Electrochemical Quartz Crystal Nanobalance Study of the Adsorption

Langmuir 2004, 20, 7547-7556

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Electrochemical Quartz Crystal Nanobalance Study of the Adsorption/Displacement Phenomena of Proteins and Lipids on Pt Craig D. Wilson and Sharon G. Roscoe* Department of Chemistry, Acadia University, Wolfville, Nova Scotia, Canada B4P 2R6 Received February 29, 2004. In Final Form: June 24, 2004 The electrochemical quartz crystal nanobalance (EQCN) was used to measure the adsorption behavior of a series of lipids (stearate, oleate, linoleate, and γ-linolenate) on a Pt surface from a phosphate buffer pH 7.0 solution at 295 K and to investigate their adsorption/displacement behavior with the proteins, β-lactoglobulin and R-lactalbumin, which are known to cause fouling during milk processing. The EQCN technique and the complementary technique of cyclic voltammetry measured simultaneously provided information on the efficiency of solubilization of the proteins by these lipids. Excellent agreement was obtained for the surface concentration of adsorbed lipid from the surface charge density from cyclic voltammetry measurements and the change in mass from the EQCN frequency measurements. The Gibbs energy of adsorption showed the lipids to have a strong affinity for the platinum surface. Addition of protein to a preadsorbed lipid layer showed R-lactalbumin to be able to coadsorb with the lipids, while β-lactoglobulin was able to desorb some of the unsaturated lipids but appeared to coadsorb with the saturated lipid, stearate. Addition of lipid to a preadsorbed protein layer showed the unsaturated lipids to be able to displace some of the protein. A comparison of the desorption ability of the lipids showed stearate to be very inefficient at removing protein, while the other three lipids were able to remove each of the proteins, with the order of efficiency for protein desorption being oleate > linoleate > γ-linolenate.

Introduction Proteins have caused problems in both industrial and medical applications because of their tendency to adhere to surfaces causing fouling.1,2 This leads to costs related to extensive cleaning and downtime of the processing equipment, the inoperability of membranes and materials which rely on clean surfaces for proper functioning, and subsequent bacterial growth on these surfaces. Lipids, such as fatty acids which commonly occur in foods, have been found to solubilize proteins and assist in their removal from surfaces.3 This study examines a technique with the electrochemical quartz crystal nanobalance (EQCN) to measure the adsorption behavior of a series of lipids on a platinum surface and to study their adsorption/ displacement behavior with proteins known to cause fouling. There are a number of techniques that have been used to study the adsorption of lipids to solid surfaces such as: surface plasmon resonance,4 X-ray photoelectron spectroscopy (XPS),4 Fourier transform infrared spectroscopy,5 photon polarization modulated infrared reflection absorption spectroscopy,6 chronocoulometry,6-9 neutron reflectivity,8-11 scanning tunneling microscopy (STM),7 * To whom correspondence should be addressed. Phone: (902) 585-1156. Fax: (902) 585-1114. E-mail: [email protected]. (1) Dejong, P. Trends Food Sci. Technol. 1997, 8, 401. (2) Changani, S. D.; Belmarbeiny, M. T.; Fryer, P. J. Exp. Thermal Fluid Sci. 1997, 14, 392. (3) Reuben, B. G.; Perl, O.; Morgan, N. L.; Stratford, P.; Dudley, L. Y.; Hawes, C. J. Chem. Technol. Biotechnol. 1995, 63, 85. (4) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden; N. Langmuir. 1997, 13, 751. (5) Cheng, Y.; Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles, P. F.; Marsh, A. Langmuir. 1998, 14, 839. (6) Horswell, S. L.; Zamlynny, V.; Li, H. Q.; Merrill, A. R.; Lipkowski, J. Faraday Discuss. 2002, 121, 405. (7) Burgess, I.; Jeffrey, C. A.; Cai, X.; Szymanski, G.; Galus, Z.; Lipkowski, J. Langmuir 1999, 15, 2607. (8) Burgess, I.; Zamlynny, V.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Smith, G.; Satija, S.; Ivkov, R. Langmuir 2001, 17, 3355.

atomic force microscopy,7,11-13 and epifluorescence microscopy.14,15 Our previous studies on the adsorption behavior on a platinum surface have included the anionic forms of the fatty acids oleic acid (C18:1ω9) and γ-linolenic acid (C18:3ω6) using cyclic voltammetry (CV) measurements16 and linoleic acid (C18:2ω6) on a stainless steel surface using CV, electrochemical impedance spectroscopy (EIS), and potentiodynamic linear polarization techniques.17 Results from these studies and additional unpublished results have found that interactions between fatty acids and the electrode, resulting in adsorption, were affected by a number of factors including length of fatty acid chain, unsaturation number of the acid, position of the unsaturated bonds, temperature of solution, and the characteristics of the electrode surface. It was found that the fatty acids in phosphate buffer at pH 7.0 self-assembled onto a platinum electrode to create lipid layers with the carboxylate heads of the fatty acids adsorbed onto the electrode surface leaving the hydrophobic hydrocarbon tails extending out from the surface.16 Since contact of the hydrocarbon tails with water is thermodynamically unfavorable, and because the lipid layer was impermeable by water and anions, it was assumed that at least a second layer of lipids self-assembled such that the carboxylate groups interacted with the aqueous solution while the hydrocarbon tails formed a hydrophobic region between (9) Zamlynny, V.; Burgess, I.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Smith, G.; Satija, S.; Ivkov, R. Langmuir 2000, 16, 9861. (10) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J. 1991, 59, 289. (11) Koenig, B. W.; Krueger, S.; Orts, W. J.; Majkrzak, C. F.; Berk, N. F.; Silverton, J. V.; Gawrisch, K. Langmuir 1996, 12, 1343. (12) Leonenko, Z. V.; Carnini, A.; Cramb, D. T. Biochim. Biophys. Acta 2000, 1509, 131. (13) Mueller, H.; Butt, H. J.; Bamberg, E. Langmuir 2000, 16, 9568. (14) Shepherd, J.; Yang, Y.; Bizzotto, D. J. Electroanal. Chem. 2002, 524-525, 54. (15) Stoodley, R.; Bizzotto, D. Analyst 2003, 128, 552. (16) Maguire, H. J.; Roscoe, S. G. Langmuir 1997, 13, 5962. (17) Omanovic, S.; Roscoe, S. G. Corrosion 2000, 56, 684.

