AC Research
`
`Accelerated Articles
`
`Anal.Chem. 1996, 68, 4081-4086
`
`Functional Integration of PCR Amplification and
`Capillary Electrophoresis in a Microfabricated DNA
`Analysis Device
`
`Adam T. Woolley,† Dean Hadley,‡ Phoebe Landre,‡ Andrew J. deMello,†,§ Richard A. Mathies,*,† and
`M. Allen Northrup‡
`
`DepartmentofChemistry,UniversityofCalifornia,Berkeley,California94720,andMicrotechnologyCenter,L-222,Lawrence
`LivermoreNationalLaboratory,Livermore,California94551
`
`Microfabricated silicon PCR reactors and glass capillary
`electrophoresis (CE) chips have been successfully coupled
`to form an integrated DNA analysis system. This con-
`struct combines the rapid thermal cycling capabilities of
`microfabricated PCR devices (10 (cid:176)C/s heating, 2.5 (cid:176)C/s
`cooling) with the high-speed (<120 s) DNA separations
`provided by microfabricated CE chips. The PCR chamber
`and the CE chip were directly linked through a photo-
`lithographically fabricated channel filled with hydroxyeth-
`ylcellulose sieving matrix. Electrophoretic injection di-
`rectly from the PCR chamber through the cross injection
`channel was used as an “electrophoretic valve” to couple
`the PCR and CE devices on-chip. To demonstrate the
`functionality of this system, a 15 min PCR amplification
`of a (cid:226)-globin target cloned in M13 was immediately
`followed by high-speed CE chip separation in under 120
`s, providing a rapid PCR-CE analysis in under 20 min.
`A rapid assay for genomic Salmonella DNA was per-
`formed in under 45 min, demonstrating that challenging
`amplifications of diagnostically interesting targets can also
`be performed. Real-time monitoring of PCR target am-
`plification in these integrated PCR-CE devices is also
`feasible. Amplification of the (cid:226)-globin target as a function
`of cycle number was directly monitored for two different
`reactions starting with 4 (cid:2) 107 and 4 (cid:2) 105 copies of DNA
`template. This work establishes the feasibility of perform-
`ing high-speed DNA analyses in microfabricated inte-
`grated fluidic systems.
`
`The development of enhanced DNA amplification and analysis
`devices and methods having improved speed and reduced reagent
`
`† University of California at Berkeley.
`
`volumes will be critical for the completion of the Human Genome
`Project and for the subsequent utilization of
`this sequence
`information. The polymerase chain reaction (PCR)1 has advanced
`DNA amplification dramatically but currently requires long cycling
`times because of the large thermal mass of typical systems with
`concomitant slow heating and cooling rates. These problems have
`been addressed in part by placing samples in small glass capillaries
`and using heated air to drive the cycling,2 but the introduction
`and removal of small sample volumes and the handling of
`capillaries can be problematic. Similarly, while the throughput
`of conventional slab gel electrophoresis can be increased by using
`thinner slab gels3 or multiplex labeling,4,5 the labor-intensive steps
`of gel preparation, as well as sample loading, are still necessary.
`We6-12 and subsequently others13,14 have shown that capillary array
`electrophoresis can be used to dramatically increase the through-
`put of DNA sequencing6-8,11-13 and fragment sizing9,10,14 separa-
`
`‡ Lawrence Livermore National Laboratory.
`§ Current address: School of Chemical Sciences, University of East Anglia,
`Norwich, NR4 7TJ, England.
`(1) Taylor, G. R. In PCR: a Practical Approach; McPherson, M. J., Quirke, P.,
`Taylor, G. R., Eds.; Oxford University Press: Oxford, England, 1991; pp
`1-14.
`(2) Wittwer, C. T.; Fillmore, G. C.; Garling, D. J. Anal. Biochem. 1990, 186,
`328-331.
`(3) Kostichka, A. J.; Marchbanks, M. L.; Brumley, R. L., Jr.; Drossman, H.;
`Smith, L. M. Bio/Technology 1992, 10, 78-81.
`(4) Church, G. M.; Kieffer-Higgens, S. Science 1988, 240, 185-188.
