Conic Electrophoretic Concentrator for Charged ... - ACS Publications

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Conic Electrophoretic Concentrator for Charged Macromolecules Victor N. Morozov,*,†,‡ Yuri M. Shlyapnikov,† Jessica Kidd,‡ Tamara Y. Morozova,† and Elena A. Shlyapnikova† † ‡

Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino, Moscow Region, Russia 142290 The National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia 20110, United States

bS Supporting Information ABSTRACT: A simple, rapid, and highly effective technique for concentrating charged macromolecules is described which employs electrophoresis in a conic cell made of a dialysis membrane. The cell is partly submerged in electrolyte solution, and the level of solution slowly moves down during the process. The electric field within the cell is at its maximum in the area that is level with the surface of the external solution. This maximum value increases and its location moves downward following the decreasing level of external solution carrying downward and concentrating charged macromolecules. It has been demonstrated that proteins can be concentrated within 12 15 min by a factor of ∼100 000 with the total yield of 60 80%. Concentrated proteins can be harvested from the nanoliter-sized cul-de-sac of the conic concentrator using chemically activated magnetic beads. The presence of certain protein molecules linked to the bead’s surface can be further revealed by specific reaction with a microarray of antibody molecules. Such “reversed magnetic array” format was applied to a cone-concentrated exhaled breath condensate (EBC) to reveal the presence of human immunoglobulin in the EBC and to estimate its concentration. The technique may be used for concentrating and detecting trace amounts of pathogens and toxins, in protein crystallization, and in many other applications.

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reconcentration is a common procedure in analysis of trace amounts of analytes in highly diluted samples like exhaled breath condensate (EBC)1 and drinking water.2 Several techniques have been suggested for concentrating diluted protein and DNA samples. These include ultrafiltration techniques used in commercial devices from Millipore and other companies,3 water removal by drying in a dialysis bag,4 or by adding dry Sephadex or other dry hydrophilic gels to a protein solution.5 7 Removal of water with the latter technique is simple to carry out but suffers from protein loss due to its adsorption onto a large surface of the gel particles. Nor can this technique provide a high level of concentration—the concentration factor rarely exceeds 1 order of magnitude. Electrophoresis-based devices reach much higher levels of concentrating for charged macromolecular analytes. Chin et al. described the first simple design of an electrophoretic concentrator for proteins which was composed of a glass tube with one end plugged with a dialysis membrane.8 Later, a few designs have been reported based on the same idea.9 12 Development of the microchannel technologies made the idea of electrophoretic preconcentration especially attractive as a means of increasing assay sensitivity. Several microelectrophoretic devices using ultrafiltration membranes or plugs made of semipermeable gels have been described.13 16 These devices exploit highly effective heat dissipation in microchannels and, therefore, cannot be used with samples larger than a few microliters. Rhodes and Yphantis suggested an electrophoretic technique which allowed an increase in the level of concentration and yield r 2011 American Chemical Society

in the plug-based electrophoretic device. This technique introduced a funnel-shaped receiving cavity in a concentrated charged polyacrylamide gel used as a plug.17 The small volume of the receiving cavity allowed an easy collection of the concentrated probe, higher concentration factor, and protein recovery. Though this design has certain advantages over the flat gel or membrane plug, it suffers from several limitations. First, special highly concentrating gels with low permeability to protein molecules should be prepared. Second, the electric field cannot be made high due to slow heat dissipation from the tube and the gel plug having the same inner diameter (i.d.) of 5 mm. To avoid overheating, concentration proceeded at a low electric field (10 15 V/cm) and took several hours to complete. Here we describe an electrophoretic method which allows extreme concentrating of macromolecular ions without overheating under conditions when the maximum of the electric field moves and substantially increases at the late stages of the concentrating process. This is achieved by using a conic concentration chamber made of a thin dialysis membrane and by changing the area of electrical contact between the concentrator and the external buffer solution. The method is intended for use with highly diluted biological samples like EBC which cannot be analyzed without a substantial preconcentration. We also suggest a unique Received: February 19, 2011 Accepted: June 1, 2011 Published: June 01, 2011 5548

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technique to harvest concentrated proteins from ultrasmall volumes of liquid by using chemically activated magnetic beads capable of making covalent bonds with protein molecules. Protein analytes bound to such beads can be further detected by their interaction with arrayed specific probe molecules like antibodies.