10.1021/la049478x CCC: $27.50 © 2004 American Chemical Society Published on Web 08/04/2004

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the two layers and even possibly formed additional layers. It is well-known that the platinum electrode is characterized by the electrocatalytic surface oxide film that develops on the electrode surface at anodic potentials in aqueous solutions.18,19 Oleate and γ-linolenate were found to form particularly tight layers that could block the oxide formation.16 Quartz crystal resonators were first used by Sauerbrey in 1959 for deposition rate monitoring of thin films in ultra-high-vacuum systems.20 The discovery that quartz crystals oscillate at a specific frequency when immersed in a liquid phase allowed the EQCN technique to be created.21 The operation of the EQCN is based on the converse piezoelectric effect. When a voltage is applied across a quartz crystal, it induces a mechanical stress, which in turn causes the quartz crystal to oscillate at a specific frequency. The deformation is elastic for quartz and quartz covered with a very thin rigid film. The frequency of oscillation of the quartz crystal is found to be proportional to the mass that is attached to its surface. Therefore, by measuring the frequency changes at various potentials, nanogram mass changes on the surface of the electrode can be measured. This enhances the information that can be obtained about processes occurring at the electrode/electrolyte surface. Recent studies in our laboratory using simultaneous CV and EQCN frequency measurements investigated the adsorption behavior of the amino acid phenylalanine,22 as well as a series of proteins.23,24 Very good agreement was obtained between results using CV, EIS, and the EQCN technique. It was therefore of interest to examine this EQCN technique for use with lipids. Since our previous publications described the use of EQCN techniques with proteins reported in the literature, we present here only those reported in the use of lipids. Hepel25 used the quartz crystal immittance technique to show that 1-R-dipalmitoylphosphatidylcholine bilayer film modified by ion channel forming molecules of gramicidin on a Au electrode are in the solid state and behave as perfectly rigid films. Ha and Kim26 studied the adsorption of lipid vesicles on a hydrophobic surface using the quartz crystal microbalance technique. They found that the frequency change resulted in an overestimation of the mass adsorbed to the surface which they attributed to the viscosity of the electrode-adjacent liquid layer being much larger than that of the bulk phase due to the presence of intact vesicles on the 1-octadecanethiol-coated surface. Stalgren et al.27 studied the adsorption of hexaethylene glycol mono-n-tetradecyl ether on different model surfaces, and they too found that the frequency shift resulted in an overestimation of adsorbed mass. They believed this to be due to either the coupling of water molecules to the adsorbed layer or water trapped within the adsorbed layer. The lipids selected for the present study were the anionic forms of the 18-member carbon chain fatty acids: stearic acid (C18:0), oleic acid (C18:1ω9), linoleic acid (C18:2ω6), and γ-linolenic acid (C18:3ω6), all with double bonds in (18) Damjanovic, A. In Modern Aspects of Electrochemistry; Bockris, J. O., Conway, B. E., Eds.; Plenum: New York, 1969; Vol. 29, p 369. (19) Vijh, A. K.; Conway, B. E. Chem. Rev. 1967, 67, 623. (20) Sauerbrey, G. Z. Phys. 1959, 155, 206. (21) Nomura, T.; Iijima, M. Anal. Chim. Acta 1981, 131, 97. (22) Wright, J. E. I.; Fatih, K.; Brosseau, C. L.; Omanovic, S.; Roscoe, S. G. J. Electroanal. Chem. 2003, 550-551, 41. (23) Wright, J. E. I.; Cosman, N. P.; Fatih, K.; Omanovic, S.; Roscoe, S. G. J. Electroanal. Chem. 2004, 564, 185-197. (24) Cosman, N. P.; Roscoe, S. G. Langmuir 2004, 20, 1711-1720. (25) Hepel, M. J. Electroanal. Chem. 2001, 509, 90. (26) Ha, T. H.; Kim, K.; Langmuir 2001, 17, 1999. (27) Stalgren, J. J. R.; Eriksson, J.; Boschkova, K. J. Colloid Interface Sci. 2002, 253, 190.

