A Miniature Analytical Instrument for Nucleic Acids Based on

MATCI is an integrated, miniaturized analytical system that uses ... Optical windows in the silicon and solid-state, diode-based detec- tion component...
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Anal. Chem. 1998, 70, 918-922

A Miniature Analytical Instrument for Nucleic Acids Based on Micromachined Silicon Reaction Chambers M. Allen Northrup*

Cepheid, 1190 Borregas Avenue, Sunnyvale, California 94089-1304 Bill Benett, Dean Hadley, Phoebe Landre, Stacy Lehew, Jim Richards, and Paul Stratton

Microtechnology Center, Lawrence Livermore National Laboratory, Livermore, California 94551

In this paper, we describe a miniature analytical thermal cycling instrument (MATCI) to amplify and detect DNA via the polymerase chain reaction in real-time. The MATCI is an integrated, miniaturized analytical system that uses silicon-based, high-efficiency reaction chambers with integrated heaters and simple, inexpensive electronics to precisely control the reaction temperatures. Optical windows in the silicon and solid-state, diode-based detection components are employed to perform real-time fluorescence monitoring of product DNA production. The entire system fits into a briefcase and runs on rechargeable batteries. The applications of this miniaturized nucleic acid analysis system include clinical, research, environmental, and agricultural analyses as well as others which require rapid, portable, and accurate analysis of biological samples for nucleic acids. This paper describes the MATCI and presents results from ultrafast thermal cycling and real-time PCR detection. Examples include human genes and pathogenic viruses and bacteria.

circuit (IC) manufacturing technology to pattern and fabricate microstructures in single-crystal silicon (SCS) wafers or other (i.e., glass) substrates. The miniaturization of chemical reaction chambers affords advantages in terms of control, speed, and efficiency. This has been shown with simple micromachined chemical reactors with integrated heaters for the polymerase chain reaction (PCR).16-21 The PCR process is well-known to be generated in vitro in abundance.22-24 Existing commercially available thermal cycling instruments limit replication and analysis of DNA samples to the laboratory because of the bulk of the instrumentation systems and their high power requirements. These constraints are dictated by the technological impediments imposed by the mass

* To whom correspondence should be directed. Formerly at Lawrence Livermore National Laboratory. (1) Petersen, K. E. Proc. IEEE 1982, 70, 420-457. (2) Manz, A.; Gaber, N.; Widmer, H. M. Sens. Actuators 1990, B1, 244-248. (3) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Ludi, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-256. (4) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (5) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642. (6) Manz, A.; Effenhauser, C. S.; Burggraf, N.; Harrison, D. J.; Sieler, K.; Fluri, K. J. Micromechan. Microeng. 1994, 4, 257-265. (7) Seiler, K.; Fan, Z. H.; Fluri, K.; Harrison, D. J. Anal. Chem. 1994, 66, 34853491.

(8) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113. (9) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (10) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680. (11) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723. (12) Harrison, J. E. Presented at the Microfabrication and Microfluidics Conference, Aug 11-13, 1996; Sponsored by International Business Communications Inc., Southborough, MA. (13) Northrup, M. A.; Ching, M. T.; White, R. M.; Watson, R. T. Transducers ‘93, Seventh International Conference on Solid State Sensors and Actuators, Yokohama, Japan, 1993; pp 924-927. (14) Northrup, M. A.; Hills, R. F.; Landre, P.; Lehew, S.; Hadley, D.; Watson, R. Transducers ‘95, Eighth International Conference on Solid State Sensors and Actuators, Stockholm, Sweden, 1995; pp 764-767. (15) Kidd, J.; Stilwell, J.; Northrup, M.; Segraves, M.; Lamerdin, J.; Strout, C.; Carrano, A. Abstract in American Society of Microbiology Conference Proceedings, New Orleans, LA, May 19-23, 1996. (16) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (17) Northrup, M. A.; Beeman, B. R. F.; Hadley, D.; Landre, P.; Lehew, S. Analytical Methods and Instrumentation, Special Issue on MicroTAS; Widmer, H. M., Ed. (Ciba Geigy); Basel, 1996; pp 153-157. (18) Northrup, M. A.; Beeman, B.; Hadley, D.; Landre, P.; Lehew, S. In Automation Technologies for Genome Characterization; Beugelsdijk, T. J., Ed.; John Wiley & Sons: New York, 1997; Chapter 9, pp 189-204. (19) Ibrahim, S.; Lofts, R. S.; Henchal, E. A.; Jahrling, P.; Esposito, J.; Weedn, V. W.; Northrup, M. A.; Belgrader, P. Submitted to Anal. Chem.. (20) Belgrader, P.; Smith, J. K.; Weedn, V. W.; Northrup, M. A. J. Forens. Sci. 1998, 43 (3), 315-319. (21) Northrup, M. A.; Beeman, B.; Landre, P.; Hadley, D.; Fisher, M.; Constantine, N.; Oldach, D. Manuscript in preparation, 1997. (22) Mullis, K.; Falonna, F. Methods Enzymol. 1987, 155, 335-350. (23) Saiki, R. K.; Gelfand, D. H.; Stoeffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Science 1988, 239, 487-491. (24) Gibbs, R. A. Anal. Chem. 1990, 62, 1202-1214.

