Anal. Chem. 2004, 76, 4426-4431
Two-Dimensional Protein Separation with Advanced Sample and Buffer Isolation Using Microfluidic Valves Ying-Chih Wang,*,†,§ Man Ho Choi,‡ and Jongyoon Han‡,§
Department of Mechanical Engineering, Biological Engineering Division, and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Methods are described to achieve more efficient multidimensional protein separation in a microfluidic channel. The new methods couple isoelectric focusing (IEF) with high ionic strength electrophoretic separations by active microvalve control in a microchip. Several experiments demonstrating independent 2D separation were performed, and critical parameters for optimal chip performance were identified, including channel passivation, electroosmosis control, and IEF linearity control. This strategy can be used for integration of different heterogeneous separation techniques, such as IEF, capillary electrophoresis, and liquid chromatography. This new device can be ideal for preseparation and preconcentration of complex biomolecule samples for a streamlined biomolecule analysis using mass spectrometry. Sample preparation is one of the most critical issues in clinical proteomics. While tandem mass spectrometry (MS/MS) is capable of identifying the structures of coeluting peptides in electrospray-mass spectrometric (ESI-MS) analyses, it may not be effective for peptide digests resulting from complex protein mixtures. As a result of the heterogeneity of proteins derived from cell populations,1 comprehensive techniques have been developed for multidimensional electrophoretic sample pretreatment to achieve adequate separation of complex protein mixtures. Also, rapid and accurate identification of proteins and their posttranslational modification is necessary because these molecular signals might be time sensitive. Although 2D gel electrophoresis has been used as the primary method for protein separation from complex mixtures, its laborious and time-consuming steps involving protein transfer and extraction from the gel can result in sample loss2,3 and make it a less favorable technique. During the past decade, microfluidics technologies have been widely applied to biomolecule analysis and separation.4 Microfluidic devices are superior to traditional tools in many aspects, * Corresponding author. E-mail:
[email protected]. † Department of Mechanical Engineering. ‡ Biological Engineering Division. § Department of Electrical Engineering and Computer Science. (1) Herbert, B. Electrophoresis 1999, 20, 660-663. (2) O’Farrell, P. H. J. Biol. Chem. 1975, 10, 4007-4021. (3) Anderson, L.; Anderson, N. G. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 54215425. (4) Auroux, P.-A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652.
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such as speed, overall dimension, small sample amount, automation, and portability. While it is highly desirable to implement multidimensional protein separation techniques on a microchip that can substitute for the bulky, time-consuming slab gel currently used, integration of two different separation techniques on a microfluidic chip has been challenging. Several groups have developed microchip- or capillary-based two-dimensional separation systems, which are an integration of micellar electrokinetic chromatography (MEKC) or isoelectric focusing (IEF) with capillary electrophoresis (CE).5-9 These previous accomplishments, however, are limited by the fact that there was no mechanism to prevent sample and buffer interdiffusion between different techniques. Such strategies could not be applied generally to the integration of different microfluidic components. More recently, a microfluidic device that couples IEF and SDS-PAGE has been demonstrated, with appropriate peak transfer between IEF and SDS-CE.10 However, in the analysis of a complex biological sample such as serum, interdiffusion between the focused protein peaks during the coupling stage could decrease the efficiency and resolution of the sample separation from the first-dimension IEF separation, especially when the sample contains both majority and minority protein species with concentrations that differ by orders of magnitudes. Here, we describe a new method to integrate heterogeneous separation techniques with incompatible buffer requirements. This new method integrates IEF, a charge-based separation, and CE or capillary gel electrophoresis (CGE) with high ionic strength buffers in a microfluidic system. A three-step process was used to achieve 2D electrophoresis on a chip. First, a high-resolution pH gradient from pH 3 to 10 for IEF was established within a short channel. As target proteins moved into the peak transfer region, one set of microfluidic valves was closed for the isolation of selected protein peaks. Second, the peak transfer region was (5) Rocklin, R. D.