Wilson and Roscoe

the natural cis configuration. Milk fat contains the fatty acids in the average yearly amounts in % weight as follows: stearic acid, 12.1%; oleic acid, 27.1%; linoleic acid 2.4%.28 Oleic acid is the most common naturally occurring fatty acid in any animal fat or plant oil and is the precursor for the production of most other polyunsaturated fatty acids (PUFAs).29 Linoleic acid, a primary product of plant PUFA synthesis, is commonly found in many seed oils. Although animals are incapable of producing linoleic acid, livestock are fed diets particularly rich in this fatty acid, and hence linoleic acid is now found in both milk and meat products.30 Although γ-linolenic acid (GLA) is produced in animals and lower plants, only minute amounts can be found in animal tissue.31 However, GLA is important both in the pharmaceutical industry and as a nutraceutical. These lipids are also present as triglycerides in most fats and oils. The globular proteins chosen for the present study were β-lactoglobulin (β-LG) (Mr 18 328 g mol-1) and R-lactalbumin (R-LA) (Mr 14 174 g mol-1), the two most common whey proteins in milk. Due to their high affinity for metal surfaces, they have been implicated in the fouling of processing equipment such as the heat exchangers used in the dairy industry.1,2 R-LA is a compact globular protein that binds calcium in a 1:1 ratio via seven oxygen atoms to form a distorted pentagonal bipyramid.31,32 Loss of calcium ion can result in formation of its molten globular state which is known to have high stability with a nativelike secondary structure but a more disordered tertiary structure.33 β-LG consists of an antiparallel β-sheet formed by nine β-strands wrapped around to form a flattened cone or calyx.34 The β-strands contain a hydrophobic cavity where hydrophobic compounds such as retinol can bind.35 Therefore β-LG is known to be a carrier of fatty acids and retinol, the latter of which is important in the development of vision in newborn calves.34 The protein exists naturally as a dimer of two noncovalently linked monomeric subunits.36 The objective of the present study was to examine the applicability of the EQCN technique for studying the surface adsorption behavior of a series of lipids with increasing numbers of double bonds as a means to examine their adsorption/displacement phenomena with proteins. CV and EQCN frequency measurements were made simultaneously which provided data on both surface charge density and mass changes as a function of potential. Measurements were first made individually on the proteins and lipids to determine (i) surface concentrations from surface charge density and mass changes, (ii) adsorption isotherms, and (iii) Gibbs energy of adsorption. Studies were then made to determine whether protein additions to an adsorbed lipid layer would result in disruption and displacement of the lipid layer by protein. Following this, to determine the feasibility of using lipids to solubilize adsorbed protein to alleviate fouling, lipids (28) Gunstone, F. D. In Food Lipids; Akoh, C. C., Min, D. B., Eds.; Marcel Dekker: New York, 2002; p 729. (29) Watkins, S. M.; German, J. B. In Food Lipids; Akoh, C. C., Min, D. B., Eds.; Marcel Dekker: New York, 2002; p 559. (30) Padly, F. B.; Gunstone, F. D.; Harwood: J. L. In The Lipid Handbook; Chapman and Hall: London, 1994; p 47. (31) Kuwajima, K. FASEB J. 1996, 10, 102. (32) Mizuguchi, M.; Nara, M.; Kawano, K.; Nitta, K. FEBS Lett. 1997, 417, 153. (33) Kuwajima, K. Proteins 1989, 6, 87. (34) Papiz, M. Z.; Sawyer, L.; Eliopoulos, E. E.; North, A. C. T.; Findlay, J. B. C.; Sivaprasadarao, R.; Jones, T. A.; Newcomer, M. E.; Kraulis, P. J. Nature 1986, 324, 383. (35) Sakai, K.; Sakurai, K.; Sakai, M.; Hoshino, M.; Goto, Y.. Protein Sci. 2000, 9, 1719. (36) Walstra, P.; Jenness, R. Dairy Chemistry and Physics; Wiley: New York, 1984; p 116

Lipid Adsorption on Pt

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were introduced to solutions containing a preformed adsorbed protein layer on the electrode surface. These measurements allowed further evaluation of the EQCN technique and provided information on the field-driven adsorption/displacement phenomena of lipids and proteins. Experimental Section Reagents and Solutions. A 0.05 M phosphate buffer solution, pH 7.0, was prepared for all measurements using 0.6805 g of monobasic, anhydrous KH2PO4 (Sigma Chemical Co.) and 29.1 mL of 0.10 M sodium hydroxide (made from concentrated volumetric solution, ACP Chemical Inc.) made up to 100 mL with conductivity water (Nanopure, resistivity 18.2 MΩ cm). Stock solutions of the proteins bovine holo-R-lactalbumin (Sigma Chemical Co., L-5385, type I) and bovine β-lactoglobulin A (Sigma Chemical Co., L-7880) were prepared by dissolving a massed amount of the solid reagent in the 0.05 M phosphate buffer solution. Stock solutions of the lipids sodium stearate (Sigma Chemical Co., S-3381), sodium oleate (Sigma Chemical Co., O-7501), sodium linoleate (Sigma Chemical Co., L-8134), and lithium γ-linolenate (Efamol Research Institute) were prepared by dissolving a massed amount of the solid reagent in the 0.05 M phosphate buffer solution. Electrochemical Equipment. A two-compartment EQCN cell was used for all measurements. The main compartment housed the counter electrode as well as the working electrode, which was mounted in the holder and attached to the cell in a horizontal position. 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 argon gas (Praxair Products Inc.) to stir as well as to remove oxygen from the solution and the 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. 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.37 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 peaks for reduction and dividing this by the known charge for monolayer coverage of H adsorbed on platinum (210 µC cm-2).38 (37) Hepel, M. In Interfacial Electrochemistry: Theory Experiment, and Applications; Wieckowski, A., Ed.; Marcel Dekker: New York, 1999; p 599. (38) Angerstein-Kozlowska, H. In Comprehensive Treatise of Electrochemistry; Yeager, E., Bockris, J. O., Eds.; Plenum Press: NewYork, 1984; Vol. 9, p 15.

Figure 1. Cyclic voltammetry and frequency response for (s) phosphate buffer and (- - -) with addition of 17 µM linoleate. The EQCN cell was then emptied, rinsed thoroughly with Nanopure water, and replaced with an accurately measured amount (∼40 mL) 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 CV 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. It is important to note that all measurements were made in a quiescent solution as bubbling caused the frequency to become unstable.

Results and Discussion Cyclic Voltammetry Measurements. Figure 1 shows a typical cyclic voltammogram in phosphate buffer in the absence and then in the presence of 17 µM linoleate. The adsorption of lipid partially blocks the platinum surface from oxide growth in the anodic (positive) going sweep and, hence, results in a smaller oxide reduction in the cathodic (negative) 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)

where QOo (C cm-2) 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 lipid or protein. Figure 2a shows the response in measured surface charge density with increase in concentration of stearate, oleate, linoleate, and γ-linolenate in the phosphate buffer solution at 295 K. The surface charge density increased rapidly with increasing lipid concentration until it reached a plateau level, which indicated that the platinum surface was saturated with lipid under these experimental conditions. The lipid or protein surface concentration, Γ (mg m-2), can be calculated using eq 2 from the surface charge density (QADS) determined directly from the cyclic voltammograms