`(5) Cherry, J. L.; Young, H.; Di Sera, L. J.; Ferguson, F. M.; Kimball, A. W.;
`Dunn, D. M.; Gesteland, R. F.; Weiss, R. B. Genomics 1994, 20, 68-74.
`(6) Mathies, R. A.; Huang, X. C. Nature 1992, 359, 167-169.
`(7) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 967-
`972.
`(8) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 2149-
`2154.
`(9) Clark, S. M.; Mathies, R. A. Anal. Biochem. 1993, 215, 163-170.
`(10) Wang, Y.; Ju, J.; Carpenter, B. A.; Atherton, J. M.; Sensabaugh, G. F.;
`Mathies, R. A. Anal. Chem. 1995, 67, 1197-1203.
`
`S0003-2700(96)00718-4 CCC: $12.00 © 1996 American Chemical Society
`
`AnalyticalChemistry,Vol.68,No.23,December1,1996 4081
`
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`
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`tions, compared to conventional slab gel electrophoresis. How-
`ever, the preparation and manipulation of large numbers of
`capillaries can be difficult, and sample introduction can be
`inefficient. It is evident that the most challenging and important
`issue in the submicroliter miniaturization of DNA analyses is the
`integration of the various amplification and analysis steps.
`Although a variety of microfabricated structures for PCR15-19
`and electrophoresis19-23 have been proposed and in some cases
`demonstrated, the integration of these two important processes
`into one functional system has not occurred. PCR amplification
`has been performed inside 4-12 (cid:237)L microfabricated Si-glass
`chambers placed in a larger thermal cycler,16,18 on a 25 (cid:237)L drop
`of solution on top of a microfabricated heater,19 and inside 20-50
`(cid:237)L microfabricated Si chambers with integrated heaters,15,17 but
`in each case the sample was removed for conventional external
`analysis. The Si chambers with integrated heaters15,17 are advan-
`tageous because they match the size of the thermal cycler to the
`sample volume, thereby reducing the time for amplification to as
`low as 30 s/cycle. Microfabricated capillary electrophoresis (CE)
`chips have been fabricated on glass substrates and used to
`perform separations of fluorescent dyes,20,22 labeled amino ac-
`ids,24,25 and short oligonucleotides.26
`In our own work, we
`demonstrated that CE chips can be used to perform very rapid
`(<120 s) separations of DNA restriction fragment digests and PCR
`products.23 We also demonstrated for the first time that DNA
`sequencing fragments could be separated on CE chips with single-
`base resolution.27 The integration of the amplification and analysis
`of DNA on-chip will require the development of methods for
`reliably fabricating multiple components on a single microdevice
`and controlling the transfer of DNA between systems. For
`example, electroosmotic flow,28,29 electrophoresis,30 and ther-
`mocapillary pumping19 have been used to move and mix solutions
`
`(11) Kheterpal, I.; Ju, J.; Radhakrishnan, A.; Brandt, G. S.; Ginther, C. L.; Clark,
`S. M.; Scherer, J. R.; Sensabaugh, G. F.; Mathies, R. A. In Ultrasensitive
`Biochemical Diagnostics; Cohn, G. E., Soper, S. A., Chen, C. H. W., Eds.;
`SPIE: Bellingham, WA, 1996; Vol. 2680, pp 204-213.
`(12) Kheterpal, I.; Scherer, J. R.; Clark, S. M.; Radhakrishnan, A.; Ju, J.; Ginther,
`C. L.; Sensabaugh, G. F.; Mathies, R. A. Electrophoresis, in press.
`(13) Takahashi, S.; Murakami, K.; Anazawa, T.; Kambara, H. Anal. Chem. 1994,
`66, 1021-1026.
`(14) Ueno, K.; Yeung, E. S. Anal. Chem. 1994, 66, 1424-1431.
`(15) Northrup, M. A.; Ching, M. T.; White, R. M.; Watson, R. T. In Digest of
`Technical Papers: Transducers 1993; IEEE: New York, 1993; pp 924-926.
`(16) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 1815-
`1818.
`(17) Northrup, M. A.; Gonzalez, C.; Hadley, D.; Hills, R. F.; Landre, P.; Lehew,
`S.; Saiki, R.; Sninsky, J. J.; Watson, R.; Watson, R., Jr. In Digest of Technical
`Papers: Transducers 1995; IEEE: New York, 1995; Vol. 1, pp 764-767.
