Anal. Chem. 1995, 67, 3676-3680
`
`Ultra-High-Speed DNA Sequencing Using Capillary
`Electrophoresis Chips
`Adam T. Woolley and Richard A. Mathies*
`Department of Chemistry, University of California, Berkeley, California 94720
`
`DNA sequencing has been performed on microfabricated
`capillary electrophoresis chips. DNA separations were
`achieved in 50 x 8 pm cross-section channels microfab-
`ricated in a 2 in. x 3 in. glass sandwich structure using
`a denaturing 9% T, 0% C polyacrylamide sieving medium.
`DNA sequencing fragment ladders were produced and
`fluorescently labeled using the recently developed energy
`transfer dye-labeled primers. Sequencing extension frag-
`ments were separated to ~433 bases in only 10 min using
`a one-color detection system and an effective separation
`distance of only 3.5 cm. Using a four-color labeling and
`detection format, DNA sequencing with 97% accuracy and
`single-base resolution to ~ 150 bases was achieved in only
`540 s. A resolution of greater than 0.5 was obtained out
`to 200 bases for both the one- and four-color separations.
`The prospects for enhancing the resolution and sensitivity
`of these chip separations are discussed. This work
`establishes the feasibility of high-speed, high-throughput
`DNA sequencing using capillary array electrophoresis
`chips.
`
`Capillary electrophoresis (CE) is a powerful technique for DNA
`analysis, which has been applied to restriction fragment sizing,
`PCR product analysis, forensic identification, and DNA sequenc-
`ing.* 1 CE separations are much faster than those in slab gels,
`because higher electric fields can be applied; however, conven-
`tional CE has the disadvantage that it only allows the analysis of
`one sample or lane at a time. Our group has addressed this issue
`by developing capillary array electrophoresis (CAE)2 34in which
`separations are performed in a bundle of parallel silica capillaries,
`and we have demonstrated the use of CAE for DNA sequencing,3 4
`restriction fragment sizing,5 and short tandem repeat analysis.6
`In CAE, the lane width is reduced from the ~1 mm typical for
`slab gels to ~100 pm. Further miniaturization of electrophoretic
`separations to increase the number of lanes, as well as the speed
`and throughput of the separations will be necessary to meet the
`needs of the Human Genome Project.78 Therefore, we have
`recently focused on the use of microfabrication techniques to
`produce miniaturized capillary arrays.
`friend and colleague Dr. Huiping Zhu (1959-1995).
`* Dedicated to our
`(1) Monnig, C. A; Kennedy. R T. Anal. Chem. 1994, 66, 280R—314R
`(2) Mathies, R. A; Huang, X. C. Nature 1992, 359, 167-169.
`(3) Huang, X. C.; Quesada, M. A; Mathies, R. A Anal. Chem. 1992, 64, 967—
`972.
`(4) Huang, X. C.; Quesada, M. A; Mathies, R A Anal. Chem. 1992, 64, 2149-
`2154.
`(5) Clark, S. M.; Mathies, R A. Anal. Biochem. 1993, 215, 163—170.
`(6) Wang, Y.; Ju, J.; Carpenter, B. A; Atherton, J. M.; Sensabaugh, G. F.;
`Mathies, R. A Anal. Chem. 1995, 67, 1197-1203.
`(7) Hunkapiller, T.; Kaiser, R J.; Koop, B. F.; Hood, L. Science 1991,254,59-
`67.
`(8) Smith, L. M. Science 1993, 262, 530—532.
`3676 Analytical Chemistry, Vol. 67, No. 20, October 15, 1995
`
`The use of microfabrication to produce electrophoretic separa-
`tion capillaries was first introduced in 1992 by Manz and Harrison.9
`Subsequently, capillary zone electrophoresis separations of fluo-
`rescent dyes10 and of fluorescently labeled amino acids1112 were
`performed in individual microfabricated capillaries on glass chips.