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 52445249. (6) 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. (7) Larmann, J. P.; Lemmo, A. V.; Moore, A. W.; Jorgenson, J. W. Electrophoresis 1993, 14, 439-447. (8) Chen, X.; Wu, H.; Mao, C.; Whitesides, G. M. Anal. Chem. 2002, 74, 17721778. (9) Michels, D. A.; Hu, S.; Schoenherr, R. M.; Eggertson, M. J.; Dovichi, N. J. Mol. Cell. Proteom. 2002, 1, 69-74. (10) Li, Y.; Bush, J. S.; Rosenberger, F.; Devoe, D. L.; Lee, C. S. Anal. Chem. 2004, 76, 742-748. 10.1021/ac0497499 CCC: $27.50
© 2004 American Chemical Society Published on Web 06/30/2004
connected to the second-dimension separation channel by opening another set of valves. Among different valve designs, we adopted the design by Unger et al.11 since it is simple to make and has a low dead volume, which is important in trace analysis. The microfluidic valves here are the key components in this device that can prevent intermixing between two separation buffers (ampholyte and CE buffer). They isolate a group of isoelectricfocused proteins, preventing peak interdiffusion and band dispersion during the peak transfer process. Finally, the trapped proteins in the peak transfer region were sent into a second-dimension capillary channel for further separation. This new strategy could be used to couple many heterogeneous separation techniques based on hydrophobic interaction and immuno/chemical affinities to be used in an integrated, multidimensional fashion, which could greatly enhance the efficiency of protein analysis from complex mixtures. EXPERIMENTAL SECTION Chip Fabrication. To isolate and control channels, multilayer microfluidic valves, previously reported by Unger et al.,11 were used as control valves for microfluidic channels, using poly(dimethylsiloxane) (PDMS) as a substrate. The fabrication includes three major processes: (1) master fabrication, (2) PDMS shaping, and (3) PDMS multilayer bonding. Further details about the fabrication can be found in the reference.11,12 The master for the PDMS device was fabricated using standard photolithography techniques, using both positive (AZ-4620, Clariant Corporation, Charlotte, NC) and negative (SU-8 50 MicroChem Corporation, Newton, MA) photoresist. The master mold for the separation channels was 150-µm wide and 12-µm high, and the mold was heated to 150 °C for 30 min to make it round-shaped. The master mold for the valve control channel was 300-µm wide and 50-µm high, with a square cross section. In order to prevent adhesion with silicone rubber, a 30 min hexamethyldisilazane (Sigma-Aldrich, St. Louis, MO) vapor treatment is required. After the silane treatment, mixed PDMS was spun onto the bottom separation channel mold with a spin speed of 2000 rpm with a target thickness of 70 µm. At the same time, the top control channel was made in ∼5-mm thick PDMS. The silicone elastomer kit (Dow Corning, Midland, MI) was mixed at a 10:1 ratio and degassed in a desiccator under an approximately 5 psi vacuum before being poured or spun onto master molds. After curing in a 65 °C oven for at least 2 h, different layers of PDMS were treated with an oxygen plasma in a plasma cleaner (Harrick Scientific Corporation, Ossining, NY) before the bonding. Oxygen plasma bonding, reported by several authors,12,13 was used to bond first the 5-mm control channel layer onto the 70-µm separation channel layer, and then together onto yet another PDMS layer spun on a flat glass, as a bottom substrate. A metal syringe needle (Hamilton Company, Reno, NV) with a 1/16-in. o.d. was used to punch holes though the end of the control channels for the connection with 1/16-in. Teflon tubing as a pressure control line. The elasticity of the PDMS assured a proper seal between (11) Unger, M. A.; Chou, H.-P.; Thorsen, T.; Scherer, A.; Quake, S. R. Science 2000, 288, 113-116. (12) Jo, B.-H.; Lerberghe, L. M. V.; Mostsegood, K. M.; Beebe, D. J. J. Microelectromech. Syst. 2000, 9, 76-81. (13) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984.
