Anal. Chem. 1999, 71, 5206-5212
Microfluidic Assays of Acetylcholinesterase Inhibitors Andrew G. Hadd,† Stephen C. Jacobson, and J. Michael Ramsey*
Oak Ridge National Laboratory, P.O. Box 2008 Oak Ridge, Tennessee 37831-6142
A microfabricated device for flow injection analysis and electrophoretic separation of acetylcholinesterase (AChE) inhibitors is described. Solutions of inhibitor, enzyme, substrate, and derivitizing agent were mixed within the channels of the microchip using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was measured in an onchip reaction of thiocholine with coumarinylphenylmaleimide, and the resulting thioether was detected by laserinduced fluorescence. Inhibitors reduced the fluorescence signal and produced a negative peak diagnostic for the type of inhibition. A Gaussian peak was observed for competitive inhibitors, whereas a broad negative peak was observed for irreversible inhibitors. From a microchip assay for tacrine, an inhibition constant, Ki, of 1.5 ( 0.2 nM was derived, which compared well with a standard cuvette assay. A flow injection assay of two irreversible inhibitors, carbofuran and eserine, was performed. With a 5-min stopped-flow reaction time, a detection limit of 10 nM carbofuran was obtained. As a potential multiplex screening device, a mixture of four cationic inhibitors, tacrine, edrophonium, and tetramethyl- and tetraethylammonium chloride, was separated and detected within 70 s. Microfabricated devices that integrate chemical reactions with rapid analysis times have significant applications for highthroughput drug screening, automated analysis, and clinical chemistry. Microchips are characterized by reduced analysis time and reagent consumption, ease of automation, and fluid control of subnanoliter volumes. Within microchannel networks, a variety of electrically driven separations have been performed.1-12 Microchips have also been developed for performing chemical † Present address: Molecular Dynamics, 928 E. Arques Ave., Sunnyvale, CA 94086. (1) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926. (2) Manz, A.; Harrison, J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Lu ¨ di, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253. (3) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481. (4) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895. (5) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114. (6) Burggraf, N.; Manz, A.; Effenhauser, C. S.; Verpoorte, E.; de Rooij, N. F.; Widmer, H. M. J. High Resolut. Chromatogr. 1993, 16, 594. (7) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2858. (8) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369.
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reactions, including arrays for solid-phase chemistry,13 reaction wells for polymerase chain reactions (PRC),14 channels with immobilized enzymes for flow injection analysis,15 and manifolds for homogeneous enzyme assays.16 Chemical reactions in organic solvents have also been demonstrated using electrokinetic transport in a microfluidic device.17 The ability to design and machine channel manifolds with lowvolume connections renders microchips suitable for combining several steps of an analytical process on one device without loss of performance. Microchips that combine chemical reactions with electrophoretic analysis have been demonstrated for pre-18 and postseparation4,19 reactions, for DNA restriction digests with fragment sizing,20 and for PCR amplification and electrophoretic sizing.21,22 The advantages of integrated microchips, computercontrolled electrokinetic fluid manipulation, and reduced solvent and analyte consumption are applicable to studying enzyme inhibition. In this work, a monolithic device was developed for flow injection analysis and electrophoretic separation of acetylcholinesterase (AChE) inhibitors. Inhibitors of acetylcholinesterase, an essential enzyme for nerve function, include organophosphorus and carbamate pesticides23 and therapeutic drugs regulating Alzheimer’s disease.24 A series of biochemical detection methods for these compounds, (9) Moore, A. W., Jr.