Concentration and Separation of Proteins in Microfluidic Channels on

were influenced by variation of parameters such as the initial pH, ionic strength, and flow rate. Transverse IEF in pressure-driven flow is demonstrat...
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Anal. Chem. 2001, 73, 1627-1633

Concentration and Separation of Proteins in Microfluidic Channels on the Basis of Transverse IEF Katerˇina Macounova´,* Catherine R. Cabrera, and Paul Yager

Department of Bioengineering, Box 352255, University of Washington, Seattle, Washington 98195

The use of microfluidic channels formed by two electrodes made of gold or palladium to perform transverse isoelectric focusing (IEF) is presented as a means for continuous concentration and fractionation of proteins. The microchannels were 40 mm long with an electrode gap of 1.27 mm and a depth of 0.354 mm. The properties of pH gradients formed as a result of the electrolysis of water were influenced by variation of parameters such as the initial pH, ionic strength, and flow rate. Transverse IEF in pressure-driven flow is demonstrated using bovine serum albumin in a single ampholyte buffer as well as in multiple-component buffers. Experimental results of protein focusing compare well to predictions of a mathematical model. Optimal conditions for efficient continuous fractionation of a protein mixture are summarized and discussed. Interest in development and fabrication of microfluidic integrated devices for analysis of chemical and biological samples has increased dramatically in the past decade. Special attention has been devoted to sample preconditioning systems whereby an analyte of interest is separated from other interferent compounds and then is concentrated to enable subsequent procedures, such as biological assays. To separate biological materials, methods based on electrokinetic effects have been popular.1,2 An advantage of these methods, including both zone electrophoresis (ZE) and isoelectric focusing (IEF), is that the separation process can be adjusted easily and rapidly by changing parameters such as the current or voltage.3 IEF, a method for separation of analytes on the basis of isoelectric point (pI), requires the development of a pH gradient between two electrodes. Amphoteric compounds migrate through the pH gradient until they reach the position where the pH is equal to their pI. Due to their zero net charge in this point, their migration stops and they concentrate. IEF has also been adapted for use in microfluidic devices4,5 and has been tested by separation of mixtures of proteins with known pI values. However, in these experiments, IEF was utilized in a batch * Corresponding author. E-mail: [email protected]. (1) Chiou, S.-H.; Wu, S.-H. Anal. Chim. Acta 1999, 383, 47-60. (2) Li, P. C. H.; Harrison, D. J. Anal. Chem. 1997, 69, 1564-1568. (3) Wehr, T.; Rodriguez-Diaz, R.; Zhu, M. In Chromatographic Science Series; Marcel Dekker: New York, 1999; Vol. 80, pp 131-233. (4) Hofmann, O.; Che, D. P.; Cruickshank, K. A.; Muller, U. R. Anal. Chem. 1999, 71, 678-686. (5) Mao, Q. L.; Pawliszyn, J. Analyst 1999, 124, 637-641. 10.1021/ac001013y CCC: $20.00 Published on Web 03/01/2001