Figure 1. Schematic drawing of the double layer PDMS channel and the four-step separation process: (a) perspective view; (b) CE buffer loading; (c) IEF ampholyte mixture loading; (d) isolation of target proteins by closing all valve sets; (e) further separation of isolated proteins.
the device and the tubing, enabling one to apply more than 50 psi of pressure without significant leakage. After this alignment-bonding step, the device should be left for at least 2 h to have the maximum bonding strength. Because PDMS is gas permeable, the top layer control channels must be filled with water before use, to prevent air bubbles in the separation channels under high-pressure operation of the valves. Otherwise, the operation of the valves will drive air bubbles into the channel, which is catastrophic in the device operation. The perspective view of the device is shown in Figure 1a. Materials and Reagents. The IEF sample solution was made of 3-10 ampholyte (Beckman Coulter, Fullerton, CA), 10% glycerol (BioRad, Hercules, CA), 1% methyl cellulose (Sigma-Aldrich, St. Louis, MO), and aqueous protein samples. Green fluorescent protein (GFP) (pI ) 5.6, MW ) 26 kDa) and three fluoresceinor Alexa Fluor 488-labeled proteins were used as samples: ovalbumin (pI ) 5.1, MW ) 45 kDa), low-density lipoprotein (pI ) 5.11, MW ) 179 kDa), and trypsin inhibitor (pI ) 4.6, MW ) 20 kDa). In the IEF linearity experiment, eight fluorescent IEF markers (Sigma-Aldrich, St. Louis, MO) with pI’s of 3.0, 4.5, 5.1, 6.2, 7.2, 8.1, 9.0, and 10.3 at a concentration of 10 µg/mL were used. These IEF markers can be detected by fluorescence microscopy with excitation wavelengths from 330 to 340 nm and emission wavelengths from 415 to 500 nm. In the CGE experiment, the liquid gel buffer used here was SDS 14-200 gel buffer (Beckman Coulter, Fullerton, CA) containing 3.0% Tris (hydroxymethyl)aminomethane and 2.0% ethylene glycol. To have better mixing in the peak transfer region, the same concentration of methylcellulose was used in all solutions including the separation gel (90% SDS 14-200 gel buffer and 1% methyl cellulose), anolyte solution (0.1 M acetic acid, pH 2.5), and catholyte solution (0.5% ammonium hydroxide, pH 10.5). As for the capillary zone Analytical Chemistry, Vol. 76, No. 15, August 1, 2004
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electrophoresis experiment, 0.375 M Tris-Cl (pH 8.6) containing 0.1% methylcellulose was used. All the fluorescent signals were imaged with an inverted epi-fluorescence microscope, which has a built-in 100 W mercury lamp with a standard FITC filter set (Olympus, Melville, NY). In this experiment, a thermoelectrically cooled CCD camera (Cooke Co., Auburn Hill, MI) was used for fluorescence imaging through the side port of the microscope. Sequences of images were taken and analyzed by IPLab 3.6 (Scanalytics, Fairfax, VA) at a rate of 1 frame/s. Surface Treatment. PDMS is sometimes considered to be an unsuitable material for microfluidic molecular separation devices, because of its nonuniformly charged, protein adsorptive surface.14 In order to obtain reliable and reproducible results, it is essential to prevent protein adsorption problems and decrease uneven EOF in either PDMS or glass microfluidic channels. Compared to glass channels, PDMS has lower EOF and less adsorption.15 However, a PDMS channel coating is still required to obtain reliable separation results. In the experiment, we used a polyacrylamide coating, which has been well-developed in glass channels,16 to prevent protein adsorption. Because the same silanol (-Si-OH) group is present in PDMS and glass, the same process can be applied for PDMS coating without special difficulty. To grow acrylamide polymer on a PDMS surface, we first coated the PDMS channel with 3-(trimethoxysilyl)propyl methacrylate (Sigma-Aldrich, St. Louis, MO) as an adhesion promoter for the polyacrylamide. Then, polyacrylamide solution was mixed with tetramethyl ethylenediamine (TEMED) and ammonium persulfate (APS) before being introduced into the microfluidic channel for polymerization. After mixing, it was put in a homemade vacuum chamber with a nitrogen purging valve to eliminate the oxygen-quenching problem in free radical polymerization.17 After 30 min, 0.5% methylcellulose solution was used as a pretreatment coating material to further minimize protein adsorption. Compared with the case when only dynamic coating by methylcellulose is used, the above process (static and dynamic coating) was better at decreasing nonspecific adsorption. Protein adsorption was reduced to a level that is not detectable using the current fluorescence microscopy setup, at least for the duration of a single 2D separation experiment. Similar surface grafting on the PDMS surface by different polymers