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184. (10) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949. (11) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11348. (12) Woolley, A. T.; Matthies, R. A. Anal. Chem. 1995, 67, 3676. (13) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767. (14) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 1815. (15) Murakami, Y.; Takeuchi, T.; Yokoyama, K.; Tamiya, E.; Karube, I.; Suda, M. Anal. Chem. 1993, 65, 2731. (16) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407. (17) Salimi-Moosavi, H.; Tang, T.; Harrison, D. J. J. Am. Chem. Soc. 1997, 119, 8716. (18) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127. (19) Jacobson, S. C.; Koutny, L. B.; Hergenro¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472. (20) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720. (21) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081. (22) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158. (23) Froede, H. C.; Wilson, I. B. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1971; Vol. V, p 87. (24) Summers, W. K.; Majovski, L. V.; Marsh, G. M.; Tachiki, K.; Kling, A. N. Engl. J. Med. 1986, 371, 1241. 10.1021/ac990591f CCC: $18.00
© 1999 American Chemical Society Published on Web 10/15/1999
based on reduction of AChE activity, have been developed.25-32 Photometric detection of AChE activity using acetylthiocholine (AThCh) and 5,5′-dithiobis(2-nitrobenzoate) (DTNB, or Ellman’s reagent)33 has been coupled to flow injection analysis of pesticides,27 to the postcolumn detection of organophosphorus nerve agents separated by high-performance liquid chromatography,32 and to microassays using immobilized AChE.28,30 Other methods for assaying AChE inhibitors include hydrolysis of R-naphthyl acetate detected with Fast Red GG salt,29 chemiluminescence detection,31,34 and electrochemical detection.27 A fluorogenic assay for AChE activity has been developed using the reaction of thiocholine with coumarinylphenylmaleimide (CPM) to produce a highly fluorescent thioether, CPM-thiocholine.35 The sequential steps required for a CPM-based AChE assay were integrated into a microfluidic device for flow injection analysis and electrophoretic separation of AChE inhibitors. Within the channels of the microchip, nanoliter volumes of the assay reagents, i.e., substrate, inhibitor, enzyme, and derivitizing agent, were manipulated using computer-controlled electrokinetic transport. AChE activity was monitored by laser-induced fluorescence (LIF) detection of CPM-thiocholine, which was formed on-chip by mixing hydrolyzed acetylthiocholine with CPM. Inhibitors reduced the fluorescence signal with a negative peak diagnostic for competitive and irreversible inhibitors. With the same microchip, a stopped-flow assay of an irreversible inhibitor and an electrophoretic separation of four competitive inhibitors were demonstrated. EXPERIMENTAL SECTION Chemicals. Acetylthiocholine chloride, 5,5′-dithiobis(2-nitrobenzoic acid), acetylcholinesterase (EC 3.1.1.7, electric eel, type VI-S, 530 units/mg), tacrine, edrophonium chloride, eserine, tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), rhodamine B, and Triton X-100 were obtained from Sigma (St. Louis, MO). Coumarinylphenylmaleimide (N-(4(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide) was purchased from Molecular Probes (Eugene, OR). Carbofuran was obtained as Furadan from FMC Corp. (Princeton, NJ). Acetonitrile (0.002% water) and methanol (Burdick and Jackson) were purchased through VWR Scientific. Tris-HCl (1 M, pH 8.0) and EDTA (0.5 M) were purchased from GibcoBRL. All reagents were used without further purification. Aqueous buffers were prepared with distilled deionized water filtered through a Barnstead Nanopure system (Dubuque, IA). Stock AThCh solutions, 100 mM, were prepared in distilled H2O, stored frozen, and remade weekly. Stock solutions of 20 mM eserine and 10 mM CPM were prepared in acetonitrile; tacrine, (25) Alfthan, K.; Kentta¨maa, H.; Zukale, T. Anal. Chim. Acta 1989, 217, 43. (26) Flentge, F.; Venema, K.; Koch, T.; Korf, J. Anal. Biochem. 1992, 204, 305. (27) Gu ¨ nther, A.; Bilitewski, U. Anal. Chim. Acta 1995, 300, 117. (28) Hammond, P. S.; Forster, J. S. Anal. Biochem. 1989, 180, 380. (29) Leon-Gonzalez, M. E.; Townshend, A. Anal. Chim. Acta 1990, 236, 267. (30) Lui, J.; Tan, M.; Liang, C.; Ying, K. B. Anal. Chim. Acta 1996, 329, 297. (31) Moris, P.; Alexandre, I.; Roger, M.; Remacle, J. Anal. Chim. Acta 1995, 302, 53. (32) Sipponen, K. B. J. Chromatogr. 1987, 389, 87. (33) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88. (34) Roda, A.; Rauch, P.; Ferri, E.; Girotti, S.; Ghini, S.; Carrea, G.; Bovara, R. Anal. Chim. Acta 1994, 294, 35. (35) Parvari, R.; Pecht, I.; Soreq, H. Anal. Biochem. 1983, 133, 450.