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S0003-2700(97)00486-1 CCC: $15.00

The applicability of silicon micromachining to manufacturing miniature physical structures was pointed out in 1982 by Petersen.1 Since then, the development of miniaturized systems for DNA analysis that use silicon microfabrication technology has become an area of active interest. It has been shown that significant advantages in speed can be gained through the application of such microelectromechanical systems (MEMS) to analytical instrumentation, especially in separation systems such as chromatography or electrophoresis.2-12 MEMS involves the use of integrated

© 1998 American Chemical Society Published on Web 02/03/1998

Figure 1. Photograph of the briefcase-sized, rechargeable, batteryoperated, portable MATCI.

and thermal conductivity of the reaction chambers in which the enzymatic amplification of nucleic acid sequences occurs. High thermal conductivity reaction chambers, which can be fabricated with MEMS technology, have advantages for reactions, in general. Silicon-based materials used in micromachining have desirable thermal characteristics (i.e., thermal conductivity constants ranging from 3.2 to 150 W M-1 K-1) that can be used in combination to allow low-power operation, high ramp speed, and thermal isolation and uniformity. Thin-film deposition processes, for example, can also be used for the integration of components such as resistive heaters and optical windows onto the reaction chambers. PCR is an excellent example of a biochemical reaction that uses instrumentation that lends itself to miniaturization through the application of MEMS technology. Silicon-micromachined reaction chambers with integrated heaters and optical windows have been incorporated into a miniature analytical thermal cycling instrument (MATCI). The application of this instrument will be shown for a variety of biological samples. EXPERIMENTAL SECTION Real-Time Detection of PCR Product by the MATCI. Direct detection of PCR product was attained during PCR by monitoring the increase of fluorescence of two dye-labeled DNA probes (Taqman assay) or by monitoring the increase of ethidium bromide fluorescence. The fluorescence signals from the sample transmit through the transparent polypropylene reaction tube and then pass through the windows etched into the reaction chamber. The optical detection apparatus, software, and control electronics consist of low-cost components and have been described.18 Detection filters were band-pass filtered and centered at 540 ( 15 and 590 ( 15 nm for the fluorescein and rhodamine (or ethidium bromide) labels, repectively. A mineral oil layer over the PCR reaction fluid prevented evaporation during thermal cycling. The complete briefcase-packgaged MATCI is shown in Figure 1. Data output is in terms of diode detector voltage on wavelengthfiltered channels, or a simple wavelength ratio is computed