including acrylamide has been recently reported by Hu et al.18 Valve Operation. Compared to another frequently used RTV 615 silicone elastomer kit (General Electric) with a tensile strength of 6.3 MPa and an elongation rate of 120%, Sylgard 184 has a tensile strength of 7.1 MPa and an elongation rate of 140%, which makes it a better material for devices with hydraulic valves. Resistance across a closed microfluidic valve indicates the integrity of electrical isolation of the microfluidic valve used in this work. By increasing the pressure from 30 to 100 psi, one can increase the closed-valve resistance from 699 MΩ to a range of over 1000 MΩ. The resistance was measured by connecting a 1-MΩ external (14) Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S. Anal. Chem. 2000, 72, 5939-5944. (15) Ocvirk, G.; Munroe, M.; Tang, T.; Oleschuk, R.; Westra, K.; Harrison, D. J. Electrophoresis 2000, 21, 107-115. (16) Hjerten, S. J. Chromatogr. 1985, 347, 191-198. (17) Zheng, J.; Mastrangelo, C.; Burns, M. A.; Burke, D. T. Proc. 2nd Int. IEEEEMBS Conf. Microtechnol. Med. Biol. 2002, 442-446. (18) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Anal. Chem. 2002, 74, 4117.
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resistance in series with the channel with a closed valve and comparing the voltage drop across the resistance as well as the channel. Isolation resistance could be further increased by a better design of the valve structure. With this isolation resistance (both fluidic and electrical), no protein intercontamination across the closed valve was observed even after 30 min. There is a dead volume in the current design of the device, which is the extra space left between two sets of valves for alignment purposes (See Figure 1c). This 0.1-nL dead volume is much smaller than that of commercially available microvalves for capillary systems (10 to ∼100 nL) and could be further reduced by a better design and alignment of the valve to the main channel. Because of the symmetric channel layout, the channels around the peak transfer region have similar fluid resistance; therefore, the opening/closing operation does not mobilize proteins in the transfer region. Chip Operation. The major operation involves two sets of valves (top control layer in Figure 1a) designed to isolate different separation media and to transfer IEF-focused analytes into the transfer region. The elastomer membrane underneath the control channel will be squeezed down under pressure and therefore block the fluid flow and electrical current in the separation channel. By closing valves with 80 psi pressure, 2D separation without cross contamination of buffer and sample was achieved. The 2D separation consisted of four major steps including CE media injection, ampholyte and sample mixture injection, IEF focusing, and CE separation. The CE media was either polymeric liquid gel or free buffer solution with relatively high pH values. First, the right valve control line (valves 3 and 4) was pressured to close the fluid channel below it. After clogging the channel, liquid gel (or CE buffer) could be injected without contaminating the IEF channel, which is very sensitive to high concentration buffer (Figure 1b). The operation pressure for the CE media injection depends on the solution viscosity, typically 20-30 psi. Pressures up to 60 psi could be applied to the main channel to inject the CE media, without affecting the closure of the valve. Then, the left valves (valves 1 and 2) were pressurized to isolate the liquid gel. After closing the left valves, the right valves were opened for the injection of the ampholyte and sample mixture (Figure 1c), followed by replacing the reservoir solutions with catholyte and anolyte buffers. The IEF separation was achieved by applying an electric potential across the anolyte and catholyte reservoirs. Due to the electroosmotic flow generated in the IEF channel, peaks were slowly mobilized from anolyte to catholyte, and focused protein peaks entered and exited the central peak transfer region sequentially based on their pI values. After the IEF of the proteins was established, one could isolate any pI region of interest from the other peaks by closing the valves (valves 3 and 4). Since the protein IEF was maintained until the very last moment of peak isolation, the resolution obtained from the IEF separation is maintained, and the interdiffusion and dispersion of peaks caused by coupling two separation techniques were minimized. Finally when valves 1 and 2 were opened, proteins isolated within this region reacquired charges and were sent to the second-dimension separation channel by electrophoresis (Figure 1d). During this migration, proteins were separated based on the separation mechanism chosen for the second dimension.