Figure 1. Schematic of the microchip used for analysis of acetylcholinesterase inhibitors. The fluid reservoirs were filled with AChE for enzyme, acetylthiocholine chloride for substrate, and coumarinylphenylmaleimide for fluorophore. The inhibitor sample was changed as indicated; the sample waste and waste reservoirs contained analysis buffer.
25 mM, was prepared in methanol. A stock solution of 50 mM carbofuran was prepared by adding 4.6 mL of acetonitrile to 0.51 g of Furadan (47% carbofuran), vortex mixing for 5 min, followed by filtering the solution from undissolved ingredients. A Tris/ EDTA buffer (TE buffer) of 50 mM Tris-HCl, pH 8.0, and 0.5 mM EDTA was prepared and used to dilute all stock solutions and for all assays. For on-chip assays, CPM was diluted to 500 µM in 2% TE-buffered acetonitrile with 1% Triton X-100 (w/w). Microchip Device. A schematic of the AChE microchip is shown in Figure 1. The chip consisted of a four-way injection cross followed by a 34.4-mm separation channel, a mixing-tee (T-1) for introducing substrate, a 10.2-mm reaction channel, a second mixing-tee (T-2) for introducing CPM, and an 8-mm detection channel. Prior to the waste reservoir, the detection channel was widened to 130 µm to allow longer reaction times between the thiocholine and CPM. Because the channel resistance is inversely proportional to the cross-sectional area of the channel, increasing the width of the channel reduced the channel resistance and consequently the electric field strength. This lower field strength resulted in lower flow rates and allowed a longer reaction time. This in turn yielded a higher fluorescence signal and better detection sensitivity. The microchip channels were fabricated into glass microscope slides (Goldseal Products, Portsmouth, NH) using photolithography and wet chemical etching. A cover plate was thermally bonded to the etched glass slide, forming a channel network.9,36 Cylindrical glass reservoirs, 140 µL (12-mm length (36) Jacobson, S. C.; Ramsey, J. M. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1996.
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and 6-mm o.d.) for the CPM solution and 70 µL (6-mm length and 6-mm o.d.) for the other reservoirs, were fitted over the termini of the channels where they extended beyond the edge of the cover plate using Epo-Tek 353ND epoxy (Epoxy Technologies, Bellerica, MA). The channels were 14 µm deep and 48 µm wide at half-height. The microchip was prepared daily by washing the channels with 0.1 N sodium hydroxide followed by analysis buffer using a vacuum line applied to one of the channel reservoirs. Platinum electrodes dipped into the solution reservoirs provided electrical contact to individual high-voltage power supplies (Ultravolt, Rokanowa, NY). Microchip Enzyme Assays. Solutions of enzyme, inhibitor, substrate, and fluorophore were mixed at channel intersections using electrokinetic flow. Unless otherwise indicated, fluid reservoirs were filled with solutions of 5.0 nM AChE for enzyme (Figure 1), 1.0 mM AThCh for substrate, and 500 µM CPM in 2% TE buffer in acetonitrile with 1% Triton X-100 (w/w) for fluorophore. Enzyme activity was monitored using laser-induced fluorescence detection. The UV lines (350.1-356.4 nm) of a Krypton ion laser (Innova 300, Coherent, Palo Alto, CA) were focused to a spot 6.5-mm downstream from the second mixing-tee (T-2, Figure 1) using a plano-convex lens and mirror to impinge the beam 50° from normal. The plasma lines of the laser were filtered with a long-pass dichroic mirror (400DCLP, Omega Optical, Brattleboro, CT). Fluorescence emission was collected with a 20× objective lens (NA ) 0.42) and measured with a photomultiplier tube (PMT, Oriel 77348, Stratford, CT). Prior to the PMT, a 2.0mm pinhole for spatial filtering and a 470DF65 band-pass filter (Omega Optical) were used. An in-house developed LabView program (National Instruments) was used to control the highvoltage power supplies and data acquisition. Inhibitor samples were introduced into the buffer stream containing AChE using a gated injection technique.19,37 Briefly, the inhibitor sample was electrokinetically transported from the inhibitor reservoir through the valve to the sample waste reservoir (see Figure 1). Simultaneously, the enzyme solution was electrokinetically pumped from the enzyme reservoir through the valve and down the separation channel with a small fraction of enzyme solution flowing into the sample waste channel to prevent inhibitor from migrating or diffusing into the separation channel. For inhibitor injection, the