`(18) Cheng, J.; Shoffner, M. A.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic
`Acids Res. 1996, 24, 380-385.
`(19) Burns, M. A.; Mastrangelo, C. H.; Sammarco, T. S.; Man, F. P.; Webster,
`J. R.; Johnson, B. N.; Foerster, B.; Jones, D.; Fields, Y.; Kaiser, A. R.; Burke,
`D. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5556-5561.
`(20) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.;
`Ludi, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-258.
`(21) Volkmuth, W. D.; Austin, R. H. Nature 1992, 358, 600-602.
`(22) Jacobson, S. C.; Hergenroeder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey,
`J. M. Anal. Chem. 1994, 66, 1107-1113.
`(23) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
`11348-11352.
`(24) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A.
`Science 1993, 261, 895-897.
`(25) Jacobson, S. C.; Hergenroeder, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal.
`Chem. 1994, 66, 4127-4132.
`(26) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994,
`66, 2949-2953.
`(27) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680.
`(28) Manz, A.; Effenhauser, C. S.; Burggraf, N.; Harrison, D. J.; Seiler, K.; Fluri,
`K. J. Micromech. Microeng. 1994, 4, 257-265.
`
`4082 AnalyticalChemistry,Vol.68,No.23,December1,1996
`
`on chips. Although many of these individual components have
`been developed, it has not been demonstrated that two funda-
`mentally different and important device functions such as PCR
`and CE can be functionally integrated onto a single device.
`We demonstrate here apparatus and methods that permit the
`direct integration of microfabricated PCR and CE components on
`a single microdevice. The functionality of this construct is
`demonstrated through PCR-CE analysis of (cid:226)-globin in under 20
`min and PCR-CE analysis of Salmonella DNA in under 45 min.
`Injection via an “electrophoretic valve” is performed, where the
`viscous hydroxyethylcellulose (HEC) sieving matrix in the inter-
`connecting channels on the CE chip prevents the flow of PCR
`reagents into the separation channel during thermal cycling, while
`still allowing us to electrophoretically drive DNA out of the PCR
`chamber for CE analysis. Finally, we show that these integrated
`devices offer the unique capability to perform real-time monitoring
`of PCR amplification. This work demonstrates the feasibility of
`fabrication and use of complex, integrated DNA analysis micro-
`devices.
`
`EXPERIMENTAL SECTION
`Microfabrication. Photolithography and etching of the CE
`chips was performed at the University of California, Berkeley
`Microfabrication Laboratory as described previously.23 Briefly,
`two CE systems consisting of an injection channel intersecting
`with a separation channel were fabricated on each device (Figure
`1A). The injection channel connected reservoirs 1 and 3 and the
`separation channel connected reservoirs 2 and 4. The separation
`channels were 100 (cid:237)m wide, 8 (cid:237)m deep, and 46 mm long, with a
`distance of 40 mm from the injection region to the end of the
`channel. The injection channels were 50 (cid:237)m wide, 8 (cid:237)m deep,
`and 12 mm long, with a distance of 2 mm from reservoir 3 to the
`intersection with the separation channel. After alignment, the
`etched bottom plate was thermally bonded to the top glass
`substrate which had access holes drilled over the etched reser-
`voirs. The drilled holes for reservoir 3 were 1.5 mm in diameter,
`while all other holes were 0.8 mm in diameter.
`The PCR chambers were fabricated at the Lawrence Livermore
`National Laboratory (LLNL) Microtechnology Center from cleaned
`1.0 mm thick double-sided polished silicon 100 wafers.17 After
`low-pressure chemical vapor deposition (LPCVD) of silicon nitride
`(1 (cid:237)m), the nitride was photolithographically patterned and
`reactive ion etched down to the silicon. The photoresist was
`removed, the silicon was etched to a depth of 850 (cid:237)m with KOH
`to form the chambers, and the wafers were cleaned again. LPCVD
`of polysilicon (3000 Å) was boron doped to a sheet resistance of
`400 ¿/square. The polysilicon was photolithographically pat-
`terned and etched, and the photoresist was removed. The
`polysilicon was again photolithographically patterned for the
`heater contacts followed by E-beam deposition of 2500 Å of gold
`(5 Å/s) on top of 100 Å of titanium (2 Å/s); after lift-off, the wafer
`was sawed. The chambers were formed by bonding two of the
`identical pieces together with polyimide (Epo-Tek 600, Epoxy
`Technology) and curing completely. The etched regions formed
`a tube of hexagonal cross section, extending the length of the
`reactor. A 30 AWG Teflon insulated thermocouple (type K,
`Omega, Stamford, CT) was affixed to one side of the reactor with
`
`(29) Seiler, K.; Fan, Z. H.; Fluri, K.; Harrison, D. J. Anal. Chem. 1994, 66, 3485-
`3491.
