Isoelectric Focusing in a Microfluidically Defined Electrophoresis

Apr 12, 2008 - Sharp Corporation. | Center for NanoBio Integration, The University of Tokyo. (1) Righetti, P. G. Isoelectric Focusing: Theory, Methodo...
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Anal. Chem. 2008, 80, 3818–3823

Isoelectric Focusing in a Microfluidically Defined Electrophoresis Channel Kiyohito Shimura,*,†,‡ Katsuyoshi Takahashi,†,§ Yutaka Koyama,† Kae Sato,†,| and Takehiko Kitamori†,‡,| Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, Kanagawa Academy of Science and Technology, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa 213-0012, Japan, Biosensing Systems Laboratories, Sharp Corporation, 1-9-2 Nakase, Mihama, Chiba, Chiba 261-8520, Japan, and Center for NanoBio Integration, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide pI markers with pI values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different pI markers was demonstrated.

(CIEF) to microchannels in planar chips has been investigated in light of the higher analytical performance of miniaturized total chemical analysis systems (micro TAS).6 A variety of materials have been investigated as possible chip substrates, including glass,7–9 quartz,10 polycarbonate,11,12 polymethylmethacrylate,13–16 polyester and other polymers,17 polydimethylsiloxane (PDMS),18–22 cyclic olefin copolymer (COC),23,24 glass and resin tape with removable silicone adhesive,25 glass and PDMS.26,27 Most investigations have used fluorescence detection of naturally fluorescent proteins or fluorescence-labeled proteins (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

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Isoelectric focusing (IEF) is a primary separation method for proteins and peptides, as it is based on a well-defined physicochemical parameter: the isoelectric point (pI).1 Adaptation of IEF to a capillary allowed evolution of IEF into a rapid high-resolution separation method.2–5 The transfer of capillary isoelectric focusing

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* Corresponding author. E-mail: [email protected]. Fax: +81-3-58417199. † Department of Applied Chemistry, School of Engineering, The University of Tokyo. ‡ Kanagawa Academy of Science and Technology (KAST). § Sharp Corporation. | Center for NanoBio Integration, The University of Tokyo. (1) Righetti, P. G. Isoelectric Focusing: Theory, Methodology and Applications; Elsevier Scientific: Amsterdam, 1983. (2) Hjertén, S.; Zhu, M. D. J. Chromatogr. 1985, 346, 265–270.

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Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 2934–2941. Rodriguez-Diaz, R.; Wehr, T.; Zhu, M. Electrophoresis 1997, 18, 2134–2144. Shimura, K. Electrophoresis 2002, 23, 3847–3857. Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 1, 244– 248. Hofmann, O.; Che, D.; Cruickshank, K. A.; Muller, U. R. Anal. Chem. 1999, 71, 678–686. Sanders, J. C.; Huang, Z.; Landers, J. P. Lab Chip 2001, 1, 167–172. Tsai, S. W.; Loughran, M.; Hiratsuka, A.; Yano, K.; Karube, I. Analyst 2003, 128, 237–244. Mao, Q. L.; Pawliszyn, J. Analyst 1999, 124, 637–641. Wen, J.; Lin, Y.; Xiang, F.; Matson, D. W.; Udseth, H. R.; Smith, R. D. Electrophoresis 2000, 21, 191–197. Li, Y.; Buch, J. S.; Rosenberger, F.; DeVoe, D. L.; Lee, C. S. Anal. Chem. 2004, 76, 742–748. Raisi, F.; Belgrader, P.; Borkholder, D. A.; Herr, A. E.; Kintz, G. J.; Pourhamadi, F.; Taylor, M. T.; Northrup, M. A. Electrophoresis 2001, 22, 2291–2295. Tan, W.; Fan, Z. H.; Qiu, C. X.; Ricco, A. J.; Gibbons, I. Electrophoresis 2002, 23, 3638–3645. Herr, A. E.; Molho, J. I.; Drouvalakis, K. A.; Mikkelsen, J. C.; Utz, P. J.; Santiago, J. G.; Kenny, T. W. Anal. Chem. 2003, 75, 1180–1187. Mok, M. L.; Hua, L.; Phua, J. B.; Wee, M. K.; Sze, N. S. Analyst 2004, 129, 109–110. Guo, X.; Chan-Park, M. B.; Yoon, S. F.; Chun, J. H.; Hua, L.; Sze, N. S. Anal. Chem. 2006, 78, 3249–3256. Chen, X.; Wu, H.; Mao, C.; Whitesides, G. M. Anal. Chem. 2002, 74, 1772– 1778. Han, J.; Singh, A. K. J. Chromatogr. A 2004, 1049, 205–209. Wang, Y. C.; Choi, M. H.; Han, J. Anal. Chem. 2004, 76, 4426–4431. Cui, H.; Horiuchi, K.; Dutta, P.; Ivory, C. F. Anal. Chem. 2005, 77, 7878– 7886. Cui, H.; Horiuchi, K.; Dutta, P.; Ivory, C. F. Anal. Chem. 2005, 77, 1303– 1309. Li, C.; Yang, Y.; Craighead, H. G.; Lee, K. H. Electrophoresis 2005, 26, 1800–1806. Das, C.; Fan, Z. H. Electrophoresis 2006, 27, 3619–3626. Fujita, M.; Hattori, W.; Sano, T.; Baba, M.; Someya, H.; Miyazaki, K.; Kamijo, K.; Takahashi, K.; Kawaura, H. J. Chromatogr. A 2006, 1111, 200–205. Yao, B.; Yang, H.; Liang, Q.; Luo, G.; Wang, L.; Ren, K.; Gao, Y.; Wang, Y.; Qiu, Y. Anal. Chem. 2006, 78, 5845–5850. Guillo, C.; Karlinsey, J. M.; Landers, J. P. Lab Chip 2007, 7, 112–118. 10.1021/ac8000594 CCC: $40.75  2008 American Chemical Society Published on Web 04/12/2008