simultaneously and plotted at each cycle. For the data here (except HIV), a three-point running boxcar average was used to smooth the graphs. The HIV data are nonsmoothed, raw voltage data. To reduce background electronic noise, a factor of 1 was subtracted from the 540/590 ratio. PCR Amplification and Detection of Hantavirus. PCR amplification and detection of hantavirus from total RNA isolated from rodent blood was done using a nested RT-PCR method (materials supplied and methods modified from T. Gutierrez, USACHPPM, Aurora, CO). The procedure and reagents are from Hjelle.25 The RT-PCR mix consisted of 1× Boehringer-Mannheim Taq buffer (10 mM Tris, 1.5 µM MgCl2); 40 mM Tris buffer, pH 8.3; 7 µM β-mercaptoethanol; 200 µM dATP, dGTP, dTTP, and dCTP; 15 pM each of forward and reverse primers (Har M + 1, Har M - 403); 10 units of AMV-RT; 2.5 units of Taq polymerase; and 5 µL of RNA sample in a 50-µL volume. Reverse-transcriptase reactions consisted of a 39 °C hold for 20 min and a 42 °C hold for 40 min. The reverse-transcriptase reaction was then thermal cycled in the MATCI for 8 cycles (97 °C for 5 s, 41 °C for 10 s, 72 °C for 10 s) and then thermal cycled for 29 cycles (97 °C for 5 s, 46 °C for 10 s, 72 °C for 10 s). The product of this PCR (403-bp PCR product) was used to seed a second PCR consisting of 1× Boehringer-Mannheim Taq buffer (10 mM Tris, 1.5 µM MgCl2); 200 µM dATP, dGTP, dTTP, and dCTP; 0.5 µM each of forward and reverse primers (Har M + 49, Har M - 369, 321 bp product); 2 µg/mL ethidium bromide (Sigma); 2.5 units of Taq polymerase; and 1 µL of PCR product from the first reaction in a 50-µL volume. The second PCR was thermal cycled in the MATCI for 8 cycles (97 °C for 5 s, 42 °C for 10 s, 72 °C for 10 s) and then thermal cycled for 29 cycles (97 °C for 5 s, 48 °C for 10 s, 72 °C for 10 s). PCR Amplification and Detection of Borrelia burgdorferi. The reagents for detection of the lyme disease-causing spirochete bacteria were kindly provided by T. Gutierrez of the USACHPPM (Aurora, CO). That PCR chemistry has been published.26 Ethidium bormide was added at 2 µg/mL for fluorescence monitoring. Cycling was quite rapid in these examples (93, 72, and 50 °C for 5 s each). PCR Amplification and Detection of Human β-actin. PCR amplification of a 294-bp human β-actin target from human genomic DNA was done using reagents designed for the Taqman assay by Perkin-Elmer/Applied Biosystems Corp. (Foster City, CA). The starting concentration was 10 ng/50 µL. Reaction conditions were 1× PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3); 3.5 µM MgCl2; 200 µM dATP, dGTP, dTTP, and dCTP; 300 nM each of forward and reverse β-actin primers; 300 nM Taqman probe; 5 units of Taq polymerase; and 0.01-10-ng starting concentration of human genomic DNA in a reaction volume of 50 mL. Thermal cycling conditions consisted of a 3-min hold at 96 °C and then 40 cycles of 15 s at 96 °C, 15 s at 55 °C, and 60 s at 72 °C. Real-time detection of Taqman fluorescence was done as described above. Fast PCR and Taqman detection of the human β-actin target were performed using a 3-min hold at 96 °C and then 40 cycles of 5 s at 96 °C, 15 s at 55 °C, and 15 s at 72 °C. PCR Amplification and Detection of HIV. A HIV isolate (tissue culture supernatant) was obtained from the NIH AIDS Repository (reference standard no. 1650). HIV control RNA was (25) Hjelle, B. In PCR protocols for Emerging Diseases; Persing, D. H., Ed.; American Society for Microbiology: Washington, DC, 1996; pp 91-99. (26) Rosa, P. A. J. Infect. Dis. 1989, 160 (6), XXX-XXX.

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Table 1. Heating and Cooling Rates and Time To Reach the Target Temperature as a Function of Input Voltage (14, 28, and 35 V dc)a cycle segment

14 V w/fan

60 f 72 °C 72 f 94 °C 94 f 60 °C

2.4 (5) 2.4 (9) 2.6 (13)

rate (°C/s) (time (min)) 28 V w/fan 35 V w/fan commercial27 15 (0.8) 18 (1.2) 3.8 (9.6)

30 (0.4) 28 (0.8) 4 (8.4)

4.8 (4) 3.4 (5) 4 (9)

a The individual rates are shown for typical denature, anneal, and extension temperatures. Rates such as these used to attain rapid thermal cycling on the MATCI. Comparative rates from the instrument of ref 27 are also shown.