© 2001 American Chemical Society

operation mode, rather than in continuous operation, which is often not convenient for use in sample preconditioning systems. Recently, continuous microfluidic transverse IEF was used successfully to concentrate a solution of vegetative bacteria.6 A first step toward the development of a continuous mode of separation was field flow fractionation (FFF), a method in which a field is applied perpendicular to the fluid flow direction under laminar conditions.7 Fractionation occurs as different particles migrate at different rates in the applied field, reaching different positions in the parabolic flow profile, thus resulting in different retention times.8 The combination of FFF with IEF was introduced in 1989 by Chmelik9 who studied separation of two components of horse myoglobin using a channel with a trapezoidal cross section (250 × 12 × 1 mm). A similar channel was used by the same author for a successful separation of cytochrome c, ferritin from horse spleen, and myoglobin from skeletal muscles.10 The generation and properties of the pH gradient formed in these channels was studied using an indicator dye.11 Continuous separation of analytes performed by free-flow electrophoresis (FFE) was reviewed by Krivankova.12 A mixture of charged particles is continuously injected into the carrier stream flowing between two (macroscale) electrode plates. When an electric field is applied, the particles are deflected from the direction of flow according to their electrophoretic mobility or pI. Examples of separation and fractionation of proteins, enzymes, membranes, DNA, and cells were cited.12 For purposes of continuous sample pretreatment, a FFE device integrated on a silicon chip was introduced to fractionate a mixture of lysine, glutamine, and glutamic acid.13 In this work, the separation chamber had to be separated from the electrodes by a groove. The use of microchannels for ZE and IEF offers several advantages. A smaller channel enables the use of lower applied voltages, lower consumption of samples, shorter analysis times, negligible effects of Joule heating, and minimization of convective disturbances due to the low Reynolds numbers. On the other hand, the definition of conditions inside the microchannels is more (6) Cabrera, C. R.; Yager P. Electrophoresis 2001, 22, 355-362. (7) Giddings, J. C. J. Chem. Phys. 1968, 49, 81-85. (8) Thormann, W.; Firestone, M. A.; Dietz, M. L.; Cecconie, T.; Mosher, R. A. J. Chromatogr., A 1989, 461, 95-101. (9) Chmelı´k, J.; Deml, M., Jancˇa, J. Anal. Chem. 1989, 61, 912-914. (10) Chmelı´k, J.; Thormann, W. J. Chromatogr. 1992, 600, 305-311. (11) Chmelı´k, J. J. Chromatogr. 1991, 539, 111-121. (12) Krˇiva´nkova´, L.; Bocˇek, P. Electrophoresis 1998, 19, 1064-1074. (13) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 28582865.

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complicated and challenging. A description of the development of pH gradients inside microchannels as monitored by acid-base indicators can be found elsewhere.9,14 In this paper, two walls of the microfluidic channels were formed by gold or palladium electrodes. The electrodes were in direct contact with the solution, so that the acid and base generated as a result of water electrolysis; OH- at the cathode, and H+ at the anode, can be exploited to form the pH gradient.14 The partial pressures of oxygen and hydrogen gases also produced by electrolysis could be kept below the threshold of bubble formation by keeping the voltages low, so no venting was required. Both acid-base indicators and protein conjugated with fluorescent dyes with experimentally determined pI values were used to monitor the formation of the pH gradients in the presence of pressure-driven flow. The transverse IEF technique was utilized to concentrate proteins, such as bovine serum albumin (BSA), streptavidin, and soybean lectin. The positions at which the BSA conjugate was focused were predicted by a mathematical model.15 An example of fractionation of a binary mixture of proteins is described. Mathematical Model. As described previously,15 a finitedifference model has been written in-house that accounts for the electrochemical and mass transport phenomena that occur in the microchannel during the IEF experiments. The model also accounts for the effects of the nonuniform flow profile characteristic of pressure-driven flow; it predicts the concentration of each species in solution at fixed points between two electrodes. Variation with the height of the electrodes is neglected, except in the calculation of the fully developed velocity profile in the channel, which is solved for a full 2-D case and then averaged along the electrode height dimension to generate average velocities. Previously, the model considered only chemical species with, at most, four protonation states, each of which was tracked individually. To model IEF of proteins, however, the model was modified to accommodate the broad range of possible protonation states associated with proteins.16 Published data on the correlation between pH and the net charge for BSA17 were used to construct a look-up table that, given a pH value, produces the corresponding net charge and degree of protonation. The charge on the protein is, therefore, allowed to vary with the local pH, which facilitates accounting for the buffering capacity of the protein during the acid-base equilibrium step of the model. This look-up table is also used to calculate electrophoretic mobility and specific conductivity. The contribution of the protein to the total solution conductivity is calculated in the standard manner, as a function of the square of the charge multiplied by the protein concentration and mobility. EXPERIMENTAL SECTION Electrochemical Cell. Gold Electrodes. The design and fabrication of the microfluidic channels formed by two gold (14) Macounova´, K.; Cabrera, C. R.; Holl, M. R.; Yager, P. Anal. Chem. 2000, 72, 3745-3751. (15) Cabrera, C. R.; Finlayson, B.; Yager, P. Anal. Chem. 2001, 73, 658-666. (16) Mosher, R. A.; Dewey, D.; Thormann, W.; Saville, D. A.; Bier, M. Anal. Chem. 1989, 61, 362-366 (17) Mosher, R. A.; Gebauer, P.; Caslavska, J.; Thormann, W. Anal. Chem. 1992, 64, 2991-2997. (18) Holl, M. R.; Macounova´, K.; Cabrera, C. R.; Yager, P. Electrophoresis. Submitted.

