Electric Field Gradient Focusing of Proteins Based on Shaped

Anal. Chem. 2004, 76, 5641-5648

Electric Field Gradient Focusing of Proteins Based on Shaped Ionically Conductive Acrylic Polymer Paul H. Humble, Ryan T. Kelly, Adam T. Woolley, H. Dennis Tolley,† and Milton L. Lee*

Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602

Electric field gradient focusing (EFGF) is a separation technique that uses an electric field gradient and an opposing hydrodynamic flow to separate and concentrate charged analytes. This work describes miniaturized EFGF devices that are used for protein analysis. These devices employ a unique ionically conductive polymer that enables the required electric field gradient to be established. This polymer has good protein compatibility and allows the transport of small buffer ions while retaining large analytes such as proteins. With the use of an EFGF device, green fluorescent protein was concentrated 10 000-fold and the separation of a protein mixture was demonstrated. The development of these ionically conductive polymer-based devices represents a step toward making EFGF a useful analytical tool for proteomics investigations.

There is great interest in developing analytical methods that can concentrate and separate proteins in complex biological mixtures. One of the motivations for this is that quantitative analysis of protein expression profiles is a useful diagnostic tool for diseases such as cancer.1,2 Currently, the most popular method for analyzing complex protein mixtures is two-dimensional (2-D) gel electrophoresis, where the first dimension corresponds to isoelectric focusing (IEF).3,4 With the use of 2-D gel electrophoresis, thousands of proteins can be separated. However, this technique is time consuming and labor intensive, requiring staining to visualize protein spots and excision of these spots for subsequent identification by mass spectrometry. Other limitations of 2-D gel electrophoresis include the inability to detect tracelevel proteins, difficulty in performing quantitative analysis, and the limited pH range of IEF. As a result of these limitations, researchers have pursued the development of alternatives to 2-D gel electrophoresis.5,6 This paper describes electric field gradient * Corresponding author. Phone: (801) 422-2135. Fax: (801) 422-0157. E-mail: [email protected]. † Department of Statistics, Brigham Young University, Provo, UT, 84602. (1) Wang, H. X.; Kachman, M. T.; Schwartz, D. R.; Cho, K. R.; Lubman, D. M. Electrophoresis 2002, 23, 3168-3181. (2) Alaiya, A. A.; Franzen, B.; Auer, G.; Linder, S. Electrophoresis 2000, 21, 1210-1217. (3) Klose, J. Electrophoresis 1999, 20, 643-652. (4) Gorg, A.; Obermaier, C.; Boguth, G.; Harder, A.; Scheibe, B.; Wildgruber, R.; Weiss, W. Electrophoresis 2000, 21, 1037-1053. (5) Wall, D. B.; Kachman, M. T.; Gong, S. Y.; Hinderer, R.; Parus, S.; Misek, D. E.; Hanash, S. M.; Lubman, D. M. Anal. Chem. 2000, 72, 1099-1111. 10.1021/ac040055+ CCC: $27.50 Published on Web 08/21/2004

© 2004 American Chemical Society

focusing (EFGF) devices that have been developed for the freezone concentration and separation of protein mixtures. EFGF,7-10 also called electromobility focusing,10-12 belongs to a family of equilibrium gradient separation techniques in which a gradient or combination of gradients causes each analyte species to seek a unique equilibrium position along a separation channel. A well-known equilibrium gradient method is IEF,13 which employs a pH gradient to focus analytes according to their isoelectric points. In contrast to the pH gradient used in IEF, EFGF utilizes a gradient in an electric field along the length of the separation channel. The electrophoretic force drives the charged analytes toward the low electric field end of the separation channel. This electrophoretic force is opposed by a constant bulk fluid flow in the channel. In EFGF, a charged analyte moves toward and focuses at an equilibrium position where its electrophoretic velocity matches that of the bulk fluid flow. Since analytes with different electrophoretic mobilities have unique equilibrium positions, EFGF separates analytes according to their electrophoretic mobilities, analogous to the way IEF separates analytes according to isoelectric point (pI). EFGF has several potential advantages over IEF for protein analysis. EFGF can be used with proteins that have inaccessible pI values, and it overcomes problems associated with the low solubilities of most proteins near their pI. In contrast to a pH gradient, the electric field can be manipulated readily during the course of a separation so that analytes can be moved or eluted from the separation channel in a controlled manner. This ability could be useful for coupling EFGF with other analytical techniques such as mass spectrometry. Also, the peak capacity of EFGF can be higher than that of IEF when a nonlinear gradient is used, and analytes are moved from one region containing a steep electric field gradient (EFG) to another region with a shallow gradient.10,11 The major challenge in implementing EFGF is the creation of a separation channel with the required EFG. EFGs have been (6) Jensen, P. K.; Pasa-Tolic, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076-2084. (7) Giddings, J. C.; Dahlgren, K. Sep. Sci. 1971, 6, 345-356. (8) Koegler, W. S.; Ivory, C. F. J. Chromatogr., A 1996, 726, 229-236. (9) Ivory, C. F. Sep. Sci. Technol. 2000, 35, 1777-1793. (10) Wang, Q. G.; Tolley, H. D.; LeFebre, D. A.; Lee, M. L. Anal. Bioanal. Chem. 2002, 373, 125-135. (11) Tolley, H. D.; Wang, Q. G.; LeFebre, D. A.; Lee, M. L. Anal. Chem. 2002, 74, 4456-4463. (12) Wang, Q. G.; Lin, S. L.; Warnick, K. F.; Tolley, H. D.; Lee, M. L. J. Chromatogr., A 2003, 985, 455-462. (13) Cao, C.-X. J. Chromatogr., A 1998, 813, 153-157.