and peptides. Ultraviolet absorption imaging has been performed using a quartz chip.10 Direct coupling of IEF to electrospray ionization mass spectrometry (ESI-MS) was demonstrated using a plastic chip equipped with a spray nozzle.11 Scanning detection with matrix-assisted laser-desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS) has been performed on pH gradients established after completion of focusing.16,17,25 Coupling of IEF and other separation processes to increase the peak capacity has been investigated by several groups, e.g., coupling with zone electrophoresis using a sieving media in the presence of sodium dodecylsulfate through multiple channels crossing the IEF channel12,18 and coupling with zone electrophoresis in a single microchannel crossing the IEF channel.15,20 However, direct coupling of microchip IEF with a pretreatment procedure or with another separation step has not been reported. This is most likely related to the specific requirement of IEF for two different electrode solutions in addition to a separation medium, the carrier ampholyte solution. Commonly used microchannels for IEF separation are made between two channel ports, on which the electrode reservoirs and electrodes are placed. The operation of this type of IEF microchip requires multiple pipetting. First, the channel is filled with sample solution containing carrier ampholytes. Second, one of the channel ports is rinsed and then filled with one of the electrode solutions. Third, the second port is rinsed and filled with the other electrode solution. Finally, IEF is started by application of high voltage between the platinum electrodes inserted in the reservoirs. Because the boundaries between the carrier ampholyte solution and the electrode solutions form the extremities of the pH gradient, ideally the pH gradient stretches throughout the channel between the reservoirs. Filling or emptying one of the reservoirs, however, could cause displacement of the boundaries or mobilization of the sample out of the IEF channel due to a change in hydrostatic pressure. To prevent sample mobilization, the anolyte and catholyte reservoirs were simultaneously filled with electrolyte solution using two pipets.7,8,15 Addition of hydrophilic polymer, e.g., hydroxyethylcellulose,8,24 agarose,18 methylcellulose,16,17,19–22 polyethyleneoxide,12 hydroxypropylcellulose,24 hydroxypropylmethylcellulose,24,26 or a commercial cIEF kit containing a polymer,27 to the sample solutions containing carrier ampholytes and/or electrode solutions may have also mitigated this problem by increasing the viscosity of the solution. In addition to the above-mentioned technical difficulties, this type of IEF chip is not suitable for direct coupling with preceding sample processing methods, such as desalting or prefractionation. These limitations discourage integration toward micro TAS. Two types of IEF chip may be coupled with pretreatment processes: those that use membranes to connect the electrolyte and carrier ampholytes10,11 and free-flow IEF in microchips.28,29 However, use of a hollow fiber membrane as the external chip connection allowed inclusion of only a small portion of the pH gradient in the chip channel due to the large volume of carrier ampholyte solution in the connection between the chip channel and hollow fiber.10 A molecular porous membrane has been used for separation of electrolyte and IEF channel in the chip with direct electrospraying from an on-chip nozzle.11 The membrane con(28) Xu, Y.; Zhang, C. X.; Janasek, D.; Manz, A. Lab Chip 2003, 3, 224–227. (29) Kohlheyer, D.; Besselink, G. A.; Schlautmann, S.; Schasfoort, R. B. Lab Chip 2006, 6, 374–380.

