The RNA template and the primer were first heated at 70 C for 5 min, to reduce any secondary structures of the RNA, and then cooled on ice. Afterward the rest of the components were added and the mixture was introduced into the RT inlet of the chip. Two protocols were used for performing RT-PCR. In one protocol (protocol A), the RT reaction mixture was pumped into the RT zone of the chip at a fast flow rate until it reached the channel intersection with the PCR reagents. At this point, the flow was stopped and the RT was allowed to incubate for 30 min at 42

(26) Verhaegen, M.; Christopoulos, T. K. Anal. Chem. 1998, 70, 4120-4125.

C. At the end of this period, the RT heating block temperature was raised to 95 C for 5 min to inactivate the reverse tran- scriptase. Subsequently, both the RT mixture and the PCR reagents were pumped continuously and simultaneously with a ratio of volumetric flow rates set at 1:9, respectively. Therefore, a 10-µL total volume of RT-PCR contains 1 µL of the RT product. The RT-PCR product was collected at the desired cycle number. It should be noted that, after mixing of the two solutions, the final concentrations of the PCR components are as above. In the second RT-PCR protocol (protocol B), the RT mixture was pumped continuously into the RT channel at a flow rate of 2 nL/s. Under these conditions, 30 min is required for the mixture to reach the intersection with the PCR channel. At this point, the pump delivering the PCR reagents is activated at 18 nL/s to carry out the amplification. Between samples, the RT channel and the PCR channel are washed once with a volume of water eaqual to the sample volume. Analysis of Amplification Products. PCR and RT-PCR products were analyzed by agarose gel (2%) electrophoresis and ethidium bromide staining. DNA markers were from a φX174 HaeIII digest with fragment sizes of 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, and 72 bp (MBI, Fermentas). The products were quantitated by densitometry of pictures taken with a Kodak DC120 digital camera while the gels were under UV illumination. The markers were used as standards for construction of calibration curve.

RESULTS AND DISCUSSION The 4 × 4.5 cm RT-PCR chip (Figure 1) enables the functional integration of reverse transcription and polymerase chain reaction using a single continuous channel 343.2 cm long. The lengths of channel segments corresponding to RT and PCR (for 40 cycles) are 54.4 and 288.8 cm, respectively, covering most of the chip. In the design process, the needs for compact device design, minimum linear thermal diffusion distance (e.g., minimum etch depth) for a given channel volume, and higher cycle count and channel volumes were balanced with microfabrication yield challenges associated with densely packed channel meanders and having only one device per substrate if the design was not sufficiently compact. Microfabrication of this chip required that particular care be paid to reduce surface damage to the glass during subsequent processing. Careful handling minimized the occurrence of significant channel defects upon etching. Microchips with smaller active areas are considerably less affected by occasional scratches or other surface damage. The 4 × 4.5 cm, 40-cycle design used afforded relatively shallow channels (55 µm), sufficient volume (5.0 and 15.5 µL for the RT and PCR sections, respectively), yet well-spaced channels and four devices per 4-in. substrate to enhance yield. The surface-to-volume ratio for the PCR channel is 57 mm -1 as opposed to a surface-to-volume ratio on the order of 1 mm -1 and a diffusion distance of 1-2 mm for a typical 25-µL macroscale PCR. The length and the volume for each PCR cycle are 7.2 cm and 0.38 µL, respectively. Reaction mixtures for RT and PCR are introduced into the chip through appropriate access holes, and the amplification products are collected from various outlets, thus allowing for cycle number selection. For 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, 7, 8, and 9 min for cycles 20, 25, 30, 35, and 40, respectively. The

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Figure 2. (Left panel) Effect of the cycle number on the concentration of amplification product