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constructed based on changing cross sections,8,14-17 concentration gradients,10,12,18 electrode arrays,19 and temperature gradients.20 Koegler and Ivory demonstrated a preparative-scale EFGF system based on a changing cross section that used a packed dialysis tube (6.4-mm diameter) mounted in the center of a fluted cooling jacket.8,14 Buffer solution was circulated through the cooling jacket to remove the heat generated in this large device. This system was described as being cumbersome to set up and giving mediocre results.19 In subsequent work, Greenlee and Ivory used a dialysis membrane and a purge channel to create a conductivity gradient along the length of a separation channel (0.8-mm wide by 0.5mm deep).18 The conductivity gradient was created as buffer ions moved from the separation channel through a dialysis membrane and into a purge channel. Without a chromatographic packing in the separation channel, the proteins formed into contiguous bands similar to those in isotachophoresis. Distortion of the electric field due to the presence of concentrated protein was suggested as a possible cause of this phenomenon. Resolved protein bands were obtained after a packing was added to the separation channel. A major drawback of this system was that it was difficult to find combinations of flow rates and buffer concentrations that resulted in the desired EFG. Reasons for this include the exponential nature of the conductivity gradient formed in this system and the electric field influencing the migration of buffer ions, which altered the gradient formed.18 Ivory and co-workers have also used a computer-controlled array of electrodes to establish an EFG.19 In this system, a dialysis membrane was used to prevent analytes in the separation channel from directly contacting the electrodes, and a purge buffer was used to remove the electrolysis products produced at the electrodes. With this multiple-electrode design, it was possible to manipulate the field during the course of a separation in order to sharpen individual protein bands and change the positions of the bands. This method of producing an EFG has produced promising results for the preparative separation of simple protein mixtures. However, this design is fairly complex, and the resolving power may be limited by the number of electrodes used. Importantly, the EFGF instrumentation described thus far was designed for preparative separations with relatively large (∼millimeter-scale) separation channels, and chromatographic packing material was required to reduce band broadening caused by laminar flow. Work has also been directed toward the development of smaller, analytical-scale EFGF devices.10,12 A hollow dialysis fiber was used to construct an EFGF system based on a conductivity gradient. The hollow fiber was inserted coaxially inside a fusedsilica capillary, and syringe pumps were used to deliver highconcentration buffer to the inside of the dialysis fiber and lowconcentration buffer to the fused-silica capillary surrounding the fiber. With this setup, protein concentration and the separation (14) Koegler, W. S.; Ivory, C. F. Biotechnol. Prog. 1996, 12, 822-836. (15) Dennison, C.; Lindner, W. A.; Phillips, N. C. K. Anal. Biochem. 1982, 120, 12-18. (16) Van Welzen, H.; Zuidweg, M. H. J. Anal. Biochem. 1974, 59, 306-315. (17) Wachslicht, H.; Chrambach, A. Anal. Biochem. 1978, 84, 533-538. (18) Greenlee, R. D.; Ivory, C. F. Biotechnol. Prog. 1998, 14, 300-309. (19) Huang, Z.; Ivory, C. F. Anal. Chem. 1999, 71, 1628-1632. (20) Ross, D.; Locascio, L. E. Anal. Chem. 2002, 74, 2556-2564.

