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Electrochemical Quartz Crystal Microbalance Study of Azurin Adsorption onto an Alkanethiol Self-Assembled Monolayer on Gold Barry D. Fleming, Slavica Praporski, Alan M. Bond, and Lisandra L. Martin* School of Chemistry, Monash UniVersity, Clayton, Victoria 3800, Australia ReceiVed August 13, 2007. In Final Form: October 3, 2007 A quartz crystal microbalance coupled with electrochemistry was used to examine the adsorption of azurin on a gold electrode modified with a self-assembled monolayer of octanethiol. Azurin adsorbed irreversibly to form a densely packed monolayer. The rate of azurin adsorption was related to the bulk concentration of azurin in solution within the concentration range studied. At a high azurin concentration (2.75 µM), adsorption was rapid with a stable adsorption maximum attained in 2-3 min. At a lower azurin solution concentration (0.35 µM), the time to reach a stable adsorption maximum was approximately 30 min. Interestingly, the maximum surface concentration attained for all solution concentrations studied by the QCM method was 25 ( 1 pmol cm-2, close to that predicted for monolayer coverage. The dissipation was monitored during adsorption, and only small changes were detected, implying a rigid adsorption model, as needed when using the Sauerbrey equation. Cyclic voltammetric data were consistent with a one-electron, surface-confined CuII/CuI azurin process with fast electron-transfer kinetics. The electroactive surface concentration calculated using voltammetry was 7 ( 1 pmol cm-2. The differences between the QCM and voltammetrically determined surface coverage values reflect, predominantly, the different measurement methods but imply that all surface-confined azurin is not electrochemically active on the time scale of cyclic voltammetry.
Introduction Adsorption of proteins at solid-liquid interfaces represents an important process that has significant applications in both nature and industry. For example, many biochemical processes within living systems rely on the adsorption of proteins to lipid layers,1 several bioseparation procedures utilize the specific adsorption of proteins to a stationary phase,2 and the fabrication and operation of biodevices such as biosensors requires bioactive protein molecules on a surface.3 Of particular interest is the controlled, selective, rapid, durable, and nondenaturing attachment of proteins, particularly enzymes, to a surface. Specifically, if the properties of the fully functional protein can be maintained, then the proteins may be used to perform analogous tasks to those they do in naturescatalyze difficult transformations, transfer electrons, sense specific molecules, harvest energy, and a host of other functions. The quartz crystal microbalance (QCM) has recently been used to examine cell and protein adsorption onto a variety of modified and unmodified solid surfaces.4-15 These studies have * To whom correspondence should be addressed. Phone: +61 3 99054514. Fax: +61 3 99054597. E-mail:
[email protected]. (1) Tamm, L. K., Ed.; Protein-Lipid Interactions: From Membrane Domains to Cellular Networks; Wiley-VCH: Weinheim, 2005. (2) Ahuja, S., Ed. Handbook of Bioseparations; Academic Press: San Diego, 2000. (3) Gorton, L., Ed. Biosensors and Modern Biospecific Analytical Techniques; Elsevier: Amsterdam, 2005. (4) Caruso, F.; Furlong, D. N.; Kinshott, P. J. Colloid Interface Sci. 1997, 186, 129-140. (5) Ho¨o¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. J. Colloid Interface Sci. 1998, 208, 63-67. (6) Ho¨o¨k, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12271-12276. (7) Fredriksson, C.; Kihlman, S.; Rodahl, M.; Kasemo, B. Langmuir 1998, 14, 248-251. (8) Ho¨o¨k, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796-5804. (9) Schenkman, J. B.; Jansson, I.; Lvov, Y. M.; Rusling, J. F.; Boussaad, S.; Tao, N. J. Arch. Biochem. Biophys. 2001, 385, 78-87. (10) Glasmastar, K.; Larsson, C.; Ho¨o¨k, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40-47. (11) Asakura, N.; Kamachi, T.; Okura, I. Anal. Biochem. 2003, 314, 153-157.