Γ ) QADSMr/nF

(2)

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Wilson and Roscoe Table 1. Comparison of Gibbs Energy of Adsorption Values Obtained from EQCN Frequency and Cyclic Voltammetry Measurements on a Pt Electrode at 295 K and pH 7.0 cyclic voltammetry ∆GADS/(1 kJ mol-1

frequency measurements ∆GADS/(3 kJ mol-1

-36 -35 -38 -37

-36 -36 -38 -38

-44 -46

-46 -50

lipid stearate oleate linoleate γ-linolenate protein R-lactalbumin β-lactoglobulin

BADS (cm3 mol-1) is the adsorption coefficient, which reflects the affinity of the molecules of interest toward adsorption to the metal surface. Therefore, if the Langmuir adsorption isotherm is valid for the system being studied, a plot of c/Γ versus bulk concentration, c, should yield a straight line with the parameters Γmax and BADS derived from the slope and intercept, respectively. Figure 2b shows the linearized adsorption isotherms for the lipids. The dashed lines in Figure 2a represent the Langmuir curves calculated from the parameters obtained from eq 3. The parameter BADS, which reflects the affinity of the absorbed molecules for the metal surface at a constant temperature, can be represented as

BADS )

Figure 2. (a) Surface charge density, QADS, in phosphate buffer (pH 7.0) at 295 K as a function of concentration of stearate (3), oleate (O), linoleate (4), and γ-linolenate (0). (b) Linearized adsorption isotherms in phosphate buffer (pH 7.0) at 295 K using eq 4 for stearate (3), oleate (O), linoleate (4), and γ-linolenate (0).

where QADS (C cm-2) is the surface charge density due to lipid or protein adsorption, Mr (g mol-1) is the molar mass of the adsorbing species, n is the number of electrons transferred (2 × the number of carboxylate groups on the molecule39-43), and F (C mol-1) is the Faraday constant. In previous studies the adsorption of proteins has been successfully described by the Langmuir adsorption isotherm,44 with the linearized form given below

1 c c ) + Γ BADSΓmax Γmax

(3)

-3

where c (mol cm ) is the equilibrium concentration of the adsorbing species in the bulk solution, Γ (mol cm-2) is lipid or protein surface concentration, Γmax (mol cm-2) is the maximum lipid or protein surface concentration, and (39) Marangoni, D. G.; Smith, R. S.; Roscoe, S. G. Can. J. Chem. 1989, 67, 921. (40) 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 319-399. (41) MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1997, 42, 1189. (42) Li, H. Q.; Chen, A.; Roscoe, S. G.; Lipkowski, J. J. Electroanal. Chem. 2001, 500, 299. (43) Roscoe, S. G. J. Colloid Interface Sci, 2000, 228, 438. (44) Phillips, R. K. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2001, 17, 2471.

1 csolvent

(

exp

)

-∆GADS 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 both the lipids and proteins (Table 1). The behavior of the proteins was similar to that described for pH 7.4 in our previous publication.24 Frequency Measurements. An experiment with the EQCN simultaneously measures the current density (j) and the frequency response (∆f) to an applied potential (V) at a sweep rate of 100 mV s-1. Using the EQCN technique, both CV and frequency measurements were measured simultaneously with each aliquot of lipid or protein that was added to the phosphate buffer. The data were normalized by setting the frequency change at -0.6 V for each addition to the same value obtained with the phosphate buffer alone. The Sauerbrey equation was used to convert the frequency response to mass changes as follows20

[ ]

∆f ) -

fo2 ∆m ) -Cf∆m 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 (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. Frequency response as a function of potential for phosphate buffer (pH 7.0) at 295 K with linoleate additions of (a) 0 µM, (b) 7 µM, (c) 17 µM, (d) 27 µM, and (e) 66 µM. Arrows indicate the direction of the potential sweep.

Previous studies in our laboratory on the amino acid phenylalanine (Phe)22 and a series of proteins23,24 have shown that the EQCN technique does not directly monitor the change in mass of the adsorbed amino acid or protein on the platinum electrode. Instead, the EQCN technique directly measures oxide growth from the aqueous solution 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.22 Also in the present study, the EQCN frequency measurements did not appear to directly monitor the adsorption of lipid onto the platinum surface during the potential sweep in the anodic region. A plot of ∆m versus QADS for oxide reduction over the potential range from 0.55 to -0.2 V (i.e., the returning sweep) provides the molar mass of the species being measured by the EQCN. Frequency measurements for surface concentrations up to the plateau level showed the molar mass to be 17 g mol-1, equivalent to the blockage of oxide due to the adsorbed lipid similar to that observed with Phe22 and the proteins.23,24 However, once the plateau level was reached with linoleate, there was an abrupt change in the curve giving a value of 94 g mol-1 which likely results from a mixture of factors and is clearly not representative of the lipid molar mass (280 g mol-1) alone. Since there is very little solvent on the electrode surface as indicated by the small amount of oxide on the surface which is being reduced in this potential sweep, and the hydrophobic nature of the lipid layer, this value probably represents the lipid layer interacting directly with the surface, similar to the behavior of amino acids in which the carboxylate groups become oxidized to the next lower aldehyde in the homologous series as shown from our previous work.39-42 Therefore, as before, the mean value for the frequency change for the forward (anodic going) and reverse (cathodic going) scans was determined at -0.20 V. This potential represents the onset of the double layer region for the anodic going sweep where neither hydrogen nor oxide species are specifically adsorbed on the surface (Figure 1). 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 lipid, and the change in mass (∆m) was then calculated using the Sauerbrey equation. It is clear from Figures 1 and 3 that the frequency change at -0.20 V increases with incremental increase in lipid to the bulk solution, indicating that a decrease in

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Figure 4. Absolute change in mass, |∆m|, phosphate buffer (pH 7.0) at 295 K as a function of concentration of stearate (3), oleate (O), linoleate (4), and γ-linolenate (0). Inset: Linearized adsorption isotherm in phosphate buffer (pH 7.0) at 295 K using frequency measurements for stearate (3), oleate (O), linoleate (4), and γ-linolenate (0).