`Our own efforts have been directed toward producing microfab-
`ricated capillary arrays on chips and using them for high-resolution
`separations of DNA restriction fragments and PCR products.13
`Separations of small,
`fluorescently labeled phosphorothioate
`oligonucleotides have also recently been performed on a micro-
`fabricated chip.14 These results suggest that if the sensitivity and
`resolution were enhanced it might even be possible to perform
`DNA sequencing on chips. To enhance the sensitivity of these
`separations, we have exploited the recently developed energy
`transfer dye-labeled sequencing primers1516 and employed confocal
`In addition,
`techniques for filling
`fluorescence detection.1316
`microfabricated channels with denaturing polyacrylamide matri-
`loading DNA sequencing samples on a chip, injecting the
`ces,
`samples, and performing sequencing separations have been
`developed. These studies show that we can achieve single-base
`resolution of DNA sequencing samples to ~200 bases on chips
`in only 10 min separations. The prospects for increasing the read
`lengths on CE chips to ~500 bases are also discussed. The
`demonstration of high-speed DNA sequencing on CE chips is the
`first step toward miniaturizing the entire DNA sequencing
`procedure on microfabricated devices.
`
`EXPERIMENTAL SECTION
`Sequencing Sample Preparation. DNA sequencing samples
`were generated using standard dideoxy sequencing chemistry and
`energy transfer dye-labeled primers;1516 the other reagents used
`to prepare the sequencing samples were obtained from Amersham
`Life Science (Cleveland, OH). The DNA sequencing fragments
`used in the one-color separations were made using 2.4 pmol of
`the fluorescently labeled F10F primer (see ref 15 for nomencla-
`ture), 10 pg of single-stranded M13mpl8 DNA template, and they
`
`(9) 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.
`(10) Jacobson, S. C.; Hergenroeder, R; Koutny, L. B.; Warmack, R. J.; Ramsey,
`J. M. Anal. Chem. 1994, 66, 1107-1113.
`(11) Harrison, D. J.; Fluri, K; Seiler, K; Fan, Z.; Effenhauser, C. S.; Manz, A
`Science 1993, 261, 895-897.
`(12) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 2637-
`2642.
`(13) Woolley, A T.; Mathies, R A Proc. Natl. Acad. Sci. U.S.A. 1994,91,11348-
`11352.
`(14) Effenhauser, C. S.; Paulus, A; Manz, A; Widmer, H. M. Anal. Chem. 1994,
`66, 2949-2953.
`(15) Ju, J.; Ruan, C.; Fuller, C. W.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad
`Sci., U.S.A. 1995, 92, 4347-4351.
`(16) Ju, J.; Kheterpal, I.; Scherer, J, R; Ruan, C.; Fuller, C. W.; Glazer, A N.;
`Mathies, R A. Anal. Biochem., in press.
`
`0003-2700/95/0367-3676S9.00/0 © 1995 American Chemical Society
`
`Downloaded via UNIV OF VIRGINIA on November 6, 2018 at 17:50:52 (UTC).
`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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`45
`
`86
`
`126
`
`185
`
`232
`
`310
`
`433
`
`—i-1-1-r-
`120
`
`160
`
`200
`Time (seconds)
`Figure 2. One-color DNA sequencing fragment separation on a CE
`chip, (top) Electropherogram of M13mp18 A sequencing fragments
`generated with the primer F10F. (bottom) Expanded view of the peaks
`corresponding to the first 100 bases, demonstrating single-base
`resolution. The sample was injected by electrophoresing it through
`the cross channel at 170 V/cm for 60 s and separated at 250 V/cm
`in a 9% Tj 0% C polyacrylamide-filled channel. Excitation was at 488
`nm and fluorescence from 515 to 545 nm was detected. The effective
`separation length ot the 50 ;<m wide and 8 «m deep channel was 3.5
`cm.
`
`240
`
`Figure 1. Schematic diagram of the electrophoresis chip indicating
`the injection procedure. The injection channel connects reservoirs 1
`and 3, and the separation channel connects reservoirs 2 and 4. In
`the inject mode, a field is applied between reservoirs 1 and 3, causing
`the DNA to migrate through the gel-filled intersection toward reservoir
`In the run mode, a field is applied between reservoirs 2 and 4,
`1.
`causing the DNA fragments in the intersection region to migrate
`toward reservoir 4 through the gel
`in the separation channel. The
`actual devices had 15 electrophoresis systems integrated on each
`chip.