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Figure 1. (A) Schematic representation of the gold-electrode microfluidic channel, (B) photograph of the palladium-electrode microfluidic channel.

electrodes have been described elsewhere.14 The microfluidic channels were 0.4 mm thick (z coordinate), 1.27 mm wide (y coordinate) and 40 mm long (x coordinate)(Figure 1). Palladium Electrodes. In a later design, the gold film electrodes were replaced by solid palladium electrodes (Figure 1B). Palladium plates (Alfa Aesar, a Johnson Matthy Co., Ward Hill, MA) of a thickness of 0.254 mm were cut with a razor blade. The fabrication of the palladium electrode device was very similar to that of the gold electrode devices. The Pd microchannel was 0.354 mm thick (z coordinate), 1.27 mm wide (y coordinate) and 40 mm long (x coordinate). An additional improvement in the design of the microfluidic channels was the inclusion of a third inlet and a third outlet. This change did not affect the geometry of the microchannel. Experimental Operation. The microfluidic channel was mounted in a rigid manifold to ensure its electrical connection to the electrodes and fluid communication via inlet and outlet ports.18 A constant potential was applied across the electrodes using a DC power supply (model 612C, Hewlett-Packard). Solutions were pumped through the device by computer-controlled syringe pumps (Kloehn Ltd., Las Vegas, NV). Visualization of the microfluidic channels was performed using an inverted optical microscope (IM 35, Carl Zeiss, Inc., Germany). A low-power objective (2.5/0.008) was used for all experiments. Images of the channel were captured using a 3-chip cooled CCD camera (ChromoCam 300, Oncor, Gaithersburg, MD) in combination with a video data acquisition card (CG-7 RGB frame grabber, Scion, Frederick, MD) and accompanying PC software (Scion Image). A mercury lamp (100W DC, Carl Zeiss, Inc., Germany) was employed in combination with different filter sets. A standard fluorescein filter set (ex. 450-490 nm, dichroic at 510 nm; em. 520-nm-long pass), rhodamine filter set (ex. 515-535; em. 590650), and dual filter set (ex. 460-490, 545-565; em. 520-540, 580-710 nm) were used for fluorescence measurements. Chemicals. Proteins. Bovine serum albumin (BSA), streptavidin (SA), neutravidin, and soybean lectin conjugated with different fluorescent dyes were used as examples of biological analytes. The BSA Bodipy FL conjugate, BSA Alexa Fluor 488