potentials at the enzyme and sample waste reservoirs were temporarily lowered and raised, respectively, to match the potential at the valve intersection. Inhibitor then electrokinetically migrated into the separation channel with no net flow observed in either the enzyme or sample waste channels. The injection process was completed by resetting the potentials at the enzyme and sample waste reservoirs to their initial values to cut off the plug of injected inhibitor and to prevent bleeding of excess inhibitor into the separation channel. For electroosmotic flow rates used in these experiments, a 1.0-s injection corresponded to a volume of 400 pL. The height of the fluorescence signal, Ho, decreased to a lower signal height, Hi, in the presence of inhibitor. Inhibition constants were derived from the difference between these two signal heights, Ho - Hi. To maintain a steady signal and electrokinetic flow rate, the CPM solution was replenished every 25 min because of evaporation. After 4 h, the chip was washed for 2 min with 0.10 N NaOH (37) Jacobson, S. C.; Ramsey, J. M. Electrophoresis 1995, 16, 481.
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followed by analysis buffer for 5 min. Typically, between chip washings, 60 assays could be performed. To reduce bubble formation in the detection channel, a 2% buffered acetonitrile solution with 1% Triton X-100 was used to dilute the stock solution of CPM. In this solution, CPM was stable for up to 6 h without a significant increase in fluorescence due to hydrolysis. Standard Enzyme Assay. AChE activity was calibrated using a standard photometric technique.33 In microcentrifuge tubes, 20 µL of 10 nM AChE was added to 0.80 mM AThCh and 500 µM DTNB for a final volume of 1.0 mL. After vortex mixing, the contents were transferred within 20 s to 1.0-mL cuvettes and placed in a thermostated Cary 1E UV-visible spectrophotometer. Substrate hydrolysis was monitored at 410 nm for 2 min. Kinetic constants for eserine and carbofuran were derived from plots of the pseudo-first-order rate constant versus inhibitor concentration.38 Capillary Electrophoresis for Inhibitor Mobilities. The electrophoretic mobilities of tacrine, edrophonium, eserine, and carbofuran were determined using a standard capillary electrophoresis instrument equipped with UV detection (270A capillary electrophoresis system, Applied Biosystems, CA). A 39 cm × 50 µm i.d. capillary (JBL Scientific) with an effective length of 20 cm was used for all experiments. The detection window was made by burning off the polyimide coating. Inhibitor samples were introduced with pressure injections and migrated through the capillary at a field strength of 510 V/cm. Migration times were determined from a minimum of three runs, and electrophoretic mobilities were referenced to the migration time of mesityl oxide. RESULTS AND DISCUSSION Channel Manifold and Electrokinetic Mixing. The channel design allowed for reaction of inhibitors with enzyme in the separation channel followed by mixing with substrate in the reaction channel. A fluorescence signal was obtained by continuously mixing in a 50:50 ratio AChE with AThCh at T-1 (Figure 1) and subsequent 50:50 mixing with CPM (in buffered acetonitrile) at T-2. Since acetonitrile inhibited the AChE activity, CPM was added postreaction to quantitate the amount of thiocholine produced in the reaction channel. The reaction time depended on the electrokinetic flow rate and transit time of the reagents through each channel section, which was controlled by the applied electric field and mixing ratio at both mixing-tees. The mixing ratio at T-1 and T-2 (Figure 1) was controlled by applying voltages derived from a circuit model of the microchip. At T-2, aqueous buffer from the reaction channel was proportionately mixed with buffered acetonitrile from the fluorophore channel. The values for the applied voltages were derived without compensating for the reduced resistivity of the organic solvent. The mixing ratio at T-2 of buffered acetonitrile to TE buffer could be increased in 25% increments from 25 to 75%. The mixing ratio was verified by adding 20 µM rhodamine B doped as a marker in the acetonitrile solution and monitoring the fluorescence signal downstream from T-2. When neat acetonitrile was used for the CPM solution, mixing of the organic and aqueous phases at T-2 resulted in the formation of bubbles up to 1 mm downstream from the intersection. The formation of the bubbles was reduced, but (38) Leytus, S. P.; Toledo, D. L.; Mangel, W. F. Biochim. Biophys. Acta 1984, 788, 74.