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of two proteins (myoglobin and bovine serum albumin) were demonstrated. However, this hollow fiber design did not work well with more complex protein mixtures. This analytical-scale device suffered from problems associated with the shape of the EFG, similar to the preparative-scale conductivity gradient device mentioned above,18 and was limited by the properties of the commercially available dialysis fibers. An alternative approach, termed temperature gradient focusing (TGF), was presented by Ross and Locascio20 who used a temperature gradient to create an EFG in a micromachined separation channel. Their device did not use membranes and thus overcame some inherent membrane-related challenges, such as the retention of small molecules. Indeed, this approach was especially suited to the concentration of small analytes. In order for TGF to work, the temperature dependence of the buffer conductivity must be different from the temperature dependence of the analyte mobility, a requirement that restricts what buffers can be used. Also, the gradient generated is constrained by the temperature limits of the buffer and analytes; thus TGF has the ability to separate and concentrate a few analytes at one time, but does not have the capability of simultaneously separating and concentrating analytes having a wide range of mobilities. If EFGF is to become a high-performance analytical tool for proteomics studies, separation channels with tailored EFGs are required to achieve the needed resolution and peak capacity. Separation channels with small cross sections are attractive in order to decrease dispersion and reduce the amount of sample required. Instrumentation that is simple to set up and operate is also desired, along with the ability to work with a wide range of buffered electrolytes. This paper reports on an EFGF design that provides a step toward achieving these characteristics. The EFGF separation channel is constructed inside an ionically conductive acrylic polymer that allows for the transport of small buffer ions but retains proteins and other large analytes. The polymer is shaped to produce the required EFG inside the separation channel. These EFGF devices are simple to use, requiring only a single syringe pump and voltage source for operation. This is a significant improvement over previous EFGF devices that required multiple pumps and, in the case of multiple electrodes,19 complex electronic circuitry. With the use of the devices described in this paper, several proteins were separated and concentrated to as much as 104 times. EXPERIMENTAL SECTION Instrumentation. Laser-induced fluorescence detection of focused proteins was accomplished in three different ways for the various experiments. In all cases, the 488-nm line from an aircooled Ar ion laser was passed into an inverted optical microscope (TE300, Nikon, Tokyo, Japan) through an excitation filter (D488/10, Chroma, Brattleboro, VT) to an objective.21 Fluorescence was collected through the same objective, passed through a 505 LD dichroic filter (Chroma) and an E515LPm long-pass filter (Chroma). Color micrographs were obtained by passing the excitation beam unexpanded into a 4×, 0.12 N. A. objective and imaging with a Nikon Coolpix 995 digital camera. For preconcentration experiments, the laser beam was expanded to ∼1.6 cm (21) Kelly, R. T.; Woolley, A. T. Anal. Chem. 2003, 75, 1941-1945.

Figure 1. Schematics showing (A) an exploded view of the components used to construct the ionically conductive polymer-based EFGF devices and (B) an assembled device; (C) shows a completed device with buffer reservoirs and tubing connected to the low-field capillary for syringe pump attachment; (D) shows a photograph of a finished EFGF device.

using a 10× beam expander (Newport, Irvine, CA) and passed through the same 4× objective, resulting in a focused beam spot with a diameter of ∼400 µm. Fluorescence data were collected as TIFF images using a cooled CCD camera (Coolsnap HQ, Roper Scientific, Tucson, AZ). To detect focused bands along the length of the entire channel, the separation channel was scanned through a fixed detection volume by connecting the microscope stage to a one-dimensional translation stage. The laser beam was expanded to 1.6 cm and passed through a 20×, 0.45 N. A. objective. After passing through the long-pass and dichroic filters, the signal was spatially filtered to remove out-of-focus fluorescence with a 200-µm-diameter pinhole. Photons passing through the pinhole were detected at a Hamamatsu HC 120-05 (Bridgewater, NJ) photomultiplier tube. The detector signal was amplified and filtered with a SR-560 preamplifier (Stanford Research Systems, Sunnyvale, CA) and then digitized with a PCI-6035E (National Instruments, Austin, TX) analog-to-digital converter controlled by LabVIEW (National Instruments) software running on a personal computer. The sampling rate for data collection was set in the software at 10 Hz. Materials and Sample Preparation. Recombinant, enhanced green fluorescent protein (EGFP) (Clontech, Palo Alto, CA) and R-phycoerythrin (R-PE) (Polysciences, Warrington, PA) were diluted in 20 mM, pH 8.7 Tris buffer (Sigma-Aldrich, Saint Louis, MO). All buffer solutions were prepared using purified water from a Barnstead EasyPure UV/UF system (Dubuque, IA) and passed through a 0.2-µm filter (Pall, East Hills, NY) prior to use. Lysozyme (Sigma) was dissolved in 20 mM, pH 7.4 Tris buffer to a concentration of 100 µM. A 10-fold molar excess of Oregon green 488-maleimide (Molecular Probes, Eugene, OR) in dimethyl sulfoxide was added to the lysozyme and allowed to react in the dark at room temperature for 2 h. To remove unconjugated label, 3 mL of Tris buffer, pH 8.7 was added to 100 µL of the fluorescently labeled lysozyme solution and placed in the upper chamber of a Microsep (Pall) centrifugal device with a molecular weight cutoff of 1000. The Microsep device was centrifuged at 4000 rpm at 4 °C for 2 h, which forced buffer and free label into

