Anal.Chem. 1996, 68, 720-723
`
`Integrated Microdevice for DNA Restriction
`Fragment Analysis
`
`Stephen C. Jacobson and J. Michael Ramsey*
`ChemicalandAnalyticalSciencesDivision,OakRidgeNationalLaboratory,P.O.Box2008,
`OakRidge,Tennessee37831-6142
`
`An integrated monolithic device (8 mm (cid:2) 10 mm) that
`performs an automated biochemical procedure is dem-
`onstrated. 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-mm-
`long capillary electrophoresis channel for sizing. Materi-
`als are precisely manipulated under computer control
`within the channel structure using electrokinetic trans-
`port. 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 (cid:2) 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 chemi-
`cal 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 se-
`quencing 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 chemis-
`try,3 reaction wells for polymerase chain reactions,4 immobilized
`enzyme reactors,5 a two-dimensional obstacle course for electro-
`phoretic 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.
`
`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 high-
`speed 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 free-
`flow 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 on-
`chip. 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.
`
`720 AnalyticalChemistry,Vol.68,No.5,March1,1996
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`0003-2700/96/0368-0720$12.00/0 © 1996 American Chemical Society
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`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 fluores-
`cence with an intercalating dye.
`
`EXPERIMENTAL SECTION
`The chips are fabricated using standard photolithographic, wet
`chemical etching, and bonding techniques as previously de-
`scribed.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 (cid:176)C. The
`reaction chamber and separation column are 1 and 67 mm long,
`respectively, having a width at half-depth of 60 (cid:237)m and a depth of
`12 (cid:237)m; 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 laser-
`induced fluorescence (LIF) using either a charge-coupled device
`(CCD) for imaging or a single-point detection scheme for produc-
`ing 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(cid:2) 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 2. Schematic of single-point detection apparatus. See text
`for details.
`
`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) hydroxy-
`ethyl 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/(cid:237)L and 4 units/(cid:237)L, respectively.
`The digestions and separations are performed at room tempera-
`ture (20 (cid:176)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 elec-
`trophoretically 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
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`Figure 4. (a) Schematic of the reaction chamber and injection cross.
`CCD images of disodium fluorescein (shaded areas) being electro-
`phoretically 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 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.
`
`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 electro-
`phoretic 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 AnalyticalChemistry,Vol.68,No.5,March1,1996
`
`Figure 6. Variation of electrophoretic mobility with fragment length
`for …X-174 RF DNA-HaeIII digest (O) and pBR322-HinfI digest (b).
`…X-174 fragments are used to size the fragments produced in the
`on-chip digestion of pBR322 by HinfI.
`
`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 (cid:24)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 electro-
`phoretically 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 polyacryla-
`mide, thus only anions migrate from the DNA and enzyme
`reservoirs into the reaction chamber with the potential distribu-
`tions 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
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`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 (cid:237)M)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 …X-174 RF DNA-HaeIII digest.
`The mobilities …X-174 fragments are used to predict the mobilities
`of the known pBR322 fragment sizes26 by interpolation of adjacent
`…X-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 …X-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 (2-
`min dwell time plus 9-s transit time) by (cid:24)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 …X-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 …X-
`174 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) …X-
`174 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 ef-
`ficiency.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 frag-
`ments 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 pro-
`cedures. 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-AC05-
`96OR22464. The authors thank Drs. M. J. Doktycz, M. R. Knapp,
`and R. S. Foote for many useful discussions.
`
`review December 20, 1995. Accepted
`Received for
`December 21, 1995.X
`
`AC951230C
`
`X Abstract published in Advance ACS Abstracts, February 1, 1996.
`
`AnalyticalChemistry,Vol.68,No.5,March1,1996 723
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