mass is being detected. The electrosorption process is known to displace strongly adsorbed solvent molecules from the metal surface,45 and therefore, if the solvent molecules at the electrode surface that are detected by the EQCN are displaced by larger electrosorbed species that are not detected by the EQCN, this process results in an increase in the frequency response (decrease in mass) of the quartz crystal nanobalance. Similar to our previously reported amino acid and protein studies,22-24 the frequency measurements represent a direct measure of displaced solvent molecules in the double layer region by the presence of lipid. Plots of the absolute change in mass, |∆m|, versus the concentration of lipid in the bulk solution (Figure 4) show similar behavior to the results obtained by CV (Figure 2). The data were again treated using the Langmuir adsorption isotherm in a manner similar to the CV 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 lipid surface concentration. By substituting |∆m| for Γ in eq 3, the following relationship is obtained

c 1 c ) + |∆m| BADS|∆mmax| |∆mmax|

(6)

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 4 (inset) shows the plots for the lipids, which do yield linear relationships indicating 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 lipid adsorption, the parameter BADS was again used to obtain the Gibbs energy of adsorption, ∆GADS, using eq 4 and the results are given in Table 1. The ∆GADS values for the lipids show a high affinity for the Pt surface and agree within the experimental uncertainty of the measurements. In terms of understanding the mechanisms related to fouling, it is important to be able to compare the present experimental results at the Pt surface with those obtained with (45) Daikhin, L.; Gileadi, E.; Tsionsky, V.; Urbakh, M.; Zilberman, G. Electrochim. Acta 2000, 45, 3615.

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Table 2. Comparison of Maximum Surface Concentation (Γmax) Values Obtained from EQCN Frequency and Cyclic Voltammetry Measurements on a Pt Electrode at 295 K and pH 7.0 cyclic voltammetry lipid stearate oleate linoleate γ-linolenate protein R-lactalbumin β-lactoglobulin a

frequency measurements

Γmax/(0.05 mg m-2

A/(0.05 nm2 molecule-1

Γmax/(0.05 mg m-2

A/(0.05 nm2 molecule-1

0.5 1.3 1.3 1.4

1.0 0.4 0.4 0.4

0.3 1.3 1.4 1.3

1.6 0.4 0.4 0.4

3.5 3.6

6.7 8.5

1.5 1.6

0.02a 0.02a

Calculated based on the molar mass of water.

industrial type stainless steel. A ∆GADS value of -43 kJ mol-1 was obtained from electrochemical impedance spectroscopy measurements of linoleate adsorption at the stainless steel surface at the open circuit potential in phosphate buffer, pH 7.0, indicating a similar affinity of linoleate toward the two surfaces.17 Chronocoulometry measurements of sodium dodecyl sulfate adsorption at the Au(111) surface gave ∆GADS ) -39 kJ mol-1,8 a value reasonably similar to that observed for stearate in the present investigation. The present values for ∆GADS at pH 7.0 for the two proteins, R-LA and β-LG, agree within the experimental uncertainty with the results obtained from previous EQCN measurements for the two proteins in a phosphate buffer pH 7.4.24 The two techniques, CV and frequency measurements, measure two very different parameters in the interfacial adsorption process, yet they give excellent agreement within experimental uncertainty for the Gibbs energy of adsorption. In CV measurements, surface charge density (QADS from eq 1) provides a measure of adsorbed analyte by directly measuring the charge associated with analyte adsorption. This charge transfer may result from an electrolyte-assisted process from the carboxylate groups on the proteins and lipids due to the potential field near the electrode surface and does not require the carboxylate group to be in direct contact with the surface. Thus, the surface charge density may provide information on multilayers of adsorbed analyte. However, frequency measurements in the double layer region give a measure of solvent displaced by the lipid or protein adsorbed directly on the electrode surface within the first monolayer (i.e., the “footprint” of the adsorbed molecule). Table 2 provides the |∆mmax| values from the frequency measurements and the corresponding surface concentrations, Γmax, calculated from the CV surface charge density measurements using eq 2. Good agreement for both the surface concentrations and the areas per molecule was obtained with the two techniques for the lipids. It is clear that the fully saturated molecule stearate occupied an area two to four times that of the unsaturated molecules, which may have been due to a strongly tilted or a horizontal orientation of the molecule on the electrode surface.46 A condensed film of stearate would give an area of 0.21 nm2 molecule-1 for vertically oriented molecules due to the strong cohesive attraction between the hydrocarbon tails.47 For example, Xing et al.48 obtained the limiting surface area of 0.21 nm2 per molecule by using the LangmuirBlogett technique to deposit a stearic acid layer onto an Fe surface. (46) Small, D. M. In The Physical Chemistry of Lipids: From Alkanes to Phospholipids; Hanahan, D. J., Ed.; Handbook of Lipid Research; Plenum Press: NewYork, 1986; Vol. 4, p 256. (47) Shaw, D. J. Introduction to Colloid and Surface Chemistry; Butterworth-Heinemann: Oxford, 1992; p 106. (48) Xing, W.; Shan, Y.; Guo, D.; Lu, T.; Xi, S. Corrosion 1995, 51, 45.