determined the amount and kinetics of adsorption as well as provided information relating to protein surface charge, specific and nonspecific interactions, and structural changes associated with the adsorption process. Changes in the fundamental resonant or overtone frequency, ∆f, of an oscillating surface (typically a gold coated quartz crystal) can be converted into a mass change, ∆m, having the units of ng‚cm-2, via the Sauerbrey equation16
∆m ) -C
∆f n
(1)
where C is a constant that depends on the physical properties of the crystal (C ) 17.7 ng‚cm-2‚Hz-1 for the crystal used in this work) and n is the overtone number. Hence, a decrease in frequency correlates to an increase in mass attached to the surface. The surface coverage of the adsorbed protein layer, Γ (mol‚cm-2), can then be readily calculated using eq 217
Γ)
∆m MW
(2)
where MW is the molar mass of the protein. The Sauerbrey relationship assumes there is no variation in the energy dissipated at the oscillating surface.18 That is, the adsorbed layer must be rigidly attached and its motion coupled to the oscillating surface, as can apply to a densely packed, rigid protein layer. Recently, a quartz crystal microbalance-dissipation or QCM-D instrument was designed which can measure the change in the (12) Beissenhirtz, M. K.; Kafka, J.; Schafer, D.; Wolny, M.; Lisdat, F. Electroanalysis 2005, 17, 1931-1937. (13) Muguruma, H.; Kase, Y.; Murata, N.; Matsumura, K. J. Phys. Chem. B 2006, 110, 26033-26039. (14) Kaufman, E. D.; Belyea, J.; Johnson, M. C.; Nicholson, Z. M.; Ricks, J. L.; Shah, P. K.; Bayless, M.; Pettersson, T.; Feldoto, Z.; Blomberg, E.; Claesson, P.; Franzen, S. Langmuir 2007, 23, 6053-6062. (15) Nakano, K.; Yoshitake, T.; Yamashita, Y.; Bowden, E. F. Langmuir 2007, 23, 6270-6275. (16) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (17) Lam, K. B.; Irwin, E. F.; Healy, K. E.; Lin, L. Sensors Actuators B 2006, 117, 480-487. (18) Rodahl, M.; Hook, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. ReV. Sci. Instrum. 1995, 66, 3924-3930.
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Figure 1. Simplified model (not to scale) of azurin adsorbed onto an alkanethiol-modified gold electrode. The molecules are shown in their idealized orientation based on the expected interactions between the hydrophobic surface residues of azurin (red) and the alkane SAM.
energy dissipation factor, ∆D, while simultaneously measuring ∆f.18 The energy dissipation factor, D, can be simply defined as shown in eq 3
D)
Edissipated 2πEstored
(3)
where Edissipated is the energy dissipated per cycle of oscillation and Estored is the total energy stored in the oscillating system. The ∆D measurement has enabled sensitive interrogation of the viscoelastic properties of the adsorbed protein and cell layers.5-7 Significant information about the adsorbed layer also can be obtained by coupling the QCM method with electrochemistry.19 In this configuration the Au-coated quartz QCM chip is also the working electrode, so direct correlation between QCM and electrochemical data can be made. Advantages in this approach were recently shown in a study of the coupling between cytochrome c3 in its different redox states and a viologen-modified electrode.11 Azurin (14.6 kDa), the protein of interest in the present study, is responsible for electron transfer in the respiratory system of several bacteria.20 It contains a single copper atom, bound by two histidine (N), a cysteine (S), methionine (S), and glycine (O) residues, that undergoes a one-electron redox process, converting it between the CuII and the CuI redox states.21 Azurin is believed to interact with its redox partners via an area of hydrophobic residues located close to the copper site.22 There are numerous reports on the exploitation of azurin adsorption onto solid surfaces, particularly on gold electrodes modified with alkanethiol selfassembled monolayers (SAMs).23-27 Figure 1 provides a simplified model of an idealized azurin monolayer formed after adsorption onto an alkanethiol SAM on gold. The majority of these investigations have been for the purpose of establishing its electron-transfer properties. The adsorbed azurin system is now regarded as a model of an essentially ideal surface-confined electron-transfer system. However, little information is available on the fundamental adsorption process. In order to address this issue we now provide the first electrochemical QCM-D study of azurin adsorption onto a SAM of octanethiol-modifed gold. (19) Bott, A. W. Curr. Sep. 1999, 18, 79-83. (20) Ryden, L. In Copper proteins and copper enzymes; Lontie, R., Ed.; CRC Press: Boca Raton, 1984; Vol. 1, pp 157-182. (21) Nar, H.; Messerschmidt, A.; Huber, R.; van de Kamp, M.; Canters, G. W. J. Mol. Biol. 1991, 221, 765-772. (22) van de Kamp, M.; Silverstrini, M. C.; Brunori, M.; Beeumen, J. V.; Hali, F. C.; Canters, G. W. Eur. J. Biochem. 1990, 194, 109-118. (23) Gaigalas, A. K.; Niaura, G. J. Colloid Interface Sci. 1997, 193, 60-70. (24) Fristrup, P.; Grubb, M.; Zhang, J.; Christensen, H. E. M.; Hansen, A. M.; Ulstrup, J. J. Electroanal. Chem. 2001, 511, 128-133. (25) Jeuken, L. J. C.; Armstrong, F. A. J. Phys. Chem. B 2001, 105, 52715282. (26) Chi, Q.; Zhang, J.; Andersen, J. E. T.; Ulstrup, J. J. Phys. Chem. B 2001, 105, 4669-4679. (27) Fleming, B. D.; Barlow, N. L.; Zhang, J.; Bond, A. M.; Armstrong, F. A. Anal. Chem. 2006, 78, 2948-2956.