of the inhibitor is minimized because the overall reaction equilibrium favors substrate binding.40 When the Km is known and the substrate concentration fixed, the Ki can be determined from the rate of reaction in the absence of inhibitor, νo, minus the rate of reaction in the presence of inhibitor, νi, by
ν o - νi )
Figure 2. Flow injection peaks showing effect of increasing tacrine concentration on the inhibition of acetylcholinesterase. Tacrine concentration was changed manually prior to injection; peaks are labeled with the indicated tacrine concentration (in nM). Conditions: 2.5 nM AChE, 1.0 mM acetylthiocholine chloride, 250 µM CPM, and 400-pL injections of tacrine.
not eliminated, by the addition of 2% buffer and 1% Triton X-100 to the acetonitrile. Flow Injection Analysis of Competitive Inhibitors. Tacrine, a potent inhibitor of AChE, was assayed by flow injection analysis. In these experiments, 400-pL injections of tacrine were made and the decrease in the peak height relative to the fluorescence signal was measured. Inhibition peaks for injections of 0, 5, 10, 20, 60, 100, and 200 nM tacrine are shown in Figure 2. The height of the fluorescence signal decreased in proportion to the concentration of tacrine injected, and a detection limit of 5 nM (1 ppb) tacrine was observed. The migration time for the tacrine peak had a 2% RSD for seven consecutive runs. A pertubation in the signal beginning at 100 s was observed for each injection. Since the injection of inhibitor introduced a “void volume” of buffer into the enzyme stream, the signal change near 100 s corresponded to the migration time of the enzyme. The decrease in fluorescence signal representing enzyme inhibition was confirmed by replacing the substrate solution with thiocholine produced off-chip and mixing with CPM in the detection channel. Injections of 400 nM tacrine in the absence of enzyme failed to decrease the fluorescence signal from the on-chip mixing of thiocholine and CPM. For competitive inhibitors, an equilibrium between inhibitor and enzyme reduces the concentration of free enzyme available for substrate binding. The inhibitor equilibrium dissociation constant, Ki, is related to the initial inhibitor concentration, [I]o, and the enzyme reaction rate, νinitial by
νinitial )
Vmax[S] (1 + [I]o/Ki)Km + [S]
(1)
where Vmax is the maximum reaction rate, [S] the substrate concentration, and Km the Michaelis constant. At zero inhibitor concentration, eq 1 reduces to the Michaelis-Menten expression for enzyme kinetics. At high substrate concentration, the effect
νo[I]o Ki(1 + [S]/Km) + [I]o
(2)
The Ki for tacrine can be derived from a nonlinear least-squares fit of eq 2 to a plot of νo - νi versus inhibitor concentration. For a standard cuvette assay, initial rates in the presence and absence of tacrine were determined from the linear portion of the absorbance versus time plot. With a microchip flow injection assay, the rate of substrate hydrolysis corresponded to the amount of thiocholine produced in the reaction channel divided by the transit time through that channel section.16 In the absence of inhibitor, the fluorescence signal height without inhibitor, Ho, was proportional to νo, and the minimum signal height in the presence of inhibitor, Hi, was proportional to νi. A Km of 75 ( 10 µM was derived from an on-chip assay by plotting the increase in fluorescence signal versus substrate concentration. The enzyme-catalyzed hydrolysis of AThCh was evaluated in the concentration range 25 µM-2 mM, by manually refilling the substrate reservoir with a new concentration of AThCh. Fluorescence at each substrate concentration was obtained and subtracted from the signal measured from the enzymecatalyzed