Electrochemical Quartz Crystal Nanobalance to Detect Solvent

The electrochemical quartz crystal nanobalance (EQCN) techniques of simultaneous measurements of frequency and cyclic voltammetry (CV) were used to ...
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Anal. Chem. 2004, 76, 5945-5952

Electrochemical Quartz Crystal Nanobalance to Detect Solvent Displacement by pH-Induced Conformational Changes of Proteins at Pt Nicholas P. Cosman and Sharon G. Roscoe*

Department of Chemistry, Acadia University Wolfville, Nova Scotia, (B4P 2R6) Canada

The electrochemical quartz crystal nanobalance (EQCN) techniques of simultaneous measurements of frequency and cyclic voltammetry (CV) were used to investigate protein adsorption behavior resulting from pH-induced conformational changes at the Pt electrode at 298 K. The adsorption behavior of holo- and apo-r-lactalbumin was studied in electrolyte solutions of pH < 2, 7.4, and 11. The EQCN frequency measurements did not directly monitor the mass of the adsorbed protein at anodic potentials, but instead, at a potential characteristic of the double layer for platinum, gave a measure of the extent of solvent displacement by the adsorbed protein (i.e., a “footprint”), which correlated well with known pHinduced conformational changes of the protein. Simultaneous CV charge transfer measurements provided information on the number of layers of protein adsorbed to the surface. This ability of the EQCN to detect solvent displacement by protein adsorption is potentially useful for biosensors to detect and to monitor protein conformational changes in the bulk and during the adsorption process. The Langmuir adsorption isotherm provided the Gibbs energy of adsorption, ∆GADS, and showed excellent agreement between the CV and EQCN frequency measurements. The interest over the years in the interfacial behavior of proteins with surfaces stems from the many important technological applications of protein interfacial behavior, such as the development of biosensors,1-5 solid supports for separation techniques,6,7 and medical implant devices.8-10 In addition, in the * To whom correspondence should be addressed. Phone: (902) 585-1156. Fax: (902) 585-1114. E-mail: [email protected]. (1) Ram, M. K.; Bertoncello, P.; Ding, H.; Paddeu, S.; Nicolini, C. Biosens. Bioelectron. 2001, 16, 849. (2) Saby, C.; Luong, J. H. T. Electroanalysis 1998, 10, 1193. (3) Olivia, H.; Sarada, B. V.; Honda, K.; Fujishima, A. Electrochim. Acta 2004, 49, 2069. (4) Quan, D.; Kim, Y.; Shin, W. J. Electoanal. Chem. 2004, 561, 181. (5) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikebukuro, K.; Karube, I. Anal. Chem. 2003, 75, 1116. (6) Puerta, A.; Jaulmes, A.; De Frutos, M.; Diez-Masa, J. C.; Vidal-Madjar, C. J. Chromatogr., A 2002, 953, 17. (7) Kandori, K.; Uoya, Y.; Ishikawa, T. J. Colloid Interface Sci. 2002, 252, 269. (8) Uniyal, S.; Brash, J. L.; Degterev, I. A. Adv. Chem. Ser. 1982, 199, 272. (9) Khan, M. A.; Williams, R. L.; Williams, D. F. Biomaterials 1996, 17, 2117. (10) Kanagaraja, S.; Lundstrom, I.; Nygren, H.; Tengvall, P. Biomaterials 1996, 17, 2225. 10.1021/ac049517+ CCC: $27.50 Published on Web 08/27/2004