`(30) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723.
`
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`merase; and 106 or 108 starting copies of (cid:226)-globin DNA template
`in 50 (cid:237)L of solution were used. The rapid PCR experiments on
`(cid:226)-globin were performed with 30 cycles at 96 (cid:176)C for 2 s, 55(cid:176) C for
`5 s, and 72 (cid:176)C for 2 s. Two-step thermal cycling of (cid:226)-globin was
`carried out with steps of 96 (cid:176)C for 30 s and 60 (cid:176)C for 30 s.
`Genomic Salmonella DNA for PCR amplification was obtained
`from Dr. William Laegreid of the USDA/ARS. Amplification was
`performed using the S18 and S19 primers with 500 ng of genomic
`Salmonella DNA per 50 (cid:237)L of solution as described previously.31
`Thermal cycling of genomic Salmonella DNA was performed with
`35 cycles at 95 (cid:176)C for 10 s, 56 (cid:176)C for 15 s, and 72 (cid:176)C for 20 s.
`Control amplifications were carried out in a Perkin-Elmer 480
`thermal cycler (Foster City, CA).
`The CE separation medium was 0.75% (w/v) HEC in 1(cid:2) TAE
`buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.3) with 1 (cid:237)M of
`the intercalating dye thiazole orange to fluorescently label the
`DNA on-column.23 The PCR-CE microdevices were filled with
`HEC buffer via reservoir 4 (Figure 1A) by forcing the solution
`through all the channels of the assembled PCR-CE microdevice
`using a syringe. The PCR chamber formed by the insert and
`microfabricated heater coupled to the CE chip was rinsed with
`water five times, primarily to remove any EDTA introduced into
`the chamber when the channels were filled with HEC buffer. The
`PCR solution (20 (cid:237)L) was introduced into the rinsed chamber,
`which was then loosely capped to reduce evaporation during
`thermal cycling. The other three buffer reservoirs were formed
`by inserting cutoff pipet tips in the drilled holes and filled with
`HEC solution. Electrical contacts were established on the CE chip
`by inserting Pt wires into the reservoirs; however, the wire in the
`PCR chamber (reservoir 3) was removed during thermal cycling
`to avoid inhibition of the PCR amplification. No sample handling
`was necessary for the subsequent analysis. After PCR, 300 V/cm
`was applied between reservoirs 1 and 3 for 10 s with (cid:226)-globin (20
`s for Salmonella) to inject the amplified DNA into the separation
`channel; separation was performed by applying 200 V/cm between
`reservoirs 2 and 4.23 Two standard DNA sizing ladders, (cid:30)X174
`HaeIII (New England Biolabs, Beverly, MA) and a 50 bp ladder
`(Pharmacia, Piscataway, NJ), were used to verify sizes of the PCR
`products.
`Instrumentation. Laser-excited confocal fluorescence detec-
`tion was performed as described previously23,27 with minor
`modifications. Briefly, the 488 nm line from an argon ion laser
`was focused within the channel using a 32(cid:2) NA 0.4 long working
`distance objective (LD Achroplan 440850, Carl Zeiss, Thornwood,
`NY). Fluorescence was collected by the objective and passed
`through a dichroic beam splitter and a band-pass filter, followed
`by spatial filtering with a confocal pinhole prior to photomultiplier
`detection. Amplified photoelectron pulses were converted to an
`analog signal (0-0.3 V) using a home-built integrator. The analog
`signal was sampled at 10 Hz with a 16 bit ADC board (NB-MIO-
`16XL-18, National Instruments, Austin, TX) controlled by a
`program written in LabVIEW running on a Macintosh Quadra 700
`computer.
