Electrophoretically Mediated Microanalysis of Leucine

Pierre-Alain Auroux, Dimitri Iossifidis, Darwin R. Reyes, and Andreas Manz ... J. Field , Richard Cisek , Virginijus Barzda , Anne W. Sylvester , Jeff...
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Anal. Chem. 2000, 72, 5731-5735

Technical Notes

Electrophoretically Mediated Microanalysis of Leucine Aminopeptidase Using Two-Photon Excited Fluorescence Detection on a Microchip Scott A. Zugel, Brian J. Burke, Fred E. Regnier,* and Fred E. Lytle*

Department of Chemistry, Purdue University, 1393 Brown Laboratory, West Lafayette, Indiana 47907-1393

Two-photon excited fluorescence detection was performed on a microfabricated electrophoresis chip. A calibration curve of the fluorescent tag β-naphthylamine was performed, resulting in a sensitivity of 2.5 × 109 counts M-1 corresponding to a detection limit of 60 nM. Additionally, leucine aminopeptidase was assayed on the chip using electrophoretically mediated microanalysis. The differential electroosmotic mobilities of the enzyme and substrate, L-leucine β-naphthylamide, allowed for efficient mixing in an open channel, resulting in the detection of a 30 nM enzyme solution under constant potential. A zero potential incubation for 1 min yielded a calculated detection limit of 4 nM enzyme. Two common goals in research today are the desire to increase sample throughput and decrease the amount of sample needed and waste generated. Simultaneous pursuit of these goals has generated great interest in the area of microtechnology. Microfabricated electrophoresis chips offer not only volumetric reduction of reagents but separation times in as little as 1 s.1,2 However, microchips are not limited to simple separations. The ability to design and construct complex channel systems has facilitated the use of microchips in biochemical analyses involving postcolumn reactions,3 DNA sequencing,4-6 and enzyme assays.7-11 Typically these assays have been performed by combining the enzyme and * Corresponding authors. F.E.R.: (phone) 765-494-3878; (fax) 765-494-0239; (e-mail) [email protected]. F.E.L.: (phone) 765-494-5261; (e-mail) flytle@ purdue.edu. (1) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118. (2) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476-3480. (3) Jacobson, S. C.; Koutny, L. B.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476. (4) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680. (5) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723. (6) Haab, B. B.; Mathies, R. A. Anal. Chem. 1996, 68, 5137-5145. (7) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18-22. (8) von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (9) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373-378. (10) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407-3412. (11) Hadd, A. G.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1999, 71, 52065212. 10.1021/ac000801k CCC: $19.00 Published on Web 10/12/2000

© 2000 American Chemical Society

substrate at a T-shaped or cross junction and then allowing the two to diffusively mix in a reaction or separation channel. An alternate method of mixing assay components, electrophoretically mediated microanalysis (EMMA), was originally developed to allow enzyme assays to be performed in a singlecapillary electrophoresis column.12,13 Differences in the electrophoretic mobilities of enzyme, substrate, and product caused the reagents to mix electrophoretically when the slower reagent was placed first on the column, followed by the faster reagent. This technique has the added advantage of using less enzyme per assay than other techniques while still achieving low detection limits. Indeed, this approach has allowed the analysis of individual enzyme molecules.14,15 Given the efficient mixing and small quantities of enzymes capable of being detected, the technique seems well-suited for use with microchips. A major obstacle in performing chip-based assays is optical detection of the product given the small path length. Typically channel depths are only 8-40 µm, depending on the etching process used for fabrication. Normal absorbance and fluorescence detection methods suffer large losses in sensitivity given their path length dependency. Two-photon excited (TPE) fluorescence is a nonlinear absorption process that has optical properties different from those observed with one-photon excitation.16 As an example, the efficiency of TPE fluorescence is inversely proportional to the area of the excitation beam. For a focused laser, this property results in a very small excitation volume centered about the focal point. Consequently, the path length dependence typically encountered in absorption/fluorescence measurements is significantly reduced. This property of TPE has made it an attractive tool for use in confocal microscopy17,18 and small-bore capillaries.19-21 However, in a recent study, round capillaries were shown to cause (12) Bao, J.; Regnier, F. E. J. Chromatogr. 1992, 608, 217-224. (13) Wu, D.; Regnier, F. E. Anal. Chem. 1993, 65, 2029-2035. (14) Craig, D.; Arriaga, E. A.; Banks, P.; Zhang, Y.; Renborg, A.; Palcic, M. M.; Dovichi, N. J. Anal. Biochem. 1995, 226, 147-153. (15) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681-683. (16) Fisher, W. G.; Wachter, E. A.; Lytle, F. E.; Armas, M.; Seaton, C. Appl. Spectrosc. 1998, 52, 536-545. (17) Diaspro, A. Microsc. Res. Technol. 1999, 47, 163-164. (18) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73-76. (19) Overway, K. S.; Duhachek, S. D.; Loeffelmann, K.; Zugel, S. A.; Lytle, F. E. Appl. Spectrosc. 1996, 50, 1335-1337. (20) Song J. M.; Inoue, T.; Kawazumi, H.; Ogawa, T. J. Chromatogr., A 1997, 765, 315-319.