’ MATERIALS AND METHODS Chemical and Materials. The following reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO): bovine serum albumin (BSA), FITC-labeled BSA (FITC BSA), avidin, human IgG (h-IgG), affinity-purified biotinilated antihuman IgG from goat serum (anti(h-IgG)), cytochrome c (Cyt c), horse hemoglobin (Hb), poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), sodium chloride, Tween-20, imidazole (Imid), glycine (Gly), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), NaBH4, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), 1-methylimidazole (NMI), N-(3aminopropyl)butane-1,4-diamine (spermidine), ethanolamine, [(cholamidopropyl)dimethylammonio]-propanesulfonic acid (CHAPS), pyridine (Py), bromophenol blue, boric acid, sodium dodecyl sulfate (SDS), and phosphate-buffered saline (PBS) buffer tablets. Dialysis membranes from regenerated cellulose were obtained from Sigma-Aldrich (MWCO of 12 14 kDA) and from Fisher Scientific (Pittsburgh, PA; Fisherbrand, MWCO of 12 14 kDA). Superparamagnetic carboxylated particles, Dynal MyOne, were acquired from Invitrogen (Carlsbad, CA, U.S.A.). Buffers Used in Electroconcentration. The following buffers have been used in concentration experiments. The first buffer (20 mM imidazole, 10 mM Gly, pH 8.5), further referred to as “Imid Gly”, has been introduced in our recent paper as a lowconducting buffer.18 It has been used in most experiments except for experiments with NHS-activated carboxylated magnetic beads. In the latter cases we used another low-conductance buffer system consisting of 33 mM Py and 3 mM H3BO3, pH 8.0 (“Py borate” buffer). These buffer components do not react with the NHSactivated carboxylated magnetic beads, unlike the buffer components of the Imid Gly buffer. For concentrating Hb having an isoionic point close to the pH of the buffers described above, 20 mM MES buffer with pH 5.0 was used instead. Fluorescence Measurements. Picofluor hand-held fluorometer from Turner BioSystems, Inc. (Sunnyvale, CA) was used in measurements of fluorescence intensity. To allow measurements in small volumes (100 120 μL), the standard 3 mL plastic cuvette that was supplied with the device was replaced with a UV vis plastic microcuvette (100 μL) (brand GMBH + CO KG Laboratory Equipment Manufacturers, Wertheim, Germany). Design of the Concentrating Device. The concentrating device consisted of three main parts: an upper electrode chamber, a lower electrode chamber, and a conic concentration cell. The latter was assembled as shown schematically in Figure 1, parts A and B. First, a piece of a wet dialysis membrane (approximately 30  30 mm2) was bent and its edges were clamped with two plastic screws between two plastic jaws of a holder as illustrated in Figure 1C. To prevent a capillary flow, a small piece of vacuum grease was placed on top of the conic concentrator where its edges were squeezed with jaws (see Figure 1B). Then 1.5 2 mL of sample was placed into the conic cell, and the latter was dipped into the lower electrode chamber so that 3 5 mm of the top edge of the dialysis film was risen over the buffer surface. The upper electrode chamber was made of a plastic tube by sealing its end

Figure 1. Design of the cone concentrating device. (A and B) A piece of a wet dialysis membrane prepared for clamping in the holder. (C) Schematic of a conic cell made of a dialysis membrane by clamping the membrane between two plastic jaws. (D) Schematic of assembling the electrophoretic system. (E) Electrophoretic concentrating in the conic dialysis membrane with a variable level of buffer in the lower chamber.

with a dialysis membrane fixed with a rubber O-ring. A platinum wire was introduced into the upper electrode chamber after filling it with 20 mL of a buffer solution as illustrated in Figure 1, parts D and E. The upper electrode chamber was placed over the conic concentration cell so that it touched the surface of the sample solution within the conic concentrator (see Figure 1E). The lower electrode chamber was equipped with a socket in its lower part for pumping out the buffer solution and a platinum wire connected to a power supply (see Figure 1, parts D and E). This chamber was filled with approximately 120 mL of buffer solution cooled to 4 °C. Concentrating Procedure. Immediately after applying constant electrical potential (200 V for Py borate buffer and 300 V for Imid Gly) to the platinum electrodes in the lower and upper electrode chambers the peristaltic pump was switched on. The voltage was chosen so that the electric power did not exceed 0.6 W. In the case of concentrating proteins having negative charges (such as FITC BSA at pH 8.5), the positive potential was applied to the lower electrode chamber. Positively charged proteins, like Cyt c, were concentrated with negative potential applied to the lower electrode chamber. The rate of pumping was chosen so that the level of the buffer in the lower electrode chamber reached the bottom of the conic cell in 10 15 min after start. Current was measured with a milliammeter and changed typically from 2 mA at the beginning to 0.4 0.5 mA at the end of the concentrating procedure. After the level in the lower electrode chamber dropped 1 2 mm below the apex of the cone, the voltage was switched off and the concentrated sample was collected from the conic tip of the cell in a series of 0.5 μL probes taken with a 1 μL Hamilton microsyringe. To avoid damaging the dialysis membrane and to reduce the diameter of the needle tip, a 20 mm long plastic tube with the i.d. equal to the outer diameter (o.d.) of the stainless steel needle of the microsyringe was slipped over the needle. To reduce the o.d. of the plastic tube to 0.2 0.3 mm, its free end of the plastic tube was heated with a small heat gun, extended, and cut 5549