Figure 1. Photograph of an IEF chip. The chip ports are numbered 1-8. The sample in the carrier ampholyte solution entered through port 1 and exited from port 2. The channels between ports 7 and 8 were used for the anodic lead and between ports 3 and 4 for the cathodic lead. Ports 5 and 6 were not used in the present experiments and were filled with water. The formation of a pH gradient and IEF separation occurred between the intersections A and B.

nection between the electrode solutions and sample/carrier ampholyte solution worked well; however, IEF separation was not fully characterized and this configuration has not been used since. Free-flow IEF devices should allow for continuous coupling with pretreatment processes. However, the arrangement is too complicated for a single-batch analysis. In the present paper, we propose an efficient and robust IEF chip that can be coupled with sample pretreatment processes by the use of an assembly composed of a glass chip and a holder having on-chip connector valves at each channel port.30 The proposed IEF chip is based on the higher electric conductivity of electrode solutions compared with that of the pH gradient formed in the microchannel. IEF separation of injected sample plugs containing different fluorescence-labeled peptide pI markers demonstrated the ability to directly couple IEF with a sample pretreatment process in one chip. EXPERIMENTAL SECTION Chemicals. The following chemicals were purchased from commercial sources: Pharmalyte 3-10 (GE Healthcare Bio-Sciences, Piscataway, NJ); hydroxypropylmethylcellulose (HPMC; Mn ) 120,000; 100,000 cps at 2 wt % in water, product number 423173, Sigma-Aldrich Inc., St. Louis, MO); 3-methacryloxypropyltrimethoxysilane (MPTS, Shin-etsu Chemical, Tokyo, Japan); and N,N-dimethylacrylamide (Wako Pure Chemicals, Osaka, Japan). Other reagents of the purest grade were obtained from Wako Pure Chemicals. Tetramethylrhodamine-labeled peptide pI markers were prepared as previously described.31 Microchips. The microchannel (200 µm wide, 90 µm deep) was formed by wet chemical etching of the top surface of the lower Pyrex glass plate (30 × 70 × 0.7 mm), and by thermal bonding with an upper plate of the same size (Figure 1). Channel ports (200 µm i.d.) were made by drilling holes in the upper plate prior to bonding. The chip was manufactured at the Institute of Microchemical Technology (Kawasaki, Kanagawa, Japan). After bonding, the wall of the microchannel was chemically coated with polydimethylacrylamide according to a published procedure.32 Scanning Detection of Fluorescence. An in-house laserinduced fluorescence (LIF) detection system for IEF chips was (30) Shimura, K.; Koyama, Y.; Sato, K.; Kitamori, T. J. Sep. Sci. 2007, 30, 1477– 1481. (31) Shimura, K.; Kamiya, K.; Matsumoto, H.; Kasai, K. Anal. Chem. 2002, 74, 1046–1053. (32) Wan, H.; Ohman, M.; Blomberg, L. G. J. Chromatogr. A 2001, 924, 59–70.

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Figure 2. Cross-section of the electrode vessel on the on-chip connector valve. (1) Platinum wire, 0.3 mm diameter; (2) silicone tube, 1 mm i.d., 3 mm o.d.; (3) Teflon tube, 0.5 mm i.d., 1/16 in. o.d.; (4) PEEK nut; (5) upper frame of the PEEK holder; (6) PEEK ferrule with stainless steel collar; (7) Teflon valve rotor; (8) stainless steel valve lever; (9) glass chip; and (10) lower frame of the PEEK holder.