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 10 7 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 are 13, 14, 15, 16, and 17 min. Sample introduction into the chip as well as product collection is accomplished through capillaries connected to the access holes. Epoxy glue is most commonly used to connect microfluidic devices with capillary tubes. However, we have found it to be both time-consuming and often unsuccessful as the epoxy seeps around the capillary into the access holes, clogging the channel, and making the chip unusable. Even if a successful connection is made with epoxy glue, it is still troublesome to remove the glued capillaries for cleaning the chip. The simple apparatus described in the Experimental Section and shown in Figure 1b ensures both an easy connection (within minutes) of the capillaries to the access holes and convenient disassembly of the chip for cleaning. The effect of the number of PCR cycles on the amount of amplification product was studied by using 10 7 molecules of DNA template (0.23 kbp of PSA DNA) in 10 µL of reaction mixture. The products were collected at 20, 25, 30, 35, and 40 cycles, and the flow rate was set at 21 nL/s. A negative control, containing no DNA template, was also subjected to PCR for 40 cycles to ensure the absence of contamination. An electropherogram of the products and a plot of product concentration as a function of the number of cycles are presented in Figure 2. In all cases, a single band was observed at the expected size (0.23 kbp). Initially the amplification is exponential, but at high cycle numbers the plateau phase of PCR was reached. The dependence of the amount of amplification product on the input DNA template molecules was studied in the range of 2.5 × 10 6 -160 × 10 6 molecules (10-µL reaction) at a constant number of 30 cycles and a flow rate of 21 nL/s. The results are presented in Figure 2. We observe a linear relationship between the concentration of accumulated product and the number of input DNA molecules. Nonspecific amplification products due either to mispriming or to primer dimers were not observed, even at a high number of template molecules.

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The reproducibility of continuous-flow PCR was assessed by running four identical mixtures containing 10 7 DNA template molecules. The products were collected at 30 cycles, analyzed by agarose gel electrophoresis, and quantified densitometrically. The calculated CV was 5.7% (n ) 4). To investigate the capability of the chip for high-throughput PCR, three samples, containing DNA templates of different sizes, were run simultaneously on the chip by continuous-flow and the products were collected at different cycle numbers. Sample 1 contained 10 7 molecules of a 0.23-kbp DNA template (PSA), and the amplification product was collected at 25 cycles. Sample 2 contained 10 9 molecules of a 4-kbp supercoiled plasmid, and the product was collected at 30 cycles. Sample 3 also contained 10 7 molecules of the 0.23-kbp template (PSA) and was collected at 35 cycles. The PCR volume was only 2 µL. The samples along with their appropriate primers were introduced at 5 nL/s from the PCR1 inlet. The rest of the PCR components were introduced at 5 nL/s from the PCR2 inlet (total flow rate of 10 nL/s). A 2-µL water plug was used as a wash between samples. A small air bubble (0.4 µL) was introduced between the sample and the wash. It was observed that the length of the bubble remained constant as the solutions were flowing through the chip. When a wash was encountered, the pump delivering the PCR mix was stopped, and the flow rate of the other pump was increased accordingly in order to maintain the 10 nL/s flow rate. After the wash was complete, the flow rate was decreased back to 5 nL/s and the PCR mix pump was activated for the next sample. The whole process from injection of samples to the collection of products after amplification took 35 min to be completed. The products (2 µL) were subjected to agarose gel electrophoresis and the results are presented in Figure 3. The three samples are amplified independently, without any evidence of cross-contamination. Therefore, the chip offers the advantage of multiple sample processing. The amplification of a single-copy gene ( -globin) from human genomic DNA was also tested by using continuous-flow PCR on

Figure 3. (a) Schematic diagram of the PCR channel of the chip containing reaction mixtures

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. S 1 , sample 1 contains 10 7 DNA molecules (PSA DNA, 0.23 kbp) and the product is collected at 25 cycles. S 2 , sample 2 contains 10 9 supercoliled plasmid DNA molecules and the product is collected at 30 cycles. S 3 , sample 3 contains 10 7 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 -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 using the QIAamp DNA blood mini kit (Qiagen, Germany).

Aliquots of 2 and 4 µL were used for PCR in a total reaction volume

of 10 µL and a flow rate of 10 nL/s. Products were collected at 40

cycles and subjected to agarose gel electrophoresis (Figure 3c).

A single band at the expected size (0.23 kbp) was observed.