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Analytical Chemistry with a razor. Before collecting samples, half of the syringe, the entire stainless steel needle, and most of the plastic tube were filled with water leaving only 2 3 mm of the plastic tip empty. The tip was then dipped into the cone apex, and 0.5 μL of concentrated sample was collected. Such a procedure avoided protein loss in contact with the large surface of the syringe and reduced variability in sample volume due to air compression and temperature changes. Sometimes 1 μM of bromophenol blue was added into the cone buffer to visualize the process of concentrating. Conductivity Measurements. Distribution of electrical conductivity in the cone was measured using a homemade microelectrode. It was made from a platinum wire, 54 μm in diameter, which was melted into a glass capillary so that only a 70 μm long end was exposed to solution, providing an area of 0.012 mm2. The latter was electrochemically coated with platinum black in the Kohlrausch’s solution. Another platinized electrode with a surface area of 4 mm2 was introduced into the buffer filling the cone. Electrical conductivity was measured with the Oakton pH/CON 510 benchtop meter (Vernon Hills, U.S.A.). Standard KCl solutions were used for the cell calibration. In measurements of the conductivity distributions the microelectrode was attached to a manual micromanipulator which allowed precise height positioning of the measuring microelectrode with visual control under a low-power stereomicroscope. Considering that the surface area of the probing microelectrode was by 2 orders of magnitude smaller than that of the macroelectrode, it was the solution conductivity in the vicinity of the microelectrode that determined the measured solution conductivity so that the measured conductivity showed no dependence on the position of the macroelectrode. Distribution of the Electric Field within the Cone. The same Pt microelectrode attached to a micromanipulator was used to measure potential distribution inside the cone during electrophoresis. The second electrode was placed into the lower electrode buffer. Voltage difference between the two electrodes was measured with a tube voltmeter with a high internal resistance to avoid errors due to polarization of electrodes. Distribution of pH within the Cone. The micro pH electrode was manufactured from iridium wire, 270 μm in diameter, melted into a glass capillary so that a 1.3 mm long free end protruded from the insulating glass. The wire was oxidized in a flame after dipping into a solution containing 1 M NaOH and 1 M NaNO3 according to ref 19. The electrode was calibrated in a series of buffer solutions with a Ag/AgCl reference electrode and displayed a nearly Nernstean slope of 59 ( 6 mV/pH. The profile of the pH distribution was measured in the cone as described above for the conductivity measurements. Concentrating of FITC BSA, Cyt c, Hb, and EBC. FITC BSA stock solution was prepared by dissolution of the commercial dry protein in water at the concentration of 1 mg/mL followed by exhaust dialysis against water to remove free FITC. The stock solution was then diluted 1000-fold with the Imid Gly buffer, and 1.5 mL of the diluted solution was placed into the cone concentrator. We have demonstrated recently that this buffer provides the best conditions for electrophoresis at slightly basic pH.18 Concentration of Cyt c was carried out using Py borate buffer in the cones made both from original and from modified (positively charged) dialysis membranes. Hb was concentrated in 20 mM MES buffer, pH 5.0, in the cone prepared from positively charged membrane at 200 V. EBC was first diluted with the Py borate buffer, and 1.5 mL of the diluted EBC was placed into the cone made from original membrane. The process of concentrating took 12 15 min for all the proteins and EBC.

