Simple Device for Multiplexed Electrophoretic Separations Using

Nov 13, 2008 - Simple Device for Multiplexed Electrophoretic Separations Using Gradient Elution Moving Boundary Electrophoresis with Channel Current ...
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Anal. Chem. 2008, 80, 9467–9474

Simple Device for Multiplexed Electrophoretic Separations Using Gradient Elution Moving Boundary Electrophoresis with Channel Current Detection David Ross* and Jason G. Kralj Biochemical Science Division, National Institute of Standards & Technology, Gaithersburg, Maryland 20899 A new microfluidic electrophoresis device and technique is described that is designed specifically for multiplexed, high-throughput separations. The device consists of an array of short (3 mm) capillaries connecting individual sample reservoirs to a common buffer reservoir. Each capillary in the array functions as both a separation channel and as a conductivity-based detection cell. The new technique is based upon the recently described gradient elution moving boundary electrophoresis (GEMBE) technique, which uses a combination of an electric field and buffer counterflow to achieve electrophoretic separations in short capillaries or microfluidic channels. A high voltage drives electrophoresis of the sample analytes through each separation channel. At the start of a separation, the bulk counterflow of buffer through the channel is high, and none of the analytes of interest can enter the channel. The counterflow is then gradually reduced until each analyte, in turn, is able to enter the channel where it is detected as a moving boundary or step. With very short capillaries, only one step at a time is present in each capillary, and the electric current through the channels can then be used as the detector signal, without any extra detector hardware. The current vs time signal for each channel is then smoothed and differentiated to produce a set of simultaneous electropherograms. Because there is no light source or other added hardware required for detection, the system is simple and can be easily and inexpensively scaled up to perform large numbers of simultaneous analyses. As a first demonstration, a 16-channel array device is used for high-throughput, time-series measurements of enzyme activity and inhibition. The ability to acquire large amounts of high quality data very rapidly is increasingly important for progress in the biological sciences (e.g., drug discovery and systems biology). Capillary electrophoresis (CE) has a number of advantages that make it well suited to provide the kind of quantitative data that is necessary.1,2 In particular, it is relatively fast, requires only small (1) Babu, S.; Song, E. J.; Babar, S. M. E.; Wi, M. H.; Yoo, Y. S. Electrophoresis 2006, 27, 97–110. (2) Song, E. J.; Babar, S. M. E.; Oh, E.; Hasan, M. N.; Hong, H. M.; Yoo, Y. S. Electrophoresis 2008, 29, 129–142. 10.1021/ac801597e Not subject to U.S. Copyright. Publ. 2008 Am. Chem. Soc. Published on Web 11/13/2008

amounts of sample and reagents, and can be applied to a wide variety of biochemical analysis problems. In addition, capillary array electrophoresis systems have been described for highthroughput operation,3-8 and their development is widely regarded as one of the key technological innovations that allowed for the earlier-than-expected completion of the human genome project.9-11 However, one obstacle to the wider application of highthroughput CE is the complexity and high cost of capillary array instruments and techniques. The advent of microchip CE12 offers one possible route to overcome this obstacle, and a great deal of research has been focused on microchip CE and related technologies over the last 15 years. Although the work on microchip CE has resulted in significantly faster analysis times, and highly multiplexed CE microchips have been described,13-15 the conversion of CE to a microchip format has usually resulted in an increase in the complexity of the technique rather than a simplification (multichannel voltage sources and additional fluid channels to form injection,12,14,16-21 specialized turn geometries required to fit long separation channels into small footprint,22-25 multispot18,20,26 or scanning13-15,17,19,27,28 laser induced fluorescence detection, etc.). Gradient elution moving boundary electrophoresis (GEMBE) is a recently described separation technique that is intended to reduce the on-chip footprint required for microchip electrophoresis, and to eliminate the need for carefully controlled injections.29 The device used for GEMBE consists of a short, straight microchannel or capillary connecting two fluid reservoirs: one for sample and one for buffer. In this regard it is similar to the “Ishaped” microchannels,30 the “well-less” capillary array chips,31 (3) Huang, X. H. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64 (April 15), 967–972. (4) Mathies, R. A.; Huang, X. C. Nature 1992, 359 (Sep 10), 167–169. (5) Huang, X. H. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64 (Sep 15), 2149–2154. (6) Gong, X. Y.; Yeung, E. S. Anal. Chem. 1999, 71, 4989–4996. (7) Ma, L. J.; Gong, X. Y.; Yeung, E. S. Anal. Chem. 2000, 72, 3383–3387. (8) He, Y.; Yeung, E. S. Electrophoresis 2003, 24, 101–108. (9) Collins, F. S.; Patrinos, A.; Jordan, E.; Chakravarti, A.; Gesteland, R.; Walters, L.; Fearon, E.; Hartwelt, L.; Langley, C. H.; Mathies, R. A.; Olson, M.; Pawson, A. J.; Pollard, T.; Williamson, A.; Wold, B.; Buetow, K.; Branscomb, E.; Capecchi, M.; Church, G.; Garner, H.; Gibbs, R. A.; Hawkins, T.; Hodgson, K.; Knotek, M.; Meisler, M.; Rubin, G. M.; Smith, L. M.; Smith, R. F.; Westerfield, M.; Clayton, E. W.; Fisher, N. L.; Lerman, C. E.; McInerney, J. D.; Nebo, W.; Press, N.; Valle, D. Science 1998, 282, 682– 689. (10) Collins, F. S.; Morgan, M.; Patrinos, A. Science 2003, 300, 286–290.