RNA amplification by RT-PCR is most often performed as a two-step reaction. First, cDNA is synthesized by reverse transcrip-

tion. Then, the cDNA is mixed with the PCR reagents for amplification. The difficulty in integrating the two processes on a single chip arises from the fact that the components of the RT mixture interfere with PCR. The Mg 2+ concentration of the RT mixture is 4 times higher than the optimum for PCR. Also, the reverse transcriptase enzyme inhibits PCR 27 and must be inacti- vated at high temperature (95 C) prior to amplification. As a consequence, the RT mixture should constitute about 10% of the total PCR volume. This was overcome by using two pumps simultaneously, for RT and PCR, with flow rates at a ratio of 1:9. We studied the influence of the cycle number on the concen- tration of amplification product in flow-through RT-PCR (protocol A) by using a reverse transcription mixture containing 10 8 RNA template molecules (1 kb)/µL. The total volume of RT-PCR was 10 µL and contained 1 µL of the RT product (cDNA). A negative control (with no RNA template) was also included. Figure 4 presents an electropherogram of the amplified products and a graph of the product concentration as a function of the number

of cycles. The product increases exponentially until the plateau

phase of PCR is reached. A single band at the expected size was observed in all cases. Nonspecific amplification products were not observed. The effect of input RNA template molecules on the amount of RT-PCR product was studied in the range of 6.25 × 10 6 -200 ×

(27) Chandler, D. P.; Wagnon, C. A.; Bolton, H. Appl. Environ. Microbiol. 1998, 64, 669-677.

10 6 molecules. Products were collected at 30 cycles. The results are presented in Figure 4. The amount of product is linearly related to the input RNA molecules. However, a plateau is reached at a high number of RNA molecules because of saturation of reverse transcription and PCR. We performed various experiments to demonstrate high- throughput RT-PCR on the chip. Four RT reaction mixtures, 0.7 µL each, were pumped in a continuous-flow manner (protocol B) into the RT channel at a flow rate of 2.6 nL/s. A 0.7-µL water plug was used as a wash between samples. Upon reaching the intersection of RT and PCR channels, the solution was mixed with the PCR reagents that were flowing at 21 nL/s. When a water plug was encountered, the PCR reagent pump was stopped, allowing the RT pump to introduce the wash. Once the next RT sample reached the intersection, the PCR reagent pump was reactivated. The products (6.3 µL) were collected after 30 PCR cycles and then were subjected to agarose electrophoresis. The results are shown in Figure 5. Samples 1 and 3 contain 4.2 × 10 7 RNA molecules and samples 2 and 4 contained 2.8 × 10 6 RNA molecules. We observe (Figure 5b) clear characteristic bands for each sample. It is well known that a major problem of PCR and RT-PCR is that of false positives arising from the contamination of the reaction mixture from previously amplified sequences. In continu- ous-flow PCR and RT-PCR, the same reaction vessel (chip) is used for amplification of various samples containing a wide range of DNA and RNA molecules. As a consequence, we were concerned that the chip might become a source of contamination, i.e., that amplification products might remain in the chip and be carried over to the next sample. To examine this, we included a negative control after a series of samples with high DNA/RNA template numbers. In all cases, no contamination was observed. Therefore, washing the chip with an equal volume of water after each sample is sufficient to avoid carryover. Generally, contamination in PCR

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Figure 4. (Left panel) Effect of the cycle number on the concentration of amplification product

Figure 4. (Left panel) Effect of the cycle number on the concentration of amplification product in RT-PCR. The input RNA (1 kb) was 10 8 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).

markers. N, negative (no RNA template in the RT mixture). Figure 5. (a) Schematic diagram of

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. S 1 , S 3 , samples 1 and 3 containing 4.2 × 10 7 RNA molecules. S 2 , S 4 , samples 2 and 4 containing 2.8 × 10 6 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 10 8 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 HNO 3 . 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 detection system requires less amplification product, thereby decreasing the possibility of contamination and enabling the use of lower ranges of starting DNA/RNA copies (or lower number of cycles). An advantage of the flow-through chip for PCR and RT-PCR is that it can be reused. No special treatment is required after the