conjugate, BSA Alexa Fluor 594 conjugate, neutravidin Alexa Fluor 488 conjugate, streptavidin Bodipy FL conjugate, and streptavidin Alexa Fluor 488 conjugate were used as purchased (Molecular Probes, Inc., Eugene, OR). Isoelectric points of protein conjugates were determined experimentally via polyacrylamide gel IEF by comparison to an IEF standard mixture (pI range from 4.45 to 9.60; BioRad Laboratories, Inc., Hercules, CA). For these purposes, a miniature IEF cell (model 111) was purchased from BioRad. Standard procedures using a carrier ampholyte Bio-LYTE (pH range 3-10) was followed for gel preparation. A constant voltage of 450 V (Power Pac 1000, BioRad) was applied on two graphite electrodes. A single band with a pI of 4.6 was found for all dye-BSA conjugates. Lectin Alexa Fluor 488 showed multiple bands in a pI range from 6.8 to 7.2. The other pI values for streptavidin (multiple bands between 5 and 6) and neutravidin (pI ) 6.3) were taken from the literature.19 Buffers. A 2-(4-morpholino)-ethane sulfonic acid (MES; Fisher Biotech, Fair Lawn, NJ) was used as a buffer in most cases. The initial pH values of the buffers were adjusted from 3.56 to 7.44 using 40% H2SO4 or 0.05 M NaOH. The pH values were measured using a pH meter (Orion, model 290A). To get a shallower pH gradient, a mixture of amphoteric buffers was employed.20,21 Constituents of a multiple-buffer solution that was used in this work were as follows: Solution A: lactic acid (Alfa Aesar, Ward Hill, MA) and propionic acid (Sargent-Welch, VWR Scientific, Buffalo Grove, IL). Solution B: MES (Fisher Scientific, Fair Lawn, NJ), bis-tris (Fisher Scientific), ACES (Mallinckrodt Specialty Chemicals Co., Paris, KY). Solution C: TES (ICN Biomedicals Inc., Aurora, OH), tricine (Fisher Scientific), bicine (Avocado Research Chemicals, Karlsruhe, Germany), glycylglycine (Fisher Scientific). Solution D: asparagine (Fisher Scientific), L-histidine (Avocado Research Chemicals), taurine (Acros Organics, NY). BRIJ 35 solution (30% w/v, Sigma Diagnostic, St. Louis, MO) was used as a surfactant in a concentration of 0.008% v/v. RESULTS AND DISCUSSION The gold electrode devices were built and used first. The results (presented below) were not satisfactory, and therefore, gold-film electrodes were replaced by solid palladium foils. The main reason for the choice of Pd was its “nongassing” character, based on the ability of Pd to catalyze the recombination of O2 and H2 to H2O.22 This allowed us to apply higher voltages without bubble formation. The higher voltages led to more efficient focusing in terms of speed and band thickness. The two-inlet configuration was changed to the three-inlet configuration to avoid any direct contact of proteins with the electrode surfaces. (The proteins are introduced into the channel through the middle stream; the both streams close to the electrodes contain only buffer solution). Experiments confirmed that the addition of the third inlet did not affect pH gradient formation in the palladium electrode devices. (19) Mori, M.; Kodama, K.; Hu, W. Z.; Tanaka, S. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 3139-3150. (20) Nguyen, N. Y.; Chrambach, A. Anal. Biochem. 1976, 74, 145-153. (21) Prestidge, L. R.; Hearn, T. W. Anal. Biochem 1979, 97, 95-102. (22) Mosher, R. A.; Thormann, W.; Bier, M. J. Chromatogr. 1988, 436, 191204.

Figure 2. Transverse IEF of BSA conjugate in gold-electrode device. Fluorescent images of the results of IEF of BSA-Bodipy FL conjugate in 1 mM MES with different initial pH values: a, 3.56; b, 4.35; c, 4.86; d, 5.75; e, 6.25. The images were taken near the end of the microchannel after 4 min of applying a potential of 2.3 V. Experimental conditions: concentration of BSA-Bodipy FL conjugate, 3 µM; current density, in a range from 0.9 (for lower pH) to 1.3 µA/ mm2; flow rate, 160µm/s; fluorescein filter set was used; signal was integrated for 0.33 s.

