Microfabricated Polycarbonate CE Devices for DNA Analysis

The authors thank David Rhine and Doug Fayden for helpful discussion and ..... Fan, Z. IEEE Electron Devices Society; Hilton Head Island, SC, 1992; 11...
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Anal. Chem. 2001, 73, 4196-4201

Microfabricated Polycarbonate CE Devices for DNA Analysis Yingjie Liu,† Dale Ganser,† Alan Schneider,† Robin Liu,† Piotr Grodzinski,† and Natasha Kroutchinina‡

Physical Sciences Research Laboratories, Motorola Labs, Motorola, Inc., and Motorola Biochip Systems, 7700 S. River Parkway, MD-ML34, Tempe, Arizona 85284

The microchip capillary electrophoresis (CE) devices were fabricated in polycarbonate (PC) plastic material by compression molding. The molded devices were enclosed utilizing thermal bonding to another PC wafer. These thermal bonds do not yield up to an applied force equivalent to 150 psi. Aqueous fluid transport inside the plastic CE devices was enhanced by UV irradiation treatment of the hydrophobic polycarbonate plastic surfaces prior to thermal bonding. In comparison to glass microchannels, electroosmotic flow (EOF) in native PC channels is low and is independent of buffer pH at pH 7 and 9. UV irradiation of PC surfaces increases surface hydrophilicity and increases EOF. CE DNA separation was demonstrated in these PC CE devices with good resolution and run-to-run reproducibility. The on-chip PCR/CE analysis of a 500-bp region of bacteriophage λ DNA was also demonstrated. Microfabricated fluidic devices are powerful tools for performing chemical and biological assays with increased speed and reliability at reduced sample consumption and cost. Chip-based capillary electrophoresis (CE) devices are among the earliest successful demonstrations of microfluidic technology.1,2 More recently, 96 radial CE channel arrays were demonstrated for highthroughput nucleic acid analysis.3 Pumping, valving, and mixing of fluids in microchip CE devices are accomplished using electrokinetics, which is the combined effect of sample electrophoresis and buffer electroosmotic flow (EOF). Glass has been the preferred material choice for the fabrication of early CE devices. Photolithographic processes used for silicon materials can be readily adapted to create interconnected channel networks in glass substrates. The chemistry of glass surfaces is well understood, and EOF can be modified or supressed in glass channels for separation applications. Finally, glass is transparent to visible light wavelengths and is nonfluorescent. Ultrasensitive fluorescence detection can be achieved in glass devices; however, glass materials do have drawbacks for microfluidic device fabrication. The enclosure of the glass microfluidic channel network relies †

Physical Sciences Research Laboratories. Motorola Biochip Systems. (1) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (2) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (3) Shi, Y.; Simpson, P.; Scherer, J.; Wexler, D.; Skibola, C.; Smith, M.; Mathies, R. Anal. Chem. 1999, 71, 5354-5361. ‡