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or the devices used for optically gated microchip electrophoresis.32,33 However, with GEMBE, operation of the device is further simplified because there is no defined injection. Instead, a combination of an electric field and controlled, variable buffer counterflow is used to achieve separations of multiple analyte species. Typically, the polarity of the applied voltage is set so that the analytes of interest are driven by electrophoresis from the sample toward the separation channel. At the beginning of a (11) Venter, J. C.; Adams, M. D.; Myers, E. W.; Li, P. W.; Mural, R. J.; Sutton, G. G.; Smith, H. O.; Yandell, M.; Evans, C. A.; Holt, R. A.; Gocayne, J. D.; Amanatides, P.; Ballew, R. M.; Huson, D. H.; Wortman, J. R.; Zhang, Q.; Kodira, C. D.; Zheng, X. Q. H.; Chen, L.; Skupski, M.; Subramanian, G.; Thomas, P. D.; Zhang, J. H.; Miklos, G. L. G.; Nelson, C.; Broder, S.; Clark, A. G.; Nadeau, C.; McKusick, V. A.; Zinder, N.; Levine, A. J.; Roberts, R. 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J.; Lopez, J.; Ma, D.; Majoros, W.; McDaniel, J.; Murphy, S.; Newman, M.; Nguyen, T.; Nguyen, N.; Nodell, M.; Pan, S.; Peck, J.; Peterson, M.; Rowe, W.; Sanders, R.; Scott, J.; Simpson, M.; Smith, T.; Sprague, A.; Stockwell, T.; Turner, R.; Venter, E.; Wang, M.; Wen, M. Y.; Wu, D.; Wu, M.; Xia, A.; Zandieh, A.; Zhu, X. H. Science 2001, 291, 1304–1351. (12) Harrison, D. J.; Manz, A.; Fan, Z. H.; Ludi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926–1932. (13) Paegel, B. M.; Emrich, C. A.; Weyemayer, G. J.; Scherer, J. R.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 574–579. (14) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91 (Nov 22), 11348–11352. (15) Aborn, J. H.; El-Difrawy, S. A.; Novotny, M.; Gismondi, E. A.; Lam, R.; Matsudaira, P.; McKenna, B. K.; O’Neil, T.; Streechon, P.; Ehrlich, D. J. Lab Chip 2005, 5, 669–674. (16) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676–3680, Oct 15. (17) Emrich, C. A.; Tian, H. J.; Medintz, I. L.; Mathies, R. A. Anal. Chem. 2002, 74, 5076–5083. (18) Shen, Z.; Liu, X. J.; Long, Z. C.; Liu, D. Y.; Ye, N. N.; Qin, J. H.; Dai, Z. P.; Lin, B. C. Electrophoresis 2006, 27, 1084–1092. (19) Huang, Z.; Munro, N.; Huhmer, A. F. R.; Landers, J. P. Anal. Chem. 1999, 71, 5309–5314. (20) Zhang, L.; Yin, X. F. Electrophoresis 2007, 28, 1281–1288. (21) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107–1113. (22) Griffiths, S. K.; Nilson, R. H. Anal. Chem. 2000, 72, 5473–5482.

