Integrating Polymerase Chain Reaction, Valving, and Electrophoresis

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Anal. Chem. 2003, 75, 4591-4598

Integrating Polymerase Chain Reaction, Valving, and Electrophoresis in a Plastic Device for Bacterial Detection Chee G. Koh, Woei Tan, Ming-qi Zhao, Antonio J. Ricco,*,† and Z. Hugh Fan*,‡

ACLARA BioSciences, Inc., 1288 Pear Avenue, Mountain View, California 94043

An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA. Detection and identification of bacteria are important for medical diagnostics, food/water safety testing, and biological warfare defense. An inexpensive, robust, and simple-to-use device to accomplish such analysis could have major societal impact. Methods to detect bacteria include immunoassay,1,2 flow cytometry,3 mass spectrometry,4 and genetic analysis.5-9 Nucleic acidbased genetic analysis has been increasingly used to identify * To whom correspondence should be addressed. E-mail: [email protected]; [email protected]. † Present address: NASA Ames Research Center, Mountain View, CA 94035. ‡ Present address: Mechanical and Aerospace Engineering, University of Florida, P.O. Box 116250, Gainesville, FL 32611-6250. (1) Ruen, C.; Yang, L.; Li, Y. Anal. Chem. 2002, 74, 4814-4820. (2) Stokes, D. L.; Griffin, G. D.; Vo-Dinh, T. Fresenius J. Anal. Chem. 2001, 369, 295-301. (3) McClain, M. A.; Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2001, 73, 5334-5338. (4) Wang, Z.; Dunlop, K.; Long, S. R.; Li, L. Anal. Chem. 2002, 74, 31743182. (5) Bradley, K. A.; Mogridge, J.; Mourez, M.; Collier, R. J.; Young, J. A. Nature 2001, 414, 225-229. (6) Belgrader, P.; Young, S.; Yuan, B.; Prinmeau, M.; Christel, L. A.; Pourahmadi, F. P.; Northrup, M. A. Anal. Chem. 2001, 73, 286-289. (7) Riffon, R.; Sayasith, K.; Khalil, H.; Dubreuil, P.; Drolet, M.; Lagace, J. J. Clin. Microbiol. 2001, 39, 2584-2589. (8) Greisen, K.; Loeffelholz, M.; Purohit, A.; Leong, D. J. Clin. Microbiol. 1994, 32, 335-351. 10.1021/ac0343836 CCC: $25.00 Published on Web 07/26/2003

© 2003 American Chemical Society

bacteria, including antharax,5 Bacillus subtilis,6 Escherichia coli,7,8 and Salmonella.8,9 The method involves DNA amplification, typically using polymerase chain reaction (PCR), which offers high sensitivity and unambiguous identification via the selective replication of genetic sequences unique to each target. PCR products (amplicons) can be analyzed using traditional slab-gel electrophoresis as shown by several groups.10-14 Several reports describe the integration of PCR with amplicon analysis on a monolithic fluidic platform, using either capillary electrophoresis (CE)15-19 or DNA hybridization.20,21 Microscale PCR devices have typically utilized silicon11,12,15,19,21 or glass13,16,17 substrate materials, or both, as do the integrated PCR-CE devices, some of which include lithographically patterned noble metal electrodes. Plastics have been employed in mesoscale PCR chambers6,10 and in one recent example of an integrated device.20 Incorporating PCR with analysis in a single disposable plastic device is attractive because of improved reliability, ease of use, and elimination of cross-contamination10 due to low cost and consequent disposability; replacing noble metal electrodes with printed inks further minimizes production costs. In addition, plastics offer a range of materials and a variety of surface properties to provide biochemical compatibility.22-25 (9) Soumet, C.; Ermel, G.; Rose, V.; Rose, N.; Drouin, P.; Salvat, G.; Colin, P. Lett. Appl. Microbiol. 1999, 29, 1-6. (10) Findlay, J. B.; Atwood, S. M.; Bergmeyer, L.; et al. Clin. Chem. 1993, 39, 1927-1933. (11) (a) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 18151818. (b) Shoffner, M. A.; Cheng, J.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 375-379. (12) (a) Northrup, M. A.; Ching, M. T.; White, R. M.; Watson, R. T. Tech. Dig. 7th Int. Conf. Solid-State Sens. Actuators (Transducers’93) 1993, 924-926. (b) Northrup, M. A.; Benett, B.; Hadley, D. Landre, P.; Lehew, S.; Richards, J.; Stratton, P. Anal. Chem. 1998, 70, 918-922. (13) Kopp, M. U.; de Mello, A. J.; Manz, A. Science 1998, 280, 1046-1048. (14) Krishnan M.; Ugaz, V. M.; Burns, M. A. Science 2002, 298, 793. (15) Woolley, A. T.; Hadley, P. L.; deMello, A. J.; Mathies, R. A.; and Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (16) (a) Lagally, E. T.; Emrich, C. A.; Mathies, R. A. Lab Chip 2001, 1, 102107. (b) Lagally, E. T.; Medintz, I.; Mathies, R. A. Anal. Chem. 2001, 73, 565-570. (17) Khandurina, J.; McKnight, T. E.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 2995-3000. (18) Waters, L. C.; Jacobson, S. C.; Kroutchininina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176. (19) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. H.; et al. Science 1998, 282, 484-487. (20) Liu, Y.; Rauch, C. B.; Stevens, R. L.; Lenigk, R.; Yang, J.; Rhine, D. B.; Grodziski, P. Anal. Chem. 2002, 74, 3063-3070. (21) Trau, D.; Lee, T. M. H.; Lao, A. I. K.; Lenigk, R.; Hsing, I.; Ip, N. Y.; Carles, M. C.; Sucker, N. J. Anal. Chem. 2002, 74, 3168-3173.

