Langmuir 2004, 20, 1711-1720
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Electrochemical Quartz Crystal Nanobalance (EQCN) Studies of Protein Interfacial Behavior at Pt Nicholas P. Cosman and Sharon G. Roscoe* Department of Chemistry, Acadia University Wolfville, Nova Scotia, (B4P 2R6) Canada Received June 27, 2003. In Final Form: December 16, 2003 The interfacial behavior of a variety of proteins ranging in molar mass from 2500 to 340 000 g mol-1 was studied at a Pt electrode surface at 298 K using the electrochemical quartz crystal nanobalance (EQCN) technique of simultaneous measurements of frequency and cyclic voltammetry (CV). It was shown that the EQCN frequency measurements did not directly monitor the molar mass of the adsorbed protein at anodic potentials but instead measured changes in the surface oxide in the absence and presence of adsorbed protein. However, at a potential characteristic of the double layer for platinum, EQCN frequency measurements gave a measure of the extent of solvent displacement by the adsorbed protein. The values for the Gibbs free energy of adsorption, ∆GADS, obtained with these EQCN frequency measurements gave excellent agreement within experimental uncertainty with those obtained from the simultaneous CV measurements for all proteins studied. The adsorption process was modeled using the Langmuir adsorption isotherm. The smallest molecules studied, chain A and chain B of insulin, have the lowest affinity for the platinum surface as indicated by their small negative ∆GADS values, while the larger proteins, such as fibrinogen, have the greatest affinity for the platinum surface.
Introduction There has been considerable interest over the years in the interfacial behavior of proteins, particularly in terms of their interactions with surfaces. This interest stems from the many important technological applications of protein interfacial behavior such as the development of biosensors, solid supports for separation techniques, and medical implant devices.1-3 In addition, there is an immense cost related to the fouling of surfaces by protein adsorption in a variety of industries including the food industry, where the heating of biological fluids often causes deposition (fouling) on the processing equipment.4,5 Fundamental studies provide a basis for investigating the features that govern this phenomenon of protein adsorption to surfaces. The mechanism for protein adsorption have been investigated by several researchers over the years, including Andrade,6-9 Hlady,6-8 Brash,10-13 * To whom correspondence should be addressed. Phone: (902) 585-1156; fax: (902) 585-1114; e-mail:
[email protected]. (1) Uniyal, S.; Brash, J. L.; Degterev, I. A. Adv. Chem. Ser. 1982, 199, 272. (2) Khan, M. A.; Williams, R. L.; Williams, D. F. Biomaterials 1996, 17, 2117. (3) Kanagaraja, S.; Lundstrom, I.; Nygren, H.; Tengvall, P. Biomaterials 1996, 17, 2225. (4) Dejong, P. Trends Food Sci. Technol. 1997, 8, 401. (5) Changani, S. D.; Belmarbeiny, M. T.; Fryer, P. J. Exp. Thermal Fluid Sci. 1997, 14, 392. (6) Andrade, J. D.; Hlady, V.; Wei, A. P.; Ho, C. H.; Lea, A. S.; Jeon, S. I.; Lin, Y. S.; Stroup, E. Clin. Mater. 1992, 11, 67. (7) Andrade, J. D.; Hlady, V.; Wei, A. P. Pure Appl. Chem. 1992, 64, 1777. (8) Andrade, J. D.; Hlady, V.; Jeon, S.-I. Adv. Chem. Ser. 1996, 248, 51. (9) Andrade, J. D. J. Intelligent Mater. Syst. Struct. 1994, 5, 612. (10) Brash, J. L.; ten Hove, P. J. Biomater. Sci. Polym. Ed. 1993, 4, 591. (11) Du, Y. J.; Cornelius, R. M.; Brash, J. L. Colloids Surf., B 2000, 17, 59. (12) Hitchcock, A. P.; Morin, C.; Heng, Y. M.; Cornelius, R. M.; Brash, J. L. J. Biomater. Sci. Polym. Ed. 2002, 13, 919. (13) Brash, J. L. In Modern Aspects of Protein Adsorption on Biomaterials; Missirlis, Y. F., Lemm, W., Eds.; Kluwer: Dordrecht, The Netherlands, 1991; 39. (14) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313.
Norde,14-22 Lyklema,15,23 Horbett,24-26 and Vroman.27,28 The present study examines the use of the electrochemical quartz crystal nanobalance to examine protein adsorption under the influence of an electric field at a polycrystalline Pt surface. Several experimental techniques have been used to study protein adsorption at solid surfaces. More recent methods include scanning transmission X-ray microscopy (STXM),12 infrared reflection spectroscopy,29 neutron reflectivity,30-33 total internal reflection fluorescence (TIRF),32,34 optical waveguide lightmode spectroscopy (OWLS),35 surface MALDI-ToF mass spectrometry,36 (15) Norde, W.; Lyklema, J. J. Biomater. Sci. Polym. Ed. 1991, 2, 183. (16) Norde, W. J. Dispersion Sci. Technol. 1992, 13, 363. (17) Norde, W. Pure Appl. Chem. 1994, 66, 491. (18) Haynes, C. A.; Norde, W. Colloids Surf., B 1994, 2, 517. (19) Norde, W. Cells Mater. 1995, 5, 97. (20) Norde, W.; Haynes, C. A. Proteins at Interfaces 2; ACS Symposium Series 602; American Chemical Society; Washington, DC, 1995; p 26. (21) Physical Chemistry of Biological Interfaces; Baszkin, A., Norde, W., Eds.; Dekker: New York, 2000. (22) Norde, W. Biopolymers at Interfaces, 2nd ed.; Surfactant Science Series 110; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; p 21. (23) Lyklema, J. In Physical Chemistry of Biological Interfaces; Baszkin, A., Norde, W., Eds.; Marcel Dekker: New York, 2000; p 1. (24) Wagner, M. S.; Horbett, T. A.; Castner, D. G. Langmuir 2003, 19, 1708. (25) Wagner, M. S.; Horbett, T. A.; Castner, David G. Biomaterials 2003, 24, 1897. (26) Wagner, M. S.; Shen, M.; Horbett, T. A.; Castner, D. G. Appl. Surf. Sci. 2003, 203-204, 704. (27) Vroman, L.; Adams, A. L. Proteins Interfaces; ACS Symposium Series 343; Amercian Chemical Society: Washington, DC, 1987; p 154. (28) Leonard, E. F.; Vroman, L. J. Biomater. Sci., Polym. Ed. 1991, 3, 95. (29) Ball, A.; Jones, R. A. L. Langmuir 1995, 11, 3542. (30) Su, T. J.; Lu, J. R.; Cui, Z. F.; Thomas, R. K.; Heenan, R. K. Langmuir 1998, 14, 5517. (31) Lu, J. R.; Su, T. J.; Thomas, R. K.; Cui, Z. F.; Penfold, J.; Webster, J. J. Chem. Soc., Faraday Trans. 1998, 94, 3279. (32) Petrash, S.; Cregger, T.; Zhao, B.; Pokidysheva, E.; Foster, M. D.; Brittian, W. D.; Sevastianov, V.; Majkrzak, C. F. Langmuir 2001, 17, 7645. (33) Nylander, T.; Tiberg, F.; Su, T.-J.; Lu, J. R.; Thomas, R. K. Biomacromolecules 2001, 2, 278. (34) Wertz, C. F.; Santore, M. M. Langmuir 2002, 18, 1190. (35) Brusatori, M. A.; Tie, Y.; Van Tassel, P. R. Langmuir 2003, 19, 5089.
