Buildup of Polyelectrolyte−Protein Multilayer Assemblies on Gold

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Buildup of Polyelectrolyte-Protein Multilayer Assemblies on Gold Electrodes. Role of the Hydrophobic Effect E Ä lisabeth Lojou and Pierre Bianco* Unite´ de Bioe´ nerge´ tique et Inge´ nierie des Prote´ ines, Institut de Biologie Structurale et Microbiologie - CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France Received July 15, 2003. In Final Form: November 17, 2003 The buildup of layer-by-layer assemblies onto gold surfaces from water-soluble charged polyelectrolytes and proteins is examined using quartz crystal microgravimetry (QCM) and electrochemical techniques. Polyelectrolytes such as poly(styrenesulfonate) and poly(ester sulfonic acid) (Eastman AQ-29D polymer) adsorb spontaneously onto gold, contrary to poly(ethyleneimine). From the modification of the gold surface with a thiol and specific adsorption of polymers under polarization conditions, it is concluded that the hydrophobicity of the gold surface seems to be a determining factor in the adsorption process. Alternate adsorption onto gold resonators first coated with AQ-29D polymer gives stable multilayer films in the case of positively charged lysozyme (pI ) 11) or polyheme Desulfovibrio vulgaris Hildenborough cytochrome c3 (pI ) 10.5). QCM frequency changes with the number of adsorption steps suggest that a linear increase in film mass occurs. Desulfomicrobium norvegicum polyheme cytochrome c3 (pI ) 7), which has a null global charge at neutral pH, is shown to give also stable multilayer AQ-29D/cytochrome c3 films, suggesting that several types of interactions, especially the hydrophobic effect, are involved in the buildup process.

Introduction Well-defined spatial organization of proteins is required to develop an optimal efficiency for most functions in biological systems. For this reason, the development of methodologies for artificial buildup of macromolecules is an important challenge in protein engineering. Techniques for the construction of ultrathin film assemblies based on stepwise adsorption in a controlled spatial arrangement (in particular layer-by-layer (LBL) assembly) from solutions have been developed increasingly in the past decade.1,2 Through these techniques, it has become possible to fabricate nanocomposite films containing various compounds such as polymers, biomolecules, nanoparticles, or molecular aggregates. One of the major reasons for interest in the LBL deposition process is that the thickness of the deposited films can be controlled at the molecular level with good precision. Another advantage of the LBL assembly technique is its simplicity. The technique of LBL assembly based on sequential adsorption by alternate immersion into aqueous solutions of anionic and cationic species has been often used for polyelectrolytes and proteins having opposite charges, thus highlighting the role of electrostatic interaction in assembly processes.3-10 Nevertheless, several experimental events have contributed to reveal the important role of * Corresponding author. Fax: (+33) 491 16 45 78. Phone: (+33) 491 16 46 10. E-mail: [email protected]. (1) Protein Architecture; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000. (2) Multilayer Thin Films; Decher, G., Schlenoff, J. B., Eds.; WileyVCH: Weinheim, 2003. (3) Decher, G. Science 1997, 277, 1232. (4) Lvov, Y.; Ichinose, I.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (5) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (6) Serizawa, T.; Hashiguchi, S.; Akashi, M. Langmuir 1999, 15, 5363. (7) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 284-285, 797. (8) Lvov, Y.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073. (9) Lu, Z.; Hu, N. J. Colloid Interface Sci. 2002, 254, 257. (10) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708.