On the other hand the unsaturated molecules, oleate, linoleate and γ-linolenate show areas that correspond to the molecules oriented in a more upright or vertical position. However, because of their cis configurations, which inhibit cohesion between the hydrocarbon chains, and the repulsive forces of the carboxylate headgroups, they require more space even in a condensed film than a saturated fatty acid. The areas determined from CV and frequency measurements correspond closely to that observed for condensed film formation of these unsaturated lipids at the aqueous/air interface, i.e., ∼0.35 nm2 molecule-1,47 and the area of the base calculated from the unit cell parameters, i.e., oleic acid, 0.45 nm2/two molecules; linoleic acid, 0.45 nm2/two molecules; and linolenic acid, 0.25 nm2/one molecule.46 Figure 3 shows a hysteresis in the change in frequency response with potential sweep for linoleate at the higher bulk concentrations corresponding to the plateau level in Figure 4. The other lipids show only moderate hysteresis or none in the case of stearate. If we therefore consider the hysteresis effects for linoleate, analysis of the data at -0.2 V as done before, but for the forward going sweep at the highest concentration used rather than the median of the forward and reverse sweeps, shows a plateau surface concentration of 0.86 mg m-2 which converts to the area occupied by the lipid of 0.54 nm2 molecule-1. Thus, in the positive-going sweep, lipids are adsorbing on the surface between the potentials of -0.6 to -0.2 V in a noncondensed form. This trend continues to 0 V. Analysis of the data at 0 V for the forward sweep gives 1.14 mg m-2 and an area of 0.41 nm2 molecule-1 approaching that of a condensed monolayer film. These measurements of increasing change in frequency correspond to decreasing ∆m, i.e., displacement of solvent at the electrode surface by the adsorbed lipid. As the potential is increased from 0 to 0.55 V, the frequency decreases slightly due to competition between a small amount of oxide formation on the surface probably from defects in the lipid layer and further growth of the lipid layer. Analysis of the data at 0.55 V for the anodic end potential gives 2.45 mg m-2 and an area of 0.190 nm2 molecule-1 approaching that almost of a double layer or bilayer on the surface (i.e., 1.8 layers based on a condensed monolayer film occupying 0.35 nm2 molecule-1). On the reverse sweep, the frequency continues to decrease to -0.4 V. Analysis of the data at 0 V for the reverse sweep gives 2.40 mg m-2, which corresponds to an area of 0.195 nm2 molecule-1 which still represents ∼1.8 layers on the surface. Continuing with the negative-going sweep: analysis of the data for the reverse sweep (i.e., lower curve) again at -0.2 V shows the maximum surface concentration as 2.05 mg m-2, which corresponds to a surface area of 0.23 nm2 molecule-1. This is still clearly more than a condensed monolayer of lipid (i.e., 0.35 nm2 molecule-1) but indicates lipids have been lost from the surface leaving 1.55 monolayers. As we proceed even further in the reverse


sweep to the potential of -0.4 V, the maximum surface concentration is 1.26 mg m-2, which corresponds to a surface area of 0.37 nm2 molecule-1 (i.e., a monolayer coverage). As the potential continues to decrease to -0.6, the frequency increases as the monolayer comes off the surface. The process is then repeated with the next cycle. These curves are identically reproducible at equilibrium at which time the measurements are made. Thus, at large negative potentials the lipids are removed from the surface. Increasing the potential to 0 V results in monolayer coverage. A continuous sweep to the highest potentials of 0.6 V results in the formation of a second layer, which gradually comes off the surface during the negative-going sweep leaving a monolayer at -0.4 V, which in turn comes off the surface at potentials of -0.6 V, very likely present in solution as micelles or aggregates as described for octadecanol behavior at high negative potentials from Au(111) by epifluorescence microscopy measurements by Bizzotto and co-workers.14 However, the median of the two values at -0.2 V (in the double layer region where there is only capacitive current, and in which the median represents ∼0 capacitive current) for the forward and reverse sweep corresponds to 0.38 nm2 molecule-1 (Table 2), which corresponds to a condensed form of the lipid monolayer. Although analysis of the hysteresis is interesting and provides insight into the potential field effects on the fluid dynamic behavior of lipid coverage, analysis for comparisons of the net equilibrium effect from each incremental addition of lipid or protein to the bulk solution and in the competitive adsorption measurements is best represented by the median values at the onset of the double layer region at -0.2 V once equilibrium has been established, i.e., for repeatable and identically reproducible cyclic voltammograms. Analysis of the anodic region by EQCN frequency measurements would provide further complications that arise from oxide formation and anion adsorption. Cyclic voltammetry measurements, on the other hand, correct for oxide and anion adsorption as described previously. Because of the hydrophobic nature of the lipid layers, the cyclic voltammetry measurements in this anodic region show charge transfer only from the first layer, and for this reason give good agreement with the EQCN analysis of the lipids for the median value at 0.2 V in the double layer region. Furthermore, in comparison, the proteins show the same amount of adsorbed mass at 0.55 V as at -0.2 V (i.e., in the double layer region) again indicating that this analysis of the frequency changes in the double layer region is valid as well for proteins. The areas occupied by the proteins calculated from Γmax from CV measurements (Table 2) indicate very close packing of the molecules. The reported values of partial molar areas for end-on orientation of R-LA (7.25 nm2 molecule-1) and β-LG (13.93 nm2 molecule-1)49 suggest that greater than a monolayer coverage is occurring at the electrode surface. The simultaneous frequency measurements of solvent displacement by protein adsorption provide the area occupied by a water molecule (Table 2, column 5). Using the O-H bond length of water (0.097 nm) as the radius of a circle representing the area occupied by a water molecule on the surface, an approximate surface area may be calculated as 0.03 nm2. This is in very close agreement with the experimental values determined from the frequency measurements, indicating that these measurements do indeed represent solvent displacement by protein. Similarly, frequency measurements of lipid (49) Al-Malah, K.; McGuire, J.; Sproull, R. J. Colloid Interface Sci. 1995, 170, 261.