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The in-channel PCR volumes of the devices reported here range from 29 to 84 nL, ∼3 orders of magnitude less than the volumes (5-50 µL) used in earlier work by Wilding11 and Northrup12 and in recent work by Manz13 and Burns.14 Reasons to use these larger volumes include PCR product analysis using slab-gel electrophoresis and loss of liquid by evaporation during PCR. Use of large volumes is sometimes necessary when samples are very dilute (there is low concentration of DNA molecules). At least one copy of the DNA sequence(s) must be present in the aliquoted sample in order to achieve DNA amplification. Nonetheless, if key technical issues including cross-contamination, sufficient volume for subsequent analysis, evaporative solution loss, and a simple integrated microvalve can be addressed, bioassay applications where nanoliter sample volumes contain adequate target DNA would be well served by the reliability and robustness of the integrated fluidic device approach. Of the challenges to integrating PCR with CE separation in low-cost monolithic form, implementation of an appropriate microvalve is the greatest.16 Valves are required (1) to separate the PCR region from the separation medium to prevent mixing and (2) to reduce evaporative loss at elevated temperatures during thermal cycling. We sought and developed a simple solution to this challenge. This report describes a plastic microfluidic device integrating PCR with electrophoresis for bacterial detection and identification. Its nanoliter in-channel PCR minimizes reagent consumption, particularly beneficial for the expensive polymerases used in PCR. The device utilizes an integrated, localized, printed ink-based heater with low thermal mass that improves the rate of thermal cycling, speeding analysis. In situ gel photopolymerization was used to create local gel plugs that function as passive valves, effectively limiting bulk flow of liquid due to thermally induced pressure differences during thermal cycling; after PCR, the negatively charged amplicons are readily driven through the plug of gel using electrophoresis, in preparation for electrophoretic separation in a polymer solution-filled microchannel. The amplicons are fluorescently labeled by in-channel mixing with an intercalating dye contained in the separation medium; detection utilizes laser-induced fluorescence (LIF). The device is made from poly(cyclic olefin), providing thermal robustness well above the upper PCR temperature of 95 °C, low lateral thermal conductivity to minimize power consumption, relatively good thermal conductivity through the thin plastic film separating the heaters from the fluid-filled channels, and a very low optical fluorescence background. Identification of bacteria was achieved by amplification of species-specific DNA sequences with unique, predefined amplicon sizes. EXPERIMENTAL SECTION Reagents and Materials. Reagent kits for PCR amplification were purchased from Applied Biosystems (Foster City, CA), and they consist of GeneAmp 10× PCR buffer II, MgCl2, AmpliTaq (22) Boone, T. D.; Fan. Z. H.; Hooper, H. H.; Ricco, A. J.; Tan, H, Williams, S. J. Anal. Chem. 2002, 74, 78A-86A. (23) Soper, S. A.; Ford, S. M.; Qi, S.; McCarley, R. L.; Kelly, K.; Murphy, M. C. Anal. Chem. 2000, 72, 642A-651A. (24) Johnson, T. J.; Ross, D.; Gaitan, M.; Locascio, L. E. Anal. Chem. 2001, 73, 3656-3661. (25) Kameoka J.; Craighead H. G.; Zhang H.; Henion J. Anal. Chem. 2001, 73, 1935-41.