in vitro transcribed from plasmid, and total RNA concentrations were determined by UV spectroscopy. PCR was performed in 20-µL reaction volumes containing 50 mM Tricine; 13.04% glycerol; 110 mM KOAc; 2.4 mM Mn(OAc)2; 5.0 units/reaction rTth DNA polymerase; 0.5 unit/reaction uracil N-glycosylase (UNG); 300 µM dATP, dGTP, dCTP; 50 µM dTTP; and 500 µM dUTP. HIVspecific primers (HIV1/HIV2) were provided by Roche Molecular Systems (Alameda, CA) and were added at the various concentrations of 0.5 µM/primer. HIV 5′-3′ exonuclease assay hybridization probes specific for the reaction were also provided by Roche Molecular Systems and were present at a concentration of 0.25 µM. Template RNA was added in a volume of 12 µL of DPECtreated H2O. Mineral oil overlays were used. The cycling parameters were performed using the following: 50 °C × 2 min (UNG step), followed by 60 °C × 30 min (room temperature step), followed by 5 cycles of 30 s each at 95, 55, and 72 °C, followed by 45 cycles of 30 s each at 91, 55, and 72 °C, with a final extension stage of 72 °C for 10 min. RESULTS Reaction Chambers. Numerous silicon reaction chambers have been designed and tested for functionality and performance in performing PCR on a variety of samples (viruses, bacteria, human DNA, plants). Previous reaction chamber versions had a single heater design with a glass top.13 However, all recent designs have two-heater chambers.14-21 Excellent thermal uniformity is achieved in the silicon chamber, allowing for highefficiency reactions. Table 1 shows examples of heating and cooling rates obtained at different input voltages on the MATCI. Voltage inpouts of 14, 28, and 35 V were able to attain heating rates of 30 °C/s from 60 to 72 °C. Cooling with the fan was typically 4.0 °C/s. For example, 35 cycles can be performed in 10 min (1 s anneal and denature, 2 s extension). A 268-bp PCR target from the β-globin gene on human cell line genomic DNA was successfully amplified with these conditions. Concerning the biocompatibilty of the chambers, the following trends have been observed: (1) silanization (APTS) of the surfaces (silicon, silicon dioxide, and silicon nitride) improves reaction fidelity; (2) chambers can be reused if they are cleaned with a combination of sodium hydroxide (10%) followed by heating and rinsing, or hydrochloric acid (1 N) followed by repetitive rinsing and heating; (3) addition of bovine serum albumin (BSA) to the reaction fluid occasionally improves reaction fidelity, but is not necessary; and (4) various polymer liners significantly enhance reaction fidelity, the best material being polypropylene (which is 920 Analytical Chemistry, Vol. 70, No. 5, March 1, 1998

Figure 2. Real-time ethidium bromide fluorescence detection of hantavirus PCR products from a total RNA extraction from rodent blood. The data represent the 540/590-nm emission ratio with a threepoint boxcar running average. These data represent the second PCR (after an intial RT step) and were thermal cycled in the MATCI for 8 cycles (97 °C for 5 s, 42 °C for 10 s, 72 °C for 10 s) and then thermal cycled for 29 cycles (97 °C for 5 s, 48 °C for 10 s, 72 °C for 10 s).