10.1021/la035154h CCC: $27.50 © 2004 American Chemical Society Published on Web 02/04/2004
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time-of-flight secondary ion mass spectrometry (ToFSIMS),24-26,37 electron spectroscopy for chemical analysis (ESCA),24-26 radiolabeling,11,24-26,38 differential scanning calorimetry (DSC),39,40 surface plasmon resonance (SPR),41 ellipsometry,41,38 ELISA,42,43 STM,44 X-ray photoelectron spectroscopy (XPS),45 and atomic force microscopy (AFM).45 Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques have been used recently in our laboratory to investigate the adsorption behavior of many globular proteins such as β-lactoglobulin,46-48 ribonuclease,47-49 lysozyme,47-48 R-lactalbumin,50,51 κ-casein,52 bovine serum albumin (BSA),51 insulin,53,54 cytochrome c,55 myoglobin,55 hemoglobin,55 and yeast alcohol dehydrogenase56 at a platinum surface. Results from these studies have found that interactions between proteins and the electrode surfaces, resulting in adsorption, were affected by several factors including temperature, pH, ionic strength, conformation of the protein in solution and its bulk concentration, as well as the surface characteristics of the material onto which adsorption occurs, consistent with the existing literature.6-28 The electrochemical techniques developed for studying protein adsorption were very sensitive to protein conformational changes in the bulk solution during the adsorption process.46-56 This research is relevant to the use of platinum as a substrate which is important in the fields of biosensors57,58 and array immunosensors.59 Quartz crystal resonators were first introduced by Sauerbrey in 1959 for deposition rate monitoring of thin films in ultrahigh vacuum systems.60 The electrochemical quartz crystal nanobalance (EQCN) technique was created as an outcome of the discovery that quartz crystals oscillate (36) Ademovic, Z.; Klee, D.; Kingshott, P.; Kaufmann, R.; Hocker, H. Biomolecular Eng. 2002, 19, 177. (37) Poleunis, C.; Rubio, C.; Compere, C.; Bertrand, P. Appl. Surf. Sci. 2003, 203-204, 693. (38) Nasir, A.; McGuire, J. Food Hydrocolloids 1998, 12, 95. (39) Larsericsdotter, H.; Oscarsson, S.; Buijs J. J. Colloid Interface Sci. 2001, 237, 98. (40) Vermonden, T.; Giacomelli, C. E.; Norde, W. Langmuir 2001, 17, 3734. (41) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796. (42) Weber, N.; Wendel, H. P.; Ziemer, G. Biomaterials 2002, 23, 429. (43) Higuchi, A.; Sugiyama, K.; Yoon, B. O.; Sakurai, M.; Hara, M.; Sumita, M.; Sugawara, S.; Shirai, T. Biomaterials 2003, 24, 3235. (44) Friis, E. P.; Andersen, J. E. T.; Madsen, L. L.; Moller, P., Nichols, R. J., Olesen, K. G.; Ulstrup, J. Electrochim. Acta 1998, 43, 2889. (45) Caruso, F.; Furlong, D. N.; Kingshott, P. J. Colloid Interface Sci. 1997, 186, 129. (46) Roscoe, S. G.; Fuller, K. L.; Robitaille, G. J. Colloid Interface Sci. 1993, 160, 245. (47) Roscoe, S. G.; Fuller, K. L. J. Colloid Interface Sci. 1992, 152, 429. (48) 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. (49) Roscoe, S. G. J. Colloid Interface Sci. 2000, 228, 438. (50) Cabilio, N.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16, 8480. (51) Rouhana, R.; Budge S. M.; Roscoe, S. G. Food Res. Int. 1997, 30, 303. (52) Roscoe, S. G.; Fuller, K. L. Food Res. Int. 1993, 26, 343. (53) Wright, J. E. I.; Cosman, N. P.; Fatih, K.; Omanovic, S.; Roscoe, S. G. J. Electroanalytical Chem. in press. (54) MacDonald, S. M.; Roscoe, S. G. J. Colloid Interface Sci. 1996, 184, 449. (55) Hanrahan, K. L.; MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1996, 41, 2469. Hanrahan, K. L. Honours Chemistry Thesis, Acadia University, 1995. (56) Phillips, R. K. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2001, 17, 2471. (57) Ram, M. K.; Bertoncello, P.; Ding, H.; Paddeu, S.; Nicolini, C. Biosens. Bioelectron. 2001, 16, 849. (58) Saby, C.; Luong, J. H. T. Electroanalysis 1998, 10, 1193. (59) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikebukuro, K.; Karube, I. Anal. Chem. 2003, 75, 1116. (60) Sauerbrey, G. Z. Phys. 1959, 155, 206.