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separation, the counterflow is high so that none of the analytes of interest will enter the channel. Over the course of the separation, the counterflow is gradually reduced so that each analyte, in turn, will enter the channel and be detected as a moving boundary or step. The time derivative of the detector signal is then equivalent to a conventional electropherogram. The mode of detection used for CE is another area where the cost and complexity could be significantly reduced. The primary detection modes that have been used for CE are optical: fluorescence and UV absorbance. Both require components to generate and detect the light as well as to focus the light first on the inside of each capillary or microchannel in an array and then onto an array of detectors. An alternative detection mode that has received increasing attention in recent years is conductivity detection (for recent reviews see refs 34-36). Conductivity detection is a universal mode of detection, because it is sensitive to changes in solution conductivity resulting from the presence of essentially any charged analyte. It has a sensitivity that is typically comparable to that of UV absorbance detection, and can be implemented either with the sensing electrodes in contact with the solution37-43 or with a capacitively coupled arrangement in which the electrodes are located outside the capillary or microchannel.44-46 The primary advantage of conductivity detection is that it is all electronic and requires no light sources, photodetectors, or optical alignment of components. Consequently, it should be well suited for use with multiplexed CE where the goal is to reduce the cost and complexity of the total system. Here we describe a new approach to multiplexed electrophoresis in which GEMBE is combined with conductivity detection with the aim of simplifying the electrophoresis device as much as possible. In this approach, short capillaries are used both as the separation channels with GEMBE and as conductivity cells for (23) Johnson, T. J.; Ross, D.; Gaitan, M.; Locascio, L. E. Anal. Chem. 2001, 73 (Aug 1), 3656–3661. (24) Molho, J. I.; Herr, A. E.; Mosier, B. P.; Santiago, J. G.; Kenny, T. W.; Brennen, R. A.; Gordon, G. B.; Mohammadi, B. Anal. Chem. 2001, 73 (Mar 15), 1350–1360. (25) Paegel, B. M.; Hutt, L. D.; Simpson, P. C.; Mathies, R. A. Anal. Chem. 2000, 72, 3030–3037. (26) Dang, F.; Shinohara, S.; Tabata, O.; Yamaoka, Y.; Kurokawa, M.; Shinohara, Y.; Ishikawa, M.; Baba, Y. Lab Chip 2005, 5, 472–478. (27) Huang, Z. L.; Jin, L. J.; Sanders, J. C.; Zheng, Y. B.; Dunsmoor, C.; Tian, H. J.; Landers, J. P. IEEE Trans. Biomed. Eng. 2002, 49, 859–866. (28) Liu, S. R.; Ren, H. J.; Gao, Q. F.; Roach, D. J.; Loder, R. T.; Armstrong, T. M.; Mao, Q. L.; Blaga, I.; Barker, D. L.; Jovanovich, S. B Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5369–5374. (29) Shackman, J. G.; Munson, M. S.; Ross, D. Anal. Chem. 2007, 79, 565– 571. (30) Inoue, A.; Ito, T.; Makino, K.; Hosokawa, K.; Maeda, M. Anal. Chem. 2007, 79, 2168–2173. (31) Kim, Y. H.; Yang, I.; Park, S. R. Anal. Chem. 2007, 79, 9205–9210. (32) Lapos, J. A.; Ewing, A. G. Anal. Chem. 2000, 72, 4598–4602. (33) Xu, X. M.; Roddy, T. P.; Lapos, J. A.; Ewing, A. G. Anal. Chem. 2002, 74, 5517–5522. (34) Kuban, P.; Hauser, P. C. Anal. Chim. Acta 2008, 607, 15–29. (35) Matysik, F. M. Microchim. Acta 2008, 160, 1–14. (36) Pumera, M. Talanta 2007, 74, 358–364. (37) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. J. Chromatogr. 1979, 169, 11–20. (38) Harrold, M.; Stillian, J.; Bao, L.; Rocklin, R.; Avdalovic, N. J. Chromatogr. A 1995, 717, 371–383. (39) Prest, J. E.; Baldock, S. J.; Bektas, N.; Fielden, P. R.; Brown, B. J. T. J. Chromatogr. A 1999, 836, 59–65. (40) Qin, W. D.; Li, S. F. Y. Electrophoresis 2003, 24, 2174–2179. (41) Shadpour, H.; Hupert, M. L.; Patterson, D.; Liu, C. G.; Galloway, M.; Stryjewski, W.; Goettert, J.; Soper, S. A. Anal. Chem. 2007, 79, 870–878.