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Concentrating of the Protein SDS Complex. A solution containing 0.1 mg/mL of dialyzed FITC BSA, 1% SDS, and 1 5% PVP in Imid Gly buffer was boiled for 3 min. Then 10 μL of the boiled mix was added to 1 mL of Imid Gly buffer and placed into the apparatus to concentrate for 15 min at 300 V. Immediately after switching the voltage off, 0.5 μL aliquots were taken from the cone apex as described above and added into 100 μL of 5 mM TAPS buffer (pH 8.5) placed into the fluorometer optical cell for fluorescence measurement. After that the cell was rinsed and refilled with a fresh 100 μL aliquot of the TAPS buffer, and then 10 μL of the stock FITC BSA (0.1 mg/mL) was added. Such control measurements were repeated three times, and the control readings were averaged. Efficiency of electrophoretic collection was calculated as a ratio of fluorescence intensity collected in the probes to the averaged control reading. Dependence of fluorescence intensity on the amounts of native and boiled FITC BSA was measured in a wide range of the protein concentrations. The calibration showed linear dependence of the fluorescence changes upon FITC BSA concentration in the range of 0 60 μg/mL. Concentration under Denaturing Conditions. FITC BSA (1 μg) was added to 1 mL of a denaturing buffer containing 8 M urea and 2% CHAPS in the Imid Gly buffer with pH adjusted to 8.6. Addition of urea changed the solution conductivity from 68 to 56 μS/cm. One milliliter of such denatured FITC BSA solution was placed into the cone. Both electrode systems were filled with Imid Gly buffer without CHAPS and urea. After concentration, 0.5 μL aliquots were taken as described above and added to 100 μL of the denaturing buffer that had been placed into the fluorometer cell for measuring fluorescence. A special calibration was performed in the denaturing solution. It showed that the fluorescence signal from the denatured FITC BSA tripled as compared to the native protein. Efficiency of collection was estimated as described above by comparing fluorescence intensity of the collected protein to that of a control containing the protein amounts equal to all the protein placed into the concentrator. Preparation of the Positively Charged Dialysis Membrane. A piece of dialysis membrane (approximately 30  30 mm2) was placed in 3 mL of 100 mM NMI buffer, pH 7.0, containing 50 mM EDC and 50 mM spermidine. The reaction was allowed to proceed at room temperature for 12 h. The membrane was then thoroughly washed with distilled water. Concentration of amino groups introduced into the dialysis membrane was estimated by titration.12 Wet dialysis membrane was weighted and then placed into 0.1 M HCl solution for 2 h at room temperature. The membrane was then thoroughly washed with a large volume of double-distilled water and then placed into 3 mL of 1 M KCl with a glass pH electrode inserted. Change in pH resulting from addition of the dialysis membrane was back-titrated with calibrated 10 mM NaOH solution. Using such titration procedure it was found that ∼5 mM of free amino groups were introduced into the dialysis membrane. Fabrication of the Anti(h-IgG) Microarray. First, an avidin microarray was manufactured by electrospray deposition on a plasma-treated dialysis membrane as described in the ref 20. Schiff’ bonds between carbonyls on the plasma-treated surface and protein amino groups were reduced in a solution containing 1% NaBH4, 1% PVP, and 1% PVA, and the array was washed with water and placed for 1 h into the blocking solution (20 mM Tris/HCl, pH 7.5, with 0.15 M NaCl, 1% BSA, 0.05% Tween-20, and 0.02% NaN3). Thus prepared avidin microarray was then reacted with biotinilated anti(h-IgG) (commercial stock solution diluted 1000-fold with 5550

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Analytical Chemistry the blocking buffer) for 1 h. The array was then washed and kept in the same buffer at 4 °C before use. Using Activated Magnetic Beads to Collect Proteins Concentrated in the Cone. prepared solution containing 50 mM EDC, 250 mM NHS, and 50 mM MES, pH 6.0. After activation for 30 min at room temperature with a constant stirring the beads were washed twice with cold 50 mM MES buffer, pH 6.0, and sonicated briefly in an ultrasound bath to disaggregate particles.20 Binding capacity of the activated MyOne particles was determined by adding 108 activated beads to 0.1 mL of Py borate buffer containing 1.25 μg of FITC BSA and 50 mM NaCl. By measuring changes in the fluorescence of free FITC BSA remaining after removal of the magnetic beads, we found that 108 activated beads bound 0.6 μg of FITC BSA. EBC was collected in 10 min from a volunteer as described in our recent paper.21 EBC was diluted with Py borate buffer, and the diluted sample was concentrated as described above. After switching the voltage off, (5 ( 1)  105 activated MyOne beads were immediately placed into the cone apex. A magnet with a sharp concentrator (see ref 18 for details) was used on the outside of the cone to pull the particles into the cone tip. After 5 min most liquid was removed from the cone leaving ∼10 μL in the cone apex. The particles were resuspended in the latter volume, collected with a pipet, and transferred to a microcentrifuge tube. Then the beads were deactivated for 1 h in 20 μL of a solution containing 250 mM ethanolamine at pH 8.0, washed with water, and finally resuspended in 2 μL of water. Detection of Human Immunoglobulin Linked to the Bead’s Surface. A piece of anti(h-IgG) microarray prepared as described above was washed with the PBS and attached into a gadget described in the Supporting Information. A rubber O-ring was pressed to the microarray so that only a limited area with a diameter of 1 mm became accessible to assay. A 2 μL suspension of freshly activated magnetic beads prepared as described above was applied to the microarray. The beads were allowed to settle and react for 10 min. Afterward, free and weakly bound beads were removed by approaching a rare earth permanent magnet with a magnetic concentrator as described in ref 20, and the images of the microarray decorated with magnetic beads were taken under a low-power microscope with a dark-field illumination. The details of the procedure have been described in our recent publications.20,21 To roughly estimate the amount of h-IgG in EBC using the new technique, special calibration experiments were performed in which known amounts of h-IgG in the Py borate buffer were concentrated in the cone, collected with activated magnetic beads, and detected on the anti(h-IgG) microarray.