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Figure 1. Schematic of the cross-shaped microchip. All dimensions are in centimeters.

beam astigmatism, affecting the sensitivity of the measurement.22,23 Because the channels on microchips are flat, beam astigmatism should not be a problem with detection on microchips. In this study, we explore two areas. First, we examine the potential of using TPE fluorescence detection on microchips by performing a calibration curve of the fluorescent tag, β-naphthylamine. Second, leucine aminopeptidase (LAP), a clinically significant enzyme found in the tissues of all organisms,24 is assayed on chip. Using a simple cross configuration, we demonstrate the feasibility of performing chip-based assays utilizing the EMMA format. EXPERIMENTAL SECTION Solutions. Glassware was prepared by an overnight soak in a concentrated H2SO4/Nochromix bath and rinsed three times with water. β-Naphthylamine (BNA), L-leucine β-naphthylamide, and LAP enzyme from porcine kidney (microsomal) were purchased from Sigma. The buffer was 10 mM sodium phosphate at pH 7.0. A stock solution of 3 × 10-5 M BNA solution was made from which concentrations ranging from 3 × 10-8 to 3 × 10-6 M were subsequently prepared. A substrate stock solution was prepared to 10-3 M by first dissolving in ∼10 mL of methanol and then diluting with buffer to a final volume of 25 mL. The substrate assay solution was prepared to 10-4 M by dilution of the stock solution with buffer. The 3 × 10-8 M enzyme solution was prepared by dilution in buffer. A molecular mass of 326 kDa was used for all concentration calculations. Microchip. Mask and microchip fabrication were performed using procedures previously described.25,26 The quartz microchip was fabricated at the Alberta Microelectronic Center (Edmonton, AB, Canada) using an anisotropic etching process. Figure 1 shows a schematic diagram of the simple cross-column configuration. The dimensions of the channels are ∼9 µm deep and 50 µm wide. Prior to use, 1 M NaOH was pulled through the columns to activate the internal surfaces. This step was followed by rinsing with buffer. (21) Wei, J.; Gostkowski, M. L.; Gordon, M. J.; Shear, J. B. Anal. Chem. 1998, 70, 3470-3475. (22) Overway, K. S.; Lytle, F. E. Appl. Spectrosc. 1998, 52, 928-932. (23) Zugel, S. A.; Lytle, F. E. Appl. Spectrosc. 2000, 54, 1203-1207. (24) Miller, K. J.; Leesong, I.; Bao, J.; Regnier, F. E.; Lytle, F. E. Anal. Chem. 1993, 65, 3267-3270. (25) He, B.; Tait, N.; Regnier, F. Anal. Chem. 1998, 70, 3790-3797. (26) He, B.; Tan, L.; Regnier, F. Anal. Chem. 1999, 71, 1464-1468.