Figure 1. Schematic of the multiplexed electrophoresis apparatus. (A) Each separation capillary assembly was made of two Luer lock fittings with a 360 µm outer diameter, 5 µm inner diamter capillary inserted through 360 µm holes drilled through the closed end of the Luer lock caps. A 1 mm thick washer was used as a spacer, and the fittings and capillary were glued together with epoxy. After the epoxy was cured, the capillary was trimmed to near the bottom of each fitting, giving a separation channel length of approximately 3 mm. (B) For the electrophoresis array, the capillary assemblies were mounted to a manifold that was connected to a 20 mL syringe that served as a common run buffer reservoir. High voltage and pressure control were applied to the buffer reservoir. The current through each electrophoresis channel was monitored with a multichannel data acquisition device by measuring the voltage drop across a 1 MΩ resistor in series with each channel.

detection. Because the channels are very short (3 mm), only one analyte step at a time is present in each channel, and the measured current through each channel can therefore be used as the detector signal, with no additional detector hardware. The technique is implemented with a 16-channel array device constructed to approximate the form of a microtiter plate. The device is demonstrated for use with high-throughput time series measurements of the activity of protein kinase A, and of the inhibition of that enzyme activity by the known inhibitor H-89 dihydrochloride.

Figure 2. Single channel, proof-of-concept demonstration of enzyme activity assay. (A) Current vs time trace for complete enzyme reaction mixture (200 µmol/L ATP, 100 µmol/L kemptide, 2 mmol/L MgCl2, and 2.5 U/µL PKA enzyme). (B) Current derivative vs time for the data shown in (A). (C) Current derivative plot for reaction mixture without enzyme (200 µmol/L ATP, 100 µmol/L kemptide, and 2 mmol/L MgCl2). (D) Current derivative plot for reaction mixture without substrate (200 µmol/L ATP, 2 mmol/L MgCl2, and 2.5 U/µL PKA enzyme). The largest peak in each plot (at 3.4 min) is due to an anionic impurity remaining from the preparation of the peptide substrate (trifluoroacetate (TFA), according to the vendor). The small peak before TFA (2.7 min) is due to carbonate from dissolved CO2 (identified by analyzing a sample spiked with carbonate).

EXPERIMENTAL SECTION47 Chemicals and Reagents. Adenosine 5′-triphosphate disodium salt hydrate (ATP), adenosine 5′-diphosphate sodium salt (ADP), 1 mol/L magnesium chloride solution, dimethyl sulfoxide (DMSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-tris), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were obtained from Sigma Aldrich (St. Louis, MO). Recombinant mouse protein kinase A catalytic subunit (PKA), kemptide, and H-89 dihydrochloride were obtained from Calbiochem (San Diego, CA). All solutions were made with water from a Barnstead (Dubuque, IA) Easypure II ultrapure water system. Device Fabrication. The construction of the capillary devices that were used is shown schematically in Figure 1(A). Two female nylon Luer lock caps (McMaster-Carr, Atlanta, GA) were used for each fitting. A 360 µm diameter hole was drilled through the end of each cap, and a length of fused silica capillary (5 µm inner diameter, 360 µm outer diameter, approximately 2 cm long; Polymicro Technologies, LLC, Phoenix, AZ) was pushed through the holes with a 1 mm thick plastic washer as a spacer. The

assembly was glued together with a two-part epoxy (Bondit B45TH; McMaster-Carr, Atlanta, GA), and cured in an oven at 65 °C overnight. A small jewelers’ file was then used to score the capillary near the bottom of each Luer lock fitting, and the extra capillary pieces were broken off, leaving approximately a 3 mm length of capillary for each device. Instrumentation. A photograph of the multiplexed electrophoresis device is shown in Figure 1(B) along with a schematic of the instrumentation used in Figure 1(C). The head space pressure of the buffer reservoir was controlled with a precision pressure controller (series 600, Mensor, San Marcos, TX) with a range of ±68.9 kPa (±10 psi). Helium was used as the supply gas for the pressure controller. The buffer reservoir was a 20 mL disposable polypropylene syringe, with a custom built plunger to allow access for the high voltage and pressure control. The syringe was connected to the manifold via 0.125 cm i.d. high purity Tygon tubing, approximately 10 cm long. The manifold was machined from Delrin with an internal volume of approximately 10 mL, and connections between the manifold and the Luer lock capillary Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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Figure 3. (A) Current vs time data for 16-channel electrophoresis array measurement of enzyme activity. Data was taken approximately 90 min after mixing the reaction solutions. The traces have been shifted vertically for clarity. From top to bottom, the enzyme concentration in each sample was zero, 0.625 U/µL, 0.825 U/µL, 1.075 U/µL, 1.45 U/µL, 1.925 U/µL, 2.55 U/µL, 3.4 U/µL, 4.525 U/µL, 6 U/µL, 8 U/µL, 10.625 U/µL, 14.125 U/µL, 18.8 U/µL, 25 U/µL, and 33.25 U/µL. (B) Derivative vs time for the data shown in (A). The traces have been shifted vertically for clarity and horizontally to line up the largest peak. The first (small) peak is due to carbonate. The second peak is due to an anionic impurity remaining from the preparation of the peptide substrate (trifluoroacetate (TFA), according to the vendor). The positions of the ATP and ADP peaks are noted on the figure.