© 2004 American Chemical Society

food and dairy industries, protein adsorption onto surfaces of processing equipment can lead to biofouling and contamination.11-16 This research reports for the first time the use of the electrochemical quartz crystal nanobalance as a tool (i.e., biosensor) to detect and monitor protein conformational changes in the adsorption of a model protein, holo- and apo-R-lactalbumin (R-LA), at the platinum electrode as a function of pH. This research is relevant to the use of platinum as a substrate, which is important in the fields of biosensors1-4 and array immunosensors.5 The R-LA molecule (Mr ) 14 174 g mol-1) is the second most prevalent whey protein in milk17 and consists of a single polypeptide chain with four disulfide bonds.18 It is a compact globular protein that binds the calcium ion in a 1:1 ratio at a specific binding site.19 A remarkable property of R-LA is the high stability of its molten globule (MG) state, which is a conformation generally defined as having a nativelike secondary structure and a more relaxed or disordered tertiary structure.20 This MG state has been extensively studied and is described as having a largely ordered R-helical domain and a more disordered β-sheet domain than the native protein.19 The hydrophobic core of the protein becomes more exposed and more hydrated in the MG state, which may be useful in allowing the protein to pass through membranes within the body.21,22 The volume of the MG state of R-LA is ∼10 to 20% larger than the volume of the native protein because of the relaxed side chains.19 This MG state has been known to occur in conditions of extreme pH, at increased temperatures, in the presence of mild denaturants, and upon removal of the calcium ion that is associated with the molecule.19 It has been postulated that the destabilization of the calcium-rich protein (holo-R-LA) upon release of the ion is the result of the unfavorable electrostatic interactions of charged groups in the calcium-binding loop.23 (11) Dejong, P. Trends Food Sci. Technol. 1997, 8, 401. (12) Changani, S. D.; Belmarbeiny, M. T.; Fryer, P. J. Exp. Therm. Fluid Sci. 1997, 14, 392. (13) Marshall, A. D.; Munro, P. A.; Tragardh, G. J. Membr. Sci. 2003, 217, 131. (14) James, B. Y.; Jing, Y.; Chen, X. D. J. Food Eng. 2003, 60, 431. (15) Velasco, C.; Ouammou, M.; Calvo, J. I.; Hernandez, A. J. Colloid Interface Sci. 2003, 266, 148. (16) Matzinos, P.; Alvarez, R. J. Membr. Sci. 2002, 208, 23. (17) Visser, J.; Jeurnink, T. J. M. Exp. Therm. Fluid Sci. 1997, 14, 407. (18) Morr, C. V.; Ha, E. Y. W. Crit. Rev. Food Sci. Nutr. 1993, 33, 431. (19) Kumajima, K. FASEB J. 1996, 10, 102. (20) Ptitsyn, O. B. In Protein Holding; Creighton, T. E., Ed.; Freeman: New York, 1992; pp 243-300. (21) Lala, A. K.; Kaul, P.; Bharata Ratnam, B. J. Protein Chem. 1995, 14, 601. (22) Banuelos, S.; Muga, A. Biochemistry 1996, 35, 3892. (23) Griko, Y. V.; Remeta, D. P. Protein Sci. 1999, 8, 554.