other factors in the buildup of multilayers, such as iondipole or dipole-dipole interactions, the hydrophobic effect, hydrogen bonding, or entropic factors related to surface-induced conformational changes.11-13 Recent experimental data have shown that polyelectrolyte multilayers are able to strongly interact with proteins whatever the sign of the charge of both the multilayer and the protein.14 In a previous paper,15 the construction of polyheme c-type cytochrome/polypeptide assemblies has been monitored through electrochemistry and quartz crystal microgravimetry (QCM) techniques. We have shown that good candidates that emerge from the polypeptide family are poly(ester sulfonic acid) ionomers (e.g., Eastman AQ29D polymer). Similar to Nafion, AQ films can bind hydrophobic cations preferentially and exclude negatively charged species.16-18 A very interesting property specific to AQ films is that they are able to incorporate basic proteins.19,20 Accordingly, they can have a dual function, as incorporating agents and as interlayer polyion “glues”.15 Moreover, we have demonstrated that AQ films may spontaneously adsorb on gold surfaces, creating stable negatively charged layers. It is one of our goals to better understand the nature of the interactions between polyions and both electrode surfaces and proteins. An important correlation has been found to exist21 between the chemical structure of the polyion, specifically the hydrophobic/ hydrophilic nature of the polymer backbone, and the partner molecules. In this paper, we attempt to explore (11) Fisher, P.; Laschewsky, A. Macromolecules 2000, 33, 1100. (12) Shimazaki, Y.; Mitsuishi, M.; Ito, S.; Yamamoto, M. Langmuir 1998, 14, 2768. (13) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717. (14) Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel, J. C. Langmuir 2001, 17, 878. (15) Lojou, E Ä .; Bianco, P. J. Electroanal. Chem. 2003, 557, 37. (16) Wang, J.; Golden, T. Anal. Chem. 1989, 61, 1397. (17) Wang, J.; Lu, J. J. Electroanal. Chem. 1989, 266, 287. (18) Gennett, T.; Purdy, W. C. Anal. Chem. 1990, 62, 2155. (19) Bianco, P.; Taye, A.; Haladjian, J. J. Electroanal. Chem. 1994, 377, 299. (20) Lojou, E Ä .; Luciano, P.; Nitsche, S.; Bianco, P. Electrochim. Acta 1999, 44, 3341. (21) Clark, S. L.; Hammond, P. T. Langmuir 2000, 16, 10206.

10.1021/la030286w CCC: $27.50 © 2004 American Chemical Society Published on Web 01/08/2004

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the relative importance of ionic interactions versus other different effects that can be suspected to be involved in multilayer buildup, in particular the hydrophobic effect. We present experimental observations on the behavior of different polymers at the gold electrode surface and buildup of LBL assemblies involving different proteins, through QCM and electrochemical techniques. One of the major reasons for using electroactive proteins is that the measure of the electrochemical activity of the immobilized enzymes can be followed concomitantly with the multilayer formation. In this report, proteins differing in structure and overall charge are tools for investigating the capability of macromolecules to associate with polyions. In particular, c-type cytochromes, that offer the advantage of displaying different properties (such as isoelectric point and charge surface distribution) and for which structures have been generally determined, are of special interest. Experimental Section Materials. Horse heart cytochrome c (type VI) from Sigma was purified by chromatography on carboxymethylcellulose (CM-52, Whatman). Cytochrome c3 from Desulfovibrio vulgaris Hildenborough (DvH) and Desulfomicrobium norvegicum (Dn) was prepared and purified in our laboratory as previously described.22-24 Lysozyme from chicken egg white and poly (L-lysine) hydrobromide (PLL, MW ) 3800 and 14 600) were obtained from Sigma. Poly(ester sulfonic acid) polymer (its trademark is Eastman AQ Polymer), in the present case Eastman AQ-29D (30% dispersion, MW ) 16 000), was a gift from Eastman Chemical Co. A diluted (1:20 v/v Eastman AQ-29D polymer/water) solution was used for modifying the electrode surfaces. Nafion coatings were prepared from a 5% alcoholic solution of Nafion (1100 equivalent weight, Aldrich); diluted solutions were obtained by adding water to the crude product. Other polyions were sodium poly(styrenesulfonate) (PSS, MW ) 70 000, from Aldrich) in water and poly(ethylenimine) (PEI, high molecular weight, average MW ) 25 000, Aldrich) in water. When necessary, gold electrodes were modified using sodium 3-mercapto-1-propane sulfonate (MPS, Aldrich). Some QCM experiments were performed at an aldrithiol-modified gold resonator using aldrithiol (i.e., bis(4pyridyl) disulfide) from Aldrich. All aqueous solutions were prepared with ultrapure (Milli-Q plus, Millipore) water. The supporting electrolyte generally was 10 mM Tris chloride buffer solution, at pH 7.6. Apparatus. Cyclic voltammetry (CV) and square-wave voltammetry (SWV) were obtained using an EG&G 6310 electrochemical impedance analyzer modulated by EG&G PAR M 270/ 250 software. CV voltammograms were generally recorded at 0.1 V s-1. SWV voltammograms were obtained using 5 Hz as the square-wave frequency, 2 mV as the scan increment, and 25 mV as the pulse height amplitude. A three-electrode system consisting of a Metrohm Ag/AgCl/NaCl(sat.) reference electrode, a gold wire auxiliary electrode, and the working electrode was used throughout. Working gold electrodes were constructed from 1 mm diameter gold wire inserted in resin casings. Unless otherwise specified, all potentials reported here refer to the Ag/AgCl/NaCl(sat.) reference electrode. Potentials versus the standard hydrogen electrode (SHE) can be obtained by adding 210 mV. Prior to each experiment, the solutions were deoxygenated by bubbling high-purity nitrogen. All experiments were carried out at room temperature (about 23 °C) under a high-purity nitrogen atmosphere. QCM studies were performed using a MAXTEK PM-710 plating monitor coupled with a MPS-550 sensor probe. The MAXTEK quartz resonators were made from AT-cut quartz crystals (resonance frequency, 5 MHz) covered by evaporated gold on both faces (apparent electrode areas of 0.316 cm2 for the “small” type and 1.37 cm2 for the “large” type). Quartz resonators (22) Bruschi, M.; Le Gall, J. Biochim. Biophys. Acta 1972, 271, 48. (23) Bruschi, M.; Hatchikian, C. E.; Golovleva, L. A.; Le Gall, J. J. Bacteriol. 1977, 129, 30. (24) Van der Westen, H. N.; Mayhew, S. G.; Veeger, C. FEBS Lett. 1978, 86, 122.