devices were made with 1/4′′-28 threaded polypropylene Luer lock couplings (McMaster-Carr, Atlanta, GA). The high voltage source was a Stanford Research Systems model PS350 (Stanford Research Systems, Inc., Sunnyvale, CA). Electrical connections to all solutions were made through high purity platinum wires (Sigma Aldrich, St. Louis, MO). The multichannel data acquisition device was a National Instruments USB-6229 (National Instruments, Austin, TX). Precision metal film resistors (1 MΩ, Mouser Electronics, Inc., Mansfield, TX), were used in series with each separation capillary to measure the current. Electrophoresis Procedures. For all the electrophoresis results, a voltage of +2000 V (applied to the buffer reservoir) was used. At the start of each separation, the pressure applied to the buffer reservoir head space was set to +40000 Pa for 15 s with the high voltage off. The high voltage was then turned on and the pressure was reduced to 35000 Pa for 10 s, after which the pressure was reduced at a rate of 150 Pa/s for 440 s (minimum pressure of -31000 Pa). The pressure was then increased to +32000 Pa and held for 10 s with the voltage on. Then the voltage was turned off until the next separation. For the time series data of Figures 3-7, the hold time between separations varied from zero to 180 min. During the hold, the pressure was held at a 9470

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Figure 4. Fraction converted [(peak areaADP)/(peak areaADP + peak areaATP)] vs time for multiplexed enzyme activity measurement. The enzyme concentrations are given in the figure legend. Final concentrations of other reaction mixture components were 200 µmol/L kemptide, 200 µmol/L ATP, 2 mmol/L MgCl2, and 10% DMSO by volume.

constant value of 30000 Pa (for hold time e60 min) or 20000 Pa (hold time >60 min). Enzyme Assay Procedures. The buffer used for all experiments was 100 mmol/L bis-tris and 100 mmol/L HEPES (pH 7.1).

For the results shown in Figure 2, the reaction mixture was in pure buffer (with no DMSO). For the multiplexed results (Figures 3-7), the reaction mixtures were in 90% buffer, 10% DMSO (by volume). The inhibitor stock solution was prepared in DMSO, and 10% DMSO was chosen for the multiplexed measurements to keep the assay conditions constant as much as possible while varying the enzyme and inhibitor concentrations. The final concentrations of the reaction mixture constituents are given below. Generally, for the multiplexed assays, the reaction mixtures consisted of 200 µmol/L ATP, 200 µmol/L substrate (kemptide), and 2 mmol/L MgCl2. Enzyme and inhibitor concentrations were varied. All of the mixture constituents other than ATP were mixed together first, and ATP was added last to initiate the reaction. The reaction times for Figures 3 and 4 were measured from the time of addition of ATP to the time midway between detection of the ATP and ADP steps. The reaction times for Figure 7 were taken as the average of the reaction times for each channel. One enzyme unit (U) is defined as the amount of enzyme that will transfer 1.0 pmol of phosphate to kemptide per min at 30 °C, pH 7.5. RESULTS The activity of protein kinase A (PKA) was chosen for the first demonstration of the new technique because of the importance of kinase activity assays in high-throughput drug discovery, and because it has been previously studied by both conventional capillaryarrayelectrophoresis8 aswellasmicrochipelectrophoresis.48,49 The peptide substrate typically used for in vitro studies of PKA activity is kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), which is phosphorylated via the reaction PKA

ATP + kemptide 98 ADP + phosphokemptide

(1)