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Quartz crystal resonators were first introduced by Sauerbrey in 1959 for deposition rate monitoring of thin films in ultrahigh vacuum systems.24 The electrochemical quartz crystal nanobalance (EQCN) technique was created as an outcome of the discovery that quartz crystals oscillate at a specific frequency when immersed in a liquid phase on the basis of the converse piezoelectric effect.25 Application of an electric field across the crystal produces a shear strain proportional to the applied potential. By monitoring the frequency changes at various potentials, nanogram changes on the electrode surface can be determined, which enhance the information that can be obtained from processes occurring at the electrode/electrolyte interface. Although the use of the EQCN has been quite widespread, very few studies have been made on proteins to date,26-29 and none have used the technique to measure protein conformational changes. Hook et al.26 studied four model proteins (myoglobin, hemoglobin, human serum albumin (HSA), and ferritin) and one antibody-antigen reaction on a hydrophobic, methyl-terminated gold surface using the quartz crystal microbalance with dissipation technique (QCM-D); however, no electrochemical measurements were made. Glasmastar et al.27 also studied the adsorption of HSA along with several other proteins on supported phospholipid bilayers using the QCM-D technique. Murray et al.28 have used the QCM technique to study the fouling of chromium and hydrophobically modified gold surfaces when heated with β-lactoglobulin at a neutral pH. Xie et al.29 described a dual impedance analysis EQCM system to investigate bovine serum albumin adsorption onto Pt and Au electrode surfaces by connecting a suitable capacitance in series with the piezoelectric quartz crystal between the QCM impedance and EIS measurement instruments. Recently in our laboratory, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques used to investigate the adsorption behavior of wide variety of globular proteins at a platinum surface30-39 were found to be very sensitive to protein conformational changes due to temperature and pH changes in the bulk solution during the adsorption process. We have extended these studies to include EQCN measurements to examine the effect of protein molar mass on the frequency measurements.39,40 (24) Sauerbrey, G. Z. Phys. 1959, 155, 206. (25) Nomura, T.; Iijima, M. Anal. Chim. Acta 1981, 131, 97. (26) Hook, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729. (27) Glasmaster, K.; Larsson, C.; Hook, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40. (28) Murray, B. S.; Deshaires, C. J. Colloid Interface Sci. 2000, 227, 32. (29) Xie, Q.; Xiang, C.; Yuan, Y.; Zhang, Y.; Nie, L.; Yao, S. J. Colloid Interface Sci. 2003, 262, 107. (30) Roscoe, S. G. In Modern Aspects of Electrochemistry; Bockris, J. O’M., Conway, B. E., White, R. E., Eds.; Plenum Press: New York, 1996; Vol. 29, p 319. (31) Roscoe, S. G. J. Colloid Interface Sci. 2000, 228, 438. (32) MacDonald, S. M.; Roscoe, S. G. J. Colloid Interface Sci. 1996, 184, 449. (33) Hanrahan, K. L.; MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1996, 41, 2469. (34) Phillips, R. K. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2001, 17, 2471. (35) Phillips, R. K. R.; Omanovic, S.; Roscoe, S. G. Electrochem. Commun., 2000, 2, 805. (36) Cabilio, N.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16, 8480. (37) Roscoe, S. G.; Fuller, K. L.; Robitaille, G. J. Colloid Interface Sci. 1993, 160, 245. (38) Roscoe, S. G.; Fuller, K. L. J. Colloid Interface Sci. 1992, 152, 429. (39) Wright, J. E. I.; Cosman, N. P.; Fatih, K.; Omanovic, S.; Roscoe, S. G. J. Electroanal. Chem. 2004, 564, 185. (40) Cosman, N. P.; Roscoe, S. G. Langmuir 2004, 20, 1711.

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We now present an investigation, using holo-R-LA (with calcium) and apo-R-LA (without calcium) as the model proteins, which examines the applicability of the electrochemical quartz crystal nanobalance (EQCN) technique to detect, monitor, and measure solvent displacement resulting directly from adsorption of protein with pH-induced conformational changes. Since the CV and EQCN measurements provide data on both current density and frequency changes as a function of potential, the objectives were to compare the two techniques by obtaining information on (i) surface charge density due to protein adsorption at anodic potentials, (ii) detection and correlation of mass of solvent displaced by adsorbed protein in the double layer region, and (iii) comparison of the Gibbs energy of adsorption for the proteins. EXPERIMENTAL SECTION Reagents and Solutions. Stock solutions of bovine holo-Rlactalbumin (Sigma Chemical Co., L-5385, type I) and bovine apoR-lactalbumin (Sigma Chemical Co., L-6010, type III) were prepared by dissolving a massed amount of solid reagent in either a 0.05 M phosphate buffer solution prepared to give a solution with a specific pH, 7.4 or 11, or 0.05 M sulfuric acid solution (pH < 2). The phosphate buffer was previously prepared using a combination of monobasic, anhydrous KH2PO4 (Sigma Chemical Co.); dibasic, anhydrous K2HPO4; and 0.10 M sodium hydroxide (made from concentrated volumetric solution, ACP Chemical Inc.), and the sulfuric acid solution was made from Baker analyzed ultrapure concentrated sulfuric acid. Conductivity water (Nanopure, resistivity 18.2 MΩ cm) was used in the preparation of all aqueous solutions. Electrochemical Equipment. A two-compartment electrochemical quartz crystal nanobalance (EQCN) cell was used for all measurements. The main compartment contained both the working electrode, which was mounted in the holder and attached to the cell in a horizontal position, and the counter electrode. Attached to this main compartment was an auxiliary compartment that contained the reference electrode. Both the electrochemical cell and the holder for the working electrode were designed by G. Jerkiewicz (Queen’s University, ON, Canada) and constructed and purchased from the University of Sherbrooke (Sherbrooke, QC, Canada).41 Both compartments were purged with purified argon gas (Praxair Products Inc.) to stir as well as to remove oxygen from the solutions and cell. The working electrode was a 3000-Å Pt/500-Å Ti, AT-cut 9-MHz quartz crystal (International Crystal Manufacturing Co., Inc., Oklahoma City, OK), and the counter electrode was constructed of high-purity platinum with attached platinum mesh (99.99%, Johnson, Matthey and Mallory). A mercury|mercurous sulfate reference electrode was constructed in-house; however, all potentials in this paper are referred to the saturated calomel reference electrode (SCE). The EQCN crystal was sandwiched between two O-rings and attached to the Teflon holder with a stainless steel bracket and screws. It was important that the stress on the crystal was minimized as much as possible during the mounting procedure. Since the hydrostatic pressure in the cell has no influence on the resonant frequency of the quartz crystal, the height of the solution, (41) Jerkiewicz, G.; Vatankhah, G.; Zolfaghari, A.; Lessard, J. Electrochem. Commun. 1999, 1, 419.