`
`were terminated using ddATP. The F10F primers are labeled with
`6-carboxyfluorescein (FAM) at the 5' end, with a second FAM
`attached to the tenth nucleotide from the 5' end of the primer on
`a modified T residue. The samples used in the four-color DNA
`sequencing experiments were generated using dITP sequencing
`chemistry with 4.8 pmol of fluorescently labeled primer and 4.8
`fig of single-stranded M13mpl8 DNA template for each of the
`four reaction mixtures. The primers used for the four-color
`reactions were F10F, F10J, F10T, and F10R. These primers are
`labeled at the 5' end as described above and have either FAM,
`2',7'-dimethoxy4\5'-dich!oro-6-carboxyfluorescein (JOE), AWV’JV'-
`tetramethyl-6-carboxyrhodamine (TAMRA), or 6-carboxy-X-rho-
`damine (ROX) attached to the tenth nucleotide from the 5' end
`the primer on a modified T residue. For the four-color
`of
`experiments, the mixtures were pooled, and then for both the
`the DNA fragments were
`one- and four-color experiments,
`precipitated, washed with ethanol, and resuspended in 2 mL of
`95% formamide/2.5 mM EDTA The samples were denatured at
`90 °C for 2 min and immediately placed on ice prior to injection.16
`Electrophoresis Procedures. Electrophoresis chips were
`fabricated and the channel surfaces were
`derivatized using
`[y-(methacryloxy) propyl] trimethoxysilane as described previ-
`ously.13 An aqueous solution of acrylamide (9% T, 0% Q in 45
`mM Tris/45 mM borate/1 mM EDTA/8.3 M urea
`(pH 8.3) was
`filtered with a 0.2 (im pore diameter filter (Millipore, Bedford,
`MA) and then degassed under vacuum for 1 h. Polymerization
`was initiated by adding 2.5 /iL of 10% ammonium persulfate and
`1.5 nL of MWV'JV’-tetramethylethylenediamine (TEMED) to a
`1-mL aliquot of acrylamide solution, which was then drawn into
`all the channels by placing the solution in reservoir 4 and applying
`vacuum to the other reservoirs (see Figure 1 for labeling). The
`acrylamide was allowed to polymerize overnight at 4 °C, and the
`channels were preelectrophoresed at 100 V/cm for 15 min prior
`to use.
`rinsed with 95%
`To perform an injection, reservoir 3 was
`formamide and then 1.0 /<L of sequencing sample was pipeted
`
`into the reservoir. The other three reservoirs were filled with 45
`mM Tris/45 mM borate/1 mM EDTA (pH 8.3);
`to establish
`electrical contacts on the chip, wires were inserted into the cutoff
`pipet tips which formed the four reservoirs.
`In the one-color
`the sample was
`"plug" injected13 by
`sequencing separation,
`applying 170 V/cm between reservoirs 1 and 3 for 60 s and run
`In the four-
`by applying 250 V/cm between reservoirs 2 and 4.
`color sequencing separation, the sample was injected for 30 s in
`the same manner and run at 200 V/cm.
`Instrumentation. The detection system used for the one-
`color sequencing separations has been described previously;13 the
`instrumentation used for the four-color sequencing separations
`has also been described.16 Briefly, the 488-nm line from an argon
`ion laser was focused within the channel using a 20 x NA 0.5
`objective. Fluorescence was collected by the objective and passed
`through a series of dichroic filters to divide the fluorescent signal
`into four spectral regions (510-540, 545-570,570—590, and 590-
`660 nm). The fluorescent signal in the first three spectral regions
`was filtered by a band-pass filter; the fourth region was filtered
`with a long-pass filter; a 100-um confocal pinhole spatially filtered
`the fluorescence in all four spectral channels before photomulti-
`plier detection. The analog output from the photomultipliers was
`filtered with a low-pass filter having a 0.2-s time constant and
`sampled at 10 Hz with a 16-bit ADC board (NB-MIO-16XL48,
`National Instruments, Austin, TX) controlled by a program written
`in LabVIEW running on a Macintosh Ilci. Additionally, a 660-
`nm short-pass filter was placed before the longest wavelength
`detector to reduce background fluorescence from the glass. The
`raw data were Fourier transformed, the high-frequency (>0.5 Hz)
`noise was removed, and then an inverse Fourier transform was
`performed. After background subtraction,
`the smoothed data
`were transformed using a multicomponent matrix transformation.17
`formed using the fluorescence
`The transformation matrix was
`
`(17) Smith, I.. M.; Kaiser, R. J.; Sanders, J. Z.; Hood, L E. Methods Emymol.