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on high-temperature annealing, which typically requires a gradual temperature ramp up to 600 °C. This is a slow, low-yield process. In addition, the cost associated with the glass photolithographic process is too high to make disposable devices on a cost-effective basis. Polymer substrates are promising alternatives for building complex microelectromechanical systems.4 Industrial interest in utilizing plastics for the production of microanalytical systems is primarily driven by the fact that these materials are less expensive and easier to use in mass production than silica-based substrates. Techniques such as plasma etching or reactive ion etching,5 laser ablation,6 imprinting,7-9 and injection molding10 are typically applicable to the fabrication of devices in plastic materials. There is also a wide variety of plastic materials with different physical and chemical properties that can be applied to different kinds of microfluidic technology. Previous works have reported on the use of polymethyl mathacrylate (PMMA) for the fabrication of CE devices using injection molding,10 in which the channel networks were sealed with lamination films. High-resolution, reproducible separation of double-stranded DNA fragments has been demonstrated in such devices. However, when additional on-chip functionalities are needed (amplification through PCR, for example), the low glass transition temperature of acrylic material (105 °C) is insufficient to support high-temperature cycling requirements. Polycarbonate plastics were also reported for the fabrication of microfluidic channels using UV excimer laser machining, and channel sealing has been achieved by thermal lamination.6 Consequently, the fluidic channels were composed of two plastics with different surface properties; this increases the difficulty of uniform surface treatment when necessary. Hybrid channel devices also suffer from band-broadening due to the EOF mismatch arising from the difference in surface charges on the different plastic materials. In addition, our experience shows that (4) Soper, S. A.; Ford, S. M.; Qi, S.; McCarley, R. L.; Kelly, K.; Murphy, M. C. Anal. Chem. 2000, 72, 642A-651A. (5) Boerner, M.; Kohl, M.; Pantenburg, F.; Bacher, W.; Hein, H.; Schomburg, W. Microsyst. Technol. 1996, 2, 149-152. (6) Roberts, M. A.; Rossier, J. S.; Bercier, P.; Girault, H. Anal. Chem. 1997, 69, 2035-2042. (7) Martynova, L.; Locascio, L. E.; Gaitan, M.; Kramer, G. W.; Christensen, R. G.; MacCrehan, W. A. Anal. Chem. 1997, 69, 4783-4789. (8) Xu, J.; Locascio, L.; Gaitan, M.; Lee, C. S. Anal. Chem. 2000, 72, 19301933. (9) Becker, H.; Dietz, W. Proc. SPIE 1998, 177-182. (10) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630. 10.1021/ac010343v CCC: $20.00

© 2001 American Chemical Society Published on Web 07/28/2001

the adhesives on most of the lamination films tend to fall into the microchannels, block the fluidic flow, and interfere with separation. In this paper, we report fabrication of microchip CE devices using polycarbonate polymer and hot compression molding. Compared to PMMA, polycarbonate has a higher glass transition temperature of 145 °C and can be appropriately used in devices requiring PCR thermal cycling. These channel networks are formed through hot compression molding and enclosed using thermal bonding with a blank polycarbonate wafer. It takes ∼3 min to compression-mold a CE device, which includes mold setup, material melting, compression, and demolding. Thermal bonding does not require the use of adhesives or lamination films. CE devices fabricated from uniform materials do not suffer from the band-broadening caused by the EOF mismatch associated with channels made from dissimilar materials.11 The robust thermal bonding was demonstrated. The electroosmotic flow characteristics of polycarbonate CE channels were studied. The effects of buffer pH on electroosmotic flow in these devices were presented and discussed. The use of these devices for CE separation of DNA markers and on-chip PCR amplification of λ DNA followed by CE analysis was also demonstrated. EXPERIMENTAL SECTION CE Microchip Fabrication. Polycarbonate CE devices were fabricated by compression molding using Carver hydraulic laboratory presses (Carver, Inc., Wabash, IN). These machines consist of two hot metal platens mounted in a hydraulic press and are capable of delivering 11 metric tons of pressure. The silicon master mold was fabricated using standard photolithographic procedures that produce a raised three-dimensional CE structure. Before molding, a 5-mm-thick glass wafer was placed on the lower platen to provide a flat, smooth foundation surface. A 5-cm separation was established between the upper and lower platens. The silicon master was then placed on the glass wafer. The system was heated to 188 °C. A predetermined amount of polycarbonate pellets (Aldrich) was placed in the center of the silicon master, and a blank nickel wafer was then placed on top of the polycarbonate pellets. The upper platen was lowered into contact with the blank nickel wafer and was then gradually compressed against the polycarbonate pellets as they melted. When the formed polycarbonate layer reached 1 mm in thickness, the two hot plates were separated, and the polycarbonate wafer and silicon master assembly were removed from the hydraulic press to air cool for ninety seconds. After cooling, the molded chip was demolded from the silicon master and the blank nickel plate. The entire molding process took approximately three minutes. Fluid access holes of 1-mm diameter were drilled at the ends of the microchannels. Both the CE wafer and blank sealing wafer were cut into 3 in. squares using a Prolight 2500 milling machine (Light Machines Inc., Manchester, NH) before being thermally bonded. During the thermal bonding process, the molded PC CE wafer and sealing wafer were sandwiched between two stainless steel plates, and the assembly was then placed inside an in-house metal bonding cartridge with vacuum and cooling capability. Once the assembly was loaded into the bonding cartridge, a vacuum of 4 × 10-2 Torr was applied to the bonding chamber. The loaded