although maintained at a constant height, could be changed with no required corrections.42 Only one side of the quartz crystal was exposed to the electrolyte solution, while the other side was exposed to the air and served only to complete the oscillator circuit. The entire electrochemical cell and oscillator circuit was shielded by a Faraday cage to reduce electromagnetic noise and stray capacitances. Experimental Methodology. The quartz crystal was first degreased in acetone for approximately 24 h and then thoroughly rinsed in Nanopure water. The Viton O-rings and quartz crystal holder were cleaned by first placing them in concentrated sulfuric acid for 20 min and then in mixed acid for 5 min. After being thoroughly rinsed with Nanopure water, the quartz crystal holder was securely mounted on the EQCN cell. The working electrode was then cleaned by potential cycling in 0.5 M H2SO4 until a reproducible cyclic voltammogram was obtained. The real surface area of the working electrode was then determined by measuring the charge for the hydrogen underpotential deposition (upd) peaks for reduction, and dividing this by the known charge for monolayer coverage of H adsorbed on platinum (210 µC cm-2).43 The EQCN cell was then emptied, rinsed thoroughly with Nanopure water, and replaced with a known amount of phosphate buffer. All measurements were carried out in an oxygen-free solution, which was achieved by continuous purging of the cell with argon gas (Praxair). Simultaneous cyclic voltammetry and frequency measurements were then made at a scan rate of 100 mV s-1 in a quiescent solution with phosphate buffer until a stable frequency response was obtained for at least 10 complete cycles. Following this, 10 cycles were made and recorded, and the frequency measurements were averaged. The anodic end potential was chosen such that the charge of the anodic oxidation region corresponded to a monolayer surface coverage. Aliquots of the protein of interest were then added to the phosphate buffer solution and allowed to mix and equilibrate by bubbling the solution with Ar for 5 min. It is important to note that all measurements were made in a quiescent solution because bubbling caused the frequency to become unstable. RESULTS AND DISCUSSION Cyclic Voltammetry Measurements. Figure 1a shows a typical set of cyclic voltammograms in phosphate buffer (pH 7.4) alone and in the presence of small amounts of holo-R-LA (calciumrich protein). Measurements were made only after identically reproducible tracings were recorded, indicating that the adsorbed protein layer is in equilibrium with the bulk solution. The adsorption of protein partially blocks the platinum surface from oxide growth in the anodic going sweep and, hence, results in a smaller oxide reduction in the cathodic going sweep. The surface charge density, QADS, was determined from the integrated areas of the cyclic voltammograms, corrected for the double layer, as described by eq 1

QADS ) [QPOo - QPOr] - [QOo - QOr]

(1)

where QOo (C cm-2) is the anodic oxidation charge density (due to oxide formation and phosphate ion adsorption), and QOr is the (42) Hepel, M. In Interfacial Electrochemistry: Theory, Experiment, and Applications; Wieckowski, A., Ed.; Plenum Press: New York, 1999; p 599.