constructed on a microscope as previously described.33 A laser beam at 532 nm was focused via a 20× (N.A. 0.5) objective into the microchannel. Fluorescence was collected through spectral filters for Cy3 with a 0.6 mm pinhole and was detected using a photomultiplier tube. The IEF chip was placed in the chip holder having on-chip connector valves and then was mounted on an electrical scan stage (OptiScan ES111, Prior Scientific Instruments Ltd., Cambridge, U.K.). The entire 24 mm IEF channel was scanned at a rate of 1 mm/s. The photomultiplier tube signal was transferred to a chromatographic data processor (Exachrom, Brechbuhler Inc.) through a 24-bit A/D converter (ULYS, Brechbuhler Inc.) at a data-acquisition frequency of 100 Hz. Operation of the IEF Chip. The valve works via rotation of a Teflon valve rotor on the chip port as previously described.30 The custom-made Teflon valve rotor, 1.5 mm high and 4 mm o.d., has a hole (0.2 mm i.d.) at its center and a radial groove (0.85 mm long, 0.2 mm wide, 0.2 mm deep) in its lower surface starting from the hole. The rotors assembled with levers were put in the sockets for the valve rotors in the chip holder that was specially designed for use with the connector valves. The valve rotors were pressed against the chip ports using commercially available tube fittings with screw nuts that are coaxial to the valve rotors and have offsets of 0.75 mm from the chip ports. Rotation of the rotor at an angle of 60° opens and closes the valve by moving the groove on the valve rotor against the chip port. The cut-out view of the valve is shown in Figure 2. For connection with the electrode, a tube of 0.5 mm i.d. was used. For other connections, smaller diameter Teflon tubing, 0.3 mm i.d. and 0.5 mm i.d., was used with appropriate tubing sleeves, with the exception of the connection between the chip and the sample injector, where a capillary PEEK tube, 0.05 mm i.d. and 80 mm long, was used. Samples of the labeled pI markers were dissolved at a concentration of 1.0-2.5 nM in a carrier ampholyte solution containing 2.5% (v/v) Pharmalyte 3-10. The anolyte solution was composed of 20 mM H3PO4 containing 0.5% (w/v) HPMC, and the catholyte was 20 mM NaOH containing 0.5% (w/v) HPMC. (33) Johnson, M. E.; Landers, J. P. Electrophoresis 2004, 25, 3513–3527.

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Port 1 of the chip (Figure 1) was connected to a 1 mL syringe pump (KD Scientific, Holliston, MA) via a sample injector (Rheodyne model 7520, Rohnert Park, CA) with an injection volume of 0.5 µL. Using the pump, the carrier ampholyte solution was delivered at a rate of 1 µL/min. The use and connections of the other ports were as follows: port 2, a waste line; port 3, a catholyte outlet and cathode; port 4, a catholyte inlet; ports 5 and 6, not used; port 7, an anolyte outlet and anode; and port 8, an anolyte inlet. All tubing and electrode assemblies were connected to the on-chip connector valves with finger-tight tube fittings (Upchurch Scientific, Oak Harbor, WA). The catholyte and anolyte solutions were delivered at a rate of approximately 1 µL/s by hand, using disposable 1 mL plastic syringes. Before starting the operation, the entire channel was filled with water and all ports were closed. The carrier ampholyte solution was introduced into the chip with ports 1 and 2 open. LIF detection was performed at a position 2 mm in front of the anodic intersection, as indicated by arrow A in Figure 1. A 0.5 µL aliquot of sample in the carrier ampholyte solution was introduced from the injector and, at approximately 10 s after detection of the top of the sample peak, when the peak top was expected to be in the middle of the separation channel, the pump was stopped and the valves on ports 1 and 2 were closed. The anolyte solution was delivered from port 8 to port 7 to reach the electrode (Figure 1). The volume between intersection A and the anode was approximately 12 µL, and around 20 µL of the anolyte solution was delivered for nearly 20 s in each run. Ports 7 and 8 were closed, and the catholyte solution was delivered from port 4 to 3 in the same manner that the anolyte solution was delivered. Port 4 was closed and then port 7 was opened. Electric focusing was started by applying +1.2 kV at the anode with the ground at the cathode. After 1 min, fluorescence scanning detection was started from intersection A to B at a scanning rate of 1 mm/s at 1 min intervals. The scanning program and the chromatographic data processor was manually started individually. RESULTS AND DISCUSSION Electric Resistance of the pH Gradient and Electrode Solutions. When capillary IEF was carried out using the carrier ampholyte solution containing 2.5% (v/v) Pharmalyte 3-10 in a 12 cm long capillary with a 50 µm i.d. at a voltage of 6 kV, the initial electric current was approximately 3.6 µA. During formation of the pH gradient, the current decreased to approximately 0.5 µA. When the capillary was filled with the anolyte, 20 mM phosphoric acid, instead of carrier ampholyte solution, the current was approximately 57 µA. The catholyte solution, 20 mM NaOH, also produced a current of approximately 57 µA. The specific resistance of the electrode solutions, the carrier ampholyte solution and the pH gradient were calculated to be 1.7 Ω m, 27 Ω m and 200 Ω m, respectively. This result indicates that the electric conductivity of the electrode solutions was 16-fold higher than that of the carrier ampholyte solution and 120-fold higher than that of the established pH gradient. This reveals the possibility that the ends of the pH gradient do not necessarily need to be located at the electrode vessels, but can be connected to the electrode vessels with lead channels containing the electrolyte solutions. When a voltage of 1 kV is applied at the two electrode vessels that are connected with a 1 cm microchannel, the voltage drop occurs almost exclusively in the channel. Thus, the field strength in the