bonding cartridge was then placed in the Carver hydraulic press, and thermal bonding was carried out at 134 °C for 10 minutes at a pressure of 4 metric tons. After bonding, the cartridge was cooled to 80 °C for 10 minutes before the bonded CE chip was taken out of the cartridge. The bonding strength was then measured on an Instron yield separation assessment instrument (Instron Corp., Canton, MS). The profiles of the compression-molded channels and the cross section of the thermally bonded channels were evaluated using a scanning electron microscope (Cambridge Instruments Ltd, Bar Hill, Cambridge, MA). The seal integrity of the bonded CE chip assembly was tested by filling the channels with 10 µM fluorescein (Molecular Probes, Eugene, OR). Vacuum was applied to one of the fluid-access reservoirs to induce the fluid into the channel network. Fluorescence images of the double T-junction in CE chip were obtained using an inverted fluorescence microscope (Nikon Eclipse, TE300), and imaged using a CCD camera. The glass CE devices were purchased either from Alberta Microelectronic Corp. (AMC) (Edmonton, AB, Canada) or from Caliper Technologies Corp. (Mountain View, CA). The glass devices from AMC have the same design as the PC CE devices. Before use, the electroosmotic mobility of the devices was minimized by covalent immobilization of linear polyacrylamide on the channel walls.12 The Caliper DNA LabChips were used as received and were run on an Agilent 2100 Bioanalyzer (Palo Alto, CA) using a DNA 500 assay. UV Surface Treatment and Contact Angle Measurement. A manual UV lamp of 4 W output (model UVGL-58, UVP, Upland, CA) was used for polycarbonate wafer surface treatment, and the contact angle between deionized water and the PC surface was measured using an angleometer (Rame-hart, Inc, Mountain Lakes, NJ). Measurement of Electroosmotic Flow (EOF). EOF was measured using the technique described by Huang.13 Briefly, the inlet/outlet reservoirs and the fluidic channels were filled with the desired sample buffer, and the contents of the inlet reservoir were then replaced with a buffer of reduced ionic strength. The electrical current in the fluidic channel was monitored after an electrical field was applied to the channel. The current decreased gradually until it reached a constant level after the content of the entire channel was replaced with the lower ionic strength buffer. The time required for the current to reach a constant level was recorded, and EOF velocity (νEOF) was calculated by dividing the channel length by the buffer replacement time. The electrophoretic mobility of the EOF was calculated as µEOF ) νEOF/E. DNA Analysis in PC and Glass CE Devices. In the CE separation of the DNA ladder experiments, DNA markers (Promega) were diluted in 0.5× TBE (44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA at pH 8.3) and then labeled with intercalating dye TO-PRO-3 (642 nm/661 nm, Molecular Probes, Inc.; a potential mutagen) with final dye concentration of 1 µM. CE channel networks were filled with sieving polymer solution made of 1 or 2% hydroxyethyl cellulose (HEC, Aldrich) in 0.5× TBE. System filling was achieved by placing the HEC solution in all but the sample reservoir and then applying vacuum (85 Torr) to the sample reservoir to draw the HEC solution into the network.

(11) Ross, D.; Johnson, T.; Waddel, E.; Baker, S.; Gaitan, M.; Locascio, L. Imaging of Electro-Osmotic Flow in Plastic Microchannels. Presented at LabAutomation2001 Conference, Palm Springs, CA, 2001.

(12) Hjerten, S. J. Chromatogr. 1985, 347, 191. (13) Huang, X.; Gordon, M.; Zare, R. Anal. Chem. 1988, 60, 1837-1838.