the lower chamber but retained the lysozyme in the upper chamber. After centrifugation, the lysozyme-containing solution in the upper chamber (approximately 100 µL) was collected. Hemoglobin (Sigma) was fluorescently labeled using a FluoroTag FITC Conjugation Kit (Sigma) according to the instructions of the manufacturer and isolated from excess label as described above for lysozyme preparation. All the monomers used to construct the ionically conductive polymer were obtained from Aldrich and used as received. The ionically conductive polymer was a UV-polymerized random copolymer hydrogel containing the following components: 27 wt % hydroxyethyl methacrylate (HEMA), 23 wt % methyl methacrylate (MMA), 19 wt % 100 mM Tris buffer, pH 8.7, 18 wt % poly(ethylene glycol) acrylate (PEGA), 12 wt % poly(ethylene glycol) diacrylate (PEGDA), and 1 wt % 2,2-dimethoxy-2-phenylacetophenone, which served as the photoinitiator. These components were mixed together to create a miscible, transparent prepolymer solution. Fused-silica capillary tubing (150-µm i.d.) was obtained from Polymicro Technologies (Phoenix, AZ). To suppress electroosmotic flow, the capillary was coated with poly(vinyl alcohol) using an established protocol.22 Device Fabrication. A 120-µm-diameter Nichrome wire was used to form the separation channel through the interior of the ionically conductive polymer. A cavity for casting the polymer slab was cut through a piece of 1.5-mm thick acrylic sheet (Acrylite OP-3, Cyro, Rockaway, NJ) using a CO2 laser cutter (C-200, Universal Laser Systems, Scottsdale, AZ). Channels for attaching sections of fused-silica capillary were also laser cut into this top piece of acrylic sheet extending from the high- and low-field sides of the cavity. The wire was threaded through two ∼5-cm long, 150-µm i.d. capillaries. The capillaries were placed in the channels with the Nichrome wire suspended through the center of the cavity used to form the polymer slab (Figure 1A). The top plate was then thermally bonded to a solid acrylic bottom plate by (22) Clarke, N. J.; Tomlinson, A. J.; Schomburg, G.; Naylor, S. Anal. Chem. 1997, 69, 2786-2792.


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clamping the two pieces together and placing them in an oven at 107 °C for 15 min. Next, the cavity in the top plate was filled with prepolymer solution and the device was placed under a UV lamp (Model 5000, Dymax, Torrington, CT) for 5 min to effect polymerization. The wire was then withdrawn from the device leaving a hollow separation channel between the two short capillaries and through the interior of the ionically conductive polymer (Figure 1B). The separation channels produced in this manner were 4-cm long and 120-µm in diameter. The short fused-silica capillaries on both ends of the separation channel facilitated the attachment of the device to pumps and buffer reservoirs. The low-field capillary was connected to a syringe pump (PHD 2000, Harvard Apparatus, Holliston, MA) (Figure 1C). The EFG was established using a Stanford Research Systems high-voltage power supply (Model PS 350) attached to platinum wire electrodes. The platinum wire that served as the anode was either placed in direct contact with the conductive polymer or placed in a buffer reservoir attached to the low-field end of the ionically conductive polymer slab (Figure 1C). The cathode was placed at the high-field end of the ionically conductive polymer slab or in a buffer reservoir attached to the capillary extending from the high-field end of the separation channel (Figure 1C). The potentials used ranged from 500 to 2000 V, and the resulting currents were generally between 10 and 60 µA. Safety Considerations. The monomers used to construct the ionically conductive polymer are irritants or toxic. Users should consult the appropriate MSDS for proper safety precautions. The high potentials used to create the EFG pose a shock hazard, so appropriate precautions, such as current-limiting settings on power supplies and isolation of electrical leads, should be taken. Sample Introduction. The proteins used in these EFGF experiments were either fluorescently labeled or natively fluorescent when excited at 488 nm. Analytes were introduced into the EFGF separation channel using electrokinetic injection or by including the analytes in the buffer being pumped through the separation channel. For electrokinetic injection, the cathode was placed in a buffer reservoir attached to the high-field capillary (Figure 1C). During injection, this buffer reservoir was replaced with a reservoir containing the analytes and +1 kV was applied for 5-30 s. After injection, the analyte reservoir was replaced with the buffer reservoir. Once proteins were observed entering the separation channel, the syringe pump was activated to create the hydrodynamic counterflow. Analytes could also be included in the buffer being pumped through the device. In this case, analytes would enter the EFGF channel at the low-field end of the device and move toward the high-field end until reaching their focused position. This technique was used for protein concentration experiments. A disadvantage of pumping analyte in from the syringe was that protein was continually being introduced into the EFGF channel, making it difficult to distinguish individual bands when multiple proteins were present. It is also possible to introduce analytes into the low-field capillary using a chromatographic injection valve, but this sample introduction technique was not used for any of the results presented here. RESULTS AND DISCUSSION Ionically Conductive Polymer. Crucial to the success of these EFGF devices was a polymeric material that could pass small buffer ions while preventing the passage of protein analytes. The 5644