channel is nearly constant at 1 kV/cm, independent of changes in channel resistance. In contrast, when the same 1 cm long separation channel is sandwiched between 1 cm long lead channels of the same cross-sectional area filled with anolyte and catholyte solutions, the field strength in each region of the channel varies according to Ohm’s law. The ratio of the separation channel resistance to that of the electrode solution would be 8 to 1 at the beginning of focusing and 60 to 1 after focusing. A voltage of 1 kV applied to the electrode vessels located at the end of the lead channels should yield a field strength of 0.88 kV/cm in the separation channel at the beginning of focusing and 0.98 kV/cm after formation of the pH gradient. The loss of voltage in the lead channel during the final stage of focusing would be approximately 2% of the applied voltage. When the same 1 cm separation channel was sandwiched between lead channels, each 2 cm in length, the voltage loss in the lead channels after focusing should be 3%. Therefore, IEF separation channels can be connected to electrode vessels with relatively long lead channels without significant loss of applied voltage. Microfluidic Definition of IEF Channels. Based on the relatively large electric resistance of the pH gradient in comparison with the resistance of electrode solutions, a new form of IEF chip was proposed (Figure 1). In principle, this form of IEF chip should have six ports with valves located at the ports or somewhere on the channels or connecting tubes. In the proposed chip, the central channel served as the separation channel, and the V-shaped lead channels on both sides were filled with electrode solutions. The electrode vessels were placed on the outlet ports of the lead channels (Figure 2). Operation of the IEF chip was started by filling the central channel with a sample containing carrier ampholytes from port 1 to port 2, and then the lead channels were sequentially filled with the anolyte solution from port 8 to 7 and with the catholyte solution from port 4 to 3. Port 5 and 6 were not used in the present application, and the channels directly connected to these ports were filled with water. Focusing was initiated by the application of +1.2 kV at the anode outside port 7 with the cathode outside port 3 grounded. The valves ensure retention of the sample solution in the separation channel during introduction of the electrode solutions. The 24 mm separation channel was defined by intersections A and B with the lead channels. To suppress displacement of the sample/carrier ampholyte solution caused by the small difference in the levels of the electrode vessels, 0.5% HPMC was added to the electrolyte solutions. The effect of the polymer on the electric conductivity of the electrolyte solutions was negligible at less than 4%. The electrodes were constructed by piercing silicone tubes (1 mm i.d.) attached to Teflon tubes (0.5 mm i.d.) fitted to the connector valves (Figure 2) with platinum wires. Because the electrode was placed at the outlet port of the lead channel, gas bubbles formed as a result of electrolysis of the water remaining on the far side of the chip. The connection between the ends of the separation channel and the electrodes, the lead assembly, was composed of a lead channel in the chip (18.4 mm), a valve rotor, a Teflon tube (0.5 mm i.d., 20 mm long) and a silicone tube (1 mm i.d., 10 mm long). Since the specific resistance of the two electrode solutions is almost the same and the structure of the lead assemblies is symmetrical, the total resistance between the electrodes, Rt, can be written as Rt ) Rs + 2Rl, where Rs and Rl are the resistance of the separation channel and that of a lead assembly, respectively. The channel shape of the