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Gold DNA polymerase, and deoxynucleotide triphosphates. Oligonucleotide primers were ordered from Integrated DNA Technologies (Coralville, IA), while genomic K562 DNA was from Promega (Madison, WI). Thermally killed E. coli O157:H7 and Salmonella typhimurium cells were from KPL (Gaithersburg, MD). Target genomic DNA was extracted from these cells using bacteria lysis buffers and “Genomic-Tips” from Qiagen (Valencia, CA). Bovine serum albumin (BSA) and 1-hydroxycyclohexyl phenyl ketone (HCPK) were purchased from Sigma-Aldrich (St. Louis, MO). HCPK powder was dissolved in 2-propanol to obtain a stock solution with a concentration of 100 mM. A 30% acrylamide/N,N′-methylenebisacylamide (bis) solution 19:1 (5% C), 1.5 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) at pH 8.8, and Precision Molecular Mass Ruler for DNA sizing were purchased from BioRad (Hercules, CA). φX174 DNA Marker digested with HaeIII was obtained from Invitrogen (Carlsbad, CA). The intercalating dye thiazole orange (TO) was purchased from Sigma-Aldrich while hydroxypropylcellulose (HPC, MW 60 000) and hydroxyethylcellulose (HEC, MW 90 000-105 000) were from Polysciences (Warrington, PA). A buffer consisting of 890 mM Tris, 890 mM boric acid, and 20 mM EDTA (10× TBE) at pH 8.4 was from BioRad. Pressure-sensitive adhesive “PCR tape” (model 9795) was obtained from 3M (Minneapolis, MN). Commercial flexcircuit-type film heaters (HK5207R42.1L12A) were ordered from Minco (Minneapolis, MN) while K-type thermocouples (unsheathed, with a diameter of 0.002 in., CHAL-002) were from Omega (Stamford, CT). Graphite ink (sheet resistance ∼30 Ω/square) and silver/graphite blend ink (sheet resistance ∼5 Ω/square) were obtained from ERCON (Wareham, MA). Power supplies (GPS-3030D) for both heater and fan were purchased from Instek (San Gabriel, CA) while the resistance temperature device (RTD, 5651PDX24A) was from Minco. Device/Valve Fabrication. Plastic microfluidic devices were fabricated using protocols described elsewhere.22,26 Briefly, the desired pattern was first created on a glass plate using photolithography and wet etching. A layer of metal (100 µm-1 mm) is then electroplated onto the surface of the glass plate, creating a molding tool (an electroform) that has inverse topology relative to the original microstructures. The electroform is mounted on an embossing or molding system to produce plastic cards that bear the same features as the original; thousands of parts can be made from a single electroform. The devices in this work were made by compression molding of poly(cyclic olefin) resin. Devices were completed by sealing the plastic substrate with a cover films made of the same poly(cyclic olefin), ∼100 µm thicksusing thermal lamination. Figure 1 shows the layout of two device designs for bacterial detection and identification. Wells were laid out with a pitch of 4.5 mm or integral multiples thereof (standards defined by the Society for Biomolecular Screening and accepted by the American National Standards Institute); this ensures compatibility with a variety of commercial fluid dispensing systems. Design ID-6, which was used for most of this work, is shown in detail in the lower part of Figure 1 and described in the caption. Integrated electrodes with contact pads function as CE driving electrodes by connecting to high voltages via “pogo” pins. The total volumes (26) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630.

Figure 1. Top: Layout of two microfluidic device designs on a single plastic card. The heated region for PCR thermal cycling is indicated by dotted lines. Bottom: The most studied of the designs, ID-6, is detailed in the expanded view, which shows gel valves in crosshatching, the PCR zone in diagonal shading, and printed ink electrodes in solid black. Channels are 120 µm wide × 50 µm deep except in the PCR zone where they are 360 µm wide. Channel lengths, measured from intersection a in the figure to wells 1-8, are 8.1, 4.1, 48.9, 9.5, 8.4, 13.1, 8.4, and 17.2 mm, respectively.