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The entire process took 2 min. The DNA sample was loaded into the sample reservoir after system filling. With the exception of the Caliper glass chips, which were run on an Agilent 2100 Bioanalyzer, all of the CE DNA separations and detections were carried out in a capillary electrophoresis system designed and manufactured by Dycor Technologies Ltd. (Edmonton, AB, Canada). The system is integrated with a confocal microscope. The light source for the laser-induced fluorescence detection is a 635-nm diode laser (8 mW). The laser line is filtered using a 640DF11.7 band-pass filter. The emission was first filtered using a sharp cut-off filter (RG665, 50% throughput at 665 nm with rapid cut-off below 650 nm) and then further filtered using a 670DF11.7 band-pass filter, and then spatially filtered through a 400-µm aperture. The DNA sample was initially loaded electrokinetically into the double T-junction injection valve. Once loaded into the injection valve, the potentials were reconfigured to inject the DNA sample from the double T-junction into the separation channel, where the DNA samples were separated and detected. The separation lengths and field strengths are indicated in each figure caption. The on-chip PCR/CE integration design was adopted from that reported on a glass microchip by Waters et al.14 The PCR reaction mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 250 µg/mL bovine serum albumin, 200 µM each deoxynucleotide triphosphate, 1.0 µM each primer, 25 units/ mL AmpliTaq, DNA polymerase (Perkin-Elmer), and DNA (10 ng/mL). A 20-µL volume of the reaction mixture was placed in the sample reservoir. All of the channels on the microchip and the other reservoirs contained sieving matrix in 0.5× TBE, and all of the reservoirs were topped with mineral oil to prevent evaporation. Amplification was carried out by thermally cycling the entire microchip in a commercial thermal cycler (MJ Research, Inc.). The thermal cycling profile was 94 °C for 2 min, 37 °C for 3 min, and 72 °C for 4 min for the first 24 cycles and held at 72 °C for 7 min in the last cycle to complete chain extension. After amplification, DNA F-174 marker and TO-PRO-3 dye were added to the sample reservoir at a final dye concentration of 1 µM. The post PCR CE separation was performed using the same protocol described in the CE DNA separation section. RESULTS AND DISCUSSIONS Compression Molding and Thermal Bonding. The polycarbonate CE chip layout is shown in Figure 1a. The insert shows the enlarged view of the double-T injection cross. All of the channels are 30 µm deep. The separation channel is 50 µm wide, and the width of the additional channels is 200 µm. This design increases the voltage drop over the separation channel and can utilize the available electrical field more effectively. The double-T injection design15 was adapted for the electrokinetic injection of the sample. Figure 1b shows a SEM picture of a compression molded channel. The trapezoidal profile shown in the channel was advantageous for structure release after molding. The channel structure was very well defined and the molded channel surfaces were very smooth. In contrast to plastic imprinting techniques in which the polycarbonate wafer is heated to temperatures slightly (14) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162. (15) Harrison, D. J.; Seiler, K.; Manz, A.; Fan, Z. IEEE Electron Devices Society; Hilton Head Island, SC, 1992; 110-113.

