Anal. Chem. 1996, 68, 720-723
Integrated Microdevice for DNA Restriction Fragment Analysis Stephen C. Jacobson and J. Michael Ramsey*
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142
An integrated monolithic device (8 mm × 10 mm) that performs an automated biochemical procedure is demonstrated. The device mixes a DNA sample with a restriction enzyme in a 0.7-nL reaction chamber and after a digestion period injects the fragments onto a 67-mmlong capillary electrophoresis channel for sizing. Materials are precisely manipulated under computer control within the channel structure using electrokinetic transport. Digestion of the plasmid pBR322 by the enzyme HinfI and fragment analysis are completed in 5 min using 30 amol of DNA and 2.8 × 10-3 unit of enzyme per run.
Important problems in biology and medicine will benefit from the ability to perform automated, rapid, and precise biochemical procedures on minute quantities of material in a highly parallel fashion. For example, genome sequencing is a mammoth chemical analysis problem under the best of conditions; the recent efficient random sequencing of the whole genome for the bacterium Haemophilus influenzae Rd involved over 30 000 sequencing reactions to determine the 1.8 million base pair sequence.1 Linear extrapolation to the human genome would predict over 50 million sequencing reactions. The generation of combinatorial libraries as an approach to drug discovery is another example where the ability to perform chemical procedures under computer control on microdevices at a massively parallel scale would be of great benefit.2 Microfabrication techniques have been employed to try to address some of these daunting problems. Microfabricated components for biological manipulations with micrometer-sized features include arrays for solid-phase chemistry,3 reaction wells for polymerase chain reactions,4 immobilized enzyme reactors,5 a two-dimensional obstacle course for electrophoretic sizing of DNA fragments,6 and a stacked module for flow injection analysis.7 There is also promise that microfabricated components can be integrated into a single device to solve a (1) Fleischmann, R. D.; Adams, M. D.; White, O.; Clayton, R. A.; Kirkness, E. F.; Kerlavage, A. R.; Bult, C. J.; Tomb, J.-F.; Dougherty, B. A.; Merrick, J. M.; McKenney, K.; Sutton, G.; FitzHugh, W.; Fields, C.; Gocayne, J. D.; Scott, J.; Shirley, R.; Liu, L.-I.; Glodek, A.; Kelley, J. M.; Weidman, J. F.; Phillips, C. A.; Spriggs, T.; Hedblom, E.; Cotton, M. D.; Utterback, T. R.; Hanna, M. C.; Nguyen, D. T.; Saudek, D. M.; Brandon, R. C.; Fine, L. D.; Fritchman, J. L.; Fuhrmann, J. L.; Geoghagen, N. S. M.; Gnehm, C. L.; McDonald, L. A.; Small, K. V.; Fraser, C. M.; Smith, H. O.; Venter, J. C. Science 1995, 269, 496. (2) Chu, Y.; Avila, L. Z.; Beibuyck, H. A.; Whitesides, G. M. J. Org. Chem. 1993, 58, 648. (3) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767. (4) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 1815. (5) Murakami, Y.; Takeuchi, T.; Yokoyama, K.; Tamiya, E.; Karube, I.; Suda, M. Anal. Chem. 1993, 65, 2731. (6) Volkmuth, W. D.; Austin, R. H. Nature 1992, 358, 600.
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complete chemical or biochemical procedure. A simple but powerful example of an integrated device for performing chemical reactions and separations have been demonstrated.8 Here, we demonstrate a monolithic integrated device for performing a biochemical analysis procedure. The advantages of integrated devices that perform chemistry and chemical analysis may be quite similar to those realized by the microelectronics industry through the integrated circuit.9 Potential advantages include low-cost, compact devices with highspeed processing while operational simplicity and reliability are improved and the added benefit of parallel architectures for solving large problems. Moreover, integration of chemical processing and analysis functions allows automated manipulation of samples and reagents at volumes orders of magnitude smaller than is feasible manually or robotically. Miniaturized devices that have been fabricated primarily involve electrically driven separation techniques including capillary electrophoresis,10-15 synchronized cyclic electrophoresis,16 freeflow electrophoresis,17 open channel electrochromatography,18 and capillary gel electrophoresis.19-21 The first devices that integrated chemical reactions with analysis included capillary electrophoresis with pre- and postseparation derivatization.8,22 These devices have exhibited the features mentioned above. To demonstrate a useful biological analysis procedure, a restriction digestion and an electrophoretic sizing experiment are performed sequentially onchip. After digestion, determination of the fragment distribution is performed by separating the digestion products using electro(7) Fettinger, J. C.; Manz, A.; Lu ¨di, H.; Widmer, H. M. Sens. Acuatators B 1993, 17, 19. (8) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127. (9) Ramsey, J. M.; Jacobson, S. C.; Knapp, M. R. Nature Med. 1995, 1, 1096. (10) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926. (11) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Lu ¨ di, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253. (12) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481. (13) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895. (14) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107. (15) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114. (16) Burggraf, N.; Manz, A.; Effenhauser, C. S.; Verpoorte, E.; de Rooij, N. F.; Widmer, H. M. J. High Resolut. Chromatogr. 1993, 16, 594. (17) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2858. (18) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369. (19) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949. (20) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11348. (21) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676. (22) Jacobson, S. C.; Koutny, L. B.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472. 0003-2700/96/0368-0720$12.00/0
© 1996 American Chemical Society
Figure 2. Schematic of single-point detection apparatus. See text for details.
