Localized Generation of Attoliter Protein Solution Droplets by

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Localized Generation of Attoliter Protein Solution Droplets by Electrofocused Liquid-Liquid Separation Mrinal Shah,†,§ Oleg Galkin,‡,⊥ and Peter G. Vekilov*,†,‡ Departments of Chemical and Biomolecular Engineering, and Chemistry, UniVersity of Houston, Houston, Texas, 77204-4004 ReceiVed: January 9, 2009; ReVised Manuscript ReceiVed: March 23, 2009

We develop a simple technique for the generation of droplets of a high-concentration protein solution of attoliter volume and a diameter of g500 nm, containing several thousand protein molecules, and their accurate deposition on micron-size electrodes, standalone or in an array. The technique is based on liquid-liquid phase separation in the homogeneous region of the phase diagram, induced by a nonuniform weak electric field, which also accurately directs the generated droplets toward the electrode. We show that the protein molecules are not chemically modified and retain their integrity, conformation, and activity post deposition. The technique is applicable to the manufacture of various types of protein/microelectrode arrays and is tunable, scalable, and applicable to a broad range of proteins. Introduction Supported 2D-arrays of clusters of protein molecules or protein solution droplets, protein chips,1,2 hold promise as high-throughput platforms for biosensors, for profiling diseaserelated proteins, and for other applications.1-5 Such arrays have been built using photolithography,6,7 self-assembly of monolayers8,9 and protein-coated colloids,4 soft lithography, microcontact printing,10,11 microfluidic networks,12,13 “dippen” nanolithography,14,15 and other methods16 on substrates such as silicon,17 glass,6 and gold.3,4,7,8 These methods rely either on elaborate manipulation of single protein molecules or on deposition of protein solution droplets. In that latter case, the smallest droplets have been of nanoliter volumes, that is, of >100 µm size, with limitations due to solution viscosity and protein denaturation at high extruding pressures.1 On the other hand, separation of a protein solution into two liquid phases, a protein-rich and a protein-lean one, has been observed with many proteins, and its thermodynamics and kinetics are reasonably well understood.18-25 While the two liquid-phase system may be metastable with respect to a solution-crystal equilibrium, Figure 1, the formation of crystals is often sufficiently slow to allow unperturbed liquid-liquid (L-L) coexistence for many hours.24,25 For many proteins, L-L separation is observed after cooling a solution below the temperature for L-L equilibrium of the given concentration; see Figure 1a; for the proteins with a retrograde phase diagram, schematically depicted in Figure 1b, L-L separation occurs upon temperature increases.18,19 An important feature of L-L phase separation is the sensitivity of the L-L coexistence temperature to the concentration of electrolyte in the solution,20,26,27 due to the participation of the ions in the interactions between the protein molecules.28-31 If a homogeneous solution is held * To whom correspondence should be addressed. † Department of Chemical and Biomolecular Engineering. ‡ Department of Chemistry. § Current address: InnoPharma LLC, 11 Deer Park Drive, Suite # 205, Monmouth Junction, NJ 08852. ⊥ Current address: Physical Optics Corporation, 20600 Gramercy Pl., Bldg. 100, Torrance, CA 90501.

Figure 1. Phase diagrams of protein solutions. Solid lines mark phase lines for liquid-solid and liquid-liquid (L-L) equilibria. The gray fill marks regions immediately beyond the L-L coexistence lines, in which a homogeneous solution is metastable with respect to a twophase system, that is, the generation of the dense liquid is hampered by a barrier and occurs by nucleation. Dashed lines mark locations of L-L coexistence at a higher precipitant concentration; for examples of similar phase diagrams and for the effects of salt concentration on the phase lines, see refs 24, 26, 27, 45, 57. The application of electric field leads to a fast increase of the concentration of counterions in the vicinity of a respective electrode, occurring simultaneously with a slow decrease of the concentration of the protein at the same electrode; see discussion in the text. The phase areas, in which this phenomenon was shown to lead to L-L separation, are hatched. (a) Normal phase diagram, in which crystallization and L-L separation occur at lower temperatures; examples include γ-crystalline and lysozyme. (b) Retrograde phase diagram, in which crystallization and L-L separation occur at higher temperatures; examples include hemoglobin A, C, and S, and peroxidase.

within a few degrees beyond the L-L coexistence temperature, the high-protein-concentration liquid phase is generated by nucleation of well-separated droplets.25 The droplets then grow by the attachment of molecules from the low-concentration solution or coalescence with other droplets until equilibration.25 The droplet nucleation and growth processes are very reproducible and can be stopped by, for example, temperature control, when the droplets reach any size above the optical detection limit of ∼0.5 µm (the volume of such droplets would be >70 attoliters).25 Such droplets contain, depending on the composition of the high-concentration liquid, from 103 to 106 protein molecules.