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above Tg, the polycarbonate pellets were heated above their melting temperature and brought to a free flow state, thus requiring less pressure for molding. Reduced molding pressure prolongs the lifetime of the silicon stampers. Furthermore, achieving PC molten flow in the molding process results in a structurally stress-free work piece following cooling. The CE channel network was enclosed by thermal bonding with another polycarbonate wafer. Bonding temperature and applied pressure are critical to the success of thermal bonding. If the applied pressure is too high, the softened polycarbonate material will be forced into the microchannels and will change the channel profile or block the channel. The efficiency of thermal sealing was tested by filling the channel network with fluorescent dye (see Figure 1c); no buffer leakage was observed. The bonds did not fail under applied forces equivalent to 150 psi of pressure. Heat Dissipation in Polycarbonate Microchannels. The ability to effectively dissipate joule heating generated from electrophoresis is essential to high efficiency separation. Compared to glass, polycarbonate (like most of the plastic materials) has poor thermal conductivity. However, because of the small channel dimensions and the high surface-to-volume ratios used in the network, heat generated by electrophoresis can still be removed efficiently. Ohm’s law plots were constructed for 10 mM borate buffer and 1× TBE buffer in the polycarbonate microchannels: 5 cm long, 50 µm wide, and 30 µm deep. For 10 mM borate buffer, the plot was linear up to 600 V/cm, with an R2 value of 0.9992. Beyond 600V/cm, the plot starts to deviate and trends to the upside. 1× TBE buffer generates less joule heating than does 10 mM borate buffer, and good linearity was observed for this Ohm’s law plot up to 800V/cm, with an R2 value of 0.9981. For 1× TBE buffer, there should not be any significant joule heating when operating below 800 V/cm. Field strengths above 800 V/cm were not tested, because of the limitation on the voltage output of the CE testing system. PC Surface Modification with UV Irradiation. Unlike glass, polycarbonate is very hydrophobic. The contact angle between deionized water and PC surfaces is 70.1°, and that for glass is only 19°. Aqueous solutions can be introduced into glass channel networks by capillary action. For DNA separation, glass surface charges have to be reduced chemically in order to reduce EOF and prevent sieving polymer solution from being pumped out of the separation channel. The polycarbonate wafer surface does not have many ionizable chemical functional groups and, therefore, exhibits very weak EOF flow.4 However, because of strong hydrophobicity, filling PC CE channel networks with aqueous solution is very difficult, especially in the wider portions of the CE channels, with width-to-depth ratios of 7 to 1. When forced into CE channels, aqueous solution usually separates into small segments that adhere to the sides of the channels. Electrokinetic pumping is impossible in such a discontinuous fluidic system. PC surface hydrophobicity has to be reduced to improve the aqueous fluid flow properties in PC channel networks. We have developed a method to increase polycarbonate surface hydrophilicity by UV irradiation using exposure to UV light. When exposed to UV light in air, the surface of PC polymer is oxidized, which leads to the introduction of functional groups and contributes to the increase of surface hydrophilicity.16-18 Some of these functional groups are peroxides, hydroxyl, carboxylic, or carbonylic groups.16-18 All of

Figure 1. (a) Schematic representation of polycarbonate microchip, (b) SEM image of compression-molded channel profile, (c) fluorescence image of the thermally bonded polycarbonate CE channel network filled with 100 µM fluorescein.

the UV-treated PC wafers exhibited no detectable change in mechanical properties. Figure 2 shows the contact angle between deionized water and UV-exposed PC surfaces. A larger contact angle is associated with higher hydrophobicity. PC surface hydrophobicity decreases with increased UV exposure time and reaches a constant level after ∼5 h of exposure. This could be due to polycarbonate surface saturation with charge-functional groups created by UV irradiation. The EOF flow velocities of different pH buffers were measured in native and UV-treated PC microchannels (Figure 3). In both cases, the EOF flow direction is from anode to cathode at the tested pH. This means that both surfaces contain negatively charged groups. EOF in UV-treated channels is higher than that in native PC channels under the same applied electric field and buffer condition. This supports literature suggesting that anionic carboxylic and carbonylic groups were generated on a PC (16) Rabek, J. F. In Polymer Photodegradation; Chapman and Hall: London, 1995, p 655. (17) Scott, G. Polymer Degradation and Stabilization; Elsevier Applied Science: London, 1990. (18) Allen, N. S.; Mckeller, J. F. Photo Chemistry of Manmade Polymers; Applied Science: London, 1979.

Figure 2. Effect of UV irradiation (220 nm) on contact angle between deionized water and UV irradiated polycarbonate wafer.

surface.16-18 As expected, EOF velocity increases linearly with the increase of applied field strength in both UV-treated and untreated PC channels. EOF mobility in native PC channels is independent of pH at the two measured pH valves of 7 and 9. This is similar to that reported for PMMA.19 EOF velocity measurements in native (19) Ford, S.; Kar, B.; Mcwhorter, S.; Davies, J.; Soper, S.; Klopf, M.; Calderon, G.; Saile, V. J. Microcolumn Sep. 1998, 10, 413-422.