Cosman and Roscoe
at a specific frequency when immersed in a liquid phase.61 These researchers used the EQCN to determine the mass of Cu(II) and Ag(I) during electrodeposition.61 Since its discovery, the EQCN has emerged as a very powerful in situ technique to complement other electrochemical techniques. The attractiveness of this technique is based on modern possibilities to measure radio frequencies with very high resolution and accuracy.60 The operation of the EQCN is based on the converse piezoelectric effect. Application of an electric field across the crystal produces a shear strain proportional to the applied potential.61 The deformation is elastic for quartz and quartz covered with a very thin rigid film. Therefore, when films are present on the crystal, assumptions can be made that the acoustic properties of the film are identical to those of the quartz. The commonly used coatings deposited on the quartz crystal include Ti, Cr, or Si (50 nm) which behaves as an adhesive for deposited layers (300 nm) of Pt or Au. The EQCN technique offers complementary information to other electrochemical techniques. 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. Hook et al.62 studied four model proteins (myoglobin, hemoglobin, human serum albumin (HSA), and ferritin) and one antibody-antigen reaction on a hydrophobic, methylterminated gold surface using the quartz crystal microbalance with dissipation technique (QCM-D); however, no electrochemical measurements were made. Glasmastar et al.63 also studied the adsorption of HSA along with several other proteins on supported phospholipid bilayers using the QCM-D technique. The adsorption of the smallest amino acid, glycine, has been studied by Lori et al.64 on gold and titanium surfaces using the electrochemical quartz crystal nanobalance (EQCN). These researchers monitored changes in mass and potential electrochemically before and after glycine was injected. Murray et al.65 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. Finally, previous studies in our laboratory investigated the adsorption of the amino acid phenylalanine (Phe) on platinum using EQCN measurements along with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurememnts.66 The present investigation was carried out to examine the applicability of the electrochemical quartz crystal nanobalance (EQCN) technique for the measurement of protein adsorption onto a platinum electrode. The EQCN measurements provided data on both current density and frequency changes as a function of potential. The objectives were to obtain information on the (i) mass and charge of oxide growth and reduction in the presence and absence of protein, (ii) charge density due to protein adsorption at anodic potentials, (iii) mass of solvent displaced by adsorbed protein in the double layer region, and (iv) Gibbs free energy of adsorption for the proteins. A series of (61) Nomura, T.; Iijima, M. Anal. Chim. Acta 1981, 131, 97. (62) Hook, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729. (63) Glasmaster, K.; Larsson, C.; Hook, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40. (64) Lori, J. A.; Hanawa, T. Corrosion Sci. 2001, 43, 2111. (65) Murray, B. S.; Deshaires, C. J. Colloid Interface Sci. 2000, 227, 32. (66) Wright, J. E. I.; Fatih, K.; Brosseau, C. L.; Omanovic, S.; Roscoe, S. G. J. Electoanalytical Chem. 2003, 550-551, 41.
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proteins were chosen to examine the effect of protein molar mass on the EQCN response. Experimental Section Reagents and Solutions. Stock solutions of bovine insulin (Sigma Chemical Co., P-8324), bovine chain A of insulin (Sigma Chemical Co., I-1633), and bovine chain B of insulin (Sigma Chemical Co., I-6383) were prepared by dissolving reagents in 0.05 M phosphate buffer (pH 7.0). Stock solutions of bovine holoR-lactalbumin (Sigma Chemical Co., L-5385, type I), bovine apoR-lactalbumin (Sigma Chemical Co., L-6010, type III), bovine β-lactoglobulin A (Sigma Chemical Co., L-7880), and β-casein (Sigma Chemical Co., C-6905), horse heart myoglobin (Sigma Chemical Co., M-1882), bovine serum albumin (Sigma Chemical Co., A-6793), and bovine fibrinogen (Sigma Chemical Co., F-8630) were prepared by dissolving the reagents in 0.05 M phosphate buffer (pH 7.4). 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.). Sulfuric acid solution (0.5 M) was made from standardized Baker analyzed ultrapure concentrated sulfuric acid and used only to clean the electrodes and to determine the real surface area of the platinum electrode in the absence of protein as described below. 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 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). 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, although maintained at a constant height, could be changed with no required corrections.67 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
Cyclic Voltammetry Measurements. Figure 1a shows a typical set of cyclic voltammograms in phosphate buffer alone and in the presence of small amounts of protein, in this case myoglobin. The adsorption of protein partially blocks the platinum surface from oxide growth
(67) Hepel, M. In Interfacial Electrochemistry: Theory, Experiment, and Applications; Wieckowski, A., Ed.; Plenum Press: New York, 1999; p 599.
(68) Angerstein-Kozlowska, H. In Comprehensive Treatise of Electrochemistry; Yeager, E., Bockris, J. O’M., Eds.; Plenum Press: New York, 1984; Vol. 9, p 15.
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 myoglobin: s, 0 g L-1; - -, 1 × 10-3 g L-1; ‚ ‚ ‚ ‚, 5 × 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. for reduction and dividing this by the known charge for monolayer coverage of H adsorbed on platinum (210 µC cm-2).68 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 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. All measurements were made in a quiescent solution as bubbling caused the frequency to become unstable. The reproducibility of the measurements was determined from triplicate experiments.
Results and Discussion
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similar trend was obtained for all proteins investigated at a neutral pH. 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 times the number of carboxylate groups on the protein), and F (C mol-1) is the Faraday constant. The adsorption of proteins onto platinum surfaces has been successfully described50 by the Langmuir adsorption isotherm given below: Figure 2. Surface charge density, QADS, as a function of concentration in phosphate buffer (pH 7.4) at 298 K of b, myoglobin; o, β-casein. Inset: Linearized adsorption isotherm in phosphate buffer (pH 7.4) at 298 K using eq 4 for b, myoglobin; o, β-casein.