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initial silanization. However, channel clogging is observed after two weeks of continuous daily usage. The clogs always occur at the high-temperature zone (95 C) and are attributed to the accumulation of denatured protein. The blockage most frequently occurs during either the extended denaturation step or during the first few PCR cycles. Perhaps the initial rapid rise of temperature in these regions contributes to the deposition to the

channel wall. Figure 5d depicts a clogged channel wall obstructing the flow. To eliminate the problem, we have developed a simple procedure for removing the clogs effectively. The chip is placed on a ceramic base into a furnace (Thermolyne, type 48000), and the temperature is raised slowly (within 1 h) to 300 C. This temperature is maintained for 1 h, and the furnace is turned off. The chip is removed when ambient temperature is reached. This process burns the organic material inside the channel. A clogged chip, after baking, is shown in Figure 5e. The heating process does not damage the chip. Subsequently, 65% HNO 3 is aspirated into the channel by applying negative pressure to remove the

burnt organic material. The acid-filled chip is left overnight at room temperature and then washed thoroughly with chloroform. Figure

5f shows a chip section after the cleaning process. The clean chip

was silanized before use. Finally, we investigated performing PCR in the flow-through microdevice by a simple hand-driven injection and transport of the reaction mixture, without using any pumps. The reaction mixture (10 µL) was aspirated into a 0.4-mm (i.d.) Teflon tubing connected to a 1-mL syringe containing water. PCR was then performed by applying moderate hand pressure to the syringe and collecting the amplification products at the desired cycle. Figure 5c presents an electropherogram of the products from two, hand-driven, continuous-flow PCRs including a negative control

and a positive containing 10 8 DNA template molecules. Products were collected at 30 cycles. The reactions were completed in 6 min (including injection and collection of 10 µL). This is 2.5 times faster than injection and collection by pump-driven PCR (21 nL/ s) for 30 cycles, which requires a total time of 15 min. Despite the possible inconsistency of the pressure in the hand-driven flow,

a clear band is observed at the expected size. We have thus

demonstrated a simple and robust system for rapid PCR, and we envisage future implementations that are based on a flow-through chip or a cartridge and that are operated by hand-driven injection

and transport of the reaction mixture without pumps. This system may be particularly useful for the rapid generation of amplified DNA in the molecular biology research laboratory. Furthermore,

it could be used in field testing, especially for detection of the

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 of 50 µL, were placed in a polypropylene tube liner that was inserted into a thermally cycled silicon cavity. A layer of mineral

oil was added over the PCR mixture to prevent evaporation during cycling. Following RT-PCR, an aliquot of the mixture was transferred manually to another tube for a second PCR (nested PCR). On the contrary, in the present work, continuous-flow RT and PCR are truly integrated for the first time on a single monolithic device. No intervention is required between the RT and PCR steps due to automated introduction and mixing of samples and reagents. Considerably faster cycling times are achieved. For instance, at a typical flow rate of 21 nL/s, the cycling time is 13 s/cycle compared to 118 (“normal” protocol) and 70 s/cycle (“fast” protocol) reported in ref 17. No mineral oil is required for PCR. Moreover, we have demonstrated that RT can be performed in final reaction volumes as low as 0.7 µL. Several RNA samples can be introduced into the chip in a continuous- flow mode for high-throughput analysis. Additionally, RT may precede PCR, or PCR can be chosen alone and to different numbers of cycles. This is a significant advantage because it allows control of the amount of amplification product depending on the abundance of starting RNA or DNA copies in the sample. It was demonstrated that multiple samples containing DNA sequences of different sizes can be run simultaneously on the chip and the products can be collected at different cycle numbers. Because it is a continuous-flow system, the sample volume can be varied in order to optimize amplification. Throughout this work, the amplification products were ana- lyzed by electrophoresis and ethidium bromide staining. It is conceivable that the chip may be coupled in-line with other separation and detection systems such as capillary electrophoresis with laser-induced fluorescence detection.

ACKNOWLEDGMENT This work was supported by a grant to T.K.C. from the Institute of Chemical Engineering and High Temperature Chemical Pro-

cesses (ICE/HT), Patras, Greece, and the General Secreteriat of Research and Technology, Greece. We gratefully acknowledge the support of the Natural Sciences and Engineering Research

Council of Canada to C.J.B

Manz for helpful discussions. We also acknowledge the financial

support of Micralyne Inc.

We thank Martin Kopp and Andreas

Received for review August 5, 2002. Accepted November 11, 2002.

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