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Figure 2. Diagram of the two-photon instrumentation. Key: M, mirror; BS, beam splitter; DM, dichroic mirror; PD, photodiode; CFD, constant fraction discriminator; MCP, microchannel plate detector; PA, preamplifier; PTD, picotiming discriminator; TAC, time-to-amplitude converter; MCA, multichannel analyzer; SCA, single-channel analyzer.

Methods for Calibration Curve. The waste reservoir was sequentially filled with the BNA solutions beginning with the lowest concentration. The remaining reservoirs were filled with buffer. Solutions were pulled through the channels and past the detection window by a pressure differential. Counts were accumulated over 1-min intervals. Prior to adding the next higher concentration, the reservoir was drained and rinsed three times with buffer. Methods for EMMA. After the calibration curves were performed, the chip was removed and cleaned using 1 M NaOH followed by buffer. Platinum electrodes were placed in each of the four reservoirs. The buffer was removed from the substrate reservoir and replaced with the assay substrate solution. A 500-V potential was applied to the substrate reservoir, and the waste reservoir was grounded in order to load the substrate on the main column. Next, the buffer was removed from the enzyme reservoir and replaced with the enzyme solution. Enzyme was loaded into the injection cross for 30 s by switching the voltage so that the positive potential was applied to the enzyme reservoir and the enzyme waste reservoir was grounded. Then, 150 V was applied to the substrate reservoir, and the waste reservoir was grounded, moving the enzyme in the cross onto the main channel. In experiments where incubations were performed, the voltage was initially applied for 10 s in order to electrophoretically mix the enzyme and substrate and then removed for the duration of the incubation. All voltage switching was performed manually. Detection Instrumentation. Figure 2 shows a schematic diagram of the instrumentation. The excitation source used in these experiments was a cavity-dumped dye laser (Coherent Series 700), synchronously pumped by a 76-MHz mode-locked, frequency doubled Nd:YAG laser (Coherent Antares 76). The dye laser was dumped at 3.8 MHz and tuned to 580 nm with approximately 120 mW of average power. The temporal width of the pulses was ∼10 ps full width at half-maximum (fwhm) as measured by an

autocorrelator (Coherent 290). An epifluorescence format was used to focus and collect the light. In short, a dichroic mirror (CVI) was used to reflect the laser into a microscope objective (Melles Griot 10×, 0.25 NA) focusing the laser onto the microchip from the bottom side. The fluorescence was collected by the same objective, transmitted by the dichroic mirror, and directed through one Corion LS500 filter and one 1-mm Schott BG-3 filter onto an uncooled microchannel plate detector (MCP, ITT F4129 Z-plate at -3000 V dc). The fluorescence was detected by time-correlated singlephoton counting running in the reverse, noninteractive mode. The signal from the MCP was sent through a preamplifier (EG&G Ortec 9306) and then to a picotiming discriminator (EG&G Ortec 9307). The output of the discriminator provided the start pulse to the time-to-amplitude converter (TAC, Tennelec TC 864 by Oxford). The stop pulse was supplied by the output of a fast photodiode (TIED 56), which was first run through a constant fraction discriminator (Tennelec TC 455). Before arriving at the TAC, the stop pulse was sent through a delay line. At this point, two different signal-processing methods were used. When performing the calibration curves of BNA, the signal was directed into a multichannel analyzer (MCA Multiport TC 864 by Oxford), which binned the voltages from the TAC and transferred the signal via GPIB to a computer. When collecting the assay data, the signal was alternatively directed into a single-channel analyzer (SCA Ortec 550), which was used to time-filter the signal via hardware. Signal was then transferred to a counter which was sampled by a computer at 10 Hz. Data Processing. All of the data were enhanced by timefiltered detection.27 For the calibration curve, the data were timefiltered in software after collection. The placement and width of the window was determined to provide the lowest detection limit with the highest S/N. The width of the detection window was reduced to 45 ns and delayed 4.9 ns from the time of the laser pulsing in order to eliminate the collection of scattered laser light. As mentioned in the Detection Instrumentation section, timefiltering for the assay data was accomplished by setting upper and lower thresholds on the SCA. These limits were determined by using the information from the calibration curve. Additionally, a running average filter was used on the assay data to remove digitization noise. RESULTS AND DISCUSSION Two-Photon Excitation with a Microchip. With nanolitersized samples, detection by one-photon excited fluorescence suffers from a reduced sensitivity, because of the shortened optical path length. In contrast, two-photon excitation with a highly focused source can create sensitivities comparable to those obtained within macroscopic sample containers. As long as the confocal parameter is much smaller than the sample path length, the total number of excited states produced with two-photon excitation is independent of the focal spot size and the sample path length.28 The microchip used in this study had 9-µm-deep, rectangular channels. This channel depth should produce a sensitivity only slightly less than a bulk measurement when used (27) Seitzinger, N. K.; Hughes, K. D.; Lytle, F. E. Anal. Chem. 1989, 61, 26112615. (28) Swofford, R. L.; McClain, W. M. Chem. Phys. Lett. 1975, 34, 455-460.