lead assembly is complicated, and the calculation of the resistance might not be precise; thus, the resistance of the lead assemblies was calculated by subtraction of the calculated resistance of the separation channel from the measured total resistance between the electrodes. The channels between the electrodes were filled with the anolyte or catholyte, and application of 1.2 kV at the electrodes yielded a current of 132 µA, i.e., a total resistance of 9.1 MΩ. The resistance of the separation channel, i.e., between A and B, was calculated from the measured size of the isotropically etched channel (206 µm wide, 92 µm deep) and the specific resistance of the solutions: 2.7 MΩ, 43MΩ and 310 MΩ for the electrolyte solution, the carrier ampholyte solution and the pH gradient, respectively. The resistance of the lead assemblies was calculated for the pair by subtraction, i.e., 9.1 - 2.7 ) 6.4 MΩ. Application of the discussion in the previous section about the voltage drops in the separation channel and lead channels yields a voltage drop in the separation channel of 1.2 kV × 43/(43 + 6.4) ) 1.05 kV at the beginning of focusing and 1.2 kV × 310/(310 + 6.4) ) 1.17 kV after focusing, corresponding to 98% of the applied voltage after focusing. The calculated electric current based on the estimated resistance of each part of the channel at 1.2 kV agreed well with those observed, i.e., 24 µA in the beginning of focusing and 3.8 µA after focusing, in comparison with the observed values of approximately 25 µA and 3 µA, respectively. Separation of Peptide pI Markers. The IEF chip was tested using tetramethylrhodamine-labeled peptide pI markers.31 Four markers were chosen to cover a pH range of 3-10 for the carrier ampholyte used. The pIs of the markers were as follows: 3.64, 5.53, 7.58, and 9.56. A 0.5 µL aliquot of sample solution containing the four markers at a concentration of 2.5 nM and 2.5% (v/v) Pharmalyte 3-10 was introduced into the chip via the carrier ampholyte solution at a rate of 1 µL/min. LIF detection was performed at a position approximately 2 mm in front of intersection A (Figure 1). The injected sample plug formed a fluorescent peak with a half-maximum width of 0.7 min corresponding to 0.7 µL. At about 10 s after detection of the peak top of the injected sample, flow was stopped and the valves on ports 1 and 2 were closed. The volume of the 24 mm separation channel was calculated to be 0.37 µL, and thus the maximal sample that can be held by the separation channel is at most approximately half of the injected sample, assuming a Gaussian peak. After freshening electrolyte solution in the lead channels, IEF was started by the application of 1.2 kV between the electrodes. Scanning LIF detection over the full length of the separation channel was started after 1 min of focusing, and was repeated every 1 min (Figure 3). Focusing was nearly complete after 2 min, although a small split in the pI 5.53 marker focused at approximately 9 mm remained. Successive scans revealed spreading of the pH gradient at both ends (Figures 3). This phenomenon can be attributed to the isotachophoretic migration of acidic and basic carrier ampholytes with the leading electrolytes, H3PO4 and NaOH, respectively.34 At both ends of the pH gradient, focused carrier ampholytes carry a residual charge to balance the protons and hydroxyl ions in the acidic and basic regions, respectively. Spreading of the pH gradient occurs at a slightly faster rate in the basic region than in the acidic region (Figure 3). This may reflect the higher velocity of boundary migration between the basic carrier ampholytes and NaOH than that between the acidic carrier ampholytes and H3PO4 when these electrolytes are (34) Mosher, R. A.; Thormann, W. Electrophoresis 1990, 11, 717–723.

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Figure 3. Chip IEF of four pI markers. A mixture (0.5 µL) of the four tetramethylrhodamine-labeled pI markers (pI 3.64, 5.53, 7.58 and 9.56) at a concentration of 2.5 nM in the carrier ampholyte solution (2.5% (v/v), pH 3-10) was introduced into the chip via the flow of the carrier ampholyte solution (1 µL/min). When the sample plug reached the separation channel, the flow was stopped, and the electrode solutions in the lead channels were freshened. Focusing was performed at a voltage of 1.2 kV. The entire separation channel from the anodic to the cathodic end was scanned every 1 min at a rate of 1 mm/s. The small peak at approximately 7 min was due to an impurity. The actual focusing times for the position located 24 mm from the anodic end were 24 s longer than the nominal focusing times due to the time required for scanning.