10.1021/jp9002388 CCC: $40.75  2009 American Chemical Society Published on Web 04/27/2009

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Below, we discuss a novel technique for generation of attoliter droplets of a high-concentration protein solution, containing several thousand protein molecules, and their deposition on microsize electrodes. The technique is based on liquid-liquid phase separation,18-21 which is induced by a nonuniform weak electric field in the homogeneous region of the phase diagram, where, in the absence of the electric field, the protein solution is the only stable phase. Results Generation of Droplets of Protein Dense Liquid by Phase Separation. We demonstrate the applicability of liquid-liquid separation for the design of protein chips with the protein peroxidase, an enzyme containing a heme close to the molecular surface,32 allowing direct electron transfer between an electrode and a protein molecule, one of the widely used proteins in biosensor research.33 Since peroxidase exchanges electrons with the electrodes in both high- and low-concentration phases, tests of the underlying mechanisms were also carried out with lysozyme. The peroxidase solutions contain from 30 to 70 mg mL-1 of protein, 12% NaCl, and 6% polyethylene glycol (PEG), with a molecular mass of 5000 or 8000 g mol-1 in 0.1 M phosphate buffer at pH 7.5. The lysozyme solutions contain 30-60 mg mL-1 of protein and 4% NaCl in 0.05 M acetate buffer at pH 4.5.24,25 The isoionic point of peroxidase is at pH 8, and for lysozyme, it is at pH 11.5,34 that is, both proteins carry net positive charges at the respective pH. Under their respective conditions, lysozyme solutions have a “normal” phase diagram, with crystallization or L-L separation occurring at a lower temperature, similar to the one schematically depicted in Figure 1a,24 while tests revealed that peroxidase has a retrograde phase diagram, with crystallization or L-L separation favored by higher temperature, as shown in Figure 1b. To determine the temperature of L-L equilibrium for a given solution composition, TL-L, a solution sample was heated (peroxidase) or cooled (lysozyme) in 1 K increments, with ∼2 min at a certain temperature setting, under continuous microscopic observation, as in ref 25, until numerous round droplets of dense liquid appeared and grew at a certain temperature, Figure 2a and b. L-L separation was reversible after a temperature change in the direction opposite to the last step. Depending on the protein concentration, the found TL-L’s for lysozyme were between 288 and 293 K (15 and 20 °C), as in ref 24, and the TL-L’s for peroxidase were in the 333-345 K (60-65 °C) range; with both proteins, the TL-L’s for each protein concentration were reproducible within 1 K. The absence of peroxidase denaturation at the relatively high temperature for L-L separation was evidenced by the lack of precipitation and by the reproducibility of TL-L after multiple cycles through it. Increasing the concentration of NaCl increases the temperature of L-L phase separation for lysozyme;26,27 tests with peroxidase revealed that using a solution with 8% NaCl leads to a TL-L higher by ∼10 K than that at the 12% NaCl used in most experiments discussed here. Thus, higher electrolyte concentration leads to lower TL-L in peroxidase solutions, corresponding, as in the lysozyme case, to a broader two-phase region. Deposition of Droplets on Electrodes. If attoliter droplets, such as the small ones in Figure 2a and b are deposited on microelectrodes, this would offer the advantage of an easy readout of a signal; this approach, with larger protein solution volumes, is utilized in electrochemical protein sensors.33 While the sizes of the droplets produced by temperature-controlled L-L separation are comparable to the ∼1 µm microelectrode spacing on such a chip, a problem is that due to the stochastic nature of nucleation, the droplets are randomly scattered