reaction. The Km obtained from a microchip assay compared reasonably well with 110 ( 20 µM AThCh measured in a cuvette using DTNB. Substrate inhibition was observed above 1.5 mM AThCh for both methods, as seen in other assays.35 The reaction rate dependence on the concentration of tacrine for a microchip and standard cuvette assay is compared in Figure 3. In this figure, the difference in peak heights, Ho - Hi for the microchip assay is shown on the left axis, and νo - νi for the cuvette-based assay is shown on the right axis. Qualitatively, both assays have a similar dependence on the tacrine concentration, with 50% inhibition near 20 nM and maximum inhibition near 200 nM. From a nonlinear regression fit of the rate differences versus tacrine concentration, a Ki of 1.4 ( 0.2 nM for the cuvette assay and a Ki of 1.5 ( 0.2 nM for the microchip assay were derived. Optimized Substrate Concentration Range. According to eq 2, the peak area due to the presence of an inhibitor is optimized by the difference between νo and νi. The rate of enzyme turnover in the absence of inhibitor increases with increasing substrate concentration, reaching a maximum above the Km of the enzyme. However, with a large excess of substrate, the equilibrium favors substrate binding, reducing the observed signal decrease in the presence of an inhibitor. Because of this relationship between substrate and inhibitor concentration, an effort was made to optimize the substrate concentration for the detection of different inhibitors. The maximum peak area was determined for tacrine and edrophonium, two competitive inhibitors, and eserine, an irreversible inhibitor. In Figure 4, a linear-log plot of normalized (39) Seiler, K.; Fan, Z. H.; Fluri, K.; Harrison, D. J. Anal. Chem. 1994, 66, 3485. (40) Engel, P. C.; Dickinson, F. M.; Cornish-Bowden, A. In Enzymology; Engel, P. C., Ed.; Academic Press: San Diego, 1996; p 77.
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Figure 3. Difference in initial and inhibited enzyme reaction rates versus tacrine concentration for microchip- and cuvette-based assays. Left axis shows values for difference in peak height (Ho - Hi) for the microchip assay (2); right axis, the difference in initial reaction rates (νo - νi) for the cuvette assay (b). Nonlinear least-squares fits to eq 2 are indicated as a dotted line for the microchip assay and as a solid line for the cuvette assay.
Figure 4. Peak area versus acetylthiocholine concentration for 20 nM tacrine (2), 2 µM edrophonium (9), and 5 µM eserine (b). Areas were calculated from the decrease in signal from 400-pL injections and normalized to the greatest area decrease for each inhibitor. The substrate concentration was changed manually, and flow injection assays were performed using 2.5 nM AChE and 250 µM CPM.
peak area versus substrate concentration is shown for 20 nM tacrine, 2 µM edrophonium, and 5 µM eserine. The substrate was increased from 125 µM to 5 mM, and the areas were normalized as a fraction of the maximum peak area for each inhibitor. The maximum area for tacrine occurred at 500 µM AThCh, for edrophonium at 250 µM AThCh, and for eserine at 2 mM AThCh. As the substrate concentration increased, the peak area for tacrine and edrophonium sharply decreased after 1.0 mM AThCh. This decrease above 1.0 mM AThCh reflected the reaction rate dependence on both the inhibitor and substrate concentrations (eq 2) and served as a diagnostic for competitive inhibition.40 5210
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Figure 5. Flow injection peaks for irreversible inhibition comparing the injections of (a) 25 µM eserine and (b) 25 µM carbofuran. Conditions: 2.5 nM AChE, 0.50 mM AThCh, and 250 µM CPM.