Figure 3. Experimental and predicted positions of focused band of BSA conjugate for different initial pH values measured in gold and palladium electrode microchannels. Experimental conditions for the gold electrode microchannel (2) are described in the caption of Figure 2. The predicted values (]) were calculated with respect to experimental conditions for the gold electrode device. Experimental conditions for the palladium electrode microchannel are described in the caption of Figure 4. Data are shown for both (0) two-inlet configuration and (×) three-inlet configuration.

Gold Electrode Microchannels. Concentration. BSA. In previous experiments in nonflowing conditions, it was observed that the positions at which proteins focused in the microchannels were strongly influenced by the initial pH of the buffer solution.14 The fact that the position of focused bands of analytes could be adjusted by modifying the starting conditions is crucial for separation and concentration of proteins in continuous-flow systems. Therefore, IEF of BSA was examined under different initial pHs in flowing buffers. Conjugates of BSA with three different fluorescent dyes were used for IEF in the microchannels. No significant difference in the behavior of BSA during IEF in microchannels or in conventional gel IEF was observed for the various conjugates. Solutions of 3 µM BSA-Bodipy FL in 1 mM MES buffer were prepared,and the initial pH was adjusted to the various values ranging from 3.56 to 7.91. The microchannel was loaded using two inlets: the buffer solution was loaded from the anode side while the solution of BSA conjugate in buffer was loaded from the cathode side. At an average velocity of 160 µm/s, the mean residence time of solutes was 250 s. Consistent with previous results in nonflowing conditions, the position of the focused BSA conjugate stream moved closer to the anode with increasing initial pH (Figures 2 and 3). In Figure 2, the positions of observed bands of focused BSA Bodipy FL conjugate are shown. These images were recorded after a steady state was reached (3-4 min of voltage application) at a location close to the outlet. For initial pH values >6.0, BSA was not fully focused. It was previously observed in static experiments that the time needed Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

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for focusing of BSA was longer than 10 min when initial pH values were >6.0.14 The residence time of protein inside the microchannels was only 4 min. There are several possible explanations why not only the position but also the time needed for focusing of proteins depended on the initial pH. The ionic strength of the solution was raised with each addition of NaOH. Raising the ionic strength of the solutions lowered the field strength across the microchannel, which reduced the efficiency of the process in terms of broadening the band and decreasing the rate of its formation.3 The distance that the protein had to traverse from its initial position in the channel to the position where it was ultimately focused was larger for higher initial pH values, which could also have contributed to the time delay required for full focusing. To be able to adjust the initial pH without also adjusting the ionic strength of the solution, a multiple buffer mixture consisting of several amphoteric buffers can be used.21 Then, proper selection of each constituent can change the initial pH of the buffer solution without changing the total buffer concentration and, therefore, the ionic strength. Components of such a multiple-buffer solution that is convenient for formation of natural pH gradients has been reported.21 A buffer (initial pH 4.93) consisting of solution A (0.1 µM of each constituent), solution C (0.05 µM of each constituent), histidine (0.2 µM) and MES (0.2 µM) for an overall buffer concentration of 0.8 mM was used for IEF in the gold electrode microfluidic device. Using this buffer, BSA-Alexa Fluor 594 conjugate was focused into one tight band after an application of 2.3 V, resulting in a current density of 0.65 µA/mm2. Comparison of Model Predictions to Experimental Data for Concentration of BSA. The positions of focused bands of BSABodipy FL conjugate in gold microfluidic channels in 1 mM MES buffer of different initial pH values were compared to the locations that were predicted by the mathematical model (Figure 3). The good correlation between experimental results (2) and model predictions (]) suggested that the phenomena in the channel were accurately modeled. The model predictions of the final position of focused BSA bands were slightly closer to the cathode, and therefore, at a slightly higher pH, than the experimental values (Figure 3). The pI of BSA, according to the pH vs ζ-potential data used in the model, was 4.8, which was slightly higher than the pI value of 4.6 determined experimentally. This difference between the pI value found in the literature and the pI value determined experimentally explains the cathodic shift (Figure 3) of the predicted values in comparison to the experiment. BSA was the only protein modeled, because it was the only protein used in the experiments for which literature data on the relationship between pH and net charge was available.23 Determining this relationship solely on the basis of protein structure can be unreliable.23 Streptavidin, Neutravidin, Lectin. IEF in the gold microchannels was also tested with other protein conjugates, such as streptavidin-Bodipy FL (or -Alexa Fluor 594) conjugate, neutravidin-Bodipy FL conjugate, and lectin-Alexa Fluor 488 conjugate. It was confirmed that the proteins focused better if the initial pH values were close to the protein pI values.14 The dependence of the position of the focused protein bands on the (23) Mosher, R. A.; Gebauer, P.; Thormann, W. J. Chromatogr. 1993, 638, 155164