Figure 3. Electropherogram for 30 nM LAP enzyme without an incubation.

in combination with high numeric aperture microscope objectives.22 The objective used in these studies had a numeric aperture of 0.25 and a working distance of 6.8 mm. In water, the focal radius is 2.2 µm and the confocal parameter is 69 µm. Thus, Gaussian optical theory predicts that only 8% of the excited states are generated compared to an infinitely long path length.22 To experimentally determine the excitation efficiency, a calibration curve of BNA in aqueous buffer was constructed. The slope of the data showed a sensitivity of 2.5 × 109 counts min-1 M-1. The lowest measured concentration was 60 nM with a signal-to-noise ratio of 4.7. A calibration curve constructed from measurements made in a 1-cm cell yielded a sensitivity of 4.14 × 1010 counts min-1 M-1. The ratio of sensitivities indicates that 6% of the total possible excited states were observed with the chip. Considering the vastly different sample formats, the agreement between experiment and theory is excellent. Use of higher numeric aperture objectives would increase the percentage of excited states generated. Unfortunately, because such lenses have short working distances, the 500-µm-thick base and cover plate of the microchip prevented their use in this study. Enzyme Assay Using Electrophoretically Mediated Microanalysis. Taking advantage of differences in electrophoretic mobilities of reactant and product species, EMMA utilizes electrophoretic transport in order to mix reagents.12,13 This method has the advantage that neither turbulence nor diffusion is required for efficient mixing, and only small amounts of reagents are needed. Traditionally, EMMA has been performed by injecting small bands of each reagent onto a capillary, placing the slowest reagent on the capillary first. When the faster moving reagent is injected, it will eventually overtake the first band and mix with it. This method is difficult to implement with enzymatic incubations because the electrophoretic flow must be stopped precisely when the two bands overlap. An experimentally simpler approach involves filling the capillary with substrate and then injecting a small plug of enzyme. The stopped-flow, zero potential incubation can be performed at any convenient time in this manner. In this study, the main channel was filled with 10-4 M tagged substrate, L-leucine β-naphthylamide, which does not fluoresce. Figure 3 shows an assay of 30 nM LAP without stopped-flow incubation. After a small plug of enzyme is injected from the cross, Analytical Chemistry, Vol. 72, No. 22, November 15, 2000