`1987, 155, 260-301.
`
`Analytical Chemistry, Vol. 67, No. 20, October 15, 1995
`
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`440
`
`460
`
`480
`
`500
`Time (seconds)
`Figure 3. Analyzed four-color DNA sequencing data from M13mp18 DNA labeled using the F10F, F10J, F10T, and F10R energy transfer
`primers and separated on a CE chip. The raw fluorescence data were transformed as described in the Experimental Section to present a plot
`of relative concentration of each of the labeled DNA fragments as a function of time. The bases are identified by the color of the peaks: blue
`(F10F) C; green (F10J) T; black (F10T) G; red (F10R) A. The three bases that were not called, T at 397 s, T at 441 s, and C at 484 s and a
`C incorrectly called at 422 s are indicated by a black asterisk. Weak signal from the C- and T-terminated peaks was most likely the cause ot
`these errors. The sample was
`injected by electrophoresing it through the cross channel at 170 V/cm for 30 s and separated at 200 V/cm in a
`9% T, 0% C polyacrylamide-filled channel. Excitation was at 488 nm and detection was at 510-540 nm for C, 545-570 nm forT, 570-590 nm
`for G, and 590-660 nm for A. The effective separation length ol the 50 /rm wide and 8 fim deep channel was 3.5 cm .
`
`520
`
`540
`
`signals in each spectral channel from known single DNA fragment
`peaks terminated at C, T, G, and A nucleotides. Transforming
`the data from the four spectral channels yields information about
`the relative concentrations of the four sets of dye-iabeled DNA
`fragments.16 The plot of these concentration data as a function
`of time was corrected for the mobility differences of the dye-
`labeled fragments by adding 2.2 s to the migration times of the
`fragments labeled with F10T and F10R, yielding the analyzed four-
`color sequencing profile.
`Safety Considerations. Microfabricated CE chips could be
`mass produced inexpensively, so a prefilled, disposable chip would
`minimize the users
`exposure to hazardous chemicals such as
`acrylamide, a neurotoxin and carcinogen. Furthermore, the small
`volumes of solutions required to fill microfabricated channels
`decrease the quantities of all reagents used (hazardous or not).
`The short length of these microfabricated channels allows separa-
`tions at 200 V/cm with a lower applied voltage (1 kV), reducing
`the hazard of electrical shock.
`
`RESULTS AND DISCUSSION
`Figure 2 presents a separation of FlOF-labeled, ddATP-
`terminated DNA sequencing fragments on a CE chip. The
`fragments were separated in a channel 50
`wide and 8 fim deep
`
`3678 Analytical Chemistry, Vol. 67, No. 20. October 15, 1995
`
`with a distance from injection to detection of only 3.5 cm. The
`peaks are visible starting at ~ 100 s, and all the peaks have been
`detected by 800 s. The peak corresponding to 433 bases after
`the primer is detected at 602 s. The lower portion of Figure 2
`presents an expanded view of the peaks corresponding to the first
`100 bases, separated in --4 min. Single-base resolution was
`obtained throughout this region; for example, bases 23 and 24,
`bases 75 and 76, and bases 82 and 83 are all resolved.
`Figure 3 presents a four-color sequencing run using a CE chip.
`These data have been analyzed by applying a matrix transforma-
`for cross
`tion to the fluorescence data to correct
`talk. Single-
`base resolution was obtained throughout the region shown; there
`four errors
`in the base calls, or 97% accuracy out to 147
`were
`bases, with a separation time of just 9 min. The signals for the
`C- and T-terminated fragments became too weak to distinguish
`from the background noise beyond this region, so the sequence
`could be read only as far as shown. However, the signal strength
`for the A- and G-terminated fragments was strong enough to
`distinguish peaks to ~400 bases at a separation time of 15 min.