Langmuir, Vol. 20, No. 3, 2004 749 were used without any washing/polishing/electrochemical pretreatment. QCM measurements were performed in two modes: either (i) one side of the resonator was covered with the polyelectrolyte (or protein) solution of interest for a selected period, washed with pure water or buffer solution, and dried with a hair-drier (without warming), and the frequency was measured; or (ii) the adsorption was monitored in situ by immersing the resonator with only one face placed in contact with the solution of interest and recording the frequency variation continuously. Preparation of the Modified Electrodes and Film Assembly. Gold electrode surfaces used for voltammetry experiments were polished with 0.05 µm alumina slurry to obtain a mirror finish and then sonicated in a water bath. Modification of the electrode surface was carried out by two methods. When no pretreatment was required, the electrodes were dipped in the polyion/protein solution of interest for a given time, rinsed with water, and then placed into the electrochemical cell containing the buffer solution. When a pretreatment was required, modification of the gold surface was accomplished by immersing the polished electrode surface (for CV and SWV) or the virgin resonator surface (for QCM) in 1 mM MPS ethanolic solution for 15 h8 or 1 mM aldrithiol aqueous solution for 5 min, followed by rinsing in pure ethanol and water (for MPS treatment) or water (for aldrithiol treatment). Films were cast on the untreated (or modified) electrode surfaces by repeating alternate adsorption for 15 min from aqueous solutions of cytochromes or PSS or 5 min from a 1:20 v/v AQ 29-D/water suspension. The deposition process was periodically interrupted, and then the electrodes were dried for QCM measurements or immersed into the electrochemical cell for CV and SWV experiments. All the measurements were performed using the sensor probe in the vertical position. During the adsorption steps, however, when using cytochrome c3 solutions (available only in small amounts), lysozyme or PLL solutions, the sensor probe was placed in a horizontal position; a drop (of about 20 µL) of protein (or polypeptide) solution was deposited on the resonator surface, a lid was placed on the top end of the crystal retainer, and contact was maintained over the selected time before recovery of the solution by suction with a micropipet and a washing step. The mass increase resulting from adsorption onto the resonator surface was estimated from the Sauerbrey equation25 using the following relationship between adsorbed mass ∆M (ng) and frequency shift ∆F (Hz), by taking into account the characteristics of the resonators:

∆M ) -17.6A∆F where A corresponds to the apparent area of the quartz microbalance electrode. In these conditions, a decrease in frequency of 1 Hz results from a mass increase of 17.6 ng cm-2 provided that shifts in frequency can be ascribed exclusively to mass effects and not to changes in solution density or viscosity.26 The thickness d (nm) of the film (adsorbed on one side of the resonator) can be estimated from the relationship d ) 0.176∆F/F where F (g cm-3) is the density of the deposited film. The values of F currently used are 1.2 g cm-3 for polymer films and 1.3 g cm-3 for protein films.27,28

Results Adsorption of Polyelectrolytes on the Gold Surface. The alternate adsorption of polyelectrolyte species is a convenient way to construct LBL assemblies. The first step in building up the structure consists of modifying the gold surface to make it negative or (possibly) positive. A well-described method for obtaining a negatively charged surface is to use thiol modification. In a previous paper,15 (25) Sauerbrey, G. Z. Phys. 1959, 155, 206. (26) Deakin, M. R.; Buttry, D. A. Anal. Chem. 1989, 61, 1147A. (27) Brandrup, J.; Immergut, E. Polymer Handbook; Wiley & Sons: New York, 1975. (28) Creighton, T. E. Protein Structure, A Practical Approach; IRL Press: New York, 1990.

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Figure 1. Structures of the three polymers studied at the QCM gold resonator. Table 1. QCM Frequency Changes at a Nonmodified/ Aldrithiol-Modified Gold Resonator with or without Polarization for Different Polymers polymer frequency change at nonmodified resonator/Hz frequency change at aldrithiol-modified resonator/Hz frequency change at polarized resonator (+0.2 V for PEI, -0.2 V for PSS and AQ-29D)/Hz

PEI

PSS

AQ-29D

0

-41

-40

-48

0

-14

0

0

-30

we have shown that another approach would be to preadsorb a polyanionic film such as AQ-29D polymer. The use of QCM for direct in situ measurements of the amount of adsorbed polymer at metal/solution interfaces and kinetics of adsorption has been well exploited.29 In the present paper, we explore the adsorption capability of three different water-soluble polyelectrolytes on a gold resonator, that is, polycationic PEI, polyanionic AQ-29D polymer, and PSS. The structure of each of these polyelectrolytes is shown in Figure 1. It arises from their structures that they have different properties ranging from hydrophilic PEI21 to hydrophobic AQ-29D polymer.16-18 Variations in QCM frequency can be followed using in situ measurements by injecting small aliquots of aqueous solutions containing increasing concentrations of the polymer of interest into pure water. Nevertheless, because of possible complications in interpreting frequency data from QCM measurements, particularly when extensive swelling occurs thus invalidating the assumption of a thin film,30 we have preferred to use the “immersion/drying” mode: the resonator was maintained for 10 min in the polymer solution, carefully rinsed with water, and then dried in an air stream before QCM measurements. When using this type of procedure, decreases in QCM frequency were detected only for PSS and AQ-29D. No variation was observed for PEI (see Table 1, line 1). From the QCM frequency changes, a film increase of 704 ng cm-2 was calculated, corresponding to a 5.4 nm thickness for PSS or AQ-29D deposited layers. (29) Xu, H.; Schlenoff, J. B. Langmuir 1994, 10, 241. (30) Borjas, R.; Buttry, D. A. J. Electroanal. Chem. 1990, 280, 73.