the same material as used in commercial thermal cyclers for PCR). The liners have the added value of making the silicon reaction chambers 100% reusable and eliminate the need for a thin-film window in order to contain the reaction fluid during real-time monitoring. All the data presented here were obtained in reaction chambers with polypropylene liners. Amplification Rates and Efficiencies. The advantages of increasing the speed of the PCR thermal cycling process have been shown in a large tabletop instrument.27,28 In the MATCI, anneal temperatures and extension times were also shown to have such effects. The average power of this instrument during thermal cycling was 1.2 W. Rapid thermal cycling was also performed. For example, by increasing the input power to 30 V (0.8 A, peak), heating rates of 30 °C/s (50 µL) have been attained. Significant productivity of PCR (human β-globin gene) have been shown with the MATCI with fast cycling conditions (i.e., 3.5 cycles/min; 1 s denature and anneal, and 2 s extension).18 The MATCI allows for true optimization due to its rapid themal cycling rates. Improvements in multiplex PCR-based identification of pathogenic bacteria with accelerated cycle times have also been shown.15 Real-Time Product Detection. Two real-time fluorescent PCR assays were employed in the MATCI: (1) monitoring doublestranded DNA production via ethidium bromide intercalation and fluorescence enhancement similar to Higuchi et al.29 and (2) specific probe with energy transfer dyes30-32 called “Taqman”. All reactions were confirmed with the appropriate negative controls. Examples of the results from ethidium bromide monitoring are shown in Figures 2 and 3. That PCR assay was for the detection of Sin Nombre Hantavirus and included a reverse-transcriptase (27) Wittwer, C. T.; Fillmore, G. C.; Garling, D. J. Anal. Biochem. 1990, 186, 328-331. (28) Wittwer, C. T.; Ririe, K. M.; Andrew, D. A.; David, D. A.; Gundry, R. A.; Balis, U. J. BioTechniques 1997, 22 (1), 176-181. (29) Higuchi, R.; Fockler, C.; Davinger, G.; Watson, R. Bio/Technology 1993, 11, 1026-1030. (30) Holland, P.; Abramson, R. D.; Watson, R.; Gelfand, D. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7276-7280. (31) Lee, L. G.; Connelly, C. R.; Bloch, W. Nucleic Acids Res. 1993, 21, 37613766. (32) Livak, K. J.; Flood, S. J. A.; Marmaro, J.; Giusti, W.; Deetz, K. PCR Methods Appl. 1995, 4 (3), 357-362.

Figure 3. Real-time ethidium bromide fluorescence detection of Borellia burgdorferi PCR products from purified DNA. The data are a simple plot of the 590-nm emission with a three-point boxcar running average. The instrument was cycled at 5, 5, and 5 s at anneal, denature, and extend. Total cycle time was about 2 cycles/min.

conversion of the RNA to DNA prior to PCR (RT-PCR) (Figure 2). In those data, the ratio 540/590 decreases due to intercalation of ethidium bromide into increasing amounts of PCR products and subsequent fluorescence emission enhancement at 590 nm. Figure 3 shows real-time ethidium bromide monitoring of a twostep dilution of a PCR assay for the cause of lyme disease or the parasite Borrelia burgdorferi. It can be seen that the signal risees above background (3×) at about 11 cycles for the concentrated sample and at 14 cycles for the 100-fold dilution, indicating the quantitative nature of this technique when run on the MATCI. The PCR product of these assays is typically carried out via agarose gel electrophoresis and subsequent staining of the gel which, with standard PCR instrumentation, could take 4-5 h. It can be seen from the real-time monitoring that the entire PCR amplification and detection took less than 20 min. In fact, the Borrelia burgdorferi samples were identified in less than 10 min. The increase in speed and the portability of the MATCI make field identification and detection of pathogens possible. The Taqman assay for the human β-actin gene on human cellline genomic DNA was performed with both “normal” (118 s/cycle) and fast (70 s/cycle) thermal cycling conditions (Figure 4). The fast ramp rates and reduced times at the extension and denature temperatures allowed for the specific detection of the 294-bp human β-actin target from human genomic DNA in less than 50 min. The quantitative nature of this assay when performed on the MATCI is illustrated in Figure 5 on a dilution series of a NIH HIV standard. This HIV standard (reference no. 1650) was diluted by 1:100, and then by 1:100 000, and run on the MATCI in a Taqman assay. The lowest concentration represents an estimated infectious viral load count of approximately 20 organisms. It appears that HIV detection systems incorporating “single-

Figure 4. Normal speed (118 s /cycle) and fast (70 s/cycle) thermal cycling results obtained with Taqman probes for the β-actin gene on human DNA. Thermal cycling conditions consisted of a 3-min hold at 96 °C and then 40 cycles of 15 s at 96 °C, 15 s at 55 °C, and 60 s at 72 °C. Fast PCR and Taqman detection of the human β-actin target were performed using a 3-min hold at 96 °C and then 40 cycles of 5 s at 96 °C, 15 s at 55 °C, and 15 s at 72 °C.