in the anodic going sweep and, subsequently, 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 ) [QOoP - QOrP] - [QOo - QOr]
(1)
-2
where QOo (C cm ) is the anodic oxidation charge density and QOr is the oxide reduction charge density in a phosphate buffer solution, while QOoP is the anodic oxidation charge density and QOrP is the oxide reduction charge density in the presence of the protein. It has been shown from previous work in our laboratory that electron transfer between the protein and the platinum surface occurs through the carboxylate groups on the acidic residues of the protein.46-56 The initial step of adsorption involves an electron-transfer process as follows:
P + nM f P(M)n + ne where P represents the protein, M represents the metal electrode, and n represents the number of sites of carboxylate interactions with the metal surface. The second step involves decarboxylation, which is the ratelimiting step:46,49
P(M)n f P′(M)n + nCO2 where P′ is the modified protein. From the number of carboxylate groups on the protein (number of acidic residues plus the C-terminal), the number of electrons transferred can be determined. The electron-transfer process appears also to be an electrolyte-assisted mechanism in the high anodic potential fields and hence does not require all the carboxylate groups to physically contact the electrode surface. Figure 2 shows the response in measured surface charge density with increase in concentration of myoglobin and β-casein 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. A
Γ)
BADSΓmax c 1 + BADSc
(3)
where c (mg mL-1) is the equilibrium concentration of the protein in the bulk solution, Γ (mg m-2) is protein surface concentration, Γmax (mg m-2) is the maximum protein surface concentration, and BADS (mL mg-1) is the adsorption coefficient, which reflects the affinity of the protein molecules toward adsorption to the metal surface. Equation 3 can be rearranged to give
1 c c ) + Γ BADSΓmax Γmax
(4)
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 myoglobin and β-casein, which shows that the Langmuir adsorption isotherm effectively describes the adsorption of these proteins onto the platinum surface. Similar results were obtained for all the proteins investigated. 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
(5)
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 our case water (cwater ) 55.5 mol dm-3), and ∆GADS (J mol-1) is the Gibbs free energy of adsorption. Using eq 5, the Gibbs free energy of adsorption was calculated for each protein and is shown in Table 1. For each protein studied, the ∆GADS values obtained using cyclic voltammetry at a sweep rate of 100 mV s-1 are within experimental uncertainty with previous results from our laboratory also obtained with CV measurements, but in a differently configured electrochemical cell, a Pt wire polycrystalline electrode rather than a Pt coated quartz crystal electrode, and at a higher sweep rate of 500 mV s-1. The higher sweep rate in previous investigations in our laboratory was chosen to avoid the problems associated with mass diffusion of protein from the bulk solution. In the present measurements, a slower sweep rate was required since the frequency measurements were recorded
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Table 1. Comparison of Gibbs Free Energy of Adsorption Values Obtained from EQCN Cyclic Voltammetry and Frequency Measurements on a Pt Electrode at 298 K and pH 7.4 ∆GADS/kJ mol-1 protein
Mr g
mol-1
EQCN cyclic voltammetrya ( 1 kJ mol-1
EQCN frequency ( 3 kJ mol-1
cyclic voltammetryb ( 1 kJ mol-1
chain An chain Bn insulinn holo-R-lactalbumin apo-R-lactalbumin
2531.6 3495.9 5733.5 14 174 14 174
-39 [53] -41 [53] -44 [53] -46 -49
-41 [53] -42 [53] -45 [53] -48 -50
-39 [53] -42 [53] -44 [53] -47 [50] -49 [50]
myoglobin β-lactoglobulin
16 952 18 328
-47 -46
-49 -51
-48 [55] -48 [46]
β-casein BSA
23 980 66 267
-47 -48
-50 -51
-48 [51]
340 000
-51
-50
-50 [69]
fibrinogen
literature values -40 [53]c -44 [53]c -43 [53]c -55 [70]d -39[71]e -40 [72]f -32 [73]g -43 [74]d -43 [75]h -33 [76]i -55 [70]d -57 [77]d -59 [78]j -48 [79]k -40 [79]l -54 [79]l -52 [80]m -52 to -55 [81]m
a Cyclic voltammetry measurements at Pt layer on quartz at sweep rate of 100 mV s-1 in phosphate buffer. b Cyclic voltammetry measurements at Pt polycrystalline wire at sweep rate of 500 mV s-1 in phosphate buffer. c Electrochemical impedance spectroscopy measurements at Pt in phosphate buffer pH 7.4 at 299 K. d Electrochemical impedance spectroscopy at stainless steel in phosphate buffer pH 7.0 at 299 K. e Ellipsometry measurements at a hydrophilic silicon surface from phosphate buffer pH 7.0 and 300 K (calculated from data in ref). f Solution depletion measurements using 304 stainless steel at pH 6.0 and 289 K (calculated from data in ref). g UV spectrophotometry applying Bradford’s protein assay methods for adsorption to synthetic hydroxyapatite in phosphate buffer solution. h Ellipsometry measurements on hydrophobic silicon surface in 0.1 M phosphate buffer pH 6.6 (calculated from data in ref). i Solutiondepletion method using stainless steel particles in 0.1 M NaCl at pH 6.85, 298 K (calculated from data in ref). j Radiolabeling and solution depletion measurements using 304 stainless steel in 0.05 M HEPES in 3% NaCl pH 7.5 at 298 K. k Cyclic voltammetry measurements at (cp) titanium in phosphate buffer pH 7.4 at 299 K. l Cyclic voltammetry measurements at (cp) titanium in phosphate buffer pH 7.4 (-40 at 295 K, -54 at 310 K). m Radiolabeling with 125I, on hydrophilic glass and various polyurethane surfaces (calculated from data in ref). n Indicates proteins studied at a pH of 7.0 for comparison with literature values.
simultaneously and 100 mV s-1 was the upper limit for reliable frequency measurements to be obtained. It was therefore of interest to compare the CV results for the two sweep rates. From Table 1 it is apparent that the present results for the ∆GADS values are in excellent agreement with results previously obtained at the higher sweep rates. In addition, these values are comparable with results reported by other investigators using a variety of surfaces and different experimental conditions. Table 2 shows the values for surface concentration, Γmax, from the cyclic voltammetry measurements at 100 mV s-1 made simultaneously with the EQCN frequency measurements. These values, which will be discussed later, are also compared with previous results from our laboratory using a Pt wire with a sweep rate of 500 mV s-1 as described above. The results show that in almost all cases (β-lactoglobulin and myoglobin being the exception), the surface concentration values were greater from cyclic voltammetry measurements using the slower sweep rate of 100 mV s-1, because of a greater charge transfer at the electrode surface. This suggests that mass transport enables a larger accumulation of protein at the surface during the slower sweep rate, compared to the higher sweep rate which measures only protein already located (69) Farcas, M.; Cosman, N. P.; Roscoe, S. G.; Omanovic, S. manuscript in preparation. (70) Cosman, N.; Fatih, K.; Roscoe, S. G. manuscript in preparation. (71) Suttiprasit, P.; McGuire, J. J. Colloid Interface Sci. 1992, 154, 327. (72) Adesso, A.; Lund, D. B. J. Food Process. Preserv. 1997, 21, 319. (73) Noshi, H. Shika Igaku 1986, 49, 531. (74) Omanovic, S.; Roscoe, S. G. J. Colloid Interface Sci. 2000, 227, 452. (75) Luey, J.-K.; McGuire, J.; Sproull, R. D. J. Colloid Interface Sci. 1991, 143, 489.