The search for the cause of preferred adsorption on the gold surface for PSS and AQ-29D and nonadsorption of PEI has been oriented toward hydrophobic/hydrophilic considerations. Since Smith’s work,31 there has been a body of evidence that a “clean” gold surface is hydrophilic but becomes rapidly hydrophobic because of “contamination”. For example, exposure to the laboratory air even for a short period is supposed to render QCM gold resonators nonwettable and thus hydrophobic. Hydrophobicity cannot be avoided even after a treatment with a piranha solution as demonstrated from the measurement of the contact angle against water.32 This would be a reason for favorable adsorption of hydrophobic colloidal AQ-29D particles and PSS on the gold surface. In contrast, hydrophilic PEI exhibits a poorer affinity toward the hydrophobic gold surface. The hypothesis that the hydrophobic effect is involved in the adsorption process is reinforced from the results obtained when using a modified gold surface. First, by diminishing the hydrophobic character of the gold surface, it can be expected that the adsorption of hydrophobic polymers might be affected. With this aim in view, we have modified the gold surface of a QCM resonator with a thiol, aldrithiol (i.e., bis(4-pyridyl) disulfide). This compound has been shown to strongly and irreversibly adsorb onto gold surfaces through S• radicals after S-S bond cleavage.33 In addition to well-known electrochemical promoting properties toward proteins (e.g., cytochrome c), it has been demonstrated that aldrithiol makes the gold electrode surface hydrophilic.34,35 In this report, when a virgin resonator was treated with 1 mM aldrithiol solution for 5 min and dried, a decrease in frequency of -16 Hz was measured, resulting from the adsorption of aldrithiol. The amount of adsorbed material calculated from the Sauerbrey equation is 281.6 ng cm-2, which corresponds to 1.28 mol cm-2 of aldrithiol. For comparison, the value of 0.98 mol cm-2 has been reported10 for another thiol, sodium 3-mercapto-1-propane sulfonate. In a second set of experiments, aldrithiol-modified resonators were immersed in polymer solutions for 5 min and dried. The corresponding frequency shifts are given in Table 1, line 2. No frequency shift is observed for PSS, and a lower decrease (-14 Hz) is detected in the case of AQ-29D, thus denoting a poor affinity of the hydrophilic gold surface toward hydrophobic polymers. The trend is reversed for PEI, with a decrease of -48 Hz that can be explained by the fixation of PEI on the resonator. However, it is important to consider that favorable electrostatic interaction does occur between PEI and the lone-pair electrons of N atoms in aldrithiol fixed onto the gold surface. In contrast, electrostatic interactions are largely disadvantaged for PSS and AQ-29D in such conditions. To overcome this problem, additional experiments were carried out by polarizing the gold resonator during the exposure to polymer solutions. Results obtained at a positively (+0.2 V) and negatively (-0.2 V) polarized resonator are given in Table 1, line 3. The frequency decrease of -30 Hz detected for AQ-29D clearly indicates that the polymer is able to adsorb even in unfavorable electrostatic conditions, thus privileging other factors such as hydrophobicity. No (31) Smith, T. J. Colloid Interface Sci. 1980, 75, 51. (32) Geddes, N. J.; Paschinger, E. M.; Furlong, D. N.; Caruso, F.; Hoffman, C. L.; Rabolt, J. F. Thin Solid Films 1995, 260, 192. (33) Taniguchi, I.; Toyosawa, K.; Yamaguchi, H.; Yasukouchi, K. J. Chem. Soc., Chem. Commun. 1982, 1032. (34) Taniguchi, I.; Isezki, M.; Yamaguchi, H.; Yasukouchi, K. J. Electroanal. Chem. 1985, 186, 299. (35) Taniguchi, I. In Redox Chemistry and Interfacial Behavior of Biological Molecules; Dryhurst, G., Niki, K., Eds.; Plenum: New York, 1988.