Langmuir, Vol. 20, No. 18, 2004 7553

adsorption give surface areas when calculated based on the molar mass of water of 0.1 nm2 molecule-1 for stearate (i.e., five water molecules displaced by the tilted hydrophobic molecule) and 0.02 nm2 molecule-1 for the other unsaturated lipids, again consistent with the displacement of water. On the other hand, when frequency measurements of lipid adsorption are calculated based on the molar mass of the lipids, the surface areas are representative of the adsorbed lipids, showing good agreement with the CV results. This suggests that the close proximity of the carboxylate groups of the lipids to the electrode surface more closely reflects the anion adsorption from the frequency measurements. However, the adsorption of protein displaces less than half the expected amount of solvent, again suggesting that more than a monolayer of protein is adsorbed. Since the EQCN technique provides useful information on the interfacial behavior of lipids and proteins, it was of particular interest to determine whether the technique could be used to investigate protein/lipid adsorption/ displacement behavior. Additions of Protein to an Adsorbed Lipid Layer. The adsorption/displacement behavior of lipids and proteins was studied by first adsorbing a lipid layer onto the platinum surface using a series of additions of analyte to the bulk solution. EQCN frequency and CV measurements were made after each addition of lipid in order to ensure reproducible behavior with the results reported in the previous two sections. The bulk solution concentration of each lipid was increased only to that which provided the onset of the adsorption isotherm plateau for that particular lipid, to avoid a large excess of lipid in the bulk solution. After a lipid layer was adsorbed to the platinum surface, additions of a protein, either R-LA or β-LG, were added to the bulk solution and frequency and CV measurements were measured in order to monitor changes on the electrode surface. Thus, the adsorption of protein to a preadsorbed lipid layer on the platinum surface was studied using the two different but complementary techniques. Figure 5a shows the surface charge density (QADS) from the CV measurements when R-LA was added to each of the absorbed lipid layers. The plateau level of the QADS for each of the lipids and proteins that were studied is shown in Table 3. Figure 5a shows that when protein was added to any of the preadsorbed lipid layers, the surface charge density reached a limiting value approximately 20-25% greater than that observed for the protein alone. This suggests the formation of a mixed layer of protein with lipids filling the voids (i.e., the geometric excess area of a square enclosing a circle is 21%). An abrupt change was observed when protein was added to either the preadsorbed γ-linoleate layer or the linoleate layer. However, when R-LA was added to a preadsorbed oleate layer or stearate layer, there was a less sensitive response to protein concentration. A similar behavior was observed when β-LG was added to the preabsorbed lipid layers (Figure 6a). Since electron transfer does not require the molecule to be in physical contact with the surface of the electrode as it can take place from an adsorbed molecule several layers away, it was of interest to compare the CV results with the simultaneously recorded frequency measurements which provide information on the absolute change in mass (|∆m|) due to solvent displacement by the adsorbed molecules directly on the electrode surface. When additions of R-LA were made to preadsorbed γ-linolenate, linoleate, and stearate layers, Figure 5b shows that the absolute change in mass increased to about 1.5 mg m-2, consistent with that normally observed with

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Wilson and Roscoe

Table 3. Comparison of the Plateau Level for Surface Charge Density (QADS) for Each of the Lipids and Proteins Studied at a Pt Surface at 295 K and pH 7.0

lipid stearate oleate linoleate γ-linolenate protein R-lactalbumin β-lactoglobulin

plateau QADS/(1 µC cm-2

∆QADS/(2 µC cm-2 with R-lactalbumin

∆QADS/(2 µC cm-2 with β-lactoglobulin

30 68 78 99

65 28 24 4

70 19 11 2

75 80

R-LA alone (cf. Table 2). However, addition of R-LA to the oleate preadsorbed layer caused a gradual increase to 1.4 mg m-2 without reaching a flat plateau. This means that as R-LA was added to the solution it was able to displace some of the preadsorbed lipid layer and was most efficient at displacing γ-linolenate, linoleate, and stearate, with oleate being the most difficult to displace. This is also confirmed by the EQCN frequency profile changing from that characteristic of the lipid (Figure 3) to that characteristic of a protein.23,24 This behavior is also consistent with the surface charge density measurements (Figure 5a). The higher surface charge densities indicate the presence of mixed protein/lipid layers. When additions of β-LG were made to the preadsorbed lipid layer (Figure 6b), the absolute change in mass decreased to about 0.8 mg m-2 for oleate, linoleate, and

γ-linolenate which suggests that the lipid layer was being removed or lifted from the surface allowing an increase in solvent adsorption to occur at the electrode surface. This is also confirmed by the EQCN frequency profile remaining as that characteristic of the lipid (Figure 3) rather than conversion to that characteristic of a protein.23,24 This is also consistent with the surface charge density results (Figure 6a) which show a rapid increase to a plateau value suggesting the presence of the β-LG above the lipid layer interacting with the preadsorbed lipid layer. This interaction is consistent with the fact that β-LG is known to be able to transport fatty acids.34 Stearate was the exception which showed the absolute change in mass increased when β-LG was added to a stearate layer, indicating that β-LG coadsorbed or displaced the adsorbed stearate. Thus the CV and frequency

Figure 5. Additions of R-lactalbumin to preadsorbed layers of stearate (3), oleate (O), linoleate (4), and γ-linolenate (0). Additions to stearate (1), oleate (b), linoleate (2), and γ-linolenate (9). (a) Cyclic voltammetry. (b) Frequency measurements.

Figure 6. Additions of β-lactoglobulin to preadsorbed layers of stearate (3), oleate (O), linoleate (4), and γ-linolenate (0). Additions to stearate (1), oleate (b), linoleate (2), and γ-linolenate (9). (a) Cyclic voltammetry. (b) Frequency measurements.


Langmuir, Vol. 20, No. 18, 2004 7555

Figure 7. Additions of lipid to preadsorbed R-lactalbumin (b). Additions of stearate (3), oleate (O), linoleate (4), and γ-linolenate (0). (a) Cyclic voltammetry. (b) Frequency measurements.