of the PCR channels in ID-4 and ID-6 are 29 and 84 nL, respectively. The solution for fabricating microfluidic gel valves was prepared in nuclease-free water, consisting of 375 mM Tris-HCl, 8% acrylamide/bis solution, and 5 mM HCPK. The entire device was filled with the monomer solution using pressure or vacuum. A photomask (or opaque black tape) was placed on top of the device to define the exposed area where microfluidic valves were desired. After an exposure of 5 min to collimated light from a filtered mercury lamp, the monomer solution in the exposed area polymerized to form a gel plug. The process took place at room temperature. The monomer solution in unexposed regions does not polymerize, and thus, given proper layout of the channel pattern, unpolymerized gel can be effectively removed by flushing and then replaced with the desired buffers and reagents. The device must be designed appropriately so that gel valves are not in the way of changing solutions in unpolymerized regions. PCR Setup and Reactions. The setup for PCR thermal cycling was built at ACLARA and consists of a heater, power supplies, RTD, a fan, and a computer. The heater and the cooling fan were connected to separate power supplies, each controlled by a signal conditioning board (National Instruments SC-2042RTD, Austin, TX) according to feedback from the RTD. The power used to maintain the temperature at 95 °C is ∼6 W. The signal conditioning board also supplied 1 mA of current to the RTD. A data acquisition board (PCI-MIO-16XE-50, National Instruments) was used for acquiring data using a custom LabView (National Instruments) program. A proportional/integral/differential module within the LabView program was used to control the cycling temperatures. The setup can control multiple PCRs simultaneously. To fabricate heating resistors and CE driving electrodes, silver/ graphite inks were screen-printed onto a poly(cyclic olefin) support film (∼100 µm thick) by Bay Area Labels (San Jose, CA). The ink-based heating resistor is 2.2 mm wide, with a length adequate

to cover all of the PCR channel; the ink CE-driving electrodes are 0.3 mm wide × 4.5 mm long. After being cured at 95 °C for 2 h, the ink pattern on its supporting film was aligned and thermally laminated onto the cover film of a device. A temperature sensor (RTD) was placed in the center of the PCR region using Kapton tape from Omega. Commercial film-based heaters were used for initial experiments as the ink heater technology was being developed. Cooling for PCR thermal cycling was achieved with a fan, which directed room-temperature air over the device. For the use of either a film-based heater or an ink resistor, the programmed temperatures must be calibrated against the actual temperatures in the solution inside the PCR channel. Very accurate control (and therefore measurement) of the temperature in the PCR channel is required for successful DNA amplification. To calibrate the temperature measurement system, a microthermocouple (K-type) was placed inside the PCR channel; it was connected to a digital thermometer via a K-type connector. The channel was filled with the PCR mixture without polymerase for thermal calibration. A linear relationship (R ) 1.000) was found between the temperature measured in the PCR solution and that measured by the card-surface RTD; the calibration slope and intercept were incorporated into the control algorithms for subsequent PCR experiments, allowing the thermocouple to be removed from the PCR channel. A PCR mixture (20 µL) was prepared containing 1× AmpGene PCR buffer II, 2 mM MgCl2, 0.5 unit/µL AmpliTaq Gold DNA polymerase, 200 µM each deoxynucleotide triphosphate, 2.5 µM each primer, 0.1 mg/mL BSA, and roughly 4.5 × 103 copies (except where otherwise specified) of each of the target templates. Half of the reaction mixture (10 µL) was amplified on a commercial thermal cycling machine from MJ Research (Waltham, MA); the result served as a positive control. About 4 µL of the remaining PCR mix was used for filling the microfluidic device; the majority of the solution was for loading two reservoirs. Prior to loading the PCR mixture, the plastic device was washed with nucleasefree water and 1% BSA solution. The PCR mixture was then pipetted into well 5; it filled the entire PCR region with the assistance of a “gentle” vacuum at well 6. After filling the other channels and wells with appropriate reagents, all wells were sealed using PCR tape to prevent evaporation. PCR was carried out using temperature cycles including an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 56 °C for 30 s, and 72 °C for 45 s, and a final extension step at 72 °C for 5 min. Separation and Instrumentation. The microchannel for amplicon separation was filled with a sieving matrix consisting of 1.5% (w/v) HPC, 0.4% (w/v) HEC, and 4 µM TO in 1× TBE buffer at pH 8.4. The separation medium was loaded into well 3, and a vacuum was applied to other wells to fill all channels. Wells 1-4 were then filled with 2.5 µL of the sieving matrix, while well 7 was loaded with 2.5 µL of 10 ng/µL Precision Molecular Mass Ruler. After PCR, amplicons were electrokinetically injected through the gel valve by applying a voltage (300 V) at well 4 while wells 7 and 8 were grounded. The injection time was 40 s to ensure amplicon’s passing through the gel valve and the injection intersection. Separation was achieved by applying a voltage of 1100 V across wells 1 and 3. Power supplies and the LIF detector for miniaturized CE were built in-house and have been described elsewhere.22,26 Briefly, a Analytical Chemistry, Vol. 75, No. 17, September 1, 2003