Figure 1. Photograph of a chip with an integrated precolumn reactor (0.7-nL volume) and 67-mm serpentine separation column. The channels and reservoirs are filled with black ink to provide contrast for the picture. The reservoirs are labeled by the various solutions normally contained.
phoresis in a sieving medium, e.g., hydroxyethyl cellulose.23 At a fixed point downstream on the separation column, migrating fragments are interrogated using on-chip laser-induced fluorescence with an intercalating dye. EXPERIMENTAL SECTION The chips are fabricated using standard photolithographic, wet chemical etching, and bonding techniques as previously described.14 Briefly, a photomask was fabricated by sputtering chrome (50 nm) onto a glass slide and ablating the microchip design (Figure 1) into the chrome film using a CAD/CAM excimer laser machining system (ArF, 193 nm). The column design was then transferred onto the substrates using a positive photoresist. The channels were etched into the substrate in a dilute, stirred HF/NH4F bath. To form the closed network of channels, a cover plate was bonded to the substrate over the etched channels by hydrolyzing the surfaces, bringing them into contact with each other, and processing thermally to 500 °C. The reaction chamber and separation column are 1 and 67 mm long, respectively, having a width at half-depth of 60 µm and a depth of 12 µm; the reaction chamber has a corresponding volume of 0.7 nL. The electroosmotic flow is minimized by the covalent immobilization of linear polyacrylamide.24 Chip performance and separations are monitored by laserinduced fluorescence (LIF) using either a charge-coupled device (CCD) for imaging or a single-point detection scheme for producing electropherograms. For CCD imaging, the argon ion laser beam (514.5 nm, 100 mW) is expanded to a 4-mm spot at the chip surface using a lens. The fluorescence signal is collected using an optical microscope, filtered spectrally (550-nm cut-on), and measured using the CCD. For single-point detection (see Figure 2), the argon ion laser (10 mW) is focused to a spot onto the chip using a lens (100-mm focal length). The fluorescence signal is collected using a 20× objective lens (NA ) 0.42), followed by spatial filtering (0.6-mm-diameter pinhole) and spectral filtering (560-nm bandpass, 40-nm bandwidth), and measured using a (23) Grossman, P. D.; Soane, D. S. Biopolymers 1991, 11, 1221. (24) Hjerten, S. J. Chromatogr. 1985, 347, 191.
Figure 3. Schematic of (a) the reaction chamber and injection cross for (b) the loading of the reaction chamber with DNA and enzyme for the restriction digestion, (c) injecting the digestion products onto the separation column, and (d) separating the product fragments. The applied potentials are listed for each step. Arrows depict direction of flow for anions.
photomultiplier tube (PMT). The data acquisition and voltage switching apparatus are computer controlled. The reaction buffer is 10 mM Tris-acetate, 10 mM magnesium acetate, and 50 mM potassium acetate. The reaction buffer is placed in the DNA, enzyme, and waste 1 reservoirs (Figure 1). The separation buffer is 9 mM Tris-borate with 0.2 mM EDTA and 1% (w/v) hydroxyethyl cellulose. The separation buffer is placed in the buffer and waste 2 reservoirs. The concentrations of the plasmid pBR322 and enzyme HinfI are 125 ng/µL and 4 units/µL, respectively. The digestions and separations are performed at room temperature (20 °C). Figure 3 shows a schematic of the reaction chamber and injection cross and depicts the sequence of loading the reaction chamber, injecting the products onto the separation column, and separating the products. First, the DNA and enzyme are electrophoretically migrated into the reaction chamber (Figure 3b). A voltage is also applied to the buffer reservoir to prevent the DNA and enzyme from migrating onto the separation column. After the reaction chamber is loaded and is at equilibrium, the digestion can be performed either dynamically with the electric potentials still applied to the chip or statically by removing all electric potentials. To perform the fragment size analysis following digestion, the products are introduced onto the separation column by applying a potential between the DNA and enzyme reservoirs Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
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Figure 4. (a) Schematic of the reaction chamber and injection cross. CCD images of disodium fluorescein (shaded areas) being electrophoretically manipulated on the chip during (b) loading of the reaction chamber, (c) injection of the sample onto the separation column, and (d) separation of the sample. The applied potentials are the same as in Figure 3. Arrows depict direction of flow for anions.