`The PCR chips were thermally cycled with an electronic
`controller based on a pulsed width modulator (PWM), designed
`and fabricated at LLNL. The PWM was controlled using a
`program written in IgorPro (Lake Oswego, OR) via an ADC board
`(National Instruments) in a Macintosh Quadra 650 computer.
`
`(31) Kwang, J.; Littledike, E. T.; Keen, J. E. Lett. Appl. Microbiol. 1996, 22,
`46-51.
`
`AnalyticalChemistry,Vol.68,No.23,December1,1996 4083
`
`Figure 1. Schematic of the integrated PCR-CE microdevice. (A)
`Laser-excited confocal fluorescence detection apparatus and an
`integrated PCR-CE microdevice. (B) Expanded view of the micro-
`fabricated PCR chamber. (C) Expanded cross-sectional view of the
`junction between the PCR and CE devices. The size of the epoxy-
`filled gaps is exaggerated for viewing clarity.
`
`thermally conductive epoxy (Tra-Bond BB2151, Tra-Con, Medford
`MA). Small wires were attached to the Au heater leads using
`conductive epoxy (Planned Products, Santa Cruz, CA). Dispos-
`able,
`thin-walled polypropylene liners for the PCR devices,
`fabricated at LLNL, were inserted down the length of
`the
`hexagonal-shaped tube in the reactor. The inserts were 2 cm tall
`narrow tubes with 1.0 mm inside diameter and 1.7 mm outside
`diameter, tapered to 1.5 mm outside diameter at the bottom, and
`expanding to a wider 4.0 mm opening at the top. The volume of
`solution held by an insert inside the PCR chamber was (cid:24)20 (cid:237)L.
`These plastic inserts helped avoid contamination of subsequent
`PCR experiments, as well as failure of PCR due to adsorption of
`reagents to the Si surface of the reactor.
`Coupling the PCR and CE Chips. The plastic liner was
`inserted in the PCR device so that it extended (cid:24)1 mm out through
`the bottom hole in the Si chamber. The protruding end was
`inserted into the drilled hole corresponding to reservoir 3 in the
`CE chip so there was a snug fit. Fast setting epoxy (Double
`Bubble, Hardman, Belleview, NJ) was applied around the con-
`necting region to form a leakproof union of the devices (Figure
`1C). Successful, leakproof integration of the devices occurred in
`19 of the 25 bondings. The PCR-CE microdevices were dis-
`assembled and then reassembled with a clean insert between
`subsequent amplifications to avoid contamination.
`PCR Amplification and Electrophoresis. PCR was per-
`formed on a 268 bp (cid:226)-globin target cloned in M13 using reagents
`provided by Roche Molecular Systems (Alameda, CA). Standard
`1(cid:2) PCR buffer (50 mM KCl and 10 mM Tris-Cl, pH 8.3) with 3
`mM MgCl2; 10% glycerol; 400 (cid:237)M dATP, dGTP, dCTP, and dTTP;
`0.5 (cid:237)M GH20 primer and PC04 primer; 2.5 units of Taq poly-
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`Figure 2.
`(top) Temperature of the PCR device as a function of
`time during a 15 min amplification of (cid:226)-globin. The temperature profile
`was 96 (cid:176)C for 2 s, 55 (cid:176) C for 5 s, and 72 (cid:176)C for 2 s, with a final 72 (cid:176)C
`extension step of 30 s. (bottom) Expanded view of the second and
`third cycles in the experiment. Typical cooling rates from melting to
`annealing temperature are 2.5 (cid:176)C/s; typical heating rates are (cid:24)10
`(cid:176)C/s, so that one cycle is completed in less than 30 s.
`
`During heating, the computer turned the PWM “full on” until the
`temperature of the reaction chamber reached the set point; then
`the PWM maintained the temperature to an accuracy of (0.5 (cid:176)C.
`When the heating cycle was completed, the PWM turned off, and
`the reaction chamber cooled passively. The PWM started to
`control again when the temperature reached the new set point.