photopolymerizable copolymer developed for this work adequately met these requirements. In this copolymer, HEMA and MMA acted as strengthening materials and increased the structural integrity of the polymer. PEGDA, with its two reactive double bonds, was used as a cross-linking agent. Both PEGDA and PEGA contain poly(ethylene glycol) chains that improved protein compatibility by decreasing protein adsorption. HEMA, PEGDA, and PEGA increased the miscibility of the aqueous buffered electrolyte with the prepolymer solution. The conductivity of the ionically conductive polymer after polymerization was approximately 100 times less than that of the 100 mM Tris buffer included in the polymer recipe. Device Description. Figure 1 shows schematics and a photograph that are representative of the EFGF devices used in this work. The ionically conductive polymer described above was cast into a thin slab with a changing cross section (wide at the low-field end and narrow at the high-field end) normal to the direction of current flow. The conductive polymer slab was 1.8mm thick, 1.8-mm wide at the low-field end, and 18-mm wide at the high-field end with a 120-µm diameter separation channel running through its center from the high- to low-field end. The shape of the conductive polymer slab was designed to create a linear EFG where the field strength increased by a factor of 10 across the length of the device. However, factors such as electrode placement, concentration gradients created by the electric field, and the high conductivity of the open separation channel relative to that of the conductive polymer also affect the shape of the EFG, such that the EFG in the separation channel of the actual devices was not exactly linear. Nevertheless, the shape of the conductive polymer slab did provide an EFG inside the separation channel that was sufficient for focusing proteins. Importantly, the shape of the polymer slab can be easily altered, enabling the production of a wide range of electric field profiles. Protein Stacking. The first experiments performed using these ionically conductive polymer-based EFGF devices involved focusing individual proteins. In these experiments, a small amount of protein was dissolved in buffered electrolyte (generally 20 mM Tris, pH 8.7) and the protein-buffer mixture (the protein concentration was in the nanomolar range) was introduced through the low-field capillary (Figure 1) using a syringe pump. The anode and cathode were placed respectively at the low- and high-field ends of the ionically conductive polymer slab, and a few drops of buffered electrolyte were placed at the points where the electrodes touched the polymer. When the high-voltage power supply was turned on, a protein band would start to form at the high-field end of the separation channel and would move toward the low-field end until reaching a stable focusing position. A stable protein band could be obtained in less than 1 min. Figure 2A shows EGFP focused into a narrow band ∼100 µm in length. In this photo, a backlight was used to illuminate the separation channel. The position of the focused protein could be moved by changing either the flow rate or the applied potential. The focused protein responded faster to changes in potential compared to changing the flow rate on the syringe pump, likely a consequence of the flexible tubing that connected the syringe pump to the EFGF device. Generally, this experimental procedure produced focused protein bands 100-200-µm wide; however, narrower bands were also observed (Figure 2B). Interestingly,

Figure 2. Fluorescence images of focused proteins. (A) EGFP. In addition to laser excitation, a halogen source illuminated the device from above to make the separation channel visible. (B) EGFP focused to a width of ∼70 µm. (C) Stacked EGFP and R-PE. (D, E) Resolved EGFP (right) and R-PE (left). Scale bars are 500 µm. The mottled appearance of the larger bands was a result of reflection and diffraction from the surfaces of the polymer slab. Additional description of all images in text.

these bands were narrower than those expected based on theory and an assumed linear EFG. The theory behind EFGF has been explained in detail by Koegler and Ivory,8,14 and Wang et al.10 The theory of EFGF can be derived starting with the flux equation

Ni ) -Di∇ci + (v + µi∇Φ)ci

(1)

where Ni, Di, ci, and µi are, respectively, the flux, diffusivity, concentration, and electrophoretic mobility of the ith species, v is convection, and Φ is the potential. From eq 1 with a linear EFG, theory predicts protein bands that are Gaussian in nature with a standard deviation (σ) given by eq 2.