Figure 1. (a) Cyclic voltammograms and (b) frequency response as a function of potential in phosphate buffer (pH 7.4, 298 K) for additions of holo-R-LA: s, 0 g L-1; - -, 3 × 10-3 g L-1; ‚‚‚‚, 8 × 10-3 g L-1; -‚-, 0.2 g L-1. The arrows indicate the direction of the potential sweep. Oo and Or denote anodic oxide formation and oxide reduction respectively.

oxide reduction charge density in a phosphate buffer solution (due only to oxide reduction), whereas QPOo is the anodic oxidation charge density in the presence of the protein (due to oxide formation, phosphate ion adsorption, and charge transfer from protein adsorption) and QPOr is the oxide reduction charge density in the presence of the protein (due only to oxide reduction).30 The mechanism for the electron transfer from the adsorbed protein at anodic potentials has been established on the basis of kinetic and product analyses of amino acid44-48 and protein30,31 adsorption studies. The first step in the mechanism involves an electrolyte-assisted quasi-reversible one-electrontransfer process at anodic potentials. At higher anodic potentials (i.e., >300 mV) decarboxylation occurs as the rate determining step, followed by a second electron-transfer process as the (43) Angerstein-Kozlowska, H. In Comprehensive Treatise of Electrochemistry; Yeager, E., Bockris, J. O’M., Eds.; Plenum Press: New York, 1984; Vol. 9, p 15. (44) Marangoni, D. G.; Smith, R. S.; Roscoe, S. G. Can. J. Chem. 1989, 67, 921. (45) Wylie, I. G. N.; Roscoe, S. G. Bioelectrochem. Bioenerg. 1992, 28, 367. (46) Petrie, R. J.; MacDonald, S. M.; Fuller, K. L.; Roscoe, S. G. Can. J. Chem. 1997, 75, 1585. (47) MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1997, 42, 1189. (48) Li, H.-Q.; Chen, A.; Roscoe, S. G.; Lipkowski, J. J. Electroanal. Chem. 2001, 500, 299.

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The adsorption of proteins onto platinum surfaces has been successfully described34-36 by the Langmuir adsorption isotherm given below in the linearized form,

c 1 c + ) Γ BADSΓmax Γmax

Figure 2. Surface charge density, QADS, as a function of concentration of holo-R-LA in phosphate buffer (pH 7.4) at 298 K. Inset: Linearized adsorption isotherm for holo-R-LA in phosphate buffer (pH 7.4) at 298 K using eq 4.

functional group is further oxidized to the next lower aldehyde in the homologous series. The formation of CO2 from adsorbed protein at the higher anodic potentials has been verified by in situ FT-IR measurements under electrochemical control.31 The physical presence of the protein at the electrode surface is sufficient to block oxide growth. This has been confirmed, not only by our analysis described above, but also by plots of ∆m versus QADS for oxide reduction over the potential range from 0.55 to -0.2 V (i.e., the returning cyclic voltammetry sweep) from which the molar mass of the species being measured by the EQCN may be determined. Analyses in the presence of the amino acid, phenylalanine,49 and a series of proteins39,40 consistently gave values in the range of 15-17 g mol-1 for the species being removed during the reduction process, which corresponds to Oads. Hence, cyclic voltammetry measurements of the electrolyte assisted charge-transfer process at anodic potentials provide a tool to quantify protein adsorption. Figure 2 shows the response in measured surface charge density with increase in concentration of holo-R-lactalbumin in the bulk solution at 298 K. The surface charge density rapidly increased with increasing bulk protein concentration to a plateau level, indicative of a saturated platinum surface under these experimental conditions. The solid line does not represent any attempt to model the data, but is shown to aid in visual presentation. From the electrochemical measurements of the surface charge density due to the adsorption of protein (QADS), the protein surface concentration, Γ (mg m-2), can be calculated using eq 2,

Γ)

QADSMr nF

(2)

where QADS (C cm-2) is the surface charge density due to protein adsorption, Mr (g mol-1) is the molar mass of the protein, n is the number of electrons transferred (2× the number of carboxylate groups on the protein30,31), and F (C mol-1) is the Faraday constant. (49) Wright, J. E. I.; Fatih, K.; Brosseau, C. L.; Omanovic, S.; Roscoe, S. G. J. Electoanal. Chem. 2003, 550-551, 41.