at the surface in the double layer region, essentially independent of mass transport from the bulk solution. Measurements of protein adsorption as a function of sweep rate in our laboratory have shown for a given bulk concentration, a sweep rate of 500 mV s-1 provides the limiting maximum surface concentration. 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. 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:60
[ ]
∆f ) -
fo2 ∆m ) -Cf∆m NFqRf
(6)
where ∆f (MHz) is the variation in frequency, f0 the frequency of the fundamental mode (8.9 MHz), N the frequency constant (167 kHz cm), Fq the density of quartz (2.648 g cm-3), Rf the ratio of the real surface area to (76) Itoh, H.; Nagata, A.; Toyomasu, T.; Sakiyama, T.; Nagai, T.; Saeki, T.; Nakanishi, K. Biosci. Biotechnol. Biochem. 1995, 59, 1648. (77) Omanovic, S.; Roscoe, S. G. Langmuir 1999, 15, 8315. (78) Hansen, D. C.; Luther, G. W., III.; Waite, J. H. J. Colloid Interface Sci. 1994, 168, 206. (79) Jackson, D. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16, 5449. (80) Leduc, C.; ten Hove, P.; Park, S.; Vroman, L.; Brash, J. L.; Leonard, E. F. J. Biomater. Sci. Polym. Ed. 1995, 7, 531. (81) Santerre, J. P.; ten Hove, P. Brash, J. L. J. Biomed. Mater. Res. 1992, 26, 1003.
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Cosman and Roscoe
Table 2. Comparison of Surface Concentrations (ΓMax) Calculated from EQCN Frequency and CV Measurements for Proteins Studied at pH 7.4 and 298 K Γmax/mg m-2 Mr g mol-1
CVa ( 0.1 mg m-2
frequency ( 0.5 mg m-2
literature valuesb
chain Acc
2531.6
12.4 [53]
2.8
chain Bcc
3495.9
10.4 [53]
1.7
insulincc
5733.5
5.7 [53]
2.4
7.0 [53] 8.7 [54] 4.2 [53] 4.9 [54] 2.0 [53] 2.9 [54] 2.9 [50]
protein
holo-R-lactalbumin
14 174
3.4
1.9
apo-R-lactalbumin
14 174
3.8
1.5
1.7 [50] 1.8 [51]
myoglobin
16 952
2.7
1.6
3.1 [55]
β-lactoglobulin
18 328
1.6
1.6
1.8 [46]
β-casein
23 980
3.3
2.3
BSA
66 267
4.7
2.8
2.7 [51]
340 000
5.0
2.0
1.8 [69]
fibrinogen
literature valuesc
calculated values flexible peptide flexible peptide
1.55 [82]d 2.9 [83]e 1.6 [14]f 3.0 [84]g 1.4 [85]h 1.2 [86]i 2.0 [71]j 1.3 [87]k 1.3 [88]l 3-3.5 [89]m 1.9 [90]n 1.4 [91]o 1.5-1.6 [92]p, [85]h 1.2 [93,94]q 3.9 [71,83]e 2.7 [85]h 2.2-2.8 [95]r 2.8 [96]s 4.3 [97]t 2.6 [98]u 5.0 [71,83]e 2.5 [79]v 2.1 [99]w 2.3[100]x 5 [101]x 4.7 [102]y 1.9, 4.2 [79]v 4.0, 4.5, 11.8 [103]z 4.0 [104]aa 5.0 [105]bb
2.7 [106] 1.9-2.0 [50] 1.7 [50]
1.9 [89] (side-on) 3.3 [89] (end-on) 2.8 [91] (side-on) 3.3 [91] (end-on) flexible protein structure 2.5 [79] (side-on) 6.7 [79] (end-on) 1.6-3.6 [79] (side-on) 11.1 [79] (end-on)
a Cyclic voltammetry measurements at Pt layer on quartz at sweep rate of 100 mV s-1 from phosphate buffer. b Cyclic voltammetry measurements at Pt polycrystalline wire at sweep rate of 500 mV s-1 from phosphate buffer. c Variety of other techniques as described below. d Ellipsometry measurements at Pt at 0.4 V/SCE. e Ellipsometry on silanized silica surfaces in 0.01 M phosphate buffer pH 7 at 300 K. f Differential scanning microcalorimetry on negatively charged lattices of polystyrene in 50 mM KCl solution at pH 7. g Stopped-flow fluorescence and circular dichroism spectroscopy on hydrophobic polystyrene nanospheres in 10 mM Tris/HCl buffer pH 7.5 containing 1 mM CaCl2. h Solution depletion on hydrophilic silanized silica surface in phosphate buffer pH 7.0. i Ellipsometry on hydrophobic chromium surface in phosphate buffered saline solution pH 6. j Ellipsometry onto a hydrophilic silicon surface in phosphate buffer pH 7.0 and 300 K. k Solution depletion measurements at AgI surface in 0.05 M KNO3 pH 7. l Reflectometry measurements at indium tin oxide (ITO) at open circuit potential in 0.01 M phosphate buffer pH 7.0. m UV spectroscopy/solution depletion adsorption onto ultrafine titania and zirconia particles in phosphate buffer pH 7.0. n UV/vis spectroscopy/solution depletion adsorption onto silica particles in a 0.1 M HEPES buffer pH 6.5. o Small-angle X-ray scattering on polystyrene latex at pH 7.2. p Solution depletion on stainless steel surface at pH 6.8 and 300 K. q Ellipsometry and radiotracer measurements on chromium hydrophobic surface in phosphate buffer saline (0.15 M NaCl) solution pH 7.0. r Neutron reflectivity measurements on adsorption onto hydrophobized silica surface in 0.02 imidazole-HCl buffered D O pH 7 with 0.05 2 M NaCl. (3.1-3.9 with 0.017 M CaCl2; 2.7-3.0 with 0.017 M MgCl2). s Time-resolved ellipsometry adsorption to hydrophobic and hydrophilic silica surfaces in 0.02 M imidazole-HCl buffer pH 7.0. t Time-resolved ellipsometry adsorption at hydrophobic and hydrophilic silica surfaces in 0.02 M imidazole-HCl buffer pH 7.0 with added 0.017 M CaCl2. u Photon correlation spectroscopy on polystyrene latex particles in pH 7.4 phosphate buffer at 295 K. v Cyclic voltammetry measurements at (cp) titanium in pH 7.4 phosphate buffer (1.9 at 295 K, 4.2 at 310 K) w Radiochemical techniques for BSA adsorption on stainless steel in cacodylate buffer pH 6.8 at 277 K. x In-situ ellipsometry and TIRF (total internal reflection fluorescence spectroscopy), adsorption of fibrinogen at methylated silica surfaces in 0.15 M NaCl, pH 7.4. y Radiotracing with 125I, adsorption of fibrinogen onto a titanium oxide surface in 10 mM PBS pH 7.4. z Ellipsometry, optical waveguide lightmode spectroscopy (OWLS), and QCM-D at titanium oxide in 10 mM HEPES at pH 7.4. aa Radiotracer measurements for adsorption onto glass beads in isotonic Tris buffer pH 7.35. bb Radiotracer measurements of adsorption onto glass from 0.24 mg mL-1 fibrinogen in plasma. cc Indicates proteins studied at a pH of 7.0 for comparison with literature values.