Figure 2. Thermally and electrochemically induced liquid-liquid separation in solutions of lysozyme and peroxidase. (a and b) Coexistence of two liquid phases in a solution of peroxidase in (a) and lysozyme in (b). Observations by transmission bright field microscopy in 5 µm thick slides. The peroxidase concentration in (a) is 30 mg mL-1, T ) 60 °C. The lysozyme concentration in (b) is 90 mg mL-1, T ) 16.4 °C. Other components of both solutions are as in the text. The bar equals 20 mm in both images. The arrow in (a) indicates a large droplet formed by coalescence of smaller droplets. In contrast, droplets of the dense phase of lysozyme in (b) do not coalesce upon contact, that is, peroxidase dense liquid retains its fluidity, while the lysozyme dense phase is viscous and likely gelled. The dense liquid lysozyme phase contains 5-10 times higher protein concentration than the low-density solution in equilibrium; for the concentration of peroxidase in dense droplets, see the text. (c) Schematic of gold electrodes on a glass substrate; the frame indicates the viewing field in d-i. (d-i) Electrochemically induced nucleation of droplets of peroxidase over the anode. Observations by reflection bright field microscopy. The electrode width is 20 µm. The same area is shown in all subfigures; the white arrow indicates a scratch on an electrode. (d) Electrodes prior to droplet deposition. (e) Thermally driven generation of peroxidase droplets in the entire solution volume; detection of the droplets deposited on the glass surface between the electrodes requires oblique illumination. (f) Dense liquid droplets are generated only at the anode. (g) Switching the polarity of the electrodes moves the location of droplet generation to a new anode. (h and i) Evolution of droplets seen in (g); droplets on the anode coalesce into bigger droplets, while droplets near the edges run toward the cathode, leaving denuded edges; for discussion, see the text.

throughout the entire solution volume. We tested the applicability of two electrochemical methods to position the attoliter droplets over electrodes after they have been generated; dielectrophoresis, which relies on the difference in dielectric constants of the two liquid phases,35 failed because of the high conductivity of a solution of ionic strength above 0.1 M, required for L-L separation, and electrophoresis failed for reasons discussed below. Note that in an aqueous environment, the maximum applicable voltage is limited by the electrolysis of water to 100 times without any detectable changes. Increasing the frequency to 200 mHz, corresponding to a period of 5 s, prevented the appearance of the droplets. Thus, the characteristic time of droplet formation was ∼10 s and was likely determined by the growth to 0.5-1 µm, at which size the droplets landed on the anode. This time is sufficiently long to allow interruption of droplet growth at a chosen size by temperature or voltage jump. The Protein Concentration in the Droplets. The dense liquid in lysozyme solutions has a concentration of 350-450 mg mL-1.24,27 However, dense liquid phases formed in polymer-protein solutions, as with peroxidase, might have protein concentrations lower or higher than the generating solution. The distribution of protein concentration in the solution in equilibrium with the droplets was determined from scans of the optical density of the solution slide at a wavelength of 405 nm, Figure 3. To account for the inevitable nonuniformity in illumination and to calibrate the optical density at this wavelength with the known concentration of the protein prior to L-L phase separation, linear intensity scans before and after phase separation were compared, Figure 3a and c. This calibration

Shah et al.

Figure 3. Peroxidase concentration in droplets and in the generating solution. The peroxidase concentration is 68 mg mL-1, and the temperature is 50 °C in (a) and 60 °C in (b), with NaCl, PEG 5000, and phosphate buffer concentrations as in the text. A 5 µm uniformly thick glass slide with solution is illuminated through a band-pass filter at 405 nm. Large-scale intensity nonuniformity is compensated for with standard image processing techniques. (a) Image of a slide containing a homogeneous solution prior to droplet generation. Red and blue lines indicate locations whose intensities are plotted in same colors in (c). The solution concentration is correlated with the mean intensity from (c), yielding a conversion factor between the local intensity and concentration. (b) Image of a slide containing the solution in equilibrium with the droplets. The larger droplets whose diameters are greater than the slide thickness touch the top and bottom glasses. This leads to uniform thickness of the dense liquid inside of such droplets. Intensity scans, converted to peroxidase concentration using the conversion factor calculated from (a) and (c), along the red and blue lines are shown in (d) in the same colors.