We chose to monitor the progress of the reaction by measuring the conversion of ATP to ADP. This was done so that the assay could be used without modification or reoptimization to measure the activity of other kinases and/or with other substrates. An additional reason for this choice was that, because they are anions, ATP and ADP migrate in the direction opposite to the electroosmotic flow of an uncoated capillary, and they can therefore be (42) Guijt, R. M.; Baltussen, E.; van der Steen, G.; Schasfoort, R. B. M.; Schlautmann, S.; Billiet, H. A. H.; Frank, J.; van Dedem, G. W. K.; van den Berg, A. Electrophoresis 2001, 22, 235–241. (43) Tay, E. T. T.; Law, W. S.; Sim, S. P. C.; Feng, H.; Zhao, J. H.; Li, S. F. Y. Electrophoresis 2007, 28, 4620–4628. (44) Zemann, A. J.; Schnell, E.; Volgger, D.; Bonn, G. K. Anal. Chem. 1998, 70, 563–567. (45) Da Silva, J. A. F.; Do Lago, C. L. Anal. Chem. 1998, 70, 4339–4343, Oct 15. (46) Guijt, R. M.; Baltussen, E.; van der Steen, G.; Frank, H.; Billiet, H.; Schalkhammer, T.; Laugere, F.; Vellekoop, M.; Berthold, A.; Sarro, L.; van Dedem, G. W. K. Electrophoresis 2001, 22, 2537–2541. (47) Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. (48) Cohen, C. B.; Chin-Dixon, E.; Jeong, S.; Nikiforov, T. T. Anal. Biochem. 1999, 273, 89–97. (49) Xue, Q. F.; Wainright, A.; Gangakhedkar, S.; Gibbons, I. Electrophoresis 2001, 22, 4000–4007.

easily separated by GEMBE with moderate applied pressures to modify the bulk counterflow velocity. Figure 1 shows a schematic of the device used for the GEMBE measurements with current detection. The short (3 mm) capillary devices were constructed out of two Luer lock fittings as shown in Figure 1(A) to provide a modular format wherein each channel could be individually replaced and/or rinsed as needed. The capillary Luer lock fittings were attached to a manifold via Luer lock ports as shown schematically in Figure 1(B), with the top half of each double Luer lock fitting serving as a sample reservoir. The manifold was connected to a buffer reservoir (disposable syringe) via another Luer lock port and a 20 cm length of tubing. The high voltage for electrophoresis and the pressure control were applied at the buffer reservoir. The manifold and buffer reservoir were filled with run buffer (100 mmol/L bis-tris HEPES, pH 7.1). Capillaries were individually rinsed with run buffer and attached to the manifold - taking care to eliminate any air bubbles at the junctions between the manifold and Luer lock fittings. Initially, a manifold with a single separation channel was used to verify that the new technique could be used to detect ATP and ADP and to measure the kinase activity. Three samples were prepared: one with a complete reaction mixture (200 µmol/L ATP, 100 µmol/L kemptide, 2 mmol/L MgCl2, and 2.5 U/µL PKA enzyme); one with no enzyme; and one with enzyme but no substrate (kemptide). Each sample was incubated at room temperature (23 °C) for 2.5 h before it was pipetted into the Luer lock reservoir and analyzed. The raw current vs time trace for the first sample is shown in Figure 2(A). The time derivative of the current for the same measurement is shown in Figure 2(B). There are 4 steps in the current trace and 4 corresponding peaks in the derivative plot. The largest peak (at 3.4 min) is due to an anionic impurity remaining from the preparation of the peptide substrate (trifluoroacetate (TFA), according to the vendor). The small peak before TFA (2.7 min) is due to carbonate from dissolved CO2 (identified by analyzing a sample spiked with carbonate). The third peak (4.3 min) is due to ATP, and the final peak (5.2 min) is due to ADP. With the complete reaction mixture (first sample, Figure 2(A,B)) peaks for ATP and ADP were detected indicating that some ATP was converted to ADP. For measurements without enzyme (Figure 2(C)) and without substrate (Figure 2(D)) no ADP peak is evident, indicating that both the enzyme and substrate are necessary for the conversion of ATP to ADP. An additional advantage of counterflow separation techniques such as GEMBE is that the counterflow can be used to exclude potentially interfering sample matrix components from the separation channel.50 When making a measurement of electrophoretically fast species (such as ATP and ADP), the parameters of the GEMBE method can be set so that electrophoretically slow species such as serum proteins or enzymes never overcome the bulk counterflow and are thus excluded from the channel. As a result, it is not necessary to use sample additives to suppress enzyme adsorption48 or to rinse the capillaries with sodium hydroxide between measurements for repeatability. Enzyme reaction samples can therefore be mixed directly in the top half of each capillary Luer lock fitting, and incubated in the device, (50) Munson, M. S.; Meacham, J. M.; Locascio, L. E.; Ross, D. Anal. Chem. 2008, 80, 172–178.