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Figure 3. Dependence of the frequency shift on the number of adsorption cycles for alternate AQ-29D polymer/PEI layers (1) in the absence of salt and (2) in the presence of 0.1 M NaCl. The solid symbols correspond to PEI adsorption steps, and the open symbols to the AQ-29D adsorption steps. Concentrations: PEI, 5 g L-1; AQ-29D, 4.7 g L-1.

frequency decrease in the case of PSS confirms that other factors must govern in fact the adsorption process. Salts present in polyelectrolyte solutions are known to regulate the substrate/layer and layer/layer interactions.36 Stepwise assemblies of ultrathin polymer films have been performed upon repetitive adsorption/drying processes6 in the presence of variable amounts of NaCl. Deposition of AQ-29D films on the gold resonator has been achieved in the presence of increasing concentrations of NaCl in the polymer solution, using repetitive adsorption (for 10 min)/drying steps. The NaCl concentration dependence of the frequency shift observed for the first adsorption cycle and a typical profile obtained in the presence of 0.1 M NaCl are shown in panels A and B of Figure 2, respectively. The frequency shifts measured for the first adsorption step increased with increasing salt concentrations. For a constant salt concentration, decreases in frequency were observed only for the first adsorption step, and then the frequency value remained constant upon repetition of cycles. Polyelectrolyte films have been shown to swell on exposure to solutions containing salt. Swelling involves adsorption of both salt and additional waters of hydration,37 thus explaining the observed decreases in frequency shift for AQ-29D films deposited in the presence of NaCl. We did not succeed in preparing salt concentrations higher than 0.3-0.4 M because of the coagulation of the polymer solution and instability of the deposited films. The choice of AQ-29D polymer to study further interactions with polypeptides and proteins can be justified because of the inherent properties of this type of polyelectrolyte, especially its capability to “incorporate” posi-

tively charged proteins.19,20 Among the attractive features of AQ-29D films are reasonably rapid rates of cationexchange or/and incorporation, even in the case of proteins such as cytochrome c and cytochromes c3 (e.g., from DvH). It is well confirmed in this work from QCM experiments that stable AQ-29D polymer films can be fixed onto the gold electrode surface, thus providing a negatively charged layer available for further binding of positively charged species. Association of AQ-29D Films with Polymers and Polypeptides. Stepwise assemblies of ultrathin films constructed with a polymer (PEI) or a polypeptide (PLL) using repetitive adsorption/drying processes have been carried out from aqueous solutions of AQ-29D polymer (4.7 g L-1) and PEI (5 g L-1) or PLL (3800 and 14 600) (0.5 g L-1). The multilayer buildup and subsequent stability of deposited films were monitored directly on gold resonators by QCM measurements. Typical profiles obtained for AQ-29D/PEI assembly are shown in Figure 3. When the assembly was constructed in the absence of salt, a nonlinear dependence of the frequency shift on cycles of adsorption was observed, exhibiting a marked scattering in the experimental data. The “serrated” profile in Figure 3 (curve 1) denotes a marked instability of the deposited films, as observed in previous works on polymers in the absence of salts.38 In contrast, a better linearity in frequency shift is noted in the presence of 0.1 M NaCl (Figure 3, curve 2). Very similar results were obtained for AQ-29D/PLL films. Multilayer assemblies become instable when the NaCl concentration is increased above 0.3 M, essentially because of the coagulation of the AQ-29D polymer suspension. Salts control the thickness increment per deposition cycle and the stability of polyelectrolyte multilayers by moderating the strength of the polymer/ polymer contacts.37 Thicker layers of AQ-29D films can be assembled in the presence of NaCl. It has been suggested that salts contribute to neutralize polyions, thus inducing coiling and thickening of the adsorbed layers. Such phenomena must occur in the case of AQ-29D films. Thus, the role of salts in the stabilization of multilayer assemblies can be explained on the basis of a balance between multilayer growth and polyelectrolyte stripping. Interaction between AQ-29D Polymer and Proteins. Lysozyme. Figure 4 gives a typical kinetic profile

(36) Dubas, S. T.; Schlenoff, J. B. Langmuir 2001, 17, 7725. (37) Schlenoff, J. B.; Dubas, S. T. Macromolecules 2001, 34, 592.

(38) Kolarik, L.; Furlong, D. N.; Joy, H.; Struijk, C.; Rowe, R. Langmuir 1999, 15, 8265.