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potential is applied for the duration of the experiment. At pH 7.0, the enzyme has a net negative charge whereas the tagged substrate and free BNA are neutral. Therefore, the faster moving substrate will travel through and mix with the plug of enzyme. Since product is continuously produced while the enzyme travels down the channel, a rectangular peak is expected. The measured plug of BNA has a relatively flat shape beginning at 45 s and lasting until 65 s, with the baseline situated at ∼160 counts s-1. The slight peakedness is attributed to the time required to manually switch the potential. The leading edge of the plug indicates that BNA has a migration rate of 311 µm s-1 (1.4 cm in 45 s). Similarly, the trailing edge yields a value of 215 µm s-1 for the enzyme. Taking into account voltage differences, these two values agree with those reported for the same assay in a capillary format.24 The average count rate, assuming a rectangular peak, is 40 counts s-1 above the baseline. Combining this value with the assay sensitivity yields an average free BNA concentration of 9.6 × 10-7 M, which is ∼1% of the substrate concentration. Nanosecond temporal resolution was used to determine that the regions from 25 to 45 and 65-90 s are due to laser scatter and to verify the presence of BNA from 45 to 65 s. The peak near 5 s is due to photohydrolysis of the substrate during the manual switching of potentials associated with the enzyme loading step. Diffusion affects the data in two ways. After reaction with the enzyme, the freed BNA will diffuse and broaden the peak while it travels down the channel. A typical linear diffusion coefficient for small molecules is 500 µm s-1.29 Considering the BNA peak starts at 45 s and ends at 65 s, this value of the diffusion coefficient predicts a root-mean-square (rms) diffusion distance of 212 µm for the leading edge of the free BNA plug and 255 µm for the trailing edge, corresponding to ∼3.7 s of the peak width. Diffusion is also responsible for loading more enzyme into the channel than predicted by the volume of the injection cross. The first step in loading the column involves transferring the enzyme from the stock to the enzyme waste reservoir for 30 s. A typical diffusion coefficient for a 326-kDa protein is 10 µm s-1.30 During this 30-s period, the enzyme will have an rms diffusion distance of 25 µm in both directions of the reaction channel. The total size of the enzyme plug can then be estimated as 4 times the rms distance plus the channel width, or 150 µm. The diffusion of substrate into the enzyme solution will not produce a large level of free BNA since electrophoretic migration will move most of it toward the enzyme waste reservoir. Using the differential migration velocity of 96 µm s-1, it can be determined that it takes 1.6 s for each substrate molecule to pass through the 150-µm plug of enzyme. During this transit time, the substrate-enzyme interaction produces 9.6 × 10-7 M free BNA. Using an enzyme concentration of 3 × 10-8 M and a 1.6 s interaction time, the turnover rate is estimated to be 20 s-1. Since not all weighed enzyme will be active in solution, this value is consistent with the range of 32-87 reported for similar leucine substrates.31 (29) Bard, Allen J.; Faulkner, Larry R. Electrochemical Methods; John Wiley & Sons: New York, 1980; p 129. (30) Marshall, Alan G. Biophysical Chemistry; John Wiley & Sons: New York, 1978; p 159. (31) Beattie, R. E.; Guthrie, D. J. S.; Elmore, D. T.; Williams, C. H.; Walker, B. Biochem. J. 1987, 242, 281-283.

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Figure 4. Electropherogram for 30 nM LAP enzyme with a 1-mim incubation. Dashed line is the sum of two Gaussian peaks (see text).

Assay with Incubation. Figure 4 shows an assay of 30 nM LAP using a 1-min incubation. Experimentally, potential was applied for 10 s to move the enzyme away from the injection cross, removed for a 1-min incubation, and reestablished to move the free BNA past the detector. Figure 4 provides additional evidence that the first peak to elute (centered around 20 s) is due to photohydrolysis and not enzyme adsorption to the channel walls. In Figure 4. the product peak has grown significantly while the initial peak has approximately the same amplitude. If the initial peak were due to enzyme adsorption, the initial peak would experience a similar increase in amplitude. In the absence of photohydrolysis and diffusion, the signal should consist of a sharp incubation peak with an amplitude of 1500 counts s-1 (40 × 60/1.6), sitting atop a rectangular plateau with an amplitude of 40 counts s-1. Considering the migration velocities, the incubation peak should be centered at 3 s (20 × 10/65) after the rising edge of the plateau. Due to BNA diffusion during the 109 s from injection to detection, the incubation peak should broaden into a Gaussian shape with a fwhm of 538 µm. Using the velocity of BNA, this can be converted into a temporal fwhm of 1.7 s. The measured value is 5 s, making the incubation peak ∼3 times wider than expected. This same type of superbroadened incubation peak was also seen in an EMMA determination of LAP with a 75-µm capillary.24 However, using a height of 200 counts s-1 and a fwhm of 6 s, the total counts within the Gaussian peak are 1200, exactly the value predicted in the absence of diffusion. Tailing of the incubation peak can be accounted for by a second bolus of enzyme. Throughout the entire experiment, enzyme is diffusing from the injection channels into the reaction channel. When the potential is continuously applied to the reaction channel, insufficient enzyme exists in any one location to produce a measurable signal. However, during incubation, each arm of the injection channel has sufficient time to inject effectively a second bolus, which can be estimated as having a width of 60 µm. Because BNA travels faster than the enzyme, the peak due to the second bolus should be 7 s behind the injected bolus. Indeed, a composite curve (shown as the dashed line in Figure 4) constructed by the addition of two Gaussian peaks (fwhm 5 s, separation 7 s, and relative heights 1:0.4) agrees remarkably well with the data in Figure 4.