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Nikon microscope (model TE200) was equipped with a highvoltage power supply and an argon ion laser (JDS Uniphase, San Jose, CA). The laser beam at 488 nm was focused via a 10× (NA 0.45) objective into the channel. The emitted fluorescence was collected through a pinhole and spectral filter (Omega 530DF30). The fluorescence signal was detected by a photomultiplier tube (Hamamatsu, Bridgewater, NJ). A computer with a LabView (National Instrument) program written in-house was used for voltage control and data acquisition. Data were collected at a sampling rate of 20 Hz. RESULTS AND DISCUSSION Plastic Devices. Figure 1 shows the layout of the integrated plastic microfluidic device for bacterial detection and identification (ID) based on nucleic acid amplification of bacterial genetic signatures. Among two device designs, ID-6 was used for the most of this work. Referring to the expanded view of ID-6 at the bottom of Figure 1, wells 5 and 6 are for the introduction of a sample into the PCR channel reactor between them. Well 7 is for the introduction of a DNA sizing ladder, which serves as a calibration standard to accurately identify the amplicons according to their sizes. Wells 8 and 4 are for loading a sample plug into the separation channel, while wells 1 and 3 are for running the separation. Well 2 was not used in this work. We compared three commonly used plastics and selected poly(cyclic olefin) for this work. Poly(cyclic olefin) has low fluorescence background, minimum water absorptivity, good transparency, and high glass transition temperature (Tg, ∼138 °C). Polycarbonate also has a high Tg (∼145 °C) that meets the temperature requirement for PCR,20 but its high fluorescence background would result in poor detection sensitivity. Acrylics have a good optical property for detection and satisfactory surface property for DNA separation, but their Tg (∼109 °C) are too low to allow them to be used for PCR. In addition, acrylics have more than 30 times higher water absorptivity than poly(cyclic olefin), resulting in significant water loss from the channel into the plastic bulk. Henion et al. also used poly(cyclic olefin) to make CE chips that are hyphenated with MS.25 The surface of poly(cyclic olefin) is negatively charged when the device was filled with the buffer used. Its electroosmotic mobility is ∼20% of a glass device. Similarly, Lukacs and Jorgenson found that there is electroosmosis in a Teflon tube,27 which has a very inert surface. PCR Thermal Cycling. Integrated heaters offer rapid thermal cycling due to localized heating combined with low thermal mass. Most integrated heaters have been made from patterned thinfilm metal resistors13,15-17,19 or Peltier heaters11,16,20 (which are “integrated” by the attachment of a discrete component, rather than processing of an integral surface film). Other heating elements include nonintegrated infrared radiation28 and heat blocks used in commercial thermal cyclers.14,18 In the case of an all-plastic device, low thermal conductivity has the advantage of minimized input power due to limited lateral (27) Lukacs, K. D.; Jorgenson, J. HRC & CC, High Resolut. Chromatogr. Chromatogr. Commun. 1985, 8, 407-411. (28) (a) Giordano, B. C.; Ferrance, J.; Swedberg, S.; Huhmer, A. F. R.; Landers, J. P. Anal. Biochem. 2001, 29, 124-132. (b) Oda, R. P.; Starusbauch, M. A.; Huhmer, A. F. R.; Borson, N.; Jurrens, S. R.; Craighead, J.; Wettstein, P. J.; Eckloff, B.; Kline, B.; Landers, J. P. Anal. Chem. 1998, 70, 4361-4368.

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Figure 2. Thermal cycling profiles of a commercial PCR machine (MJ PTC-200 DNA engine) and an ink heater. The expanded view of one cycle is shown in the inset on the right. The temperature rampup speed using the ink heater is ∼4 times faster than that using the MJ thermal cycler.

heat conduction, but it can also slow response time due to poor conductivity between the heating element and the PCR solution. This problem was mitigated by covering the PCR channels with a plastic film just 100 µm thick and printing the heaters directly on a film laminated directly against this cover layer, providing a very short thermal path between heater and liquid. We previously reported integration of screen-printed ink resistors or the attachment of a commercial film-based strip heater with a plastic microfluidic device and found these two implementations have similar time-temperature profiles.29 The screen-printed ink heater is based upon very low cost mass production technology and provides intimate thermal contact (no interfacial adhesive layer) and minimum thermal mass. In addition, screen-printed ink resistors can also function as temperature sensors (via their temperature coefficient of resistivity, which is positive) or driving electrodes for CE. Feasibility was shown for the use of ink traces as driving electrodes (solid blocks in the bottom part of Figure 1) for on-chip electrophoresis, and it was shown that they have the potential to achieve fast and efficient separations with improved standard deviation for multiple runs than noble metal wires.29 The ink heater was compared with a commercial thermal cycler, and the results are discussed as follows. Figure 2 shows the thermal cycling profiles obtained using a MJ PTC-200 DNA engine and the ink heater. Both temperatures were measured by immersing a thermocouple into a PCR mixture in either a tube or a channel. The data show that the temperature ramp-up speed using the ink heater is ∼12 °C/s, which is roughly 4 times faster than the MJ thermal cycler. The higher speed allows less time (29) Zhao, M.; Ricco, A. J.; Nguyen, U.; Crooks, R. M.; Zhu, Q. In Micro Total Analysis Systems 2001; Ramsey, J. M., van den Berg, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2001; pp 193-194.