σ)

x

DT |µib|

(2)

In eq 2, DT is the total dispersion caused by diffusion and other factors such as laminar flow, µi is the electrophoretic mobility of the analyte, and b is the slope of the EFG. Assuming laminar flow, as opposed to electroosmotic flow, in the open separation channel, DT can be calculated using the correlation for Taylor dispersion shown in eq 3.18

D ) Di +

u2d2 192Di

(3)

Here, Di is the diffusion coefficient of the analyte, u is the average flow velocity, and d is the diameter of the EFGF separation channel.

An electrophoretic mobility of 1.7 × 10-4 cm2/(V‚s) for EGFP in 20 mM Tris at pH 8.7 was measured in our laboratory using free-zone capillary electrophoresis in a 50-µm i.d., poly(vinyl alcohol)-coated capillary. A diffusion coefficient of 8.7 × 10-7 for EGFP has been measured by Terry et al.23 A flow rate of 0.05 µL/min and an applied potential of 800 V were used in the experiment shown in Figure 2A. The applied potential of 800 V corresponds to b ) 50 V/cm2 when a linear EFG across the 4-cmlong separation channel is assumed. With the use of these values, eq 2 predicts that the focused EGFP band should have a standard deviation of 0.75 mm corresponding to a bandwidth of approximately 3 mm. This theoretical value is more than 10 times larger than the actual experimental bandwidths. Even assuming there was no dispersion due to flow (in this case the dispersion coefficient is just the diffusivity), the experimental bandwidth was 4 times smaller than theory predicts. Bandwidth is related to the slope of the electric field (b) with steeper EFGs producing narrower bands. The narrower-thanexpected bandwidth was caused by the focused protein altering the local EFG. The presence of the concentrated protein band increased the local conductivity inside the separation channel, creating a local minimum in the electric field and a steep EFG at the concentrated protein-buffer interface. This phenomenon was somewhat surprising since the buffer ion concentration was in the millimolar range and the protein entering the separation channel had a concentration in the nanomolar range. The concentration of the focused band increased with time to the micromolar range, since protein was continually being introduced into the separation channel. With this buffer ion-to-protein ratio, (23) Terry, B. R.; Matthews, E. K.; Haseloff, J. Biochem. Biophys. Res. Commun. 1995, 217, 21-27.


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the conductivity inside the separation channel should have been relatively constant. However, in the EFGF experiments shown in parts A and B of Figure 2, the experimental setup created a situation in which buffer ions were depleted from the separation channel, as described below. Equation 1 applies not only to analytes but also to the cations and anions of the buffered electrolyte. In the presence of an electric field, cations are pulled toward the negative electrode and anions are pulled toward the positive electrode. However, the requirement of electroneutrality prevents anions and cations from moving independently of each other, which can create a situation in which the high-mobility ions essentially drag the less mobile counterions in the direction they are being pulled. This is the same type of phenomenon that creates diffusion potentials, and it is present to some degree in all electrophoretic processes. A discussion of transport phenomena in electrolytes and the interplay between electric fields and concentration gradients can be found elsewhere.24 In the case of Tris buffer, the chloride ion has the higher mobility and diffusivity.14 In this situation, buffer ions (both anions and cations) concentrate toward the anode which is at the low-field end of the EFGF device. The buffer ion concentrations inside the separation channel and in the ionically conductive polymer are thus time-dependent and also a function of the electric field, starting concentration, and size of the buffer reservoirs. In the experimental setup used in obtaining parts A and B of Figure 2, the buffer reservoirs consisted of a few drops of buffered electrolyte (∼50 µL) placed on top of the ionically conductive polymer. Due to the small size of the anodic buffer reservoir, buffer ions were quickly depleted from the high-field side of the polymer slab and separation channel when voltage was applied to the device. This depletion of buffer ions created the situation in which the focused protein induced a large local EFG, resulting in the narrow peak widths shown in parts A and B of Figure 2. With the use of this same experimental set up, the focusing of protein mixtures was also attempted. Figure 2C shows the focused protein band that resulted when a mixture of EGFP and R-PE were introduced into the EFGF device. These proteins focused into a continuous band with the more mobile R-PE on the low-field (left) side of EGFP. When the applied potential or flow rate was changed the two proteins would move as a single band, and the proteins could not be separated. This same phenomenon was observed by Greenlee and Ivory in their conductivity gradient EFGF device without chromatographic packing.18 As described above, a local minimum in the electric field and a steep EFG can be produced when the presence of a concentrated protein band decreases the local electric field. If large enough, this local minimum can trap other proteins, producing stacked protein bands. This protein stacking is similar to isotachophoresis in that proteins form into contiguous bands according to their mobilities. This stacking phenomenon makes it difficult to distinguish individual analytes and potentially limits the usefulness of EFGF as an analytical tool. However, protein stacking may be useful for concentrating proteins into narrow bands prior to performing capillary electrophoresis, chromatography, or other analytical separations. (24) Newman, J. S. In Electrochemical Systems, 2nd ed.; Prentice Hall: Englewood Cliffs, NJ, 1991; pp 241-264.