Figure 5. Electropherogram for diluted LAP enzyme and a 50min incubation. Dashed line is the sum of two Gaussian peaks (see text).

To verify the presence of a second bolus, the enzyme solution was diluted and incubated for 5 min. The result, shown in Figure 5, clearly shows two peaks. A composite curve (shown as the dashed line in Figure 5) using two Gaussian peaks (fwhm 6 s, separation 8.5 s, and relative heights 1:1.8) gives the same general shape as that in Figure 5. The increased peak separation is most likely an artifact due to manually switching the potentials and initiating the timing sequence. One additional contribution to the superbroadening of the peaks that should be considered is the possibility of unwanted flow resulting from slight differences in the fluid levels in the reservoirs. If the unwanted flow was counter to the electroosmotic flow, the result would be effectively broader peaks. This phenomenon may also explain the slight variance in elution time of the peaks. Instrumental Improvements. One concern in the present study is the high concentration of free BNA required for quantitation. This value is 1% the substrate concentration, resulting in a narrow linear dynamic range. A simple change in the detection optics should improve the measurement. By fabricating a chip with a cover plate thinner than 240 µm, a 1.25 NA objective could be used. In water the focal radius would be 0.45 µm, corresponding to a confocal parameter of 2.86 µm. Using this objective would result in an 81% excitation efficiency compared to the value of 8% in the present study. Additionally, the light-gathering power of an optical system is proportional to the square of the numeric aperture. Thus, it is tempting to predict that the combination of (32) Zugel, S. A.; Lytle, F. E. Appl. Spectrosc. 2000, 54, 1490-1494.

these two parameters would increase the measurement sensitivity by a factor of 250. Unfortunately, this expectation is counter to experience obtained from a previous study using epi-excitation with a 75-µm capillary format.32 On the basis of this previous study, a realistic improvement would be a value from 10 to 20. The extrawide injection bolus of enzyme and the second, diffusion, bolus can both be eliminated by computer control of the potential applied to each of the four channel segments. During the injection step, the potential on the reaction channel needs to be adjusted to create a small flow from both the substrate and waste reservoir into the enzyme waste reservoir. This will “pinch” the enzyme flow and prevent diffusion into the reaction channel. Likewise, during incubation, the potential on the injection channel needs to be adjusted to create a small flow into the enzyme and enzyme waste reservoirs. The resultant flow will prevent enzyme and free BNA from accumulating in the reaction channel. Computer control of the electric potential might also reduce the width of the incubation peak. In both this work and the referenced capillary experiment, the potential was disconnected from the solution during incubation. If there were any capacitive effect due to built up surface charge, the extra width might be due to migration of the negatively charged enzyme. If such charging is occurring, it could be reduced or eliminated by adjusting the potentials such that both the center of the injection cross and the reaction channel waste reservoir are grounded during incubation. CONCLUSIONS The utility of performing TPE fluorescence detection with microchips was demonstrated. Additionally, an alternative assay format, EMMA, was successfully performed for the first time on a microfabricated chip. Despite the current optical limitations caused by the microchip used in this study, this first-generation instrumental arrangement has already yielded excellent detection limits with ∼5% reproducibility. The combination of these two techniques with microchips allows for efficient detection of enzyme molecules with the added advantages of reduced analysis time (less 3 min) and minimal chemical consumption. ACKNOWLEDGMENT This research was supported in part by NIH Grant 035421.

Received for review July 11, 2000. Accepted September 11, 2000. AC000801K

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