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initial pH was observed for streptavidin and lectin (results not shown, observed for multiple buffer solutions). Precipitation of neutravidin occurred during focusing at an initial pH of 5.41. Addition of Brij. For each protein tested, it was observed that the focused band was broader near the exit of the microchannel than it was in the middle. This band broadening may be an artifact associated with parasitic electroosmotic pumping in the microchannels. Addition of the surfactant Brij 35 reduced the protein band broadening. The effect of electroosmotic pumping will be a subject of future studies. Separation. Different mixtures of two of the above-mentioned proteins were prepared to examine whether the gold microfluidic channel could be used for fractionation. No separation was observed for a mixture of the BSA-Alexa Fluor 594 conjugate and the streptavidin-Bodipy FL conjugate, which have a net difference in pI of one pH unit. Minimal fractionation also was obtained using mixtures of BSA with neutravidin or lectin conjugates, which have a net difference in pI closer to two pH units. In all cases, when 1 mM MES was used as a buffer, both of the proteins were focused into one stream (a voltage of 2.3 V, resulting in a current density of 0.65 µA/mm2). When a multiple buffer mixture was used, it was possible to see two protein bands of different color, which indicated protein separation. Better results were obtained when the initial pH of a multiple buffer mixture was increased from 5.56 (mixture of solution A, solution C, MES, and histidine; each constituent concentration was 0.01 mM; total buffer concentration, 0.8 mM) to 6.65 (mixture of solution C, solution D, MES; total buffer concentration, 0.8 mM); however, the protein bands were too close to each other to be effectively separated. Two distinct streams of individual proteins were observed at the device outlet only when the polarity of the electrodes was switched after steady-state IEF conditions were reached. This experiment was performed using a mixture of BSA and neutravidin conjugates that was introduced to the channel together with the multiple buffer mixture. A potential of 2.3 V was applied, resulting in a current density of 0.77 µA/mm2. The polarity of the electrodes was switched after a stable appearance had been achieved, whereupon two distinct streams (with a nonfluorescent gap between them) were rapidly formed. The streams remained separate for about 30 s, then began to merge over the next 2 min. The same experiment was carried out with a mixture of the BSA and lectin conjugates, with a similar result. These polarity-switching experiments confirmed that two proteins could be separated under the proper conditions. Unfortunately, the high current density that was in part responsible for the efficient separation caused protein precipitation, especially in the case of neutravidin. The experiments inside the gold electrode microfluidic channels confirmed that higher voltages are, as expected, more efficient at focusing and separation of proteins. Unfortunately, at voltages >2.3 V, bubbles of gas were formed at the electrode surfaces; therefore, the flow cell was redesigned to use palladium foil, because Pd electrodes have been described in the literature as “nongassing”.22 Palladium Electrode Microchannels. Concentration of Proteins. The effect of switching to palladium electrodes was tested by repeating the experimental conditions previously tested in a