Figure 8. Additions of lipid to preadsorbed β-lactoglobulin (9). Additions of stearate (3), oleate (O), linoleate (4), and γ-linolenate (0). (a) Cyclic voltammetry. (b) Frequency measurements.

measurements show two distinctly different behaviors of the proteins with the preadsorbed lipid layers; i.e., R-LA coadsorbs by a displacement process, while β-LG adsorbs on top of the oleate, linoleate, and γ-linolenate layers and removes the lipids from the surface without adsorbing directly on the surface. However, β-LG appears to be able to displace a stearate layer or coadsorb directly on the surface. Desorption of Protein with Lipids. After studying the adsorption behavior of the proteins, R-LA and β-LG, to a preadsorbed lipid layer, it was of interest to determine the ability of the lipids to desorb or solubilize preadsorbed protein and to determine whether the two simultaneous techniques of CV and EQCN frequency measurements would be able to detect and monitor these changes on the electrode surface. A layer of protein was adsorbed to the surface of the platinum electrode by making additions of protein to the bulk solution and monitoring the adsorption process with the CV and frequency measurements described previously. The concentration of protein in the bulk solution was increased until the onset of the isotherm plateau was reached, which corresponded to bulk concentrations of 5.0 × 10-2 and 8.0 × 10-2 g L-1 for R-LA and β-LG, respectively. Additions of one of the lipids were then made to the bulk solution and the simultaneous measurements were made in order to monitor surface changes. When lipid was added to a preadsorbed layer of R-LA, the surface charge density (QADS) increased by about 20

µC cm-2 with oleate, linoleate, and γ-linolenate and reached a value of ∼87 µC cm-2 (Figure 7a). However, stearate showed only a small increase in the surface charge density suggesting little interaction with the adsorbed protein. A similar response was observed in Figure 8a when β-LG formed the preadsorbed layer, but this time the response with linoleate was significantly less sensitive to bulk concentration than the other two lipids. The interaction of γ-linoleate with the preformed β-LG layer was greater than that of oleate and reached a value of ∼97 µC cm-2. Since the surface charge densities were greater than either the lipids or proteins alone, with the exception of γ-linoleate, this indicates the presence of coadsorbed lipid/protein layers. Stearate again showed little interaction with the adsorbed protein. The EQCN frequency measurements for the addition of oleate, linoleate, and γ-linolenate to a preformed R-LA layer (Figure 7b) and a preformed β-LG layer (Figure 8b) showed the absolute change in mass (|∆m|) to decrease to levels similar to those observed for the plateau levels for the lipids in the absence of protein (see Table 2). This indicates that the protein has been desorbed or solubilized from the electrode surface, that the surface is covered essentially by lipids, but that some protein remains sufficiently near the surface to cause the surface charge density increase observed with the CV measurements. Oleate was the most efficient lipid to solubilize both proteins since the absolute change in mass decreased

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rapidly to that observed in the absence of protein. The results indicated that linoleate and then γ-linolenate were the next most efficient, approaching the values normally observed in the absence of protein, but with stearate the level did not decrease. These results are also confirmed by the EQCN frequency profile changing from that characteristic of a protein23,24 to that characteristic of the lipid (Figure 3) for oleate and linoleate. This change in frequency profile was not observed for stearate with either protein or γ-linolenate with R-LA. Thus the results from both CV and frequency measurements are consistent in showing that oleate, linoleate, and γ-linolenate are able to solubilize proteins from the platinum surface but stearate was unable to effectively desorb either of the proteins. However, an independent method such as XPS, time-of-flight secondary ion mass spectrometry, or radiolabeling measurements would be required to unequivocally verify that the surface is essentially covered by lipids. Conclusions CV and EQCN frequency measurements were used to investigate the role of double bonds on the surface adsorption behavior on a platinum surface of a series of lipids containing 18 carbon atoms; stearate (C18:0), oleate (C18:1), linoleate (C18:2), and γ-linolenate (C18:3). In addition, the adsorption behaviors of the two milk proteins, R-LA and β-LG, were characterized using the EQCN technique in order to subsequently study their adsorption/ displacement behavior with the lipids. The EQCN frequency changes in the double layer region were used as a measure of solvent displacement by the adsorbed protein or lipid and served as an indirect measure of the amount of adsorbed analyte. The Langmuir adsorption isotherm successfully described the adsorption process for both CV and double layer EQCN frequency measurements for each of the proteins and lipids studied. The ∆GADS values (Table 1) obtained from the frequency measurements gave excellent agreement within the experimental uncertainty with the ∆GADS values calculated from CV measurements for all of the proteins and lipids. The high negative values indicate a high affinity of both types of species for the platinum surface. The areas occupied by the proteins (Table 2) calculated from the surface concentration (Γmax) values from CV

Wilson and Roscoe

measurements indicated very close packing of the molecules. The areas calculated from the frequency measurements correspond closely to that of water, indicating that these measurements do indeed represent solvent displacement by protein. The high Γmax values for the lipids with double bonds indicate that the molecules are oriented in an upright condensed state on the electrode surface. On the other hand, the very low Γmax value observed for stearate suggests the molecule occupies a large surface area, perhaps in a strongly tilted or horizontal orientation with little cohesion between the hydrocarbon tails. The addition of R-LA added to a preadsorbed lipid layer showed the protein to coadsorb with the lipid by the increase in the surface charge density from CV measurements and the increase in the absolute change in mass from the frequency measurements. However, when β-LG was added to a preadsorbed lipid layer of oleate, linoleate, and γ-linolenate, the protein was able to desorb some of the lipid, consistent with its known ability to transport lipids. The exception was stearate where the protein appeared to coadsorb with the lipid in a manner similar to that observed with R-LA. A study of the solubilization of preadsorbed milk proteins on the Pt surface by lipids showed that the proteins were readily desorbed with the order of efficiency by lipid being oleate > linoleate > γ-linolenate. However, stearate was very ineffective in removing protein. The results of the present investigation indicate that the EQCN is a valuable tool in monitoring the adsorption processes of individual species at a Pt surface as well as the adsorption/displacement phenomena of two very different species (i.e., protein and lipids). These results also help us to understand the adsorption behavior of these lipids and proteins in terms of their roles in surface fouling and as potential corrosion inhibitors of metal surfaces. 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. LA049478X

Electrochemical Quartz Crystal Nanobalance Study of the Adsorption

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