`Figure 4 presents an analysis of the resolution in the four-
`color sequencing separation. The figure shows a plot of resolution
`as a function of base number, with a least-squares exponential fit
`to the data. A resolution of at least 0.5 between adjacent peaks is
`
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`fragments would have been approximately the same as for the A-
`if a 2-fold higher
`and G-terminated fragments (~400 bases)
`concentration of the C- and T-terminated fragments had been used
`in this run or if the detection sensitivity were improved.
`In order to achieve adequate signal, 12 times more primer and
`DNA template were used for sequencing on the CE chip than for
`conventional capillaries.16 The reduced sensitivity with chips is
`due to the smaller injection and detection volumes; for example,
`the cross-sectional area of a 50 x 8 /un channel is 20 times smaller
`than that of a 100-um-i.d. capillary. The detection sensitivity could
`be improved and the amount of reagents could be reduced
`through several modifications to the experiments. For example,
`< 10% of the channel width was illuminated by the laser in the
`current setup. By using a cylindrically focused beam and a
`confocal slit matching the channel width,
`the signal could be
`increased by a factor of 10. Also, making the channels 20 wm
`deep instead of 8 um would raise the signal by a factor of 2.5
`more. These two improvements alone would produce a 5-fold
`increase in the signal-to-noise ratio. Finally, improved matching
`of the volume of sample placed on the chip (1 /*L) to the volume
`of sample actually injected (~100 pL) would significantly reduce
`the quantities of reagents used. The reagent volumes could be
`reduced through integration of the sequencing reactions onto the
`chip. These improvements should allow DNA sequencing to be
`performed on CE chips with sensitivity and quantities of reagents
`comparable to other techniques.
`Although the resolution was adequate to call only ~200 bases
`there are several possible ways to extend the
`in these runs,
`resolution to 400-500 bases.
`Increasing the separation distance,
`decreasing the length of the injected plug,19 or using a sieving
`medium that is better optimized for longer DNA fragments20 can
`all increase the range of acceptable resolution. Using the peak
`variances and published values for diffusion coefficients of single-
`stranded DNA in denaturing polyacrylamide gels,19 we calculated
`the length of the injected plug to be 101 ± 6 um from the 50-um-
`wide injection channel. By using a 20-30-nm-wide injection
`channel or controlling the potentials at all reservoirs during
`injection,10 the plug length could be decreased to <50 um. With
`a 50-«m plug length and a 7-cm effective separation distance, the
`resolution for adjacent fragments would be at least 0.5 out to ~320
`the limit of resolution for 9% T, 0% C
`bases, which is near
`the limit of
`polyacrylamide in longer capillaries.4 However,
`resolution in capillaries can be extended to ~500 bases by using
`cross-linked polyacrylamide at lower concentrations.16 The in-
`creased length would double the time required for separation;
`nevertheless, these separations would still be significantly faster
`than the current methodologies. These considerations suggest
`that it should be possible to obtain single-base resolution to ~500
`bases on CE chips by optimizing the channel length, injection,
`and sieving medium.
`
`CONCLUSIONS
`The demonstration of high-speed DNA sequencing on micro-
`fabricated capillary electrophoresis chips paves the way to a variety
`of important applications. Once the resolution and run length are
`improved, and a more facile method of loading samples is devised,
`it is reasonable to think that we could make a 2-in. x 3-in. CAE
`
`(19) Luckey, J. A.; Norris, T. B.; Smith, L. M./. Phys. Chem. 1993, 97, 3067-
`3075.
`(20) Figeys, D.; Dovichi, N. J./. Chromatogr. 1993, 645, 311-317.
`
`Analytical Chemistry, Vol. 67, No. 20, October 15, 1995
`
`3679
`
`0
`
`100
`
`300
`
`400
`
`200
`Base number
`Figure 4. Plot of resolution as a function of base number for the
`four-color sequencing run. The solid line is a least-squares fit of an
`exponential function to the data:
`(y = 1.0762e'00038603*, R = 0.94)
`Resolution was normalized to single-base spacing when the bases
`analyzed were not adjacent. Peak parameters for the resolution
`calculations were determined by fitting a Gaussian function to peaks
`in the raw data. The resolution of two peaks was calculated by taking
`the difference of the migration times and dividing by 4 times the mean
`of the variances of the peaks. The resolution of nonadjacent bases
`was normalized by dividing the calculated resolution by the base
`spacing between the fragments.