`To speed the cooling step, a fan circuit was enabled and air was
`drawn along the heater surfaces of the reaction chamber. Prior
`to use, each PCR chamber was thermally calibrated by filling it
`with water and measuring the temperature of the reactor’s exterior
`and the temperature of the liquid inside the insert using a
`calibrated thermocouple for temperature set points from 45 to 95
`(cid:176)C at 10(cid:176) C intervals. This allowed the computer program to
`compensate for any difference between the solution temperature
`and the measured reactor exterior temperature. Additionally, the
`thermal cycling soak times were optimized to compensate for the
`small lag time between solution temperature and reactor exterior
`temperature, due to the reduced thermal conductivity of the insert.
`
`RESULTS AND DISCUSSION
`Figure 2 documents the reactor temperature as a function of
`time for the rapid PCR-CE analysis of (cid:226)-globin cloned in M13.
`The time for performing 30 cycles of amplification is 900 s. The
`lower panel shows the temperature profile for cycles 2 and 3 in
`this experiment in greater detail. Cooling from the melting
`temperature (96 (cid:176)C) to the annealing temperature (55 (cid:176)C) takes
`(cid:24)16 s, corresponding to a cooling rate of 2.5 (cid:176)C/s; the heating
`rates with the microfabricated PCR device are even faster, (cid:24)10
`
`4084 AnalyticalChemistry,Vol.68,No.23,December1,1996
`
`Figure 3. High-speed analysis of (cid:226)-globin on the integrated PCR-
`CE microdevice. (top) Chip CE separation of the (cid:226)-globin PCR product
`performed immediately after the 15 min thermal cycling shown in
`Figure 2. The primer-dimer peak (light gray) is visible at 60 s, and
`the PCR product peak (dark gray) appears at 83 s. Total time for
`analysis in the integrated PCR-CE microdevice was less than 20
`min. (bottom) CE chip sizing of the (cid:226)-globin PCR product (1:100
`dilution) after external mixing off-chip with a 50 bp ladder (1 ng/(cid:237)L).
`The primer-dimer peak and the 50 bp peak in the ladder overlap in
`this separation. The sizing was performed in a different CE chip that
`did not have a PCR chamber affixed to it.
`
`(cid:176)C/s. The time required for one complete cycle of melting,
`annealing and extension is between 25 and 30 s, compared to
`typical heating and cooling rates of (cid:24)1 (cid:176)C/s 1 and 2-6 min cycle
`times for conventional thermal cyclers.
`The top panel of Figure 3 presents the results of a high-speed
`the (cid:226)-globin target on the integrated PCR-CE
`analysis of
`microdevice. After thermal cycling, the sample was immediately
`injected electrophoretically; no pipetting or manual transfer of the
`mixture was required. The smaller primer-dimer peak was
`detected at 60 s, and the PCR product peak was detected (cid:24)83 s
`after injection. The entire time for integrated PCR-CE analysis of
`the (cid:226)-globin target was less than 20 min. The bottom portion of
`Figure 3 presents an electropherogram of the same PCR product
`mixture performed on a separate CE chip following external 1:100
`dilution of the product in water and spiking with a 50 bp sizing
`ladder. The PCR product peak and primer-dimer are detected
`along with the peaks of the 50 bp ladder. The (cid:226)-globin PCR
`product was sized to 266 bp using the known peaks of the standard
`ladder, confirming that the desired target was amplified (actual
`size 268 bp). The fragment migration times differ between these
`two runs because the separations were performed on different
`CE chips.
`Figure 4 demonstrates that the PCR-CE microdevice can also
`be used for the analysis of genomic Salmonella DNA.
`Im-
`mediately following thermal cycling, the sample was electro-
`phoretically injected for 20 s, and the separation shown in the
`
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`Figure 4. High-speed integrated PCR-CE microdevice assay of
`genomic SalmonellaDNA. (top) Chip CE separation of the Salmonella
`PCR product was performed immediately following a 39 min PCR
`amplification in the integrated PCR-CE microdevice. The primer-
`dimer peak (light gray) appears at 51 s and the PCR product peak
`(dark gray) appears at 61 s. Total analysis time for the Salmonella
`sample using the integrated PCR-CE microdevice was under 45 min.
`(bottom) Sizing of the SalmonellaPCR product (1:100 dilution) using
`(cid:30)X174 HaeIII DNA (1 ng/(cid:237)L) in a separate CE chip.