Figure 2. Electropherogram of GFP and fluorescent pI markers in a 1-cm long 100-µm by 12-µm PDMS microfluidic channel.
RESULTS AND DISCUSSION Here, we first present the linearity of the first-dimension IEF and ways to achieve better separation. Then, on-chip 2D separation including IEF-CGE and IEF-CE are shown and compared. Several experiments are presented to demonstrate both the ability to choose the target protein in IEF and the independence of the second-dimension separation from the first-dimension separation. Last, the relation between IEF and CE are discussed to have a better understanding about the chip-based 2D separation system. IEF of Proteins in Ultrashort PDMS Channels. In this experiment, fluorescent IEF markers were used to test the ability of the system to establish a wide pH gradient from pH 3 to 10 (Figure 2). Different from other experiments, the sample ampholyte mixture here used 3% methylcellulose and 5% ampholyte. The higher methylcellulose concentration increases the viscosity, which helps visualize high pI number markers. The whole channel image was taken after applying a 500 V/cm electrical field for 30 s. During the focusing, the completion of the IEF (reaching the steady state) was checked by monitoring the current through the channel. The overall 10-mm length of the channel makes the required separation potential smaller, compared with the 20 to 30 kV needed in capillary isoelectric focusing (CIEF). Moreover, the focusing time is about 2 orders of magnitude faster than that typical in CIEF (about 30 min), where proteins have to travel a long capillary (25-50 cm).19-21 Compared with the short fusedsilica CIEF by Wu et al.,22 which take 280-360 s to reach equilibrium, the presented ultrashort IEF on a microfluidic PDMS chip has a better signal-to-noise ratio and separation properties. Moreover, since the IEF resolution depends only on the pH gradient established, which is an inherent property of ampholyte mixture, decreasing the channel length will not affect the separation resolution. To achieve a relatively high-resolution dynamic gradient by using carrier ampholyte, it is critical to decrease the electroosmotic flow by controlling the ampholyte-sample mixture viscosity, ampholyte concentration, and electrical field in the ultrashort IEF channel. As discussed in previous sections, the electroosmotic flow can be decreased by coating with polyacrylamide and increasing the methylcellulose concentration. Raising the methylcellulose concentration also increases the viscosity of the ampholyte-sample mixture, which also contributes to a decrease in electroosmotic flow mobility. The control of EOF is crucial in attaining a reliable IEF. If the EOF mobility is too high, focused (19) Shen, Y.; Smith, R. D. J. Microcolumn Sep. 2000, 12, 135-141. (20) Shen, Y.; Berger, S. J.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2154-2159. (21) Shimura, K. Electrophoresis 2002, 23, 3847-3857. (22) Wu, X.-Z.; Pawliszyn, J. Anal. Sci. 2001, 17, 189-192.