Figure 6. Variation of electrophoretic mobility with fragment length for ΦX-174 RF DNA-HaeIII digest (O) and pBR322-HinfI digest (b). ΦX-174 fragments are used to size the fragments produced in the on-chip digestion of pBR322 by HinfI.
Figure 5. Electropherogram of products from the digestion of the plasmid pBR322 by the enzyme HinfI. The separation field strength is 380 V/cm, and the separation length is 67 mm. The numbers correspond to the fragment lengths in base pairs.
fluorescein bleeds onto the separation column. After the reaction chamber is filled with reagents, the potential applied to the chip can be temporarily removed, if necessary, to allow for a digestion period. The fluorescein is injected onto the separation column by applying a potential between the DNA and enzyme reservoirs and the waste 2 reservoir with potentials at the buffer and waste 1 reservoirs removed for ∼4 s (Figure 4c). To break off the injection plug of fluorescein onto the separation column and to transport the plug down the separation column electrophoretically, the potentials at the buffer and waste 1 reservoirs are reapplied (Figure 4d). Also, as the analysis is being performed, the reaction chamber is reloaded for a subsequent digestion and analysis. Similar to the fluorescein, the DNA and enzyme are electrophoretically loaded into the reaction chamber from their respective reservoirs. Due to the electrophoretic mobility differences between the DNA and enzyme, the loading period is made sufficiently long to reach equilibrium. The electroosmotic flow is minimized by the covalent immobilization of linear polyacrylamide, thus only anions migrate from the DNA and enzyme reservoirs into the reaction chamber with the potential distributions used in Figures 3 and 4. The reaction buffer, which contains cations required for the enzymatic digestions, e.g., Mg2+, is also placed in the waste 1 reservoir. This enables the cations to propagate into the reaction chamber countercurrent to the DNA and enzyme during the loading of the reaction chamber. Due to the relatively short transit time of the DNA through the reaction chamber, longer digestion times and, consequently, better results are achieved performing the digestion statically by removing all electric potentials after loading the reaction chamber (see below). Following the digestion period, the products are migrated onto the separation channel for analysis as illustrated in Figure 4c. The injection has a mobility bias where the smaller fragments are injected in favor of the larger fragments. In these experiments the injection plug length for the 75-base pair (bp) fragment is estimated to be 0.34 mm whereas for the 1632-bp fragment only 0.22 mm. The entire contents of the chamber are not injected for sizing as the injection plug length would severely impact resolving power. The fragments are resolved using 1.0% (w/v) hydroxyethyl cellulose as the sieving medium. Figure 5 shows
and the waste 2 reservoir with potentials at the buffer and waste 1 reservoirs removed for a brief period of time, 1-10 s (Figure 3c). To break off the injection plug and to perform the electrophoretic separation, the potentials at the buffer and waste 1 reservoirs are reapplied (Figure 3d). RESULTS AND DISCUSSION In Figure 4, disodium fluorescein (shaded areas) is imaged using the CCD to mimic the flow path of DNA into the reaction chamber, through the injection region, and onto the separation column. The arrows indicate the migration direction for anions. Figure 4b shows the electrophoretic transport of “DNA” through the reaction chamber and into the waste reservoir. The electric potential applied to the enzyme reservoir during this period would also transport enzyme from its reservoir into the reaction chamber. Because a voltage is also applied to the buffer reservoir, no 722 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
an electropherogram of the restriction fragments of the plasmid pBR322 following a 2-min digestion by the enzyme HinfI. To enable efficient on-column staining of the double-stranded DNA after digestion but prior to interrogation, the intercalating dye TOTO-1 (1 µM)25 is placed in the waste 2 reservoir only and migrates countercurrent to the DNA. As expected, the relative intensity of the bands increases with increasing fragment size because more intercalation sites exist in the larger fragments. The unresolved 220/221- and 504/517-bp fragments have higher intensities than adjacent single-fragment peaks due to the band overlap. In Figure 6, the mobilities of the pBR322 fragments are compared with fragments of a ΦX-174 RF DNA-HaeIII digest. The mobilities ΦX-174 fragments are used to predict the mobilities of the known pBR322 fragment sizes26 by interpolation of adjacent ΦX-174 fragments. The differences between the estimated and observed pBR322 fragment mobilities range from 0.36 to 0.96% relative standard deviation (% rsd). The separation exhibits typical behavior in that the linear Ogston region27 for