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with periodic measurements of the ATP and ADP concentrations giving a time course of the reaction. For the first multiplexed measurement, 15 different reaction mixtures were prepared with different final enzyme concentrations ranging from 33.25 U/µL to 0.625 U/µL (plus one mixture with zero enzyme). The concentration of the other species in the reactions mixtures was left constant (200 µmol/L kemptide, 200 µmol/L ATP, 2 mmol/L MgCl2, and 10% DMSO by volume). The samples were mixed and incubated in the device. First, 40.7 µL of each enzyme solution (at a concentration 1.47× the final concentration) was pipetted into the each Luer lock reservoir. A 19.3 µL aliquot of the remaining reaction mixture components (at 3.11× final concentration) was then pipetted into each reservoir at 15 s intervals starting with the reservoir containing the lowest enzyme concentration and progressing to reservoirs with higher enzyme concentration. As the reaction mixture components were added to each sample, they were mixed by drawing the solution up into the pipet tip and dispensing it into the sample reservoir several times. Loosely fitting lids were then placed over each sample reservoir and platinum electrodes were placed into each sample. The first GEMBE measurement was started as quickly as possible after the insertion of the electrodes. A total of 11 GEMBE measurements were run at increasing time intervals over the next 6 h to construct a time series for the enzyme reaction in each of the 16 channels. The raw current data from all 16 channels is shown in Figure 3(A) for the seventh GEMBE measurement (approximately 90 min after mixing). As with the data of Figure 2(A) the largest step is due to the TFA impurity from the preparation of the peptide substrate. The positions of the ATP and ADP peaks relative to the TFA peak are the same as in Figure 2(A). The current traces are spread out vertically for clarity and arranged in order from the lowest enzyme concentration at the top to the highest enzyme concentration at the bottom. The current for each channel was approximately 1.1 µA at the beginning of the measurement, and the TFA step height was approximately 0.035 µA. The mean position for the TFA peak was 3.2 min, with a standard deviation of 0.6 min. The channel-to-channel variability was primarily due to variations in the electroosmotic mobility (rinsing each capillary with 0.1 mol/L NaOH was later found to improve the channelto-channel reproducibility). The derivative data for the same 16-channel GEMBE measurement is shown in Figure 3(B) with each trace shifted vertically for clarity and horizontally to line up the TFA peaks. Again, the traces are arranged with the lowest enzyme concentration at the top and the highest enzyme concentration at the bottom. The data show that at this time point, the enzyme concentrations used for the multiplexed assay span the range from complete conversion of ATP to ADP (bottom traces) to little or no conversion (top traces). The time series data for the 16 GEMBE channels is shown in Figure 4, with the fraction converted calculated as [(peak areaADP)/ (peak areaADP + peak areaATP)]. Again, the enzyme concentrations used in these experiments spanned the range from fast, complete conversion to slow, incomplete conversion. For the higher enzyme concentrations (>6 U/µL), the measured initial reaction rates were between 32% and 56% of the rates predicted using the unit definition (one enzyme unit (U) is defined as the amount of 9472

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Figure 5. Fraction converted [(peak areaADP)/(peak areaADP + peak areaATP)] vs time for multiplexed enzyme inhibition measurement. Final concentrations of the inhibitor H-89 dihydrochloride are given in the figure. The reaction mixture for the bottom data set contained no enzyme and no inhibitor, and showed no detectable ADP peak at any time point. Final concentrations of other reaction mixture components were 200 µmol/L kemptide, 200 µmol/L ATP, 2 mmol/L MgCl2, and 10% DMSO by volume.