yielded the product of the protein concentration, extinction coefficient, and slide thickness. Since only the protein concentration distribution changes after the droplets are generated, this calibration factor was used to convert scans of optical density after droplet generation to the distribution of the protein concentration inside of the droplets and in the generation solution, Figure 3b and d. The results in Figure 3d show that although the protein concentration in the dense droplets is lower than that in the generating solution, the peroxidase concentration inside of the droplets is significant, 20-30 mg mL-1. Tests with different PEG and NaCl concentrations showed that these two components can be used to tune the protein concentration in the dense-phase droplets. The Electrochemical Reaction. We compared the cyclic voltammograms recorded during deposition of lysozyme droplets at the anode, Figure 4a, to those corresponding to direct electron transfer from the anode to a dissolved protein.36,37 The voltammograms in the presence of lysozyme are within the error range of those recorded in the absence of lysozyme, Figure 4a. Local minima or maxima of ∼1-3 µA at voltages of -(0.3-0.5) V, characteristic of direct electron transfer,36,37 are not seen in Figure 4a. The cyclic voltammograms in Figure 4a are very similar to those recorded during anodic dissolution of gold into Au(I) at potentials below 0.8 V and Au(III) at potentials above 1.1 V.38 The presence of Cl- enhances this dissolution.38,39 A black residue of gold oxide/ hydroxide forms on the anode surface near 1-1.2 V,38,40 and this explains its darker color in Figure 2g. Voltages above 1.3 V destroy the electrodes. We conclude that the electrochemical reaction does

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Figure 4. Tests for the preservation of the structural, conformational, and charge integrity of the protein after electrochemical generation of dense liquid droplets. (a) Cyclic voltammograms of lysozyme solutions and controls on gold electrodes. Solutions contain 4% NaCl (w/v) in 50 mM NaAc buffer with pH 4.5. The lysozyme concentration is 60 mg mL-1 or 0. Two cyclic voltammograms were recorded with each composition. Lines are just guides for the eye. (b, c, and d) MALDI-TOF mass spectra of lysozyme with 4% NaCl (w/v) in 50 mM NaAc buffer (pH ) 4.5) and peroxidase with 12% NaCl and 6% PEG 8000 in 0.1 M phosphate buffer (pH ) 7.5). Lysozyme: (b) native and (c) postdeposition. The molecular mass of lysozyme prior and post deposition is 14.3 kg mol-1. Peroxidase: (d) The molecular mass of peroxidase is ∼42.6 kg mol-1. The peak at 8000 g mol-1 in (d) corresponds to PEG 8000. (e) Native polyacryl amide gel electrophoresis for native and processed lysozyme as compared to standard molecular weight markers. Migration plots indicating molecular weight standard proteins in 103 g mol-1. The numbers on traces for native and processed lysozyme indicate (1) the lower molecular weight marker, (2) the system peak of the instrument, (3) lysozyme at 14300 g mol-1, (4) 18000 g mol-1 contaminant, (5) 24000 g mol-1 contaminant, and (6) the upper molecular weight marker. (f and g) The activity of native and postdeposition lysozyme in peroxidase. For the lysozyme in (f), the rate of lysis of Micrococcus luteus cells measured as a decrease in optical density OD. Two determinations with each type of lysozyme are shown. (g) The peroxidase activity is assayed from the rate of oxidation of phenol by H2O2 catalyzed by this protein; the products of phenol oxidation are detected at 510 nm.

not involve the protein and likely consists of gold dissolution of the anode and deposition on the cathode. Peroxidase in solution readily oxidizes at voltages of -(0.3-0.5) V.36,37 However, this oxidation does not affect the L-L separation and droplet deposition with this protein. The only difference with lysozyme is that the deposition of the peroxidase droplets on the anode perturbs the current and voltage in the electrochemical cell. The low current values in Figure 4a explain the lack of electro-osmotic flows in the cell, as evidenced from observations of suspended dust particles. The low current also leads to a negligible ohmic heating of the solution; for a solution mass of ∼0.03 g, ∆T ∼ 0.03 K is expected after 10 min of electric current. The Integrity of the Protein in the Droplets. Gold dissolution from the anode releases Au+ and Au3+ ions in the solution. To test if these ions bind to the protein,41 we carried out mass spectroscopy characterization of lysozyme and peroxidase before and after multiple repetitions of electrochemically induced L-L phase separation. Comparison between panels b and c of Figure 4 shows with a resolution of a few g mol-1 the reproducibility of the molecular mass of lysozyme of ∼14330 g mol-1; the binding of gold ions with an atomic mass of 197 g mol-1 would be easily detectable. The spectra for peroxidase before and after

multiple electrochemically assisted phase separation are compared in Figure 4d; while they are identical, the resolution does not allow detection of Au ions, which may or may not be bound to peroxidase. Native polyacryl amide gel electrophoresis is an adequate test for conformational and charge consistency. The results for native and deposited and dissolved lysozyme in Figure 4e show no differences in these two characteristics. For further tests of the preservation of the integrity of the protein inside of the droplets deposited on the anode, the biological activity of lysozyme was assessed using Micrococcus luteus (ML) cells,42 Figure 4f. The activity of peroxidase was evaluated from the rate of catalytic oxidation of phenol by H2O2,43 Figure 4g. The activities of lysozyme and peroxidase, estimated from the initial slopes of the curves in Figure 4f and g, were reduced by ∼16 and 17%, respectively, that is, significant activity of both proteins was retained after numerous cycles of deposition and dissolution. Discussion The Mechanism of Droplet Deposition: Electrofocused L-L Separation. The observations of the formation of the droplets and the evidence for the preservation of the