Figure 4. Transverse IEF of BSA conjugate in palladium electrode device. Fluorescent images of the results of IEF of BSA-Alexa Fluor 594 conjugate in 1 mM MES with different initial pH values in microchannels: a, pH 4.10; b, pH 4.98; c, pH 5.89; d, pH 6.88; e, pH 7.44. The images were taken near the end of the channel after a potential of 2.1 V was applied for 4 min. Experimental conditions: three-inlet configuration; concentration of BSA-Bodipy FL conjugate, 0.3 µM; current density was calculated to be between 1.0 (lower pH value) and 2.4 µA/mm2; flow rate, 324 µm/s; a rhodamine filter set was used; signal was integrated for 0.67 s.

gold electrode device: 0.3 µM BSA Alexa Fluor 594 conjugate in 1 mM MES buffer. The initial pH of the solution was adjusted from 4.10 to 7.44 (the buffer concentration and other conditions were kept the same as in the case of gold microfluidic channels). As can be seen in Figure 3, the final position of the focused BSA did not vary with the type of metal used for an electrode. The results of focusing of the BSA conjugate at an applied voltage of 2.1 V are shown in Figure 4. Current density was typically 5 times higher than with the gold electrodes. As predicted, and as seen in the gold electrode devices, the focused BSA bands were located closer to the anode for higher initial pH. In Figure 4, it is possible to see that the bands of focused BSA were narrower in comparison to their appearance in gold microchannels. BSA was also focused when the initial pH was 7.44, in contrast to the situation with gold electrodes in which no focusing was observed for initial pH values >6. The presence of a higher current density at lower applied voltages could explain this more rapid focusing with Pd electrodes. The addition of a third inlet was also tested during these experiments. As discussed, the addition of the third inlet enabled loading the protein solutions into the microchannel while not allowing contact of those proteins with the electrode surfaces. The three-inlet configuration prevented possible changes of protein structure due to electrochemical processes on the metal surface and possible fouling of the electrodes by the proteins. No changes in the positions of focused bands of BSA compared to two-inlet experiments were observed, so it was concluded that the development of pH gradients was not influenced by the addition of the third inlet. The positions of the BSA conjugate bands for two- (0) and three-inlet (×) configuration are compared in Figure 3, which shows that the IEF results are reproducible with an experimental error 2.3 V were avoided, even if the BRIJ solution was used. At flow rates >647.5 µm/s, the pH gradient was not fully developed, even at the device exit ports, and the separation process was not efficient. Several parameters, such as concentration and composition of buffers, flow rate, and applied voltage, were varied to obtained Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

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Figure 6. Transverse IEF of BSA conjugate and lectin conjugate in a palladium electrode device. Fluorescent images near the exit at various times after the application of 2.0 V. Brij was added as a surfactant. Experimental conditions: 0.3 µM BSA Alexa Fluor 594 conjugate and 0.3 µM lectin Alexa Fluor 488 conjugate in multiple buffers with initial pH of 5.48; three-inlet configuration in which flow rate of adjacent electrode streams was 0.084 µL/s; concentration of single buffer constituent, 0.2 mM. A flow rate of 0.042 µL/s was employed for the middle stream with a buffer constituent concentration of 1mM. A mean flow velocity was calculated to be 539 µm/s. An applied potential of 2.0 V resulted in a current density of 2.86 µA/mm2.