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(3)

where c (mol cm-3) is the equilibrium concentration of the protein in the bulk solution; Γ (mol cm-2) is protein surface concentration; Γmax (mol cm-2) is the maximum protein surface concentration; and BADS (cm3 mol-1) is the adsorption coefficient, which reflects the affinity of the protein molecules toward adsorption to the metal surface. Thus, if the Langmuir adsorption isotherm is valid for any system, a plot of c/Γ versus concentration, c, should yield a straight line with the parameters Γmax and BADS derived from the slope and intercept, respectively. Figure 2 (inset) provides an example of a linearized adsorption isotherm for holo-R-LA at pH 7.4, which shows that the Langmuir adsorption isotherm effectively describes the adsorption of holo-R-LA onto the platinum surface. This is consistent with our previously reported result,s32-40 which have shown proteins to exhibit Langmuir adsorption behavior over a wide range of temperature, including those that have been denatured by heat, causing agglomeration in the bulk solution, and that subsequently appear to adsorb as a monolayer of agglomerates onto the surface.33,37 We have shown that cyclic voltammetry measurements of adsorbed proteins follow the association/denaturation behavior of the molecules in the bulk solution.32,33,37 In addition, protein adsorption behavior under equilibrium conditions sometimes reveals a dynamic rearrangement of the adsorbed species on the surface as a function of increasing bulk concentration of protein.36 Although there will presumably be some interaction among the adsorbed species on the surface, the driving force for the adsorption process is clearly the affinity of these species (i.e., monomers or aggregates) for the electrode surface. The parameter BADS, which reflects the affinity of the absorbed molecules for the metal surface at a constant temperature, can be represented as

BADS )

(

)

- ∆GADS 1 exp csolvent RT

(4)

where R (J mol-1 K-1) is the gas constant; T (K) is the temperature; csolvent is the molar concentration of the solvent, in the present case, water (cwater ) 55.5 mol dm-3); and ∆GADS (J mol-1) is the Gibbs energy of adsorption. Using eq 4, the Gibbs energy of adsorption was calculated for holo- and apo-R-LA and is discussed later. Frequency Measurements. A typical experiment with the electrochemical quartz crystal nanobalance (EQCN) simultaneously measures the current density (j) and frequency response (∆f) to an applied potential (V) at a sweep rate of 100 mV s-1 in a quiescent solution after bubbling with argon to remove oxygen and provide a well-mixed solution. Thus, using the EQCN, both cyclic voltammetry (CV) (Figure 1a) and frequency measurements (Figure 1b) were obtained for each incremental addition of each protein studied.

The Sauerbrey equation was used to convert the frequency response to mass changes as follows:24

[ ]

f02 ∆m ) - Cf∆m ∆f ) NFqRf

(5)