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Figure 5. Schematic of electrochemically induced generation of dense liquid droplets of protein solution over microelectrodes. (a) The geometry of the system; dashed lines denote electric field lines; single protein molecules, and a droplet are shown. (b) The time-dependent distribution of protein and chloride ions. At t ) 0, the system is at a temperature in the homogeneous phase region several degrees away from the liquid-liquid separation temperature. Application of potential causes electromigration of the ions and protein molecules. At the anode, this leads to a gradual increase of the concentration of the counterions, Cl-, and a ∼10× slower decrease of the concentration of positively charged protein. When the increased counterion concentration shifts the temperature for liquid-liquid separation and places the protein solution in the two-phase region, dense liquid droplets nucleate. The migration of the protein away from the anode ensures that the nucleation occurs at a certain distance from the anode.

structural, conformational, and charge integrity of the proteins postdeposition suggest the mechanism of electrochemically induced L-L phase separation, illustrated in Figure 5. The electric field causes eletromigration of the positively charged protein molecules toward the cathode and of the counterions, Cl-, toward the anode. Since the field strength is insufficient to oxidize the protein or to reduce the Cl-, both species accumulate at the respective electrodes. At the anode, the local increase in Cl- concentration is faster by about an order of magnitude than the decrease in protein concentration because of the higher mobility of Cl-. The increase in Clconcentration causes a local increase (for lysozyme) or decrease (for peroxidase) of TL-L. The shift of TL-L places the solution in the metastable two-phase region (see Figure 1a for lysozyme or Figure 1b for peroxidase), and droplets of the dense liquid nucleate and grow. When they reach a size of ∼0.5-1 µm, the gravity force overcomes the thermal energy of Brownian motion, and they land on the anode. Since they contain a high concentration of positively charged protein, they are driven away from the positively charged anode toward the cathode and dissolve as soon as they leave the Cl--rich vicinity of the anode. The dissolution of the droplets upon approach to the Cl--poor cathode explains the failure of the earlier trials to deposit preformed droplets using electrophoresis. This mechanism was supported by tests in an upturned slide assembly, in which the patterned electrodes were at the top. We observed in an inverted microscope the formation of ∼0.5-1 µm droplets at ∼100-120 µm below the anode. As they grew, they moved downward, away from the anode, and dissolved. Further tests, in which L-L separation with peroxidase was achieved with polyethylene glycol only, in the absence of NaCl, showed no localized phase separation in the homogeneous region of the phase diagram. If L-L separation with peroxidase was induced by lower (2 or 3%) concentrations of NaCl, at which conditions TL-L is insensitive to the NaCl