`
`necessary to obtain peak information from the data;18 from the
`least-squares fit, the resolution in this run was above 0.5 out to
`200 bases. Similar results were obtained for the one-color run
`with a resolution greater than 0.5 out to 215 bases. The maximum
`number of theoretical plates for a band was 1.1 x 106, correspond-
`ing to 3.1 x 10' plates/m.
`We have demonstrated that high-speed DNA sequencing can
`be performed using microfabricated CE chips. DNA sequencing
`with 97% accuracy has been demonstrated, and single-base
`resolution to >200 bases has been demonstrated in only 7 min
`(1700 bases/h per lane), using an effective separation distance of
`only 3.5 cm. DNA sequencing fragments as long as 433 bases
`can be detected in ~10 min separations (2400 bases/h per lane).
`By comparison, DNA sequencing using slab gel electrophoresis
`yields 500 bases of sequence in ~8-10-h (50-60 bases/h per
`lane), and sequencing with capillary electrophoresis yields 500
`bases of sequence in ~l-2 h (250-500 bases/h per lane). This
`comparison makes it clear
`that
`the use of microfabricated
`capillaries may lead to a significant improvement in DNA sequenc-
`ing technology.
`The ability to read the sequence past ~200 bases was limited
`in the first two spectral
`in these experiments by the signal
`channels. The raw signal in these channels was a factor of ~2
`less than in the other channels because the sensitivity of these
`two photomultipliers was less than that of the others by a factor
`of 2-3. The dyes that primarily fluoresce in the wavelength range
`collected in the first two spectral channels, F10F and F10J, were
`used to label the C- and T-terminated fragments, hence the lower
`sensitivity for these fragments. The length of the read for these
`
`(18) Best, N.; Arriaga, E.; Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1994, 66,
`4063-4067.
`
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`chip that would perform parallel DNA sequencing of 32 or more
`different samples in ~15 min. The detection region for 32 closest-
`packed channels would be ~3 mm wide, which could be detected
`in a scanning format with a duty cycle per channel of ~2%.
`Alternatively, by using a multiple array detector, nearly a 100%
`duty cycle could be achieved. Assuming a read length of 400
`bases in 20 min for 32 parallel channels yields a raw DNA
`sequencing rate of ~40 000 bases/h for a single chip. Further-
`recent work has demonstrated that simple chemical
`more,
`reactions can be combined with free zone electrophoresis separa-
`tions on chips.21'22 Coupling more complex reactions involving
`DNA sample preparation with our high-speed, high-resolution
`separation technology could drastically simplify DNA analysis.
`This integration on a large scale could lead to low-cost, disposable
`microfabricated devices which combine DNA sample preparation,
`amplification, injection, separation, and detection with analysis
`times of just a few minutes. The development of these microfab-
`ricated integrated DNA analysis systems will be an important step
`in the Human Genome Project.
`
`(21) Jacobson, S. C.; Koutny, L. B.: Hergenroeder. R.; Moore, A. VV., Jr.: Ramsey,
`J. M. Anal. Chem. 1994, 66, 3472-3476.
`(22) Jacobson, S. C.; Hergenroeder, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal.
`Chem. 1994, 66, 4127-4132.
`
`ACKNOWLEDGMENT
`We thank Jingyue Ju for supplying the energy transfer primers,
`Indu Kheterpal for expert assistance with the data analysis and
`the four-color detection system, and the other members of the
`Berkeley High-Sensitivity DNA Analysis Project for their advice
`and suggestions. We also thank Amersham Life Science Corp.
`for providing DNA sequencing kits and Rich Roy of the J. M. Ney
`Co. for loaning us a programmable furnace. Microfabrication was
`performed at the University of California, Berkeley Microfabri-
`cation Laboratory. This research was supported by the Director,
`Office of Energy Research, Office of Health and Environmental
`Research of the U.S. Department of Energy under Contract DE-
`FG-91ER61125. AT.W. was supported by a predoctoral fellowship
`from the Fannie and John Hertz Foundation.
`
`review June 5, 1995. Accepted July 18,
`
`Received for
`1995.®
`
`AC9505379
`
`® Abstract published in Advance ACS Abstracts, September 1, 1995.
`
`3680 Analytical Chemistry, Vol. 67, No. 20, October 15, 1995
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