Figure 2. (A) Dependence of the frequency shift on the NaCl concentration for the first deposition of AQ-29D polymer on the QCM gold resonator. (B) Dependence of the frequency shift on the number of adsorption cycles for successive depositions of AQ-29D polymer in the presence of 0.1 M NaCl.

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Figure 4. Time dependence of the QCM frequency shift for the adsorption of lysozyme onto a gold resonator preequilibrated with an AQ-29D polymer solution at 4.7 g L-1. The arrows correspond to the successive additions of a stock solution of lysozyme. The lysozyme concentrations are (a) 1 µM, (b) 6 µM, and (c) 12 µM, respectively.

Figure 5. Dependence of the frequency shift on the number of adsorption cycles for alternate AQ-29D polymer/lysozyme: (b) lysozyme adsorption steps and (O) AQ-29D polymer adsorption steps. Concentrations: lysozyme, 2.8 µM; AQ-29D, 4.7 g L-1.

obtained at a AQ-29D-modified resonator for QCM frequency change in dependence on time, for successive additions of aliquots of a lysozyme stock solution. Prior to the first addition, the gold resonator has been equilibrated with an AQ-29D polymer solution (containing 4.7 g L-1), dried, and immersed in water. Each addition of lysozyme solution (corresponding to 1, 6, and 12 µM, respectively) results in a decrease in frequency which can be related to the adsorption of protein on the resonator surface. In a second set of experiments, films of negatively charged AQ-29D polymer and positively charged lysozyme were assembled on an AQ-29D-pretreated gold resonator by alternate adsorption followed by intermediary drying. QCM monitoring of film assembly shows linear changes of the frequency with the number of adsorption steps (Figure 5). This suggests a linear increase in film mass with increasing number of layers. For every layer of adsorbed lysozyme, the decrease in QCM frequency is of -30 Hz. This change corresponds to a film mass increase of 530 ng cm-2 and a 4 nm thickness for each deposited lysozyme layer. Taking into account crystallographic data on the lysozyme molecule (3 × 3 × 4.5 nm),39 it can be (39) Dickerson, R.; Kopna, M.; Weinzierl, J.; Warnun, J.; Eisenberg, D.; Margoliash, E. J. Biol. Chem. 1967, 242, 3015.

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concluded that monomolecular layers are fixed at every step of the assembly process. For every layer of AQ-29D adsorption, a QCM frequency decrease of about -30 Hz was measured, corresponding to a thickness increase of 4 nm. This value accords well with the deposit of monomolecular layers of polymer, based on the estimation described in a previous paper.15 This behavior is different from that observed in the case of AQ-29D and PEI or PLL. The capability of lysozyme to form a composite Lys/PSS bilayer has been well established.7 In this paper, it is demonstrated that stable multilayer assemblies are obtained also in the case of AQ-29D polymer and lysozyme. Cytochrome c. Electroactive species can serve as probes to detect the extent of the incorporation inside the partner/ host layers and to monitor the stability of associated films. Because of its high isoelectric point (pI 10.5) and its ability to incorporate in AQ-29D polymers, cytochrome c is a good candidate for taking part in the multilayer buildup. Unfortunately, we were not able to obtain stable multilayer AQ-29D/cytochrome c films. The dependence of the frequency shift on the cycles of adsorption displays a serrated profile like those obtained previously in the case of PEI and PLL (see above). It can be assumed that strong interactions exist between AQ-29D polymer and cytochrome c. Stripping off of AQ-29D polymer from the gold surface on exposure to cytochrome c solution can be explained on the basis of a kinetically reversible adsorption process.40 An argument which supports this hypothesis is that relatively more stable films are obtained, when constructing for example PSS/cytochrome c multilayer assemblies by alternate adsorption, on a MPS-pretreated gold surface.15 In this case, MPS is retained by the gold surface via covalent bonding and yields a stable negatively charged layer on the resonator (or electrode) surface. When using this modification procedure, a reversible pair of reduction-reoxidation peaks are detected (curves not shown), with an average midpoint potential of +30 mV (i.e., +240 mV versus SHE), in agreement with previous data on immobilized cytochrome c.19 Integration of the CV peaks obtained at a MPS-modified gold electrode treated with a solution of cytochrome c gives an average surface concentration of 2.4 × 10-11 mol cm-2. Assuming that monolayer coverage is attained, the area occupied by one molecule would be 10 nm2. This result matches well with the crystallographic data for the cytochrome c molecule (2.5 × 2.5 × 3.7 nm).41 Because of differences in charge distribution and density and hydrophobicity at protein surfaces, there is usually an interplay between several factors for an efficient interaction between proteins to occur. An alternative to facilitate the LBL assembly of proteins based on the partial compensation of protein surface charge resulting from premixing with polyions can be proposed.42 If release of AQ-29D film from the gold surface is induced by cytochrome c (as suggested above), it can be interesting to attempt to lessen the interaction between AQ-29D polymer and cytochrome c by using solutions containing various ratios of cytochrome c/AQ-29D polymer. In such conditions, it can be expected that the globular cytochrome c molecule is wrapped up by the more flexible AQ-29D polyelectrolyte and that a more or less extensive charge compensation is attained. It has been established in our previous works19,20 that anionic AQ-29D polymer has the capability to improve (40) Sui, Z.; Salloum, D.; Schlenoff, J. B. Langmuir 2003, 19, 2491. (41) Wahlgren, M.; Arnebrandt, T. J. Colloid Interface Sci. 1993, 142, 503. (42) Ariga, K.; Onda, M.; Lvov, Y.; Kunitake, T. Chem. Lett. 1997, 25.