In contrast to tacrine and edrophonium, the peak area did not sharply decrease with increasing substrate concentration for eserine, an irreversible inhibitor. For irreversible inhibitors, the lack of change in the peak area reflected the mechanism of inhibition. For eserine, covalent binding of the enzyme and inhibitor occurred in the separation channel, which permanently inactivated a zone of enzyme. Since the inhibited zone of enzyme was covalently deactivated, the observed inhibition peak was unaffected by increases in the substrate concentration. The inhibition peak for two irreversible inhibitors is described below. Flow Injection Analysis of Irreversible Inhibitors. Relative inhibition of acetylcholinesterase was determined from a flow injection assay of carbofuran and eserine. Inhibition peaks of fluorescence signal versus time are compared in Figure 5 from injections of 25 µM eserine (a) and 25 mM carbofuran (b). The peak width measured at half-height for these inhibitors was significantly broader than the peaks for tacrine, e.g., 44 s for eserine compared to 8 s for tacrine. The width of the peak also depended on the inhibitor, ranging from 44 s for eserine and 28 s for carbofuran. The peak observed for irreversible inhibitors depended on the reaction kinetics and electrophoretic mobilities of enzyme and inhibitor. In contrast to tacrine, these inhibitors inactivate AChE by covalent phosphorylation or carbamylation of the active site serine.27,29 When the concentration of inhibitor is in large excess over the enzyme concentration, this reaction can be simplified to
E + I f E-I*
(3)
where E is the enzyme concentration, I is the inhibitor concentration, and E-I is the concentration of the inactivated enzymeinhibitor complex. As the zone of inhibitor migrated through the separation channel, the inhibitor mixed and reacted with enzyme, forming E-I*. Since the inactivated enzyme had a lower mobility than free inhibitor, a trailing product zone of E-I* was formed. The start of the E-I* product zone, seen as the initial decrease in signal in Figure 5, corresponded to the migration time of the
Figure 6. Stopped-flow assay of carbofuran comparing an 800-pL injection of 10 nM carbofuran (solid line) to an injection of buffer (dotted line). The increase in signal at 325 s corresponds to restored analyte flow past the detection region after the stop-flow time. Conditions: 2.5 nM AChE, 500 µM AThCh, and 250 µM CPM; stopflow time of 300 s.
inhibitor and the end of the product zone to the migration time of the E-I* complex. Broad product peaks due to differences between reactant and product mobilities have been observed for electrokinetically controlled enzyme reactions41,42 and in affinity capillary electrophoresis.43 Similarly, the microchip flow injection assay allowed irreversible inhibitors to be characterized by their peak shape. Stopped-Flow Assay and Detection Limit for Carbofuran. Since irreversible inhibition depends on the reaction time for inhibitor and enzyme, a stopped-flow method was used to increase the detection sensitivity. A sample zone of 800 pL of carbofuran was introduced into the separation channel in a 50:50 mixing ratio with enzyme-doped buffer. After the sample migrated downstream for 4 s, fluid flow through the separation channel was stopped. Since the injected sample occupied half of the separation channel during the injection, diffusional mixing of the inhibitor with enzyme was improved. To stop the flow of fluid through the separation channel, the voltages applied at each reservoir were set such that 100% flow from the substrate channel through T-1 was maintained. Constant flow downstream from T-1 was necessary to prevent excessive buildup of product in the reaction and detection channels of the microchip. After 5 min, fluid flow was returned to normal assay conditions, and the signal change relative to an injection of buffer under the same conditions was compared. A stop-flow assay of 10 nM carbofuran compared to a blank injection for a 5-min stop time is shown in Figure 6. The buffer and E-I zones separated from one another during electromigration to the detection channel, allowing for detection of carbofuran. The decrease in fluorescence between 365 and 400 s relative to the buffer run (dotted line) indicated inhibition of AChE and detection of 10 nM carbofuran. Although the sensitivity was increased with a stop-flow assay, for a 5-min stop time, a significant change in signal was not (41) Bao, J.; Regnier, F. J. Chromatogr. 1992, 608, 217. (42) Avila, L. Z.; Whitesides, G. M. J. Org. Chem. 1993, 58, 5508. (43) Busch, M. H. A.; Kraak, J. C.; Poppe, H. J. Chromatogr., A 1997, 777, 329.