fragments less than 300 bp and a nonlinear reptation region28 for the larger fragments are observed in Figure 6. Also, the reproducibility of the migration times and injection volumes from the 118-bp ΦX-174 fragment are 0.55 and 3.1% rsd, respectively, for five replicate analyses. The digestion times ranged from 9 to 189 s. The 9-s reaction period corresponds to the transit time of the plasmid through the reaction chamber and is the minimum reaction time. For other reaction times, the voltage to the chip is removed to allow digestion to occur. The intensity of the 1632- and 504/517-bp fragment peaks increases for reaction times from 9 to 129 s (2min dwell time plus 9-s transit time) by ∼10 times, but for a 189-s reaction period (3-min dwell time plus 9-s transit time), no further increase in fragment yield is observed. This suggests that either the digestion is near completion or losses of the DNA or enzyme to the walls prevent generation of more product. To test for adsorption losses of DNA to the walls of the reaction chamber during the digestion period, the ΦX-174 fragments are analyzed immediately and with a 2-min dwell time in the reactor. No losses are observed in these experiments. The high surface-to-volume ratio of the reaction chamber could influence the activity of the enzyme due to association of the enzyme with the surface, and this is under further investigation. Diffusional losses from the reaction chamber during the digestion period are small due to the small diffusion coefficients of the DNA, the viscosity of the sieving medium, and the short reaction times. As a first demonstration for on-chip restriction fragment analysis, the results are extremely promising. However, to perform routine analysis, the separation efficiency requires improvement. Separation conditions were tested using the ΦX174 fragments mentioned above. In Figure 7, the variation of the peak widths for the 72- and 603-bp fragments with separation field strength is plotted. For the three field strengths used, the peak width for the 72-bp fragment is larger than the 603-bp fragment due to the electrophoretic bias of the injection scheme. Also, smaller peak widths are observed at 380 V/cm for both fragments. At lower field strengths, e.g., 190 V/cm, diffusion contributes more (25) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.; Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1992, 20, 2803. (26) Bolivar, F.; Rodriguez, R. L.; Greene, P. J.; Betlach, M. C.; Heyneker, H. L.; Boyer, H. W.; Crosa, J. H.; Falkow, S. Gene 1977, 2, 95. Roberts, R. J. Nucleic Acids Res. 1983, 11, R135. (27) Ogston, A. G. Trans. Faraday Soc. 1958, 54, 1754. (28) Lumpkin, O. J.; DeJardin, P.; Zimm, B. H. Biopolymers 1985, 24, 1573. (29) McGregor, D. A.; Yeung, E. S. J. Chromatogr. 1993, 652, 67.
Figure 7. Variation of peak width for 72- (b) and 603-bp (9) ΦX174 fragments and mobility for the 72- (O) and 603-bp (0) fragments with separation field strength.
to band dispersion, and at higher field strengths, e.g., 690 V/cm, both Joule heating causing thermal gradients and orientation of the fragments with the applied electric field degrade the efficiency.29 Consequently, the chip is operated in this intermediate range for the separation field strength. In order to improve the separation efficiency, the primary modifications to new designs would be to increase the separation column length and to use narrower channels to decrease the band dispersion from the corners.14 Also observed in Figure 7 is a nonlinear increase in the mobility of the fragments with the separation field strength. Again, this is due to Joule heating of the chip, decreasing the viscosity of the separation medium and orientation of the fragments with the electric field. Both cases reflect negatively on the separation performance of the chip. In conclusion, this demonstration of a microchip device that performs plasmid DNA restriction fragment analysis indicates the possibility of miniaturizing more sophisticated biochemical procedures. There are remaining issues to be addressed for devices that could perform useful analyses on more complex samples, for example, automated genetic analysis of whole blood. On-chip procedures will need to be developed to reduce the complexity of the sample (extraction of genetic material) before integration with the techniques presented here. In addition to genetic analysis, miniature devices that perform immunochemistry and enzymatic assays will be of interest. ACKNOWLEDGMENT This research is sponsored by Oak Ridge National Laboratory (ORNL) Laboratory Directed Research and Development Fund. ORNL is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under Contract DE-AC0596OR22464. The authors thank Drs. M. J. Doktycz, M. R. Knapp, and R. S. Foote for many useful discussions. Received for review December 20, 1995. December 21, 1995.X
Accepted
AC951230C X
Abstract published in Advance ACS Abstracts, February 1, 1996.
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