Figure 3. (a) Gel valve precisely located at the edge of a T intersection. (b) Images of the interface between a gel valve and the PCR region filled with 10 mM fluorescein, before the 1st cycle and after the 30th cycle of PCR. Fluorescein diffused ∼2 mm into the gel after 30 cycles (for scale, the width of the channels shown here is 350 µm).

for mismatching that can occur between primers and targets; thus, fewer false positive results should be obtained.30 This quick response is mainly a consequence of the small thermal mass of the heating area. The cooling speed by forced air is ∼2 °C/s, which is very similar to that in the MJ thermal cycler. Our results, in terms of ramp-up and cool-down speeds, are comparable to microscale PCR systems reported by others.6,11,20,21 Durations of 45, 30, and 45 s were used for the denaturation, annealing, and extension steps in PCR according to the literature7-9 and for easy comparison with the MJ cycler. More rapid PCR analysis can be achieved by reducing the duration in these steps to 1-5 s.15,30 The PCR time is also influenced by the amount (the copy number) of starting materials. For instance, the threshold cycle number to achieve the same amplicon concentration when starting with 103, 105, and 107 copies of 18S rRNA is 33, 26, and 20 cycles, respectively31 (PCR monitored in real-time using TaqMan chemistries). Evaluation of the effects of duration and cycle number led to the appropriate parameters for the applications detailed below. Microfluidic Valves. To integrate PCR and amplicon separation/identification, microfluidic valves are needed to prevent the PCR mixture from flowing convectively due to pressure differences into the separation medium during PCR.16,20,32 It was verified experimentally that thermal heating and cooling result in convective mixing between the PCR mixture and the separation medium if there is not a valve separating the two. In addition, diffusion at PCR temperatures is significantly more rapid than at room temperature. We explored the concept of using an in-channel plug of gel as a closed valve during amplification and as an open valve (30) (a) Wittwer, C. T.; Garling, D. J. Biotechniques 1991, 10, 76-83. (b) Wittwer, C. T.; Herrman, M. J.; Gundry, C. N.; Elenitonba-Johnson, K. S. J. Methods 2001, 25, 430-442. (31) Real-time PCR Goes Prime Time; Tech Notes No. 8;Ambion, Inc., 2002. (32) Anderson, R. C.; Bogdan, G. J.; Puski, A.; Su, X. Technical Digest of SolidState Sensors and Actuators Workshop; 1998; pp 7-10.

during injection after PCR, made feasible by the fact that charged amplicons are readily electrophoresed through the gel. Localized gel polymerization has also been explored for other applications including flow control inside microfluidic channels33 and forming microreactors.34 The gel valves were found to cause little, if any, inhibition of PCR. Comparison of chemical polymerization with photopolymerization showed the latter to be an easier way to define a gel valve at a precise location without undesirable residue. The initiators in chemical polymerization are tetramethylethylenediamine and ammonium persulfate, whereas that in photopolymerization is a UV-curing agent, HCPK. To make a gel valve at a precise location, acrylamide monomer solution containing HCPK was first filled into all channels and wells. After the device had been covered by a photomask, photopolymerization was conducted by exposure to a UV lamp. The solution in the region blocked by the photomask did not polymerize; nonpolymerized solution was removed by flushing of the channels at the end of the process. The solution in the exposed region polymerized to form a plug of gel. The predefined boundary shifted slightly (∼70 µm) relative to the mask edge, possibly because photogenerated radicals diffused and polymerized monomers nearby. By slightly shifting the photomask in subsequent experiments, it was possible to accurately define the location of a gel valve within a few micrometers. Figure 3a shows a gel valve with a boundary at a T intersection. The gel is dyed for easy visualization. The performance of the gel valve was evaluated by measuring the degree of leakage. A device with a gel valve was subjected to thermal cycling with fluorescein added to the solution; the length of fluorescein that diffused through/into the gel was taken as an indication of the potential for leakage of a relatively small molecule. To see the fluorescence image, a high concentration (10 mM) of (33) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; et al. Nature 2000, 404, 588-590. (34) Zhan, W.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2002, 74, 4647-4652.