’ SAFETY CONSIDERATIONS The pyridine-containing buffer is toxic due to pyridine evaporation and requires efficient ventilation. It is also recommended to place the electrophoretic unit on a nonconductive table and avoid touching its part while running electrophoresis. ’ RESULTS AND DISCUSSION Evolution of the field distribution during electroconcentration in the cone was experimentally measured with a microelectrode. component maximum at the level of the buffer surface in the lower electrode chamber. The maximum grows and becomes narrower when it moves downward to the cone apex following the buffer level in the lower electrode chamber. It is obvious from Figures 1E and 2 that the small cross section area of the cone at

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Figure 2. Distribution of the electric field inside the cone cell during electrophoresis with the buffer level in the lower electrode chamber being (A) 5.0, (B) 12.5, and (C) 20.0 mm as measured from the top of the cone. The electrical potential, j, was measured along the cone axis. The corresponding electrical field strength E was calculated by differentiation of the smoothed dependence of the potential on the depth (shown as the solid line). Total voltage applied to the Pt electrodes was 200 V.

the level of contact with the buffer in the lower electrode chamber provides a bottleneck for the current, resulting in the highest resistance with the highest voltage drop and the highest electric field at this level. When the buffer level decreases, the cross section reduces even further increasing the electric field. Thus, electrophoretic concentration in the conic cell combines advantages of free electrophoresis (large choice of buffers) and those of isotachophoresis (moving the maximum of the electric field brushing charged macromolecules). Though certain small voltage changes were recorded when the probing microelectrode was moved perpendicularly to the cone axis the field component directed normally to the cone axis was much smaller than the vertical component of the field directed along the cone axis at the same heights; namely, the horizontal component of the field at the level of the contact with the lower buffer did not exceed 0.1, 0.5, and 2 V/mm for the external buffer levels, measured from the top of the cone, of 5.0, 12.5, and 20.0 mm, respectively (as shown in Figure 2A C). Several important benefits of the technique should be mentioned. First, a much higher level of concentration can be reached due to higher heat dissipation from the thin cone apex at the final stage of concentrating. Hence, a much higher electric field can be applied. Second, as seen from the field distribution in Figure 2, the area of the intensive electrophoresis is submerged into the cooled lower electrode buffer, providing effective heat dissipation. Thus, protein molecules are moved and kept in the cooled buffer throughout the concentrating process. Third, unlike concentrating on the flat dialysis membrane used as a plug,9 12 where concentrated protein cannot be effectively collected, the 5551

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Figure 3. Concentration in a dialysis cone and polarization effects. (A) Image of 1 μg of FITC BSA concentrated in the cone apex. (B) Solution conductivity as a function of depth in the cone after electroconcentration of 0.5 μg/mL FITC BSA for 15 min in Py borate buffer. Depth at the cone apex corresponds to 25 mm in the panels A and B. (C) Efficiency of collecting FITC BSA as a function of the total volume of aliquots taken from the cone apex. Concentrating was performed in the Imid Gly buffer. (D) Efficiency of collecting FITC BSA samples boiled in the presence of 1% SDS, 5% PVP in Imid Gly buffer before dilution and electroconcentration. Two independent experiments are presented with squares and circles. In both cases electroconcentration was performed at constant voltage of 300 V and current of 0.7 1 mA with the positive potential applied to the lower electrode buffer.

cone enables an extreme localization of the concentrated sample so that even submicroliter samples can be easily collected. Finally, with heating starting from the top part of the cone, the upper layers of the solution in the cone are subjected to longer heating. Being warmer, they additionally stabilize the solution against convection. Thus, no additional gradient stabilization is required. Visual Observation of Protein Concentrating. FITC BSA was chosen for experimentation because of its high solubility, easy visualization of the concentrating process due to the protein color, as well as quantitation of the collection efficiency by fluorescence. FITC BSA is negatively charged at pH 8.5 and moves downward if positive potential is applied to the lower electrode solution. It was noted that yellow color appeared at the level of contact with the surface of the lower buffer and its intensity increased when the level of the lower buffer decreased. As illustrated in Figure 3A, the concentrating process results in the apex of the concentrator becoming bright yellow indicating a very high concentration of the collected protein. Concentrating of two other colored proteins, Hb and Cyt c, was also visually observed in the cones made of the positively charged modified dialysis membrane. Simple estimates, based on the size of the colored zone (with the height of 0.5 mm and half-cone angle of 15°), show that the volume of the colored zone is ∼14 nL. This demonstrates that the concentrating level reaches ∼100 000 with the FITC BSA, provided no substantial loss of the protein happens. Its concentration in the apex should reach ∼100 mg/mL. We cannot exclude that certain aggregation and precipitation of the protein molecules occurs at such high concentration, though this should be reversible at least for FITC BSA, since in our experiments no