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Figure 7. Dependence of the frequency shift on the number of adsorption cycle for alternate AQ-29D polymer/ cytochrome c using mixed (AQ-29D + cytochrome c) solution at the ratio of 0.2. (*) (AQ-29D + cytochrome c) adsorption steps, and (O) AQ-29D polymer adsorption steps.

Figure 6. (A) SWV curves obtained at a gold electrode in 10 mM Tris chloride buffer at pH 7.6: (1) in the absence of modification; (2) after a treatment for 5 min with (4.7 g L-1) AQ-29D solution and then for 10 min with a 50 µM cytochrome c solution; (3) after a treatment for 5 min with (4.7 g L-1) AQ29D solution and then with mixed [AQ-29D]/[cytochrome c] (at the ratio of 0.3) solution. (B) Dependence of the SWV peak current on the [AQ-29D]/[cytochrome c] ratio at a gold electrode pretreated for 5 min with a (4.7 g L-1) AQ-29D solution, in 10 mM Tris chloride buffer at pH 7.6.

the electrochemistry of basic cytochrome c. Advantage has been taken here of this property to monitor the electrochemical behavior of mixtures of AQ-29D and cytochrome c, and thus their assembly. First, a polished gold electrode was pretreated for 5 min with a (4.7 g L-1) AQ-29D polymer solution, carefully rinsed, and then immersed for 10 min in cytochrome c solutions containing various amounts of AQ-29D. After this treatment, the modified electrode was transferred into 10 mM Tris chloride buffer solution at pH 7.6. Cyclic and square-wave voltammetry were used to control the presence of immobilized cytochrome c and to estimate the extent of the fixation. SW curves obtained at a gold electrode for different conditions of pretreatment are shown in Figure 6A. A very slight wave is observed when using a polished electrode modified first for 5 min with (4.7 g L-1) AQ-29D polymer solution and then for 10 min with cytochrome c and transferred into the electrochemical cell containing 10 mM Tris chloride buffer at pH 7.6. In contrast, a wellshaped curve with a peak at +26 mV (i.e., +236 mV versus SHE) corresponding to the reversible reduction-reoxidation potential of cytochrome c is observed when the polished gold electrode is submitted to [(4.7 g L-1) AQ29D polymer/(AQ-29D + cytochrome c)] double treatment. The dependence of SWV peak current on the [AQ-29D polymer]/[cytochrome c] ratio