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Protein Separation. Protein stacking was eliminated by placing a large buffer reservoir at the high-field end of the EFGF channel. Generally, this was accomplished by placing the platinum wire cathode in a 0.25-mL buffer reservoir attached to the fusedsilica capillary extending from the high-field end of the EFGF device. Alternatively, placing a (0.25-mL) buffer reservoir in contact with the conductive polymer slab at the high-field end of the separation channel also eliminated protein stacking. The larger cathode buffer reservoir acted as a source of buffer ions and prevented the buffer ion concentration in the separation channel from becoming too low under normal operating conditions. However, buffer ion depletion and protein stacking was observed in some instances when the EFGF device was operated for very long periods of time (>5 h) without changing the buffer in the cathode reservoir. Changes in buffer pH were also observed at the anode of the EFGF device after long periods of operation. This change in pH was eliminated by placing a 0.25-mL buffer reservoir on top of the conductive polymer slab at the low-field end of the separation channel during the polymerization step of device fabrication. Figure 2D shows two focused protein bands that were observed when a mixture of R-PE and EGFP were introduced into the EFGF separation channel using electrokinetic injection. These bands were approximately 0.5-mm wide, considerably wider than the stacked protein bands described previously, and almost completely resolved. In this experiment, the cathode was placed in a buffer reservoir attached to the capillary extending from the high-field end of the device. The buffered electrolyte consisted of 5 mM Tris, pH 8.7, the applied potential was +2 kV, and the flow rate was 0.03 µL/min. Due to the drop in potential through the fused-silica capillary, the potential at the high-field end of the separation channel was not known precisely. For an estimated EFG of 60 V/cm2 inside the separation channel, a standard deviation of 0.42 mm corresponding to a bandwidth of 1.7 mm is calculated from eqs 2 and 3. The calculated bandwidth exceeds the observed one by a factor of ∼2 which can be rationalized by two possible explanations. The first possibility involves a steeper EFG inside the separation channel than that estimated for the calculations. An EFG greater than 200 V/cm2 would be needed for bands narrower than 1 mm. Such a high EFG could not be present if a linear EFG is assumed but would be feasible with a nonlinear EFG. The second explanation involves the presence of electroosmotic flow. Compared to pressure-driven flow, electroosmotic flow is less dispersive due to its plug-flow nature. If the flow inside the separation channel was somewhat electroosmotic in nature, narrower bands would result. It is also possible that a combination of electroosmotic flow and a nonlinear EFG are responsible for the narrower-than-expected bandwidths shown in Figure 2D. Protein separation experiments using higher buffer concentrations produced wider bands. For instance, Figure 2E shows EGFP and R-PE bands that were focused using 20 mM Tris, 1 kV, and a flow rate of 0.02 µL/min. These bands were approximately 1.5mm wide and agree well with the theoretical bandwidth calculation. More complex protein mixtures were also separated. Figure 3 shows the six peaks that were observed when a mixture containing Oregon green lysozyme, FITC hemoglobin, R-PE, and

Figure 3. EFGF of a four-protein mixture. A moving stage fluorescence detection system was used, with a travel speed of 25 mm/ min. Much of the noise in this electropherogram was due to mechanical scanning.

Figure 4. Calibration curve used to determine the degree of preconcentration of EGFP from an 18 pM solution. CCD image from concentrated EGFP (inset) indicated a focused concentration of 180 nM.