insoluble material was noticed in the aliquots taken for the fluorescence measurements. It was also noticed that the size of the colored zone expands very quickly after switching the voltage off. By taking a series of images, the expansion of the colored zone was plotted with respect to the square root of time and showed perfectly linear character which is described by a diffusion coefficient of 1.4  10 9 m2/s (see the Supporting Information for details). The latter considerably exceeds the diffusion coefficient of FITC BSA molecules in water, 6.8  10 11 m2/s.22 It was closer to the diffusion coefficient of boric acid, D = 9.8  10 10 m2/s,23 which was the major anion in the Py borate buffer used in this experiment. We speculate that the protein molecules follow the expansion of the polarization layer, consisting mainly of the borate and pyridinium ions, under these experimental conditions. Role of the Membrane Polarization in Concentration. Membrane polarization plays an important role in inhibition of convection in the cone. Though dialysis membrane is generally considered as electrically neutral, it includes ∼10 mM acidic groups12 making it a mild cation-exchanger. With a positive potential at the lower electrode, negative ions are accumulated in the cone apex due to their lower permeability through the negatively charged membrane. A certain amount of cations is accumulated here to provide electroneutrality, thus forming a salt layer with higher electric conductance and reducing the local electric field. Figure 3B shows that the solution conductivity at the end of the concentrating process in the vicinity of the cone tip is ∼20 times higher than the initial buffer conductivity. Similar dependence has been recorded for the buffer alone and for diluted 5552

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Figure 4. Detection of collected proteins via beads array interaction. Carboxylated magnetic beads are activated using EDC/NHS chemistry and placed into the cone apex to bind electroconcentrated proteins. A magnetic bead bearing h-IgG together with other proteins collected from the cone apex is tethered to a biotinilated anti(h-IgG) linked to arrayed avidin.

protein solutions. Attempts to concentrate Cyt c in a cone made of the original dialysis membrane in the Imid Gly or in the Py borate buffers with negative voltage applied to the lower electrode were unsuccessful: the colored zone was first formed but soon smeared and disappeared. We attribute this to the absence of stabilizing polarization inside the cone: at such reversed polarity, salt is accumulated at the external surface of the cone apex. It was only after the cone was made of positively charged dialysis membrane that concentration of this protein became possible with the negative potential at the lower electrode (see the Supporting Information for details). At this polarity, anions become the leading ions in the anion-exchange membrane and now cations are accumulated in the apex forming a stabilizing salt layer. Switching off the potential resulted in a rapid fall of the solution conductivity in the cone apex. Plotted versus square root of time the conductivity changes display a linear behavior (see the Supporting Information for details), like the expansion of the colored zone described above. It is worth noting that a 2-fold decrease in the conductivity happened in approximately 80 s, whereas a 2-fold increase in the size of the colored zone took ∼200 s. The difference shows that the protein “cloud” expands less rapidly than the expansion of the polarization zone but still much more rapidly than the free protein molecules should diffuse as we emphasized above. Surprisingly, despite much our efforts we could not find any notable changes in pH to accompany the polarization in the cone apex (see the Supporting Information for details). Small changes in pH upon the polarization process on cation-exchanging membranes at high current density have been well-documented in the literature.24 At high current density, the transmembrane current, supported by the motion of salt ions, is replaced by the overlimiting current due to protons generated upon water dissociation in the strong electric field. These protons readily move through the cation-exchanging membrane and neutralize the hydroxide ions incoming to the cone apex. However, the transition between ohmic (“normal” cationic current) and overlimiting current modes is shown to be accompanied with the steplike rise in electrical resistance and, consequently, a sharp break in the volt ampere characteristic of the cell.24 We do not see any notable deviations of our volt ampere characteristics from ohmic behavior in a wide voltage range (see Figure S-6B in the Supporting Information), which makes this explanation doubtful. An extensive study of the electrochemical processes within the conic concentrator presents a subject for future research going beyond the scope of the present paper. In addition to suppression of convection, polarization greatly reduces the electric field in the cone apex, thus preventing the