tube” reactions for both RT and DNA amplification (with single primer pairs) can be used as two-step RT/cDNA amplification strategies utilizing nested primers. Rapid, quantitative, homogeneous assays such as this, coupled with the portability, allows for portable detection of specific nucleic acids or genetic diseases in the field, in doctors’ offices, or in medical clinics. DISCUSSION Some groups have developed simple silicon reaction chambers for PCR,33-35 but without integration of functionalities such as heaters. Micromachined, thin-film heaters such as those used in the MATCI allow for extremely fast thermal response at low power. However, integration of heaters onto the bulk silicon can result in differences of thermal response and unformity.17 In fact, due to thermal inhomogeneities of micromachined reactors with integrated heaters on a large substrate, large variations (factor of 4) in PCR production can occur.36 Particular attention needs to be paid to both thermal uniformity and isolation from conducting bulk substrates in order to get high-quality production without mispriming artifacts. A large variety of DNA and RNA targets (33) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 18151818. (34) Cheng, J.; Shoffner, M. A.; Hvichia, G. E.; Kricka, L. J.; Wilding. P. Nucleic Acids Res. 1996, 24, 380-385. (35) Shoffner, M. A.; Cheng, J.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 375-379. (36) Burns, M. A.; Mastrangelo, C. H.; Sammarco, T. S.; Man, F. P.; Webster, J. R.; Foerster, B. N.; Jones, D.; Fields, Y.; Kaiser, A. R.; Burke, D. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5556-5561.

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Figure 5. Taqman detection of a quantitaitve series of dilutions (1: 1000; and 1:100 000) of NIH HIV reference standard no. 1650. Taqman probes and primers were provided by Roche Molecular Systems. The initial reverse-transcription step was performed homogeneously in the same reaction mixture. The cycling parameters were performed using the following: 50 °C × 2 min (UNG step), followed by 60 °C × 30 min (RT step), followed by 5 cycles of 30 s each at 95, 55, and 72 °C, followed by 45 cycles of 30 s each at 91, 55, and 72 °C, with a final extension stage of 72 °C for 10 min.

have been amplified and/or detected in the MATCI, including human immunodeficiency virus (HIV) and HCV RNA, other pathogenic viruses and bacteria, plants, and human genetic diseases and fingerprints.13-21 PCR product analysis is typically carried out as an end-point analysis via fluorescence staining and imaging of an agarose electrophoresis gel. This has been miniaturized, as represented by the microelectrophoresis work.2-12 An alternative PCR analysis method, electrochemiluminescence, has also been miniaturized with micromachined components.37 Direct coupling and integration of the PCR chamber onto a microelectrophoresis system have also been performed.16 In that work, fast PCR (35 cycles in 20 min) and fast electrophoretic (less than 2 min) separations with intercycle electrokinetic injections were able to show real-time, exponential accumulation of PCR products. The “Taqman” ap(37) Hsueh, Y.-T.; Smith, R. L.; Northrup, M. A. Sens. Actuators 1996, B33, 110114.

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proach, however, is both highly specific and quantitative but requires longer thermal cycles. Rapid detection of blood-borne pathogens could be extremely valuable in particular clinical contexts, such as organ transplantation and occupational blood exposure, where difficult decisions are frequently required, often without the benefit of data derived from traditional time-consuming laboratory assays. New fluorescence-based, real-time PCR analysis approaches are emerging which have been demonstrated with the large commercial systems. The results shown in this work on the MATCI indicate that nucleic acid analysis can be performed in a truly portable format with battery operation, fast thermal cycling, and true real-time quantitative detection with solid-state components. These are a unique set of advantages which present commercial systems do not have. For the first time, quantitative PCR analysis has been performed in a briefcase without the need for an external power supply. Replacement of high-power and high-cost components such as lasers and photomultipliers or high-power heaters and cooling mechanisms with low-cost, high-efficiency components as demonstrated also indicates that less expensive analytical tools for nucleic acids are feasible. ACKNOWLEDGMENT The authors would like to acknowledge the following persons for their valuable input, expertise, and collaboration: David Oldach and Neil Constantine of The University of Maryland Medical School for the HIV samples; John Sninsky, Robert Watson, and Mary Fisher of Roche Molecular Systems for the HIV primers and probes; and Tony Gutierrez, Judy O’Brien, and Mark Randolph of the U.S. Army Center for Health Promotion and Preventative Medicine (USACHPPM) for the hantavirus and Borellia burgforeri PCR reagents. The work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory, Contract No. W-7405-ENG-48. Received for review May 12, 1997. Accepted December 1, 1997. AC970486A