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fluorescein was used in this experiment. The actual leakage for amplicons should be significantly lower due to their larger size and much lower concentrations (0.1-100 µM). Figure 3b shows two images taken before the 1st cycle and after the 30th cycle of the PCR. Other images taken between them illustrate the trend of slow, thermally enhanced diffusion of fluorescein. The data show ∼2 mm of fluorescein diffusion into the gel after 30 cycles, which can be managed by designing other fluidic components slightly more than 2 mm away. The diffusion of fluorescein through the gel without thermal cycling was characterized as well: the length of diffusion is less than 1 mm after 3 h at room temperature. The degree of gel cross-linking has a large effect on the performance of the valve; the degree of leakage in 4% crosslinked gel is about twice that in 10% cross-linked gel. In six trials of PCR experiments in the ID-6 device, the gel valve remained in place every time. These gel valves are able to withstand hydrostatic pressures up to 100 psi without moving, at which pressure the cover film delaminates. That pressure is much higher than the water vapor pressure (12 psi) at 95 °C, the denaturation temperature for PCR. For those applications that require even higher withholding pressure, tethering the gel valves to the channel surfaces may be considered. For comparison, the pressure required to move viscous polymer solutions was also determined; such solutions might have been considered as both valve and separation medium. However, various polymer solutions including 3% poly(ethylene oxide) with a viscosity of 445 cP were examined, and in no case was a pressure differential greater than 5 psi sustained without appreciable flow. It is expected that there is a bias in the amount of DNA sample injected through the gel valve if the injection time is not long enough. This is the same with electrophoretic injection in CE when there is only separation buffer without gel valves. However, Harrison et al. demonstrated that the amount of a sample injected through a channel junction of a microfluidic device can be defined by the geometry.35 When the injection time is long enough to ensure that the slowest moving component has passed the intersection, the sample in the intersection might be expected to be representative of the original sample. As a result, the bias in the amount of injected samples is reduced or eliminated. DNA Fragment Separation. Slab-gel electrophoresis was the traditional method for analysis of amplicons, although capillary electrophoresis and other methods are now increasingly employed. CE separation of DNA is now commonly implemented in linear polymers, as reflected by their use in the commonest commercial DNA sequencers. In glass and poly(dimethylacrylamide) devices, Mathies’ and Landers’ groups used HEC to demonstrate the separation of DNA fragments.36,37 Landers et al. also investigated HPC and the buffer matrix using neural network simulation.38 After studying separation efficiency by varying matrix composition, we found an appropriate separation matrix consisting of a mixture of 1.5% HPC and 0.4% HEC in a buffer that contains 50 mM Tris, 50 mM boric acid, and 2 mM EDTA at pH 8.4. This (35) Shultz-Lockyear, L. L.; Colyer, C. L.; Fan, Z. H.; Roy, K. I.; Harrison, D. J. Electrophoresis 1999, 20, 529-538. (36) Woolley, A. D.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (37) Tian, H.; Landers, J. P. Anal. Biochem. 2002, 309, 212-223. (38) Sanders, J. C.; Breadmore, M. C.; Kwok, Y. C.; Horsman, K. M.; Landers, J. P. Anal. Chem. 2003, 75, 986-994.

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Figure 4. Representative electropherogram of 1 ng/µL φX174 DNA Marker (HaeIII digest) separated in a poly(cyclic olefin) microfluidic device. The separation medium consisted of 1.5% HPC, 0.4% HEC, and 4 µM TO in 1× TBE buffer, pH 8.4. The field strength was 193 V/cm and the effective separation distance, from the injection point to the detection point, was 4 cm.