collected proteins from overheating. We measured the temperature by introducing a microthermocouple (0.2 mm diameter, WPI, Sarasota, FL) into the apex at the final stage of the concentration process in the Imid Gly buffer with 1 μM bromophenol blue added. With a total constant voltage of 300 V applied to the platinum electrodes the current dropped from the initial 1 to 0.4 mA and the apex temperature increased by 4 5 °C by the time the buffer level in the lower electrode chamber dropped to 1 2 mm below the apex. This measurement, together with the fact that no protein denaturation resulting in irreversible aggregation was observed in the concentrated samples of three different proteins, confirms that no substantial overheating accompanied the electrophoretic process in our conditions. Collection of the Concentrated Protein with a Microsyringe. Though protein is visually concentrated into a volume as small as 10 20 nL we found it difficult to collect such a small probe. Instead, 0.5 μL aliquots were taken as described in the Materials and Methods. It was found that the first 0.5 μL probe taken from the cone apex contained only ∼30% of the total FITC BSA placed in the cone. Further 3 4 probes increased the yield to 60 70% as seen in Figure 3C. Thus, the protein concentration in the first probe was ∼600 times higher than in the initial solution. Direct measurements of the solution fluorescence in the probes taken from the top of the cone indicated that not more than 10% of FITC BSA was left in the solution, presumably due to contamination upon taking probes and/or convection or desorption from the cone surface after switching off the current. No fluorescence was detected in the upper and lower electrode chambers after the concentrating procedure. Efficiency of collection of the concentrated protein can be visually controlled by adding small amounts of bromophenol blue dye which concentrates together with protein. In contrast to proteins, only 20 30% of the dye was found in multiple probes taken from the cone apex, presumably due to penetrability of the dialysis membrane to the dye molecules. Collection in the Presence of SDS. As a preparatory step in extraction of proteins from gels after SDS polyacrylamide gel electrophoresis (SDS PAGE), we tested if protein SDS complexes could be effectively concentrated from diluted extracts. It was seen that the presence of SDS in the diluted sample after boiling does not interfere with the concentration procedure. With the positive potential at the platinum electrode in the lower chamber, the fluorescent substance was readily accumulated at the apex of the cone as described above for the native FITC BSA. As seen in Figure 3D, approximately 30 40% of all the FITC BSA was collected in the first 0.5 μL probe taken from the cone apex. Further 3 4 probes increased the overall yield to 80%. Thus, boiled 5553

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Figure 5. Illustration of microarray-based detection of proteins collected on a magnetic bead’s surface after electrophoretic concentration. (A) Images of the anti(h-IgG) array after contact with magnetic beads having collected h-IgG electroconcentrated in a cone. The specified amount of h-IgG shown at the top of each image was dissolved in a 2 mL volume of Py borate buffer and concentrated in the cone. Concentrated h-IgG was collected in the cone apex with 5  105 activated magnetic beads which were further analyzed for interaction with the anti(h-IgG) microarray. The area marked by the white rectangle corresponds to a defect on the microarray surface. (B) Decoration of the anti(h-IgG) microarray spots with the same number of magnetic beads which were used to collect proteins electrophoretically concentrated from human EBC. Before the electroconcentration procedure EBC was diluted with Py borate buffer by a factor indicated on the top of each image. Images have been taken under a low-power microscope with a dark-field illumination.

denatured FITC BSA can be collected with a better yield as compared to the native protein. The larger negative charge of protein SDS complexes, resulting in higher electrophoretic mobility, protection of the proteins from adsorption to the surface of the cone by SDS, as well as inhibition of protein aggregation, may be thought of as possible explanations for the observed difference in the collection efficiency. Collection in the Denatured State. Protein molecules boiled in the presence of SDS and then diluted may be concentrated as partly or completely renatured molecules. As we saw in the previous section, they were collected as efficiently as native protein molecules. Concentrating in the completely unfolded state, which was maintained by the presence of urea in the cone, resulted in notably lower yields. Although 78% of native FITC BSA was found after concentration in the cone made from dialysis membrane with MWCO 12 14 kDa, this amount dropped to 6.5% when the same protein was collected under the denaturing conditions in the same cone. We speculate that the dialysis membranes used here were penetrable for the unfolded FITC BSA molecules. Even though diffusion might be slow and incomplete, once a part of the unfolded protein molecule penetrates into the membrane, the whole molecule becomes stuck and cannot slide along the membrane surface to the cone apex. This explanation is supported by direct observation of such stuck molecules. After opening the cone and rinsing the membrane, a cone-shaped fluorescence pattern was seen under UV illumination on the dialysis membrane in the apex area. Collection of Proteins on Activated Magnetic Beads. Considering that proteins are concentrated into an extremely small final volume of 10 20 nL, we suggest here a new technology of protein sampling from such a tiny volume. It is based on use of EDC NHS-activated magnetic beads capable of chemically binding any protein molecules containing free amino groups. The presence