peaks would be flushed out of the ultrashort channel, possibly within a few minutes. This problem can be managed as long as the operation of peak selection and isolation is performed before the complete flushing of samples out of the device. In this work, the equilibration time was reduced to less than 30 s, therefore allowing time to manipulate and isolate the focused peaks by the microfluidic valves. However, the main problem related with EOF is not its magnitude, but its pH sensitivity. During the focusing, different channel regions are subject to different pH values, leading to different surface charge density and different EOF mobility. This will lead to a pressure gradient between the high and low pH regions, leading to additional broadening of the peaks focused. Achieving a stable coating over a wide pH range is still actively pursued by many researchers.23 The uneven buffering capacity (conductivity) and EOF of the commercially available ampholyte mixture makes the pH gradient in the channel nonlinear, especially in the high pH side (Figure 2). One of the reasons is that these ampholyte mixtures were optimized for a coated, fused-silica capillary, with different EOF characteristics than those of the PDMS surface. It is believed that, by balancing the concentration distribution for each ampholyte component to match the local EOF condition and passivating the channel surface better, one could improve the linearity of the pH gradient. For example, when 8.1-9.8 ampholyte was added to the wide range 3-10 ampholyte to suppress the EOF of the high pH side of the IEF column, we observed that peaks were shifted in the high pH side of the IEF channel. However, further study and characterization should be done to make the pH gradient in the PDMS microchannel more linear. In addition, nonlinearity of the IEF separation in this device does not critically affect the overall operation of the 2D separation of proteins, as long as they are separated with high-enough resolution so that the microfluidic valves can physically isolate them from one another. Also, shorter focusing time and sharper bands can be obtained by higher electrical field strength, which can be easily achieved in a short IEF channel. IEF Coupled with Second-Dimension Capillary Gel Electrophoresis. The four-step separation and transfer process described in the chip operation session was used to perform 2D separation with a protein mixture (Figure 3). In the first step, a 500 V/cm electric field was applied on the 1-cm IEF channel, which was isolated from the second-dimension CGE channel by the microfluidic valve. Because the fluorescein and Alexa Fluor dyes were designed to be attached to the lysine group, proteins could acquire different numbers of dyes and could have multiple pI values.24 The broadened IEF bands of the labeled proteins were mostly coming from the labeling process, while the local nonuniformity of the EOF could also be a possible reason for the dispersion. After applying the electric field for 130 s (Figure 3a), the target proteins were mobilized (by EOF) into the transfer region and trapped by closing both valve sets. Then, the valves to the CGE channel were opened, and buffer ions were allowed for 30 s before applying the CE field, to prevent an abrupt concentration change between the distinct separation media (Figure 3b). Soon after opening the valves, protein molecules will be rendered negatively charged, by the high pH, high concentra(23) Horvath, J.; Dolnik, V. Electrophoresis 2001, 22, 644-655. (24) Richards, D. P.; Stathakis, C.; Polakowski, R.; Ahmadzadeh, H.; Dovichi, N. J. J. Chromatogr., A 1999, 853, 21-25.
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Figure 4. Electropherograms of (a) four proteins after IEF and (b) further separation of ovalbumin and GFP by CGE as four proteins were loaded at 100 µM each.
Figure 3. 2D protein separation CCD camera images: (a) IEF done 130 s after applying 500 V/cm to a 1-cm long, 12-µm deep, and 150µm wide channel; (b) 30 s after trappingspeaks gradually diffuse and reacquire charges; (c) stacking effect shown when trapped proteins were being sent into the second dimension; (d) proteins further separated by different mass-to-charge ratios.
tion CGE buffer. The protein diffusion during this waiting period (30 s) could disperse the focused bands from the first dimension. However in this device such diffusion does not affect the separation resolution in the second dimension, because they get stacked at the interface between the CGE buffer and the ampholyte buffer. Also, protein samples have a much lower diffusion constant than buffer ions, and it would take a long time for protein peaks to diffuse out of the peak transfer region. The existence of a small amount of ampholyte in the CGE channel does not affect the resolution of the CGE separation. Most likely, the ampholyte molecules coisolated with proteins in the peak transfer region will be eluted out by electrophoresis quickly, due to their smaller MW than that of most proteins. Finally, a 250 V/cm electric field was applied to the proteins for CGE separation. Proteins were stacked when low-conductivity ampholyte solution mixed with 0.4 M high-conductivity Tris-Cl gel buffer (Figure 3c). Because the higher-conductivity environment (lower resistance) has a lower field strength, proteins will encounter a sudden decrease in electrophoretic velocity at the buffer boundary. Also, the highly viscous gel for CGE has a sieving effect that contributes to this stacking mechanism. This stacking of proteins decreases the width of the launching band for the second-dimension separation, which leads to better separation resolution. Also, by re-collecting all the IEF-focused peaks in the peak transfer region and making all of them start from the same launching band, this stacking process makes sure that the firstdimension separation and the second-dimension separation are 4430 Analytical Chemistry, Vol. 76, No. 15, August 1, 2004