`
`top panel was obtained. The PCR product peak is visible at 61 s,
`along with the primer-dimer peak at 51 s. This integrated PCR-
`CE analysis for Salmonella required less than 45 min from initiation
`of PCR to the completion of the separation. The lower panel in
`Figure 4 shows an electropherogram of the PCR product mixture,
`diluted 1:100 in water and spiked with (cid:30)X174 HaeIII DNA, which
`was performed in a separate CE chip. The PCR product peak
`and the primer-dimer are visible among the peaks of the (cid:30)X174
`DNA standard. The excellent resolution and high speed in these
`CE chip separations are evidenced by the near-baseline resolution
`of the 271 and 281 bp fragments in under 75 s. The PCR product
`peak was sized to 164 bp versus the peaks of the standard, which
`confirms that amplification of the desired target occurred (actual
`size 159 bp).
`One of the unique capabilities of our integrated PCR-CE
`microdevice is the ability to perform real-time monitoring of PCR
`amplification. Figure 5 presents sequential electropherograms
`obtained of the same reaction mixture after 15, 20, 25, and 30
`cycles of amplification of the (cid:226)-globin target. After 15 cycles, the
`PCR product peak is rather small, and no primer-dimer is visible.
`The target peak continues to grow after 20 and 25 cycles, and
`the primer-dimer peak also is detected. By 30 cycles of
`amplification, the product peak growth has plateaued, and in
`addition to the primer-dimer peak, an artifact peak at 57 s is also
`detected. Figure 6 plots the logarithm of the PCR product peak
`area versus cycle number for this experiment, as well as for a
`similar experiment with 100-fold fewer starting copies of the
`(cid:226)-globin template. The signal approaches a plateau region as the
`reagents are consumed at higher cycle numbers for both 4 (cid:2) 107
`
`Figure 5. Real-time analysis of a (cid:226)-globin PCR amplification using
`an integrated PCR-CE microdevice. Chip CE separations of the
`same sample were performed sequentially in the integrated PCR-
`CE microdevice after 15, 20, 25, and 30 cycles at 96 (cid:176)C for 30 s and
`60 (cid:176)C for 30 s. The PCR product peak is shaded with dark gray and
`the false amplification and primer-dimer peaks are shaded with light
`gray.
`
`Figure 6. PCR amplification of the (cid:226)-globin target as a function of
`cycle number measured in an integrated PCR-CE microdevice. The
`logarithm of PCR product peak area as a function of cycle number
`for one sample containing 4 (cid:2) 107 starting copies ((cid:2)) and another
`sample containing 4 (cid:2) 105 starting copies (+) of the (cid:226)-globin DNA
`template is plotted.
`
`and 4 (cid:2) 105 starting copies of template. The plateau region occurs
`at a higher cycle number and lower signal strength for 4 (cid:2) 105
`starting copies, in agreement with previous work.32
`A fundamental step in the development of complex micro-
`fabricated DNA analysis systems is the demonstration that DNA
`amplification and analysis can be functionally integrated on a single
`microdevice. This successful integration is in many ways similar
`to the fabrication of the DNA analog of an integrated circuit. In
`this work, we have coupled a microfabricated PCR chamber and
`
`(32) Higuchi, R.; Fockler, C.; Dollinger, G.; Watson, R. Bio/Technology 1993,
`11, 1026-1030.
`
`AnalyticalChemistry,Vol.68,No.23,December1,1996 4085
`
`Agilent Exhibit 1270
`Page 5 of 6
`
`

`

`CE chip and shown that PCR and CE can be directly integrated
`(1) to perform rapid, hands-off DNA analysis, (2) to amplify targets
`from genomic DNA, and (3) to analyze PCR amplification in real
`time.
`These integrated PCR-CE microdevices have several clear
`advantages. Microfabricated PCR chambers perform fast thermal
`cycling of samples so amplification can be completed in as little
`as 15 min, and microfabricated CE chips perform electrophoretic
`analysis of the PCR product in under 120 s; therefore, these
`integrated devices have the potential to dramatically speed up PCR
`analysis of targets. The ability to amplify and detect PCR products
`from genomic DNA, such as Salmonella, demonstrates that
`integrated PCR-CE microdevices have the potential to be used
`for fast, remote diagnostics. Furthermore, our PCR-CE microde-
`vices are applicable to all types of PCR amplifications, because
`no complicated or expensive target-specific probe chemistry is
`required for fluorescence detection of the PCR product, which is
`distinguished from false amplification by electrophoretic separa-
`tion. This allows informative real-time monitoring of PCR, with
`considerably less expensive and less complex instrumentation than
`that previously used for real-time PCR monitoring.32 Finally,
`integration of the PCR amplifier and CE analyzer eliminates the
`manual transfer of liquid that normally occurs between amplifica-
`tion and separation, thereby simplifying the procedure, eliminating
`manual pipetting errors, and reducing opportunities for contami-
`nation.