Figure 7. Electrophoretic separation of 8 mM tetramethylammonium chloride (1), 2 mM tetraethylammonium chloride (2), 20 nM tacrine (3), and 4 µM edrophonium (4), detected by inhibition of AChE. The dotted line indicates the fluorescence signal versus time of thiocholine (produced off-chip) for an injection of the same inhibitor mixture. The thiocholine peak is offset by 5% for comparison. Conditions: 0.2-s injection, 2.5 nM AChE, 500 µM AThCh, and 250 µM CPM.
observed until the concentration of carbofuran was increased to 400 nM, suggesting a high limit of quantitation. The microchip stop-flow assay requires further refinement for improved detection limits and increased throughput. For comparison, a detection limit of 8 nM carbofuran using a 35-s stop-flow time and immobilized AChE has been reported.29 Electrokinetic Separation of Cationic Inhibitors. An electrophoretic separation of four AChE inhibitors, 8 mM TMAC, 2 mM TEAC, 20 nM tacrine, and 2 µM edrophonium, is shown in Figure 7. The inhibitors were detected as a reduction in AChE activity. Inhibitor peaks were identified by the migration times of individual compounds analyzed by flow injection analysis. The relative mobilities of tacrine and edrophonium were verified by a standard capillary electrophoresis experiment. The inhibitor concentration ranged from 8 mM TMAC to 20 nM tacrine, thereby demonstrating the detection and isolation of tight-binding inhibitors from a potentially complex mixture. Since the concentration of TMAC and TEAC was nearly equal with the concentration of the analysis buffer, an experiment was performed verifying that the decrease in fluorescence signal corresponded to modulated enzyme activity. In this experiment, AChE was replaced by analysis buffer and the substrate replaced with 20 µM thiocholine. A fluorescence signal from the on-chip mixing of thiocholine and CPM was obtained. An injection of the same inhibitor mixture under these conditions is shown as the dotted line in Figure 7. For TMAC, a perturbation occurred at 43 s, near the migration time of TMAC; a similar, but smaller, perturbation occurred near the TEAC peak. No distortion of the thiocholine signal was observed for tacrine and edrophonium. The sharp increase and decrease in the CPM-thiocholine fluorescence corresponded to stacking of the analytes at the acetonitrile-buffer interface due to the increase in the conductivity of the analyte zone relative to the analysis buffer.44 Since TEAC was only 2 mM, Analytical Chemistry, Vol. 71, No. 22, November 15, 1999
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the effect was not as pronounced, and unobservable for tacrine and edrophonium. The perturbation for 8 mM TMAC was observed as a shoulder preceding the TMAC peak detected by enzyme inhibition. Because the signal change occurred with a different migration time (43 versus 46 s), the observed decrease in the fluorescence signal was due to enzyme inhibition and not due to indirect fluorescence or displacement of the product zone. In conclusion, a microfluidic device capable of comparing, screening, and determining the inhibition constants for acetylcholinesterase inhibitors was demonstrated. All reagent mixing was performed on-chip and additional steps of the assay, such as inhibitor dilution, could be integrated with additional channels to control dilution. The electrokinetic mixing of CPM in acetonitrile with the enzyme reaction products in aqueous buffer demonstrated the ability to integrate a wide variety of on-chip chemistries. Although limited presently to flow injection analysis of irreversible inhibitors, this microchip represents the ability to screen com(44) Shihabi, Z. K. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1996; p 457.
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pounds or complex samples for relative decreases in enzyme activity. By miniaturizing the components of an enzyme assay, less expensive and versatile biochemical detection systems can be developed for drug screening, clinical diagnostics, and fieldready applications. ACKNOWLEDGMENT Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under Contract DE-AC05-96OR22464. This research was supported in part by an appointment of A.G.H. to the ORNL Postdoctoral Research Associates Program administered jointly by the Oak Ridge Institute for Science and Education and ORNL. We thank Dr. Bill Shamiyeh from the University of Tennessee Department of Entomology and Plant Pathology for donating the Furadan. Received for review June 3, 1999. Accepted September 2, 1999. AC990591F