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Figure 3. Effects of applied electric field strength and buffer pH on EOF of UV-irradiation-treated and native polycarbonate microchannels: (a) pH, 9; channel wall was UV-treated for 3 h; (b) pH, 7; channel wall was UV-treated for 3 h; (c) pH, 9; native channel wall; (d) pH, 7; native channel wall. Each data point in the plots is the average of three individual measurements.

PC channels at pH 4 were also attempted, but the EOF was too weak to obtain any statistically meaningful measurements. This could mean that the pKa of the ionizable groups on PC surfaces is above pH 4. The EOF mobility in native PC channels at pH 7 is 7 × 10-5 cm2/Vs. This is about 58% of the value reported for injection molded PMMA channels.19 EOF in UV-treated PC channels showed pH dependence, a characteristic that resembles that of glass channels. However, the EOF mobility of 2.7 × 10-4 cm2/Vs in UV-treated channels at pH 9 is still only about 29% of the value reported for glass. DNA Analysis in PC CE Chip. Figure 4a shows the separation of a DNA 100-bp marker on PC CE chips with 3 h of UV treatment prior to thermal bonding. Applying high electric field strength to DNA separation will result in poor separation of longer DNA fragments. Longer fragments tend to coelute because of the phenomenon of biased reptation with stretching.20 Therefore, only moderate electric field strength of 120-160 V/cm was used for DNA separation, although joule heating in 1× TBE buffer can be dissipated in our PC devices, in the range of 100-800 V/cm. All 11 fragments of the marker were very well resolved in 2% HEC sieving solution in 8 min. A few unknown peaks were also resolved from the marker mix (labeled with question marks). The additional unknown peaks could have originated from contamination by the original sample, because these unknown peaks were also observed while running the same sample on the Agilent bioanalyzer 2100 (500 DNA assay) and on the Caliper DNA LabChip. The CE chips used in this demonstration were 4 weeks old, and no signs of chip performance deterioration were observed. This means that the UV-irradiation-modified PC surface is stable under normal storage conditions. The separation efficiency for the PC CE devices was evaluated by the number of theoretical plates N. The N values for 300-bp and 500-bp fragments in a 1% HEC filled PC device at a field strength of 120 V/cm are 30 770 and 21 374, and those in glass CE devices under similar conditions are 71 795 (20) Slater, G. W.; Noolandi, J. Biopolymers 1985, 24, 2181.

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Figure 4. (a) DNA separation on PC CE devices: sample, 100-bp ladder; sieving matrix, 2% HEC; electric field strength, 160 V/cm; separation length, 2.8 cm; sample concentration, 3 µg/mL for the 500bp fragments and ∼1 µg/mL for the remaining 10 fragments. (b) Electrophoregrams of 7 consecutive separations of DNA pGEM marker: sieving matrix, 1% HEC; electric field strength, 120 V/cm; separation length, 2.8 cm; sample concentration, 10 µM.

and 25 455. The separation efficiency in PC devices is lower than that in glass devices. The decrease in efficiency is moderate for 500-bp fragments, but more dramatic for 300-bp fragments. The difference in efficiency between PC and glass devices was examined by comparing individual band-broadening contributors between these two types of devices. The contributions to the band broadening (σ2total) for both PC and glass devices are longitudinal diffusion (σ2diff), injection plug length (σ2inj), detector observation length (σ2det), and wall adsorption (σ2ads).21 The effects of joule heating were not considered, because heat was efficiently removed from these devices. Because both the PC and glass devices had the same channel design and were operated under the same conditions on the same CE detection system, the σ2diff, σ2inj and σ2det contribution to σ2total should be seen to be comparable. This suggests that the loss of efficiency could come from the DNA or DNA/dye complex adsorption onto the channel wall. If this were the case, then one would observe increases in PC autofluorescence due to dye adherence to the wall and significant tailing in the CE peaks. However, the peak shape in PC and glass devices was carefully examined, and no tailing or peak asymmetry was observed, neither were increases in autofluorescence. Therefore, DNA or DNA/dye adsorption onto PC channel walls was not considered to be likely. Further investigation will be carried out in the future to determine the causes of reduced efficiency in PC devices. The limit of detection on PC CE devices was also evaluated. The lowest sample concentration tested was 3.6 µM pGEM (21) Kenndler, E. In High Performance Capillary Electrophoresis; Khaledi, M. G., Ed.; Wiley-Interscience: New York, 1998, pp 25-76.