geometric surface area (i.e., surface roughness factor) of the electrode, ∆m the variation of mass per unit area in g cm-2, and Cf the sensitivity factor (Cf-1 ) ∼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 3 shows for the phosphate buffer that as the potential is swept to higher anodic potentials, species adsorb to the platinum surface, resulting in a correspond-
ing decrease in the change in frequency (∆f) of the quartz crystal. This phenomenon is in accordance with the Sauerbrey equation (Equation 6), 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 species are 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
Electrochemical Quartz Crystal Nanobalance
Figure 3. Cyclic voltammetry and frequency response for phosphate buffer (pH 7.4) at 298 K. Horizontal arrows indicate the direction of potential sweep. Vertical arrows indicate the potentials chosen for the frequency analysis. These potentials correspond to the end potentials used to measure the area of the oxide reduction region (Or).
Figure 1b. The data was normalized by setting the frequency change for each 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. 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. This result is not unexpected as the electrosorption process displaces strongly adsorbed solvent molecules from the metal surface.107 Therefore, if solvent molecules are (82) Razumas, V.; Kulys, J.; Arnebrant, T.; Nylander, T.; Larsson, K. Electrokhimiya 1988, 24, 1518. (83) Al-Malah, K.; McGuire, J.; Sproull, R. J. Colloid Interface Sci. 1995, 170, 261 (84) Engel, M. F. M.; van Meirlo, C. P. M.; Visser, J. W. G. J. Biol. Chem. 2002, 277, 10922. (85) Krisdhasima, V.; Vinaraphong, P.; McGuire, J. J. Colloid Interface Sci. 1993, 161, 325. (86) Arnebrant, T.; Barton, K.; Nylander, T. J. Colloid Interface Sci. 1987, 119, 383. (87) Galisteo, F.; Norde, W. J. Colloid Interface Sci. 1995, 172, 502. (88) Bos, M. A.; Shervani, Z.; Anusiem, A. C. I.; Giesbers, M.; Norde, W.; Kleijn, J. M. Colloids Surf., B 1994, 3, 91. (89) Kondo, A.; Mihara, J. J. Colloid Interface Sci. 1996, 177, 214. (90) Buijs J.; Ramstrom, M.; Danfelter, M.; Larsericsdotter, H.; Hachansson, P.; Oscarsson, S. J. Colloid Interface Sci. 2003, 263, 441. (91) Mackie, A. R.; Mingins, J.; Dann, R. In Food Polymers, Gels and Colloids; Royal Society of Chemistry: Cambridge, 1991; p 96. (92) Kim, J. C.; Lund, D. B. In Fouling and Cleaning in Food Processing; Kessler, H. G., Lund, D. B., Eds.; Druckerei Walch: Ausburg, Germany, 1989; p.187. (93) Arnebrant, T.; Ivarsson, B.; Larsson, K.; Lundstrom, K. I.; Nylander, T. Prog. Colloid Polymer Sci. 1985, 70, 62. (94) Arnebrant, T.; Nylander, T. J. Colloid Interface Sci. 1986, 111, 529. (95) Nylander, T.; Tiberg, F.; Su, T.-J.; Lu, J. R.; Thomas, R. K. Biomacromolecules 2001, 2, 278. (96) Nylander, T.; Tiberg, F.; Wahlgren, N. M. Int. Dairy J. 1999, 9, 313. (97) Kull, T.; Nylander, T.; Tiberg, F.; Wahlgren, M. N. Langmuir 1997, 13, 5141. (98) Fair, B. D.; Jamieson, A. M. J. Colloid Interface Sci. 1980, 77, 525. (99) Van Enckevort, H. J.; Dass, D. V.; Langdon, A. G. J. Colloid Interface Sci. 1984, 98, 138. (100) Malmsten, M. J. Colloid Interface Sci. 1994, 166, 333. (101) Malmsten, M.; Lassen, B. J. Colloid Interface Sci. 1994, 166, 490. (102) Liu, F.; Zhou, M.; Zhang, F. Appl. Radiat. Isot. 1998, 49, 67. (103) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf., B 2002, 24, 155. (104) Wojciechowski, P.; ten Hove, P. Brash, J. L. J. Colloid Interface Sci. 1986, 111, 455.
Langmuir, Vol. 20, No. 5, 2004 1717
Figure 4. Dependence of mass change on the oxide reduction charge (QOr) at pH 7.4 and 298 K for b, myoglobin; o, β-casein.