EGFP was introduced into the EFGF separation channel. These separation experiments were very repeatable, and similar electropherograms to that shown in Figure 3 have been obtained in multiple experiments using ∼10 different devices. The identity of each peak was determined by comparing the mixture separation to experiments in which single proteins and mixtures of two or three proteins were focused in the EFGF channel. EGFP and lysozyme each resolved into two peaks; multiple peaks have been reported for both of these proteins in capillary electrophoresis separations.25, 26 The protein peaks shown in Figure 3 are roughly 5-mm wide, and the peaks corresponding to hemoglobin, R-PE, and EGFP are not completely resolved. These peaks are somewhat wider than those observed in previous experiments (e.g., Figure 2); however, they are symmetrical and the widths seem reasonable when compared to theory. The resolution of these proteins could be improved by using a shallower EFG to move adjoining peaks away from each other. According to theory this would produce broader peaks but higher resolution.11 Decreasing the peak width without altering the EFG would also improve resolution. This could be accomplished by creating a narrower separation channel or by including a packing or monolith in the separation channel to reduce band dispersion. In the context of protein analysis, both good resolution and high peak capacity are desired. Tolley et al. showed how the peak capacity and resolution of EFGF can be improved when a nonlinear EFG is used along with the ability to move analytes from a region containing a steep EFG to a region with a shallow gradient.11 One of the promising features of the EFGF devices described in this paper is that the EFG can be easily tailored by altering the shape of the ionically conductive polymer slab. Sample Concentration. An important attribute of EFGF that it shares with other focusing techniques such as IEF is the ability to concentrate analytes. To determine the degree of concentration that could be achieved using the EFGF devices described in this paper, a calibration curve was created using EGFP samples of known concentration between 37 nM and 1.5 µM. Each sample was pumped through the EFGF device at a flow rate of 100 nL/ min, and fluorescence images were obtained at three positions

along the channel using the cooled CCD camera with an integration time of 50 ms. The intensity values of the 20 brightest pixels in the images were averaged to provide a data point on the calibration curve. The signal intensity increased linearly between 150 nM and 1.5 µM, which was the concentration range used in constructing the calibration curve (Figure 4). Preconcentration experiments were performed by operating the device as described above, with an applied potential at +2 kV and a counterflow rate of 30 nL/min. Electrokinetic injection was not used for these experiments; instead, the syringe pump that provided the EFGF counterflow was filled with 18 pM EGFP, which was continuously pumped into the channel. The highest observed signal gains were obtained after the EGFP continuously focused into a narrow band for 40 min, resulting in an EGFP concentration of 180 nM and an enrichment factor of 104. The enrichment factor reported here is approximately equivalent to the concentration performance published by Ross and Locascio20 using TGF, which reportedly surpassed any other sample concentration methods. Taken together, these two demonstrations show the tremendous power of analytical equilibrium gradient methods for sample enrichment. Enrichment factors beyond those reported here should be readily attainable with this design, simply by allowing dilute protein to focus at its equilibrium position for longer periods of time. Because the rate of sample concentration is determined by the counterflow, which introduces the dilute protein into the focusing channel, similar levels of concentration could be achieved more quickly by increasing the counterflow rate and correspondingly increasing the applied voltage. When multiple proteins are simultaneously separated and concentrated, care should be taken to ensure that the local conductivity in the channel is not altered by the focused protein bands, as analyte stacking could occur (see above).

(25) Radko, S. P.; Stastna, M.; Buzas, Z.; Kingsley, D.; Chrambach, A. Anal. Biochem. 1999, 274, 146-148. (26) Gilges, M.; Kleemiss, M. H.; Schomburg, G. Anal. Chem. 1994, 66, 20382046.

CONCLUSIONS This paper describes miniaturized EFGF devices based on an ionically conductive polymer. The polymer was cast into a thin slab, shaped to produce a suitable EFG, with an open separation channel running through the interior of the slab. These devices were simple to operate and produced promising results for the separation and concentration of protein mixtures. This work represents a step toward making EFGF a useful tool for protein Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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analysis. However, improvements in peak capacity and resolution are needed. Future work with these EFGF devices will include optimizing the shape of the electric field and decreasing dispersion. Also, techniques for eluting protein peaks past a point detection system need to be developed in order to realize the performance improvements that will come with using a bilinear electric field gradient.11 With these advancements and improvements, EFGF should become a powerful tool for proteomics research.

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ACKNOWLEDGMENT The authors gratefully acknowledge the National Institutes of Health (Grant No. 1 RO1 GM0645547-01A1) for funding.

Received for review March 29, 2004. Accepted July 5, 2004. AC040055+

Electric Field Gradient Focusing of Proteins Based on Shaped

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