marker. At this concentration, the S/N ratio for the 126-bp fragments is ∼8 and that for the 2645-bp fragments is ∼49. The amount of TO-PRO-3 dye bonded to the DNA molecule is proportional to its size; therefore, larger DNA fragments can be detected with larger S/N ratios. We also observed that the runto-run reproducibility in terms of migration time and peak intensity improved with an increased number of CE runs. Figure 4b shows seven consecutive injections and separations of DNA pGEM marker from run 22 to run 28. All of the corresponding peaks overlap. For the 676-bp fragments, the relative standard deviations of the run time and peak intensity are 0.18 and 2.3%, respectively. The glass transition temperature for polycarbonate is about 145 °C; therefore, the devices made of this material are well-suited for PCR reactions, which require thermal cycling up to 94 °C. To test PCR/CE integration on polycarbonate devices, we adapted PCR/CE integration designs reported by Waters et al.14 Briefly, the sample reservoir of the CE device was used as the PCR chamber, and the entire chip was thermally cycled. CE separation was performed after PCR amplification. The targets were 500-bp fragments on λ DNA. The amplification reaction was cycled 25 times and analyzed using 2% HEC sieving matrix along with DNA f174 sizing marker. Coelectrophoresing PCR product with DNA marker eliminated the electrophoretic mobility variations caused by the variation of separation field strength, buffer strength, temperature, and sieving matrix. Both the 500-bp PCR product and the primer dimer (24 bp) were observed in the electropherogram (Figure 5a). The sizing curve (Figure 5b) was constructed by least-squares fitting of mobility data of f174 fragments from Figure 5a. The measured mobilities of the 500-bp PCR product and the 24-bp primer-dimer product are 9 × 10-5 and 18.3 × 10 -5 cm2/(V.s), respectively, and the measured values are within 3% of those predicted from the sizing curve. In conclusion, we have demonstrated the feasibility of CE DNA separation and PCR/CE analysis of DNA molecules in compression-molded polycarbonate microdevices. The cost associated with the PC microdevices could be further reduced using injection molding techniques. Because of relatively stronger intrinsic fluorescence background, PC CE devices do not match the sensitivity of glass devices; however, the use of the confocal microscope detection setup helps to reduce the out-of-focus PC fluorescence background. Surface treatment by UV irradiation

Figure 5. (a) Electrophoregram of PCR product mixed with a sizing ladder ΦΧ174-HaeIII digest: sieving matrix, 2% HEC; electric field strength, 120 V/cm; separation length, 2.8 cm. (b) Electrophoretic mobilities of the PCR product (2), primer-dimer product (9), and the sizing ladder ΦΧ174 vs fragment size. The solid cure is the leastsquares fit of the sizing ladder.

increases PC surface hydrophilicity. Thermal bonding was performed after this surface treatment, and aqueous fluid transport inside the treated PC microchannels was seen to improve. The modified surfaces retain good hydrophilic properties, even after exposure to elevated temperature. The optimal UV irradiation time is 3 h using a manual UV lamp (4 W). UV lamps or plasmas of higher output power will be tried in the future to shorten the time required for surface treatment. ACKNOWLEDGMENT This research is sponsored in part by DARPA BioMEMS program under contract F30602-98-2-0197 and ATP, NIST under contract 70NANB9H3012. The authors thank David Rhine and Doug Fayden for helpful discussion and technical assistance. Received for review March 22, 2001. Accepted June 26, 2001. AC010343V

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