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. Previous studies in our laboratory on the amino acid phenylalanine (Phe) have shown that the EQCN technique does not directly monitor the change in mass of the adsorbed amino acid at the platinum electrode.66 Instead, the EQCN technique 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. In the present investigation, the resulting frequency changes again appeared to correlate with the oxide growth and removal processes. In the anodic region, the potential sweep in the positive going direction is a measure of oxide formation, adsorption of protein, and adsorption of phosphate ions, whereas the cathodic or negative going sweep measures only oxide reduction. Therefore, the frequency change was measured for the cathodic going sweep from 0.4 V to the double layer region at -0.2 V and hence is a direct measure of only the oxide reduction (Or) process, as indicated by the peak in the cyclic voltammogram profile. Figure 3 shows both the CV and frequency responses in phosphate buffer and provides an example of the method of analysis carried out for each protein investigated. The oxide reduction charge density (QOr) was obtained by integration of the peak between these two potentials, below the charge due to the double layer region (i.e., below the dotted line). From the Sauerbrey equation, the change in the mass (∆m) was calculated from the difference in the measured ∆f values over the same potential range, as indicated by the vertical arrows. A plot of ∆m versus the oxide reduction charge density (QOr) from the simultaneous CV and frequency measurements yields a linear relationship as shown in Figure 4. From eq 2 for the reduction of oxide, the molar mass of the species causing the frequency change was 17.0 and 17.2 ((10%) g mol-1 for myoglobin and β-casein, respectively, which is in excellent agreement with the displacement of adsorbed oxide, OHads or Oads, from the electrode surface. This is also in excellent (105) Cornelius, R. M.; Wojciechowski, P. W.; Brash, J. L. J. Colloid Interface Sci. 1992, 150, 121. (106) Arnebrant, T.; Nylander, T. J. Colloid Interface Sci. 1988, 122, 557. (107) Daikhin, L.; Gileadi, E.; Tsionsky, V.; Urbakh, M.; Zilberman, G. Electrochim. Acta 2000, 45, 3615.
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Table 3. EQCN Frequency Results for Molar Mass of Desorbed Species Calculated Using Eq 2 for Each Protein at pH 7.4 and 298 K
protein
Mr of protein g mol-1
Mr of desorbed species g mol-1 (( 10%)
chain Aa chain Ba insulina holo-R-lactalbumin apo-R-lactalbumin myoglobin β-lactoglobulin β-casein BSA fibrinogen
2531.6 3495.9 5733.5 14 174 14 174 16 952 18 328 23 980 66 267 340 000
15.5 [53] 17.5 [53] 18.2 [53] 16.6 16.9 17.0 17.2 17.2 18.0 16.7
a
Indicates proteins studied at a pH of 7.0.
agreement with our previously obtained value of 16.9 ((10%) g mol-1 for Phe66. Table 3 shows the consistent results obtained for each of the proteins studied, confirming that EQCN measurements are unable to directly measure protein adsorption at Pt in the anodic region but instead measures the strongly adsorbed surface oxide film over these potentials. 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.108 Since the EQCN measurements did not appear to directly monitor the adsorption of protein onto the platinum surface during the potential sweep in the anodic region, a different approach was taken to investigate protein adsorption. 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). Thus, 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 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. A plot of the absolute change in mass, |∆m|, versus the concentration of protein in the bulk solution (Figure 5) 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|
(7)
Consequently, a plot of c/|∆m| versus concentration should yield a linear relationship with the parameters BADS and |∆mmax| obtained from the slope and intercept, respectively.
Figure 5. Plot of the absolute change in mass of solvent molecules displaced as a function of the concentration in the bulk solution at pH 7.4 and 298 K of b, myoglobin; o, β-casein. Inset: Linearized adsorption isotherm at pH 7.4 and 298 K using frequency measurements from the EQCN technique and eq 7 for b, myoglobin; o, β-casein.
Figure 5 (inset) shows, as an example, the plots for myoglobin and β-casein, which do yield linear relationships. This indicates that the mass change determined from the frequency change in the double layer region does exhibit Langmuir adsorption behavior. To correlate this absolute mass change to protein adsorption, the parameter BADS was again used to obtain the Gibbs free energy of adsorption, ∆GADS, using eq 5. The values obtained from the frequency measurements for Gibbs free energy of adsorption are shown in Table 1. At high protein bulk concentrations and just before the onset of the plateau values for maximum surface concentration, the frequency response increases to positive values relative to those observed at -0.6 V, similar to that shown for the highest concentration of myoglobin in Figure 1b. This is indicative of increasing amounts of solvent displacement due to even higher protein adsorption which increases with increasing potential up to 0.2 V. At higher potentials, oxide formation, Oads, occurs on the surface with a corresponding increase in mass and decrease in ∆f. On the reverse sweep, Oads is removed from the surface over the potential range 0.5 to -0.2 V, reaching a value of ∆f similar to the maximum value attained from protein adsorption in the forward sweep (i.e., at 0.2 V). Finally, as the potential decreases between -0.3 and -0.6 V, the frequency decreases because of loss of protein and increased solvent adsorption with a corresponding recorded mass increase. This behavior is consistent with all the proteins studied. Effect of Protein Size on Gibbs Free Energy of Adsorption (∆GADS) from EQCN Frequency and CV Measurements. The proteins chosen for this study cover a wide range in molar mass (2500-340 000 g mol-1) to investigate the influence of protein size on adsorption behavior at a platinum surface and the applicability of the analytical technique of the EQCN for measuring protein adsorption on surfaces. A comparison between the ∆GADS values obtained for the cyclic voltammetry and frequency measurements for all proteins investigated is shown in Table 1. The two techniques measure two very different parameters in the interfacial adsorption process, yet they (108) Zolfaghari, A.; Conway, B. E.; Jerkiewicz, G. Electrochim. Acta 2002, 47, 1173.
Electrochemical Quartz Crystal Nanobalance
Figure 6. Dependence of Gibbs free energy of adsorption on molar mass of protein at pH 7.4 (7.0) and 298 K obtained from cyclic voltammetry (ο) and frequency (0) measurements.