`
`PROSPECTS
`The good results obtained with the junction we have fabricated
`here between the PCR and CE devices indicate the great potential
`for microfabricated integrated DNA analysis systems. A more
`sophisticated bonding between the devices, such as anodic
`bonding or polyimide, should lead to a system that is even easier
`to fabricate and use. Such improvements should also allow the
`fabrication of arrays of PCR chambers coupled to capillary arrays6
`on chips, which would increase the throughput of these micro-
`devices. In addition, although the “electrophoretic valve” works
`well in our application, more sophisticated devices with active
`microfabricated valves such as bistable polymer diaphragms33
`would improve injection efficiency. Simple improvements in the
`placement of the fan on our integrated microdevice could increase
`cooling rates to 5 (cid:176)C/s and thereby decrease the cycle times to
`below 20 s, so a 30 cycle amplification would require just 10 min.
`Further reduction in the size of the PCR chamber would allow
`even faster heating and cooling, eventually making the processivity
`of the DNA polymerase the limiting factor in the speed of PCR.
`An ideal PCR-CE microdevice would also integrate a method for
`spiking the separation of the PCR-amplified mixture with an
`external standard, thus eliminating any ambiguity caused by
`
`(33) Goll, C.; Bacher, W.; Bustgens, B.; Maas, D.; Menz, W.; Schomburg, W.
`K. J. Micromech. Microeng. 1996, 6, 77-79.
`
`variations in migration times. One way to address this issue would
`be to modify the current layout of the CE device by adding an
`additional channel for simultaneous electrophoretic loading of a
`standard DNA ladder. Analogous mixing devices based on
`electroosmotic flow25 and electrophoresis30 have been demon-
`strated.
`In summary, an integrated PCR-CE microdevice has been
`fabricated and shown to perform amplification and electrophoretic
`DNA analysis much faster than conventional techniques, with a
`complete absence of manual sample transfer. The demonstration
`of a functional integrated PCR-CE microdevice is an important
`step toward complete integration of DNA analyses on chips. The
`next challenges will be the integration of sample preparation and
`detection components on a single microdevice. These micro-
`fabricated integrated DNA analysis systems, the DNA analogs of
`integrated circuits, have the potential to significantly impact the
`Human Genome Project and molecular biology, just as integrated
`circuits have revolutionized electronics and computers.
`
`ACKNOWLEDGMENT
`We thank Stacy Lehew, Bill Benett, Bart Beeman, and Paul
`Stratton of
`the LLNL Microtechnology Center for technical
`assistance. We also thank Dr. William Laegreid of the USDA/
`ARS for providing the Salmonella DNA samples and Roche
`Molecular Systems for providing (cid:226)-globin PCR reagents. PCR
`chips, control electronics, and plastic inserts were designed and
`fabricated at the LLNL Microtechnology Center. CE chip fabrica-
`tion was performed at the University of California, Berkeley
`Microfabrication Laboratory. M.A.N. acknowledges the support
`of Dr. Ken Gabriel of the MEMS program of the Advanced
`Research Projects Agency and the collaboration of Roche Molec-
`ular Systems. Work at Lawrence Livermore National Laboratory
`was performed under the auspices of the U.S. Department of
`Energy, Contract W-7405-ENG-48. Work at U.C. Berkeley was
`supported in part by Grant 70NANB5H1031 from the Advanced
`Technology Project of the National Institute of Standards and
`Technology to Affymetrix, Inc. and Molecular Dynamics. Ad-
`ditional support was provided by the U.S. Department of Energy
`under Contract DE-FG-91ER61125 and by the National Institutes
`of Health (Grant HG01399). A.T.W. gratefully acknowledges th