where ∆f (MHz) is the variation in frequency, f0 is the frequency of the fundamental mode (8.9 MHz), N is the frequency constant (167 kHz cm), Fq is the density of quartz (2.648 g cm-3), Rf is the ratio of the real surface area to geometric surface area (i.e., surface roughness factor) of the electrode, ∆m is the variation of mass per unit area in g cm-2, and Cf is the sensitivity factor (C-1 ) f ∼5.56 ng cm-2 Hz -1 × Rf). The Cf value was calculated for each experiment using the measured f0 for the buffer solution and the real surface area of the electrode, Rf, measured from the hydrogen reduction peak in a 0.5 M sulfuric acid solution. Figure 1b shows for the phosphate buffer that as the potential is swept to higher anodic potentials, there is a decrease in the change in frequency (∆f) of the quartz crystal due mainly to oxide growth on the platinum surface. This phenomenon is in accordance with the Sauerbrey equation (eq 5), in that a decrease in the frequency change corresponds to an increase in the mass of species adsorbed to the surface. Subsequently, in the cathodic sweep, there is an increase in the frequency change as oxide is reduced and desorbed from the metal surface. Additions of incremental amounts of protein to the phosphate buffer result in a shift in the frequency change of the quartz crystal to more positive values, as shown in Figure 1b. The data were normalized by setting the frequency change, which fluctuated randomly with each protein addition, at the same value obtained with the phosphate buffer alone at -0.6 V, which represented the lowest potential used before the onset of hydrogen evolution, in accordance with the method described by Jerkiewicz et al.41 At these very low potentials, it is assumed that the molecules have desorbed, because potential cycling of a protein fouled platinum electrode transferred to a clean phosphate buffer solution showed the platinum electrode to be rapidly cleaned as a result of protein desorption.50 Since the frequency change over the anodic potential range increases with increasing protein in the bulk solution, this implies a decrease rather than an increase in the recorded mass on the electrode surface. Previous studies in our laboratory on the amino acid phenylalanine (Phe)49 and a series of proteins40 have shown that the EQCN technique does not directly monitor the change in mass of the adsorbed amino acid or protein at the platinum electrode but, instead, directly measures oxide growth during the anodic sweep and oxide removal by reduction during the cathodic sweep. The adsorption of amino acid was determined indirectly by the ability of the adsorbed Phe to block oxide growth. It is also important to note that it has been very recently established that the oxide growth involves direct adsorption of O (i.e., Oads) rather than a two-step adsorption process with OH species.51 Since the EQCN measurements did not appear to directly monitor the adsorption of protein onto the platinum surface during (50) Fuller, K. L.; Roscoe, S. G. In Food Proteins: Structure-Function Relationships; Yada, R. Y.; Jackman, R. L.; Smith, J. L., Eds.; Blackie Academic and Professional: London, 1994; pp 143-162. (51) Zolfaghari, A.; Conway, B. E.; Jerkiewicz, G. Electrochim. Acta 2002, 47, 1173.

Figure 3. Plot of the absolute change in mass of solvent molecules displaced as a function of the concentration of holo-R-LA in the bulk solution at pH 7.4 and 298 K. Inset: Linearized, adsorption isotherm for holo-R-LA at pH 7.4 and 298 K using frequency measurements from the EQCN technique and eq 7.

the potential sweep in the anodic region, the mean value for the frequency change for the forward (anodic going) and reverse (cathodic going) scans was determined at -0.20 V, which represents the onset of the double layer region for the anodic going sweep. The double layer (DL) region represents a potential region for the Pt electrode where neither hydrogen nor oxide species are specifically adsorbed on the surface. However, it was apparent that the ∆f values recorded in the double layer region changed with each incremental addition of protein (Figure 1b). The mean value for the frequency change at -0.20 V in phosphate buffer was then subtracted from each similar measurement in the presence of protein. The change in mass (∆m) was then calculated using the Sauerbrey equation. It is clear from Figure 1b that the frequency change at -0.20 V increases with incremental increase in protein to the bulk solution, indicating that a decrease in mass is being detected. This would, therefore, represent a direct measure of displaced solvent molecules in the double layer region by the presence of protein. This result is not unexpected because the electrosorption process is known to displace strongly adsorbed solvent molecules from the metal surface.52 Thus, if solvent molecules are strongly adsorbed, displacement by weakly adsorbed (i.e., physisorbed) species results in an increase in the frequency response (decrease in mass) of the quartz crystal nanobalance. A plot of the absolute value of the change in mass, |∆m|, versus the concentration of protein in the bulk solution (Figure 3) shows similar behavior to the results obtained by cyclic voltammetry (Figure 2). The data were again treated using the Langmuir adsorption isotherm in a manner similar to the cyclic voltammetry results. The absolute change in mass, |∆m|, obtained from the frequency measurements was used under the assumption that the direct measure of solvent displacement is an indirect measure of protein surface concentration. Thus, by substituting |∆m| for Γ in eq 4, the following relationship is obtained.

1 c c ) + ∆m BADS∆mmax ∆mmax

(6)

Consequently, a plot of c/|∆m| versus concentration should yield Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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Table 1. Comparison of Surface Concentrations (Γmax) for Proteins at a Pt Electrode from Solutions of Various pH at 298 K Calculated from CV and EQCN Frequency Measurements Γmax for holo-R-LA CVa

pH