give excellent agreement within experimental uncertainty for the Gibbs free energy of adsorption. From cyclic voltammetry measurements, surface charge density (QADS) provides a measure of adsorbed protein by directly measuring the charge associated with protein adsorption, after correcting for the charge due to adsorbed oxide (eq 1). Therefore, the surface charge density gives information on the blockage of adsorbed oxide by the adsorbed protein. On the other hand, frequency measurements in the diffuse double layer give a measure of the extent of solvent displacement by the adsorbed protein (eq 7). The results indicate that the EQCN technique provides useful complementary information for the study of protein interfacial behavior. A plot of the Gibbs free energy of adsorption (∆GADS) as a function of the molar mass (Mr) of the protein is shown in Figure 6 for both cyclic voltammetry and frequency measurements. The observed trend showed that the smallest molecules studied, chain A and chain B of insulin, have the least affinity for the platinum surface as indicated by their small negative ∆GADS values, while the larger protein molecules, for example, BSA and fibrinogen, have greater affinity for the platinum surface. Table 1 also shows a comparison of the present results with those presented in the literature using a variety of techniques and surfaces. The values for ∆GADS show a good consistent agreement for each of the proteins despite the difference in techniques, surfaces, and experimental conditions. Surface Concentrations from EQCN Frequency and CV Measurements. A comparison of surface concentrations is shown in Table 2 for cyclic voltammetry measurements, as discussed previously, and frequency measurements from |∆mmax| values as a result of solvent displacement from the surface by protein adsorption. In addition, the right-hand column shows the theoretical or maximum surface concentration for monolayer coverage of protein on the basis of the dimensions of the molecule in the native state. The excellent agreement between the surface concentrations from frequency measurements and the calculated values suggest that the |∆mmax| values from solvent displacement reflects the first monolayer of protein coverage and in all cases with protein adsorption in a side-on configuration. These results are also in excellent agreement with the cyclic voltammetry measurements made with the fast sweep rate of 500 mV s-1 (Table 2, column under b) which measures charge transfer from
Langmuir, Vol. 20, No. 5, 2004 1719
surface protein essentially independent of mass transport. On the other hand, the cyclic voltammetry measurements made simultaneously with the frequency measurements at the slower sweep rate of 100 mV s-1 (Table 2, column under a) give higher surface concentrations ranging from approximately 1.4 to 2.2 layers of protein. As described previously, the slower sweep rate may allow a greater accumulation of protein on the surface accompanied by electrolyte assisted charge transfer. However, the frequency measurements of |∆mmax| values results only from solvent displacement directly at the electrode surface and therefore detects only the first monolayer of adsorbed protein. It is interesting also to compare our results with those reported in the literature using a variety of different surfaces, techniques, and experimental conditions. In general, there is very good agreement among the results for each protein. A comparison of these values indicates that the orientation of the proteins on the surface or multilayer formation may vary with the types of surfaces and experimental conditions. Our EQCN results show multilayer formation (∼1.5-2 layers) for all proteins, with the exception of β-lactoglobulin, with no direct evidence of complete protein surface denaturation at 298 K. It is interesting to compare the results from the present investigation with those using the quartz crystal microbalance with dissipation technique (QCM-D).62,63,103 In these measurements, the absolute dissipation factor, D, of the QCM can be obtained with high accuracy and repetition rate. The principle behind the measurement is to drive the crystal to oscillation with a signal generator, disconnect the generator, and then record how the crystal oscillation decays on a digitizing oscilloscope. The recorded decay curve is numerically fitted to an exponentially damped sinusoidal whereby the resonant frequency and the dissipation factor D of the crystal are obtained simultaneously. A study by Hook et al.103 report a value of 11.8 mg m-2 for fibrinogen adsorption to TiO2 coated surface using this technique, significantly higher than their results using OWLS (4.5 mg m-2) and ellipsometry (4.0 mg m-2) measurements or our EQCN results (2.0 mg m-2). They also observed higher values for the proteins human serum albumin and hemoglobin obtained from QCM-D measurements in comparison with their reported values using OWLS and ellipsometry.103 In consideration of the discrepancy between the results for fibrinogen from QCM-D and EQCN, there are significant differences, not only between the two techniques of QCM-D and that used in the present investigation (EQCN), but also the substrates (TiO2 and Pt) and experimental conditions which differ substantially. However, the QCM-D result suggests that fibrinogen adsorbs end-on on TiO2, while the present EQCN results suggest fibrinogen adsorbs side-on on Pt from the frequency measurements, and with multilayer formation from the simultaneous cyclic voltammetry measurements. The present EQCN results show good agreement for the proteins in comparison with the results from other researchers using a variety of techniques. Conclusions Cyclic voltammetry (CV) and electrochemical quartz crystal nanobalance (EQCN) measurements were used to investigate the adsorption of insulin, chain A and chain B of insulin, holo-R-lactalbumin (R-LA), apo-R-lactalbumin (apo-R-LA), myoglobin, β-casein, β-lactoglobulin (β-LG), bovine serum albumin (BSA), and fibrinogen at a platinum electrode. This series of proteins were chosen to examine the effect of size and conformational changes in protein structure on the surface adsorption behavior as measured by the EQCN technique. The EQCN frequency measure-
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Langmuir, Vol. 20, No. 5, 2004
ments in the anodic region did not directly monitor the molar mass of the adsorbed protein but rather measured the decreasing change in the surface oxide as a result of adsorbed protein blocking the surface oxide growth. The molar mass of the desorbed species measured simultaneously by both frequency and surface charge density gave an average value of 17 ((10%) g mol-1 for all the proteins investigated which was consistent with the measurement of either OHads or Oads on the electrode surface at the anodic potentials. On the other hand, CV measurements recorded simultaneously with the frequency measurements provided a measure of surface charge density from protein adsorption after correcting for the presence of oxide. This allowed determination of ∆GADS for protein using the Langmuir adsorption isotherm. Since the EQCN measurements did not appear to directly monitor the adsorption of protein onto the platinum surface during the potential sweep in the anodic region, a different approach was made, which was to investigate the effect of the frequency change in the double layer (DL) region with each incremental addition of protein. These observed frequency changes gave a measure of the extent of solvent displacement by the adsorbed protein. The ∆GADS values obtained from the |∆m| values calculated from the frequency measurements gave excel-
Cosman and Roscoe
lent agreement within experimental uncertainty with the ∆GADS values calculated from the QADS values from the CV measurements for all proteins studied. All proteins had high negative values for ∆GADS indicating that spontaneous adsorption occurs at the platinum surface. The smallest molecules studied, chain A and chain B of insulin, have the least affinity for the platinum surface as indicated by their small negative ∆GADS values, while the larger protein molecules such as fibrinogen have the greatest affinity for the platinum surface. While CV measurements provide information about protein surface concentration (Γ) from a direct measure of the surface charge density, EQCN frequency measurements give a measure of nanogram changes (∆m) occurring on the metal surface. Hence, EQCN provides complementary techniques to measure protein surface concentration to study interfacial behavior of proteins at the electrode/electrolyte surface. Acknowledgment. Grateful acknowledgment is made to the Dairy Farmers of Canada and the Natural Science and Engineering Research Council of Canada for support of this research. LA035154H