used at the same concentrations.34 The detection limit was calculated to be 3 pM (1.5 amol) for the pI 5.53 peak after 4 min of focusing using a signal-to-noise ratio of 3 to 1. This value can be reduced by 50% if the entire sample is trapped in the IEF channel. The reproducibility of the focusing position of the pI markers for 11 runs of IEF in the chip is summarized in Table 1. Although the reproducibility of the peak-to-peak interval was relatively high, that of absolute peak position was not satisfactory. We suspect that this is attributable to a low level of leakage of solutions at the valve faces of Teflon valve rotors during the introduction of the electrode solutions. This small component of the valve is currently made using a cutting-and-shaving process that results in a face with small scratches that cause low levels of oozing of solutions. Lowering the flow rate of electrolyte solutions, approximately 1 µL/s at present, would decrease the level of this leakage and could help increase reproducibility. Scanning detection employed in this report takes 24 s for the entire separation channel of 24 mm. This means that there is a time difference depending on the position in the channel. When

the bands do not move after the completion of focusing, this time difference would not affect the determination of pI values of an unknown band using pI markers but would do so when they move as in the case presented in Figure 3. We estimated this effect on the determination of the pI value of an assumed peak that would appear in the middle of two pI markers, pI 7.58 and pI 9.56, using the data from 3 min focusing presented in Figure 3. The distance of the two pI markers was 8.10 mm and increased at a rate of 0.72 mm/min. When the pI of the peak that appears in the middle is determined by assuming a linear relationship between peak position and pI values, the pI would be calculated to be (9.59 - 7.58) × 4.05/8.10 + 7.58 ) 8.585. However, actual distance of the two peaks should be smaller by 0.72 × 4.05/60 ) 0.05 mm than 8.10 mm, when the middle peak is detected. A better estimation would be (9.59 - 7.58) × 4.05/8.05 + 7.58 ) 8.591. The estimated value would be lower than the actual value by 0.006 pH unit. Although the difference is negligible, imaging detection has an apparent advantage on this point.10,15,20,24,26 Stability of the Coating after Exposure to the Catholyte. In capillary IEF, stability of the coating has been an issue. In particular, the polyacrylamide coating is sensitive to alkaline conditions.35 The IEF chip proposed in the present study requires exposure of the coated lead channel to the catholyte solution: 20 mM NaOH. Degradation of the coating may increase electro-osmosis, which, in turn, affects the stability of the pH gradient and the reproducibility of IEF separation. Fortunately, there was no evidence of degradation in the polydimethylacrylamide coating after 15 IEF runs. We tested the effect of 20 mM NaOH on the polydimethylacrylamide-coated fused-silica capillary. The coated capillary was filled with 20 mM NaOH and allowed to stand for 1 h at room temperature (∼25 °C). The results of capillary IEF runs before and after NaOH treatment were not significantly different (unpublished result). The stability of the polydimethylacrylamide coating under alkaline conditions is compatible with the proposed IEF chip. To minimize the effects of the catholyte on the coating, the catholyte in the lead channel between the intersection and the cathode vessel was replaced with carrier ampholyte solution after each IEF run. Resolution of the IEF Chip. The two peaks at neutral pH were apparently sharper than those in the acidic and basic regions (Figure 3). This phenomenon also was observed in capillary IEF as peak heights for neutral pI markers were higher than acidic and basic pI markers at pH values ranging from 3 to 10.31 This observation may be related to the flattening of the pH gradient at the acidic and basic regions by the isotachophoretic migration of the carrier ampholytes at the extremities of the pH gradient, as mentioned above. However, this phenomenon warrants further investigation. The peak capacity of the IEF chip was calculated using the peak width of each peak and a separation length of 24

Table 1. Reproducibility of Focusing Position of Peptide pI Markers in IEF Chipa position from the anodic end pI average (mm) SD (mm) RSD (%)

3.64 3.41 0.86 3.6b

5.53 10.19 0.83 3.5b

7.58 15.20 0.89 3.7b

interval between two pI markers 9.56 22.42 0.92 3.8b

5.53-3.64 6.78 0.24 3.5c

7.58-5.53 5.01 0.08 1.6c

9.56-7.58 7.23 0.07 1.0c

a Isoelectric focusing was carried out as described in the legend of Figure 3. Peak positions after 2 min focusing were summarized for 11 runs of chip IEF. b The relative standard deviation of the focusing position against the total length of the channel (24 mm), i.e., SD/24 × 100. c The relative standard deviation of the interval of two peaks against its average, i.e., SD/average × 100.