of any particular protein in the sample can be further probed by the bead’s interaction with specific antibodies. A similar principle has been suggested by Espins et al. under the name of “reverse phase microarray” for microarrays of lysate spots.25 The combination of concentration in the cone and collection with EDC NHS-activated magnetic beads solves several problems. First, due to high protein concentration in the cone apex, the competition between NHS ester hydrolysis and its reaction with proteins shifts dramatically in favor of the reaction with protein molecules to allow complete protein binding before all the active groups on the magnetic beads are hydrolyzed. Second, nanoliter volumes become easily accessible for exhaustive collection of proteins. Third, magnetic beads can further be used as extremely sensitive active labels20 to recognize specific proteins immobilized on the beads surface by their interaction with antibody microarray as illustrated in Figure 4. As a “proof-of-concept” we analyzed human EBC for the presence of immunoglobulins. EDC NHS-activated magnetic beads bind any protein molecule in EBC which possesses free amino groups. Contacting these beads with antibody microarray revealed the presence of immunoglobulin molecules on their surface by decorating of anti(h-IgG) spots on the microarray. Binding of beads was recorded using a dark-field microscope as described in ref 20. In a series of control experiments with known amounts of h-IgG added to buffer solution we estimated the limit of detection (LOD) of our method. Images in Figure 5 demonstrate that, under our experimental conditions employing a low number of beads and small microarray surface, the LOD for h-IgG reached 0.5 pg/mL. Although all the experiments with an h-IgG concentration of 0.5 pg/mL gave a positive signal, those with 0.05 pg/mL gave a positive signal (shown in Figure 5) in only one of three attempts. 5554

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Analytical Chemistry Figure 5 clearly demonstrates the presence of immunoglobulins even in highly diluted EBC samples: decoration of anti (h-IgG) spots on the microarray became visible when the EBC sample was diluted 1:10 000 (see Figure 5). Further dilutions gave no visible signal. Using the obtained LOD value we estimated the concentration of h-IgG in the volunteer’s EBC as ∼5 ng/mL. This estimate seems reasonable considering natural variability of the protein content in EBC collected from different persons. Although some other authors report total protein in the EBC as high as 40 μg/mL,26 most authors give values that are 1 2 orders of magnitude lower than that.27,28 Taking 400 ng/mL as a lower limit for the total protein concentration in the EBC and the above found estimate for the LOD of our technique, we conclude that proteins which are present in as low as ∼1  10 4 % fraction of the total protein content in EBC can be detected.

’ CONCLUDING REMARKS A simple device described here can be easily reproduced in any laboratory possessing a power supply for electrophoresis and a machine shop to manufacture a few plastic details. We believe that the device would be especially effective with highly diluted samples to be investigated by methods requiring tiny sample volumes, like capillary electrophoresis, matrix-assisted laser desorption ionization (MALDI)- and electrospray ionization (ESI)based mass spectrometry (MS). Due to its ability to concentrate protein molecules to a high extent, the technique could be useful in studies of crowding effects and protein behavior at high concentrations as well. When combined with harvesting concentrated proteins on activated magnetic beads, this technique may exploit the benefits of another powerful technology based on antibody microarrays with magnetic beads as carriers and as active labels. We demonstrate here that tiny amounts of proteins in EBC can be readily detected using such a combined technique. We also anticipate that the technique could be used for removal of unwanted proteins and impurities from samples, for crystallization of proteins, and for detection of trace amounts of toxins and pathogens in a drinking water, foods, and biological fluids. ’ ASSOCIATED CONTENT

bS

Supporting Information. (i) Design of the electrophoresis device, (ii) design of a gadget for detection with magnetic beads, (iii) data on diffusion of proteins from the cone apex after switching off the voltage, (iv) data on diffusion of electrolytes from the cone apex, (v) data on the concentrating of positively charged proteins, (vi) data on pH gradients in the conic concentrator, and volt ampere characteristic of the concentrating system. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: 7-496-733-0553.

’ ACKNOWLEDGMENT The authors gratefully acknowledge support from DOE Grant DE-F C52-04NA25455. ’ REFERENCES (1) Griese, M.; Noss, J.; Bredow, C. Proteomics 2002, 2, 690–696. 5555

dx.doi.org/10.1021/ac201146w |Anal. Chem. 2011, 83, 5548–5555