Figure 6. Fraction converted [(peak areaADP)/(peak areaADP + peak areaATP)] vs inhibitor concentration for the seventh time point of Figure 5 (approximately 90 min after mixing). The line is a fit to the form A + [B/(1 + c/IC50)], where c is the inhibitor concentration, and A, B, and IC50 are the adjustable parameters for the fit. For the best fit, A ) 0.031 ( 0.015, B ) 0.792 ( 0.019, and IC50 ) 1.13 µmol/L ( 0.14 µmol/L.

enzyme that will transfer 1.0 pmol of phosphate to kemptide per min at 30 °C, pH 7.5). For those solutions, a significant portion of the ATP had already been converted before the second measurement in the time series, so the measurement was probably an underestimate of the actualinitial rate. The initial reaction rates measured for the solutions with moderate enzyme concentrations (1.45 U/µL to 4.525 U/µL) were approximately 67% of the rates predicted from the unit definition. The slightly slower rate is not surprising given that the reaction mixtures were incubated at 23 °C rather than 30 °C. For the sample with zero enzyme, there was no detectable ADP peak at any time point. For all but the 4 highest enzyme concentrations, the fraction of ATP converted to ADP reached a plateau value less than 1, and the plateau value decreased with decreasing enzyme concentration, indicating that enzyme activity was lost over time.51 The reaction rates for most (51) Assay Guidance Manual Version 5.0, 2008 Eli Lilly and Company and NIH Chemical Genomics Center. Available online at http://www.ncgc.nih.gov/ guidance/manual_toc.html (last accessed September 2008).

Figure 7. IC50 vs time for the data of Figure 5. At each time point of Figure 5, the conversion vs inhibitor concentration data was fit to the form given in eq 2 to give the IC50 as a function of time. The error bars are the asymptotic standard errors resulting from the fit of eq 2 to the data.

of the measured samples varied smoothly from low to high enzyme concentration. However, the time series trace for one channel (6 U/µL enzyme) was lower than expected based upon the results for the other channels, and the traces for two other channels (3.4 U/µL and 4.525 U/µL) were slightly higher than expected (see Supplemental Figure 1 in the Supporting Information). There were no obvious indications of problems in the electropherograms from those channels (high noise level, extra peaks, etc.), and the next time series data set measured with those channels (see Figures 5 and 6) was quantitatively consistent with data from the other channels. So, the most likely explanation was a pipetting or mixing error with either the enzyme solutions or the final reaction mixtures. With 11 repeated measurements on the same set of samples, there could be some concern that the total amount of ATP and ADP removed from each sample for analysis could significantly effect the concentrations of the analytes in the sample. However, because very small capillaries were used, only a very small fraction of the ATP and ADP was removed from each sample, even over the course of 11 measurements. For example, the separations continue for at most 200 s after the ATP step crosses the capillary. The volume of ATP solution removed from the sample in that time is equal to the cross sectional area of the capillary (1.9625 × 10–5 mm2) multiplied by the time integral of the ATP velocity through the capillary over those 200 s. The ATP velocity is zero at the beginning of that time and increases with a constant acceleration, a ) [r2/(8ηL)] · (∂P/∂t), where r is the internal radius of the capillary, η is the viscosity (of water), L is the channel length, P is the applied pressure, and t is time. The volume of ATP solution that is removed from the solution during each separation is then approximately 17 nL, which is approximately 10-4 times less than the total sample volume. The second multiplexed measurement was of the inhibition of the PKA enzyme using a range of different concentrations of a known inhibitor, H-89 dihydrochloride. For this measurement, 14 reaction mixtures were prepared with final inhibitor concentrations ranging from 0.3 nmol/L to 1 mmol/L. The concentrations of the other species in the 14 reaction mixtures were constant (200 µmol/L kemptide, 200 µmol/L ATP, 2 mmol/L MgCl2, 5.42 U/µL PKA enzyme, and a volume fraction of 10% DMSO). Two additional reaction mixtures were prepared, one containing no inhibitor and

the last containing no inhibitor and no enzyme. Again, the samples were mixed and incubated in the device. First, 44 µL of 1.36× kemptide, MgCl2, and enzyme was added to each reservoir. Then, 6 µL of 10× inhibitor solution was added. To initiate the reaction in each reservoir, 10 µL of 6× ATP solution was added to each well at 15 s intervals starting with the reservoir containing the highest inhibitor concentration and progressing to reservoirs with lower inhibitor concentrations. Again, the reaction mixture components were mixed as the ATP solution was added. After adding ATP to each reservoir, the electrodes were inserted into each reservoir, and the first GEMBE measurement was started. A total of 16 multiplexed GEMBE measurements were run at increasing time intervals over the next 16 h to construct the time series shown in Figure 5. As with the variable enzyme experiment of Figure 4, the different reaction mixtures spanned the range from little or no conversion for the highest inhibitor concentrations to nearly complete conversion for the lowest inhibitor concentrations. The initial reaction rate measured for the samples with low inhibitor concentrations (