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Figure 4. Analysis of three different sample plugs successively injected into the IEF chip. (A) Three aliquots (0.5 µL each) of the mixture of different pI markers were injected into the chip through the outside sample injector at a 2 min interval, and the fluorescence signal was recorded at a position 2 mm in front of the anodic intersection. (B) After identical injections to those in (A), the flow of the pump was stopped after each peak was detected, and the sample in the IEF channel was subjected to IEF analysis. The electropherograms for a 2 min focusing period are presented. Samples 1 to 3 each contained the four pI markers used in the experiment shown in Figure 3, i.e., pI equal to 3.64, 5.53, 7.58 and 9.56, at a concentration of 1 nM. Specific markers (2.5 nM) added to each sample were as follows: pI 4.50 and 4.99 for sample 1; pI 6.18 and 6.86 for sample 2; pI 8.21 and 8.77 for sample 3. The numbers on the peaks are the pI values of the markers. The connection between the sample injector and port 1 was changed to a Teflon tube, 0.25 mm i.d. and 86 mm long, with a larger volume to hold three samples injected over 4 min.

mm as follows: pI 3.64 marker, 50; pI 5.53 marker, 150; pI 7.58 marker, 135; and pI 9.56 marker, 100. The minimal pI differences for separation were as follows: pI 3.64, 0.14; pI 5.53 and pI 7.58, 0.05; and pI 9.56, 0.07. The smallest value, 0.05, was still considerably larger than that reported for capillary IEF: 0.02.36 Since the minimum difference is inversely proportional to the root of the applied voltage,37 a shorter separation distance negatively (35) Chiari, M.; Micheletti, C.; Nesi, M.; Fazio, M.; Righetti, P. G. Electrophoresis 1994, 15, 177–186. (36) Zhu, M.; Rodriguez, R.; Wehr, T.; Siebert, C. J. Chromatogr. 1992, 608, 225–237. (37) Rilbe, H. Ann. N.Y. Acad. Sci. 1973, 209, 11–22. (38) Wu, J.; Pawliszyn, J Anal. Chem. 1995, 67, 2010–2014.

affects resolution when the field strength is constant. When the lower voltage is taken into consideration, the resolution obtained using the IEF chip should be reasonable in comparison with that obtained using CIEF. Analysis of Consecutively Injected Samples. To demonstrate the ability of the IEF chip to perform downstream separation of prefractionated samples, samples injected consecutively were analyzed individually. Three sample plugs that each contained two unique pI markers at a concentration of 2.5 nM, in addition to the four common pI markers at 1 nM, in 2.5% (v/v) Pharmalyte 3-10 were injected at 2 min intervals via a flow of the carrier ampholyte solution at 1 µL/min (Figure 4A). After each sample plug entered the IEF channel, the pump was stopped and the sample in the IEF channel was subjected to IEF. The electropherogram of each sample plug represented its unique composition (Figure 4B). Thus, the IEF chip would easily be integrated with other sample pretreatment processes from which the sample is transferred via pressurized flow. Unexpected small peaks were detected, e.g., the four small peaks between the pI markers 5.53 and 9.56 in the electropherogram 1 of Figure 4B. These peaks are a carryover from previous analyses. Such a carryover was not observed when a labeled protein was used as a sample in preliminary experiments. There might be a low level of adsorption of fluorescence-labeled peptide pI markers on the glass surface underneath the coating with linear polydimethylacrylamide. The protein would not be accessible to a glass surface due to its size or would simply be less adsorptive because of a more hydrophilic surface. The source of the carryover is under investigation. A focusing time of 2 min is sometimes inadequate for some pI markers, resulting in split peaks. However, other times a 2 min focusing period is adequate for the same pI markers. CONCLUSION By selective operation of valves on the port of the IEF chip, carrier ampholyte-sample mixture and electrolytes (catholyte and anolyte) can be independently loaded into their respective channels without interfering with one another while electrical contact between them is kept. As a result, the procedure of IEF was simplified with minimal loss of samples. The proposed IEF chip is ideal for coupling with other sample treatment processes on the same chip. Desalting is a pretreatment process that is often required for IEF analysis of biological fluids.38 Combination of IEF with other separation processes, such as affinity chromatography, is under development in our laboratory. Such combinations would allow precise identification of proteins in complex biological sample matrices by discrimination of unrelated proteins using IEF separation. This combination would be as useful as Western blotting for protein separation and analysis. Integration of IEF with pretreatment processes on a microscale would significantly reduce the required sample size and analysis time. ACKNOWLEDGMENT Part of this work was a project of The Research Association of Micro Chemical Process Technology (MCPT).

Received for review January 10, 2008. Accepted March 5, 2008. AC8000594

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