separation medium is effective for sizing DNA fragments between 500 and 1500 bp in poly(cyclic olefin) microchannel devices. An intercalating dye, thiazole orange, was used to facilitate detection.36 All double-stranded DNA complexes with TO and thus can be detected by laser-induced fluorescence. Using intercalating dye can yield higher sensitivity than labeling amplicons by covalent attachment of a single fluorescent molecule (e.g., fluorescein), because many dye molecules are intercalated by a single DNA molecule. Furthermore, direct addition of TO to the separation buffer can be used to induce and maintain intercalation without creating excessive background fluorescence: the effective fluorescence quantum yield increases up to 2000 times when a TO-intercalated 25-mer DNA fragment is compared to unintercalated TO.39 Separation efficiency and related parameters were evaluated by performing separations of φX174 DNA Marker (HaeIII digest) and Precision Molecular Mass Ruler in plastic microfluidic devices. A representative electropherogram of φX174 DNA Marker is shown in Figure 4 while that of the Precision Molecular Mass Ruler is in Figure 5. The range of the ladder and the size of each DNA fragment are indicated in both figures. The φX174 ladder has 11 DNA fragments; those at 271 and 281 bp have a small difference in size (10 bp), and their separation is therefore a good indicator of separation efficiency and resolution. The Precision Molecular Ruler covers a range of 100-1000 bp; it has ample “open space” in the electropherogram for multiple amplicons produced by multiplexed PCR. Initial experiments revealed inconsistency in the migration times for consecutive runs, very possibly a result of interactions between reagents and wall surfaces in plastic channels. The migration time became much more consistent when a preconditioning step was performed between runs: buffer was electrophoresed through the separation channel for ∼5 min under typical separation conditions without injection of a sample. Other additives or methods may also be used to improve the reproducibility of the migration times as described in the CE literature.40 Device Integration for Bacterial Detection and Identification. Integrating all microfluidic components in one device and realizing all the desired functions was the goal for the device to be used for bacterial detection and identification. Devices of design ID-6 were employed to demonstrate the functionality of the (39) Nygren, J.; Svanvik, N.; Kubista, M. Bioplymers 1998, 46, 39-51. (40) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons: New York, 1995; pp 172-175.

Figure 5. Electropherograms of amplicons (bottom), DNA sizing ladder (middle), and the mixture formed by simultaneously injecting amplicons and the ladder (top). The separation medium and the field strength were the same as in Figure 4. The effective separation distance was 3 cm.

integrated device concept and the interoperability of all microfluidic components. The steps to integrate all components were implemented as described in the Experimental Section. Figure 5 shows the electropherograms obtained for amplicons from E. coli and Salmonella; all expected amplicons were produced with a gain of 8.5 × 105-8.0 × 106. To correctly size the amplicons, the DNA sizing ladder was added to the sample during electrophoresis as in traditional slabgel measurements. From the migration times of each of the ruler’s fragments, a calibration line (log-log plot) relating fragment size to migration time was obtained, and interpolation along this line yields the size of each amplicon. The data, summarized in Table 1, show that the discrepancy between the actual size and the determined size is less than 10%, a typical result.41 Accurate identification of amplicons permits genetic specification. E. coli O157 and S. typhimurium were chosen as analytes because they are dangerous food-borne pathogens. Where genetic engineering of a bacterium or spore may change some of its DNA or surface antigens, nucleic acid sequences responsible for toxic effects must be preserved if toxicity is maintained. As shown by Legace et al., the primer set designed for E. coli O157 and the resultant amplicon of 232 bp is very specific,7 allowing differentiation of E. coli O157 from other serotypes of E. coli. Primer sets designed for Salmonella are similarly specific, with an amplicon at 559 bp being specific to S. typhimurium, while an amplicon at 429 bp is common to several serotypes of Salmonella, according to Soumet et al.9 Negative PCR results were observed for both amplicons at 429 and 559 bp when the sample contained only E. coli O157. The sequences of all primers and the size of the corresponding amplicons are listed in a table in the Supporting Information. To avoid false negatives due to potential PCR failure or other inhibition, a positive control was implemented by adding a primer set for genomic DNA K562 along with the corresponding template (data not shown). Whenever PCR was successful, the DNA K562 (41) Mueller, O.; Hahnenberger, K.; Dittmann, M.; Yee, H.; Dubrow, R.; Nagle, R.; Ilsley, D. Electrophoreisis 2000, 21, 128-134.

Table 1. Size and Migration Time of the DNA Ladder and Amplicons fragment ladder

E. coli amplicon Salmonella amplicons

DNA size (bp)

migration time (s)a

calcd size (bp)b

discrepancy (%)

100 200 500 700 1000 232 429 559

114.4 ( 1.5 122.7 ( 2.0 141.0 ( 2.6 146.6 ( 2.5 150.0 ( 2.3 125.1 ( 2.5 137.0 ( 2.7 142.6 ( 2.6

212 437 600

-8.4 1.9 7.5

a The standard deviation of the migration time was calculated from five repeat experiments. b The calculated size is interpolated from the amplicon’s migration time using the calibration line determined by the ruler fragments.

amplicon at 320 bp was observed for samples with or without genomic DNA from E. coli O157 or S. typhimurium cells. The detection limit for target DNA was measured for the integrated device. Samples were prepared with 4.5 × 105, 4.5 × 104, 4.5 × 103, and 4.5 × 102 copies of genomic E. coli and Salmonella DNA in a total of 20 µL of PCR mixture, and then PCR was conducted in integrated devices of design ID-4 (see Figure 1), which has a PCR channel volume of ∼29 nL. Therefore, these samples correspond to about 600, 60, 6, and