optimal conditions for separations. It was experimentally confirmed that the main problem was the steepness of the pH gradients. As previously discussed, higher applied voltage (and, therefore, higher current density) led to a more efficient focusing of proteins. However, higher current density corresponds to a higher rate of production of H+ and OH- and, therefore, a steeper pH gradient, which is, as stated, not ideal for fractionation of similar molecules. For fractionation, a main effort has to be devoted to the consumption of produced OH- and H+ to obtain the shallower pH gradient. A three-inlet configuration offers two possible solutions to these issues: (1) The buffer concentration in streams adjacent to electrodes is high so that excess H+ and OH- will be consumed by the buffers. The middle stream contains a lower concentration of buffer, enabling the formation of shallower pH gradients. (2) The buffer concentration in streams adjacent to the electrodes is lower than that in the middle stream. If the initial pH of buffer in the middle stream were between the pI values of proteins, successful separation could occur. The latter configuration proved to be more effective. The best separations of lectin and BSA conjugates were found when the mixture of proteins had a higher buffer concentration and slower flow rate than the two flanking electrode-adjacent streams. For the best results, the flow rate in the middle stream was one-half of that of both adjacent electrode streams, so the entering proteincontaining flow stream occupied the central approximately 20% of the fully developed flow stream. The parabolic nature of the flow profile in the channel results in a higher velocity at the center of the channel than along the side walls. Therefore, the center stream occupies a slightly smaller cross-sectional area than if the flow profile were blunt, because it is traveling slightly faster than the surrounding fluid. A multiple buffer mixture of initial pH 5.48 was employed; concentration of single constituents in the middle stream was 1 mM (overall buffer concentration was 8 mM) but 0.2 mM (overall buffer concentration 1.6 mM) in the electrode adjacent streams. The higher buffer concentration in the central stream prevented protein precipitation3 and helped to flatten the pH gradient. The mixture of BSA and lectin separated within the first minute of application of 2.2 V (Figure 6) and remained 1632 Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

separate for the next two minutes. After 3 minutes, the two protein bands began to coalesce. The minimum retention time of the proteins was approximately 1 min. As a protein stream remained in the center of the channel, it experienced the highest fluid velocities, due to the parabolic flow profile, and therefore, a minimum retention time is a more accurate description than mean residence time. As seen in Figure 6, the intensity of the green lectin fluorophore decreased dramatically as the protein progressed from the inlet to outlet and over time at the outlet. However, when the polarity of the electrodes was flipped, the intensity of the green fluorophore increased greatly. The experiment was repeated in nonflow conditions with the same result. This showed that there was no permanent bleaching of the lectin conjugate. The reduction in green fluorescent signal could be due to sensitivity of the fluorescent dye to changes in pH or O2 concentration; however, the presence of a second protein-dye conjugate also appears to contribute to the decreased signal, because when only lectin was focused, the intensity of the green signal was higher than in the BSA-lectin mixture. A bubble seen in Figure 6 was caused by partial delamination of the microfluidic device and did not affect the process of protein separation. CONCLUSIONS The experimental results showed that the use of gold for the electrodes in the microchannel had some disadvantages for continuous separation and concentration of proteins. The major problem was that the low maximum current (limit set to avoid bubble generation) did not allow concentration of proteins at higher initial pH values. This problem was removed by changing the electrode material to palladium, enabling the use of higher currents without bubble formation. However, higher currents often led to precipitation of proteins, so the surfactant BRIJ 35 was added to suppress protein precipitation. As expected, the higher current obtained in the palladium device led to improved concentration of single protein solutions and separation of binary mixture of proteins.

Simultaneously with the change of electrode material, the third outlet and inlet were added to avoid contact of protein with the electrode surface and, thus, prevent electrochemical reaction of proteins at the electrode surface; however, the IEF results were the same for both two- and three-inlet experiments under identical conditions, suggesting that protein reactions at the electrodes were not a concern in these experiments. The addition of a third inlet also allowed varying the buffer concentration and flow rate of the fluid proximal to the electrodes relative to the flow rate at the center of the channel. Varying these parameters significantly affected IEF results. It was shown that a shallower pH gradient was formed in multiple buffers as compared to single buffer solutions. The

optimal separation conditions were discussed, and an example of separation was shown for a mixture of BSA and lectin conjugates. ACKNOWLEDGMENT We thank Prof. Wolfgang Thormann for his helpful suggestions and comments. We also thank Andrew Evan Kamholz for valuable discussions. This work was supported by DARPA contract N660001-97-C 8632.

Received for review August 23, 2000. Accepted January 15, 2001. AC001013Y

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