Experimental Section Chemicals. All chemicals were commercially available and used as supplied, including 1-octanethiol (98%, Lancaster Synthesis), ethanol (Merck), sodium chloride (Fluka), and isopropanol and sodium acetate (Sigma). A stock solution of purified Pseudomonas aeruginosa azurin (0.93 mM) was diluted into acetate buffer (0.02 M, pH 4.0, 0.1 M NaCl) to the desired concentration. Ultrapure water (18.2 MΩ) was used to prepare all solutions (Sartorius Arium 611 system). QCM. QCM measurements were performed with a Q-sense E4 instrument (Q-Sense, Sweden) using gold ‘chips’ that consisted of a thin quartz crystal coated with a gold layer (Q-Sense, f ) 5 MHz, C ) 17.7 ng‚cm-2‚Hz-1 geometric area of gold working surface ) 0.78 cm2). The gold chips also served as the working electrode in the electrochemical measurements. The chips were housed in a specially designed temperature-controlled cell that served as a chip holder and as an electrochemical cell (volume ≈ 0.7 mL; Q-sense; Echem module, QEC401, Sweden). All experiments were performed at a temperature of 22.00 ( 0.05 °C. Prior to each experiment, chips were cleaned in a NH4OH (28%):H2O2 (30%):H2O (1:1:3 v/v/v) mixture at 70 °C for 15 min, washed several times with water and ethanol, and dried under a stream of nitrogen. The cleaned chip was immediately immersed into an octanethiol solution (1 mM in isopropanol), and the SAM developed on the gold surface over a period of at least 12 h. The chip was rinsed with isopropanol to remove any physisorbed thiol molecules, dried under a nitrogen stream, and then immediately positioned and sealed in the QCM cell. A peristaltic pump was used to introduce solutions into the cell. Following inclusion of the sample into the measurement chamber, the ∆f and ∆D changes associated with four overtone frequencies (15, 25, 35, and 45 MHz) were recorded. These frequencies correlate with the third, fifth, seventh, and ninth harmonics. The ∆f and ∆D changes associated with the fundamental frequency (5 MHz) were sensitive to changes in bulk solution. The Q-sense E4 instrument provides the overtone-normalized ∆f data, that is ∆f/n, and this is the value that is generally reported throughout this paper. This allows us to compare directly the frequency change for each harmonic. To help distinguish this, we refer to the normalized ∆f data as ∆fN. The stability of the QCM response was first tested in pure water, and if stable after 10 min, the cell contents were exchanged with buffered electrolyte solution. Again, a period of time (10-15 min) was allowed to check for stability in the ∆fN and ∆D responses. Following this, approximately 1 mL of azurin solution was slowly pumped (50 µL/min) into the cell and ∆fN and ∆D were monitored with time. After a maximum ∆fN value had been reached (5-40 min) the flow was stopped and the response monitored for at least 15 min. Buffered electrolyte solution containing no azurin was then pumped (300 µL/min) into the cell for 5 min to remove unadsorbed or weakly bound azurin. The flow was stopped and ∆fN and ∆D were monitored with time. The frequency change observed at this stage was designated to be ∆fmax, the maximum change in frequency. Electrochemistry. The QCM cell was coupled to a µ-Autolab (EcoChemie, Utrecht, Netherlands) or an Epsilon (BAS, West Lafayette, USA) electrochemical workstation. Cyclic voltammetry experiments were performed using a conventional three-electrode configuration. The gold chip was used as the working electrode, a
Study of Azurin Adsorption
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Figure 3. Change in ∆fN observed prior to the high flow rinse step for azurin concentrations of (9) 0.035, (b) 0.35, and (2) 2.75 µM. Data shown are from the ninth overtone frequency with a flow rate of 50 µL/min.
Figure 2. Change in (a) ∆fN and (b) ∆D observed for the third (9), fifth (b), seventh (2), and ninth (1) overtones during a QCM experiment when azurin is adsorbed onto an octanethiol SAM on gold. The timing of flow and solution changes is indicated with dotted lines. The plots shown are from an experiment using an azurin concentration of 0.35 µM. platinum disc served as the counter electrode, and a “no leak” Ag/ AgCl (3 M KCl) electrode (Cypress Systems, Chelmsford, MA) was used as the reference electrode. All potential values are quoted with respect to the standard hydrogen electrode (SHE), where SHE is 0.207 V versus Ag/AgCl (3 M KCl) at 22 °C. The working electrode area was calculated electrochemically to be 0.87 cm2 (geometric area ) 0.78 cm2) based on the peak current obtained from a 0.75 mM potassium ferricyanide solution and applying the RandlesSevcˇick equation.28 Voltammetric scans were performed in 0.1 M NaCl solution containing 0.02 M acetate buffered at pH 4.0. The potential was cycled between 0.4 and -0.1 V vs Ag/AgCl (3 M KCl). The scan rate ranged from 0.01 to 100 V‚s-1. The uncompensated cell resistance was