independent of each other (See Figure 5). This is yet another advantage of using two different buffers, which could be crucial in enhancing separation resolution in the second dimension. Electropherograms were obtained at the end of the 25-mm second-dimension channel (Figure 4) by measuring the fluorescence intensity. In this CGE separation (without SDS) ovalbumin (45 kDa) has a higher electrophoretic mobility than GFP (26 kDa), because the electrophoretic mobilities of nondenatured proteins should depend on their charge-to-mass ratios rather than their molecular sizes. (It has been shown by Herr et al.,6 in a 1D onchip CE, that FITC-ovalbumin has a mobility of -1.3 × 104 cm2/ V‚s while GFP has a mobility of -1.0 × 104 cm2/V‚s.) The overall 2D separation was achieved within 10 min. IEF Coupled with Second-Dimension Capillary Zone Electrophoresis. Integration between IEF and CE was also achieved in a similar fashion. The second-dimension CE shows higher resolution and faster separation compared with that of CGE. The CGE experiment was performed mainly to demonstrate the ability to couple separation media with distinct viscosities. Moreover, CGE can separate proteins based on molecular size when they are denatured by SDS. In these CE experiments, the same stacking effect still occurs at the boundary even without a polymeric gel matrix, because proteins are entering a highconductivity buffer region from low-conductivity ampholyte. However, the liquid-liquid interface mixing occurs much faster than that of the liquid-gel mixture, mainly due to the lower viscosity of the CE buffers. We tested the independence between the two separation steps by changing the orientation of the first-dimension IEF. In one experiment (Figure 5a), catholyte and anolyte reservoirs were arranged in such a way that the ovalbumin moves into the seconddimension separation channel earlier than the GFP, while in another experiment (Figure 5b), the order of the peaks are reversed by switching the catholyte and anolyte reservoir. In both cases, the separation results in the second dimension were not affected by the order of the peaks in the peak transfer region, mainly due to the sample stacking that occurs at the interface
Figure 5. Isolation strength of the 2D separation device: (a) electropherogram of the second-dimension CE (proteins were sent into the second-dimension CE with the ovalbumin-GFP sequence); (b) same second-dimension CE electropherogram acquired while proteins were sent into the second dimension with a reversed GFP-ovalbumin sequence; (c) isolate and send only the GFP into the second dimension by valve operation.
between the ampholyte and the high ionic strength CE buffer. This result clearly demonstrates that the separation of peaks in the first dimension does not have remaining effects on the seconddimension separation, which makes the interpretation of the result much easier. The isolation of protein peaks was highly controllable and repeatable. As shown in Figure 5c, certain peaks can be selected or unselected in the sample transfer process in a straightforward manner. Therefore, this device is well suited for “picking out” a single peak from the IEF separation channel and analyzing it further, either by MS or a second-dimension separation. Also, by integrating more valve sets into the system, the remaining region can be transferred simultaneously into the second-dimension channel for follow-up analysis.
separation on-chip in 20 min. Also, the presented multidimensional protein separation strategy might be useful for the integration of different heterogeneous electrophoretic and/or liquid chromatographic techniques. In this work, detection was achieved by fluorescent precolumn labeling, which is not ideal for the analysis of real clinical samples. One could adopt on-column25 or postcolumn labeling,26 for example, to analyze protein samples without fluorescent prelabeling. Since the ability to prefractionate a certain pI range out of a complex protein sample has been shown to be a promising way to facilitate proteomics data-based searches in MS analysis,27 we plan to couple this device with an ESI-MS/MS system in order to decrease the sample complexity of the peptide samples.
CONCLUSION AND FUTURE DIRECTION With the use of monolith valves, we have successfully integrated chip-based IEF with CE or CGE and achieved 2D protein
ACKNOWLEDGMENT This research was partially supported by the DARPA BIM (Bio: Info: Micro) program and the Whittaker Foundation, as well as the MIT Ferry Fund. Fabrication of the device was mainly done in the microsystems technology laboratories of MIT.
(25) Jin, L. J.; Giordano, B. C.; Landers, J. P. Anal. Chem. 2001, 73, 49944999. (26) Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S. Anal. Chem. 2000, 72, 4608-4613. (27) Tan, A.; Pashkova, A.; Zang, L.; Foret, F.; Karger, B. L. Electrophoresis 2002, 23, 3599-3607.
Received for review February 13, 2004. Accepted May 27, 2004. AC0497499
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