Shah et al. concentration, again localized phase separation in the homogeneous region of the phase diagram did not occur. The mechanism illustrated in Figure 5 relies on nonuniform concentration of protein and precipitant in the aqueous solution, induced by a nonuniform electric field. The concentration nonuniformity leads to a local increase in the free energy of the homogeneous mixture and makes the two-phase system more favorable. In this respect, it is similar to the recently reported demixing of two-component mixtures of dielectric liquids in a nonuniform electric field; because of the weaker response of the dielectric liquids to the applied potential, the latter phase separation required significantly higher voltages.44 The redistribution in an electric field of the ionic third component makes the phase separation in protein solution more robust; (a) it is induced by electric fields as weak as ∼1 V, a must if water electrolysis is to be avoided, and it is thus applicable to protein solutions; and (b) the phase separation occurs at temperatures as far as 5° away from the two-phase coexistence line into the homogeneous solution region. Scalability and Broad Applicability of Protein Deposition by Electrofocused L-L Separation. This mechanism provides for the deposition of droplets of a size of 0.5-1 µm over electrodes of similar size. The only requirement for the operation of this mechanism with any protein is that the protein should exhibit L-L separation for which the equilibrium temperature depends of the concentration of an ion smaller in size than the protein. With some proteins, such as lysozyme27 and lens proteins,45 L-L phase separation occurs in solutions containing electrolyte, with other proteins, such as the human hemoglobins A, S, and C in oxy and deoxy states,22,46 apoferritin,47 brome mosaic virus,48 and others; including peroxidase, discussed above, it requires the addition of 5-10% polyethylene glycol. With most of these proteins, it was found that the temperature of L-L separation depends on the concentration of an electrolyte. These findings suggest that the combination of polyethylene glycol of appropriate molecular weight and concentration and an electrolyte, such as NaCl, would result in temperaturesensitive L-L phase separation. Thus, the method for electrochemically induced nucleation of attoliter droplets of protein solution can be used with many proteins. The use of polyethylene glycol offers an additional advantage; it significantly lowers the protein concentrations of both phases in L-L equilibrium.46,49 Thus, by varying the molecular weight and concentration of polyethylene glycol, the protein concentration and the number of protein molecules in the deposited droplets can be fine-tuned. The fixation of the droplets deposited at the electrodes that would ensure their stability in flowing solutions or in air is still an open issue. The scalability of the method of localized deposition is underscored by the following considerations. In the above experiments, this method was tested with 1 × 1 cm2 electrode areas, and microscopic observations revealed that the droplets population is uniform in size and number density throughout the whole surface of the electrodes. Since electrochemically assisted nucleation occurs within a temperature range of a several degrees, its outcome is not affected by the inevitable temperature nonuniformity over larger areas. Thus, the method is easily scalable for application in the design of large protein arrays, and biochips. On the other hand, none of the processes comprising the mechanism of droplet generation are affected by a reduction of the scale from 10 or 20 µm, as in Figures 2d-i, to 1 or 0.5 µm. Although we have not tested it, a reasonable conclusion is that this method would allow generation and deposition of a single

Generation of Attoliter Protein Solution Droplets droplet of submicron size on electrodes of similar width. On an array of such electrodes, droplets of different proteins could be deposited in sequence, creating a multichannel analytical protein microchip with electrochemical detection. Another possible application is to microfluidic devices with embedded electrodes,50 where this method could be used for generation of high-concentration protein droplets at desired locations. The nonuniformity of precipitant concentration induced by an electric field could be used for the localized generation of crystals on electrodes. Since this nonuniformity is transient, the nucleation of crystals of a protein of interest and their growth to a dimension of several hundred nanometers need to occur within the time scale of existence of the nonuniformity; for electrodes of spacing 100 µm, with the typical diffusion constant of inorganic ions of 10-5 cm2s -1, this time scale is 10 s. Nucleation and growth of protein crystals, sufficiently fast to fit within these time scales, while rare, are not impossible; lumazine synthase forms crystals detectable with a microscope, that is, of size ∼1 µm, within a few seconds of solution preparation.51 Methods Section Solution Preparation. Hen egg white lysozyme (six times recrystallized, lyophilized) purchased from Seikagaku Corporation, Japan (Lot # E99303), was used without further purification. All other chemicals used were reagent grade. Lysozyme stock solutions were prepared following the procedures in ref 52. Horseradish peroxidase (HRP) purchased from Toyobo Co. Ltd., Japan (Lot # 24160), was used without further purification. HRP stock solution containing ∼150 mg mL-1 of the protein was prepared in 0.1 M phosphate buffer, pH 6.0, and stored at 4 °C. The concentration of the stock solution was determined by measuring the optical density of the solution at 403 nm using a Beckman DU-68 UV spectrophotometer and using an extinction coefficient of 2.3182 mL mg-1 cm-1.53 Electrochemically Induced Phase Separation. Interdigitated gold electrodes (see schematic in Figure 2c), deposited on a microscope glass slide (5 cm × 5 cm), were kindly provided by the group of Dr. P. Gascoyne at M.D. Anderson Cancer Center. They were cleaned with Liqui-Nox critical cleaning detergent and deionized water. Protein solutions (∼30-35 µL) were pipetted into an open Teflon cell (∼1 cm × 1 cm × 300 µm), resting over the electrodes, and covered with a coverslip. The slide-Teflon cell-coverslip assembly was placed on a Peltier element connected to a Marlow SE 5010 temperature controller. The gold electrodes were connected to a BK Precision 1787 DC power supply in series with a Fluka 45 dual display multimeter to measure the DC current flowing through the solution. AC potentials were applied using a Hewlett-Packard 33120A function generator connected to a Tektronix 2205 oscilloscope. To induce L-L phase separation at a temperature several degrees into the homogeneous single-phase region, a DC potential of 0-1.2 V in increments of 0.1 V was applied to the protein solution. At each step, the current was allowed to reach a steady value; this took ∼30 s. Droplet formation was observed with a Leica DML microscope working in reflection mode with a 20× Leica objective. Determination of Concentration Distribution in Slides. To determine the concentration of peroxidase in dense liquid droplets formed in a solution containing PEG, we observed by bright field transmission microscopy solution samples before and after phase separation, Figure 3a and b. The solution was held in slides of uniform thickness maintained by suspending

J. Phys. Chem. B, Vol. 113, No. 20, 2009 7345 glass spheres (Duke Scientific) with diameters of 4.9 ( 0.5 µm in the solution.25,54,55 The larger droplets have diameters greater than this thickness and touch the bottom and top glasses of the slide. In this way, a uniform region of thickness equal to that of the slide exists for such droplets. In such regions, the concentration of peroxidase can be quantified. The slides were illuminated through a narrow band-pass filter (Edmund Industrial Optics, Inc.) at a wavelength of 405 ( 10 nm, the Soret region of plant peroxidases.56 Acknowledgment. We thank O. Velev and P. Atanasov for critical suggestions on this work, J. Vykoukal, P. Gascoyne, B. Jackson, N. Tucker, and R. C. Wilson for assistance with essential equipment, and the Mass Spectrometry Laboratory, University of Houston, for the use of the Applied Biosystems Voyager systems. This work was supported by the National Science Foundation (Grants CBET 0609387 and MCB 0843726) and The Welch Foundation (Grant # E-1641). References and Notes (1) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (2) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101. (3) Abad, J. M.; Ve´lez, M.; Santamarı´a, C.; Guisa´n, J. M.; Matheus, P. R.; Va´zquez, L.; Gazaryan, I.; Gorton, L.; Gibson, T.; Ferna´ndez, V. M. J. Am. Chem. Soc. 2002, 124, 12845. (4) Gleason, N. J.; Nodes, C. J.; Higham, E. M.; Guckert, N.; Aksay, I. A.; Schwarzbauer, J. E.; Carbeck, J. D. Langmuir 2003, 19, 513. (5) Zhu, H.; Klemic, J. F.; Chang, S.; Bertone, P.; Casamayor, A.; Klemic, K. G.; Smith, D.; Gerstein, M.; Reed, M. A.; Snyder, M. Nat. Genet. 2000, 26, 283. (6) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12287. (7) Yang, Z.; Frey, W.; Oliver, T.; Chilkoti, A. Langmuir 2000, 16, 1751. (8) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (9) Neto, C. Phys. Chem. Chem. Phys. 2007, 9, 149. (10) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. AdV. Mater. 2000, 12, 1067. (11) Bernard, A.; Fitzli, D.; Sonderegger, P.; Delamarche, E.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Nat. Biotechnol. 2001, 19, 866. (12) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276, 779. (13) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363. (14) Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702. (15) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661. (16) Belegrinou, S.; Mannelli, I.; Sirghi, L.; Bretagnol, F.; Valsesia, A.; Rauscher, H.; Rossi, F. J. Phys. Chem. B 2007, 111, 8713. (17) Lee, C.-S.; Lee, S.-H.; Park, S.-S.; Kim, Y.-K.; Kim, B.-G. Biosens. Bioelectron. 2003, 18, 437. (18) Thomson, J. A.; Schurtenberger, P.; Thurston, G. M.; Benedek, G. B. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7079. (19) Muschol, M.; Rosenberger, F. J. Chem. Phys. 1997, 107, 1953. (20) Casselyn, M.; Perez, J.; Tardieu, A.; Vachette, P.; Witz, J.; Delacroix, H. Acta Crystallogr., Sect. D 2001, 57, 1799. (21) Velev, O. D.; Kaler, E. W.; Lenhoff, A. M. Biophys. J. 1998, 75, 2682. (22) Galkin, O.; Chen, K.; Nagel, R. L.; Hirsch, R. E.; Vekilov, P. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 8479. (23) Asherie, N.; Lomakin, A.; Benedek, G. B. Phys. ReV. Lett. 1996, 77, 4832. (24) Petsev, D. N.; Wu, X.; Galkin, O.; Vekilov, P. G. J. Phys. Chem. B 2003, 107, 3921. (25) Shah, M.; Galkin, O.; Vekilov, P. G. J. Chem. Phys. 2004, 121, 7505. (26) Broide, M. L.; Tominc, T. M.; Saxowsky, M. D. Phys. ReV. E 1996, 53, 6325. (27) Muschol, M.; Rosenberger, F. J. Chem. Phys. 1997, 107, 1953. (28) Tardieu, A.; Verge, A. L.; Malfois, M.; Bonnette, F.; Finet, S.; Ries-Kaut, M.; Belloni, L. J. Cryst. Growth 1999, 196, 193. (29) Muschol, M.; Rosenberger, F. J. Chem. Phys. 1995, 103, 10424.

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J. Phys. Chem. B, Vol. 113, No. 20, 2009

(30) Leckband, D.; Israelachvili, J. Q. ReV. Biophys. 2001, 34, 105. (31) Petsev, D. N.; Vekilov, P. G. Phys. ReV. Lett. 2000, 84, 1339. (32) Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nat. Struct. Biol. 1997, 4, 1032. (33) Ghindilis, A. L.; Atanasov, P.; Wilkins, M.; Wilkins, E. Biosens. Bioelectron. 1998, 13, 113. (34) Sophianopoulos, A. J.; Rhodes, C. K.; Holcomb, D. N.; VanHolde, K. E. J. Biol. Chem. 1962, 237, 1107. (35) Yang, J.; Huang, Y.; Wang, X.-B.; Becker, F. F.; Gascoyne, P. R. C. Anal. Chem. 1999, 71, 911. (36) Guo, Y.; Guadalupe, A. R. Chem. Commun. 1997, 15, 1437. (37) Razumas, V.; Arnebrant, T. J. Electroanal. Chem. 1997, 427, 1. (38) Frankenthal, R. P.; Siconolfi, D. J. J. Electrochem. Soc. 1982, 129, 1192. (39) Frankenthal, R. P.; Thompson, D. E. J. Electrochem. Soc. 1976, 123, 799. (40) Oesch, U.; Janata, J. Electrochim. Acta 1983, 28, 1237. (41) Yamashita, M.; Wesson, L.; Eisenman, G.; Eisenberg, D. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 5648. (42) Pfeiffer, J. E. The Cell; Time-Life Books: New York, 1972. (43) Mesulam, M.-M. Tracing Neural Connections with Horseradish Peroxidase; John Wiley & Sons: New York, 1982. (44) Tsori, Y.; Tournilhac, F.; Leibler, L. Nature (London) 2004, 430, 544.

Shah et al. (45) Broide, M. L.; Berland, C. R.; Pande, J.; Ogun, O. O.; Benedek, G. B. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 5660. (46) Chen, Q.; Vekilov, P. G.; Nagel, R. L.; Hirsch, R. E. Biophys. J. 2004, 86, 1702. (47) Tanaka, S.; Ataka, M. J. Chem. Phys. 2002, 117, 3504. (48) Casselyn, M.; Tardieu, A.; Delacroix, H.; Finet, S. Biophys. J. 2004, 87, 2737. (49) Bloustine, J.; Virmani, T.; Thurston, G. M.; Fraden, S. Phys. ReV. Lett. 2006, 96, 087803. (50) Velev, O. D.; Prevo, B. G.; Bhatt, K. H. Nature (London) 2003, 426, 515. (51) Gliko, O.; Neumaier, N.; Pan, W.; Haase, I.; Fischer, M.; Bacher, A.; Weinkauf, S.; Vekilov, P. G. J. Am. Chem. Soc. 2005, 127, 3433. (52) Petsev, D. N.; Wu, X.; Galkin, O.; Vekilov, P. G. J. Phys. Chem. B 2003, 107, 3921. (53) Delincee, H.; Radola, B. J. Eur. J. Biochem. 1975, 52, 321. (54) Galkin, O.; Vekilov, P. G. J. Mol. Biol. 2004, 336, 43. (55) Galkin, O.; Nagel, R. L.; Vekilov, P. G. J. Mol. Biol. 2007, 365, 425. (56) Welinder, K. G.; Mauro, J. M.; Norskov-Lauritsen, L. Biochem. Soc. Trans. 1992, 20, 337. (57) Katsonis, P.; Brandon, S.; Vekilov, P. G. J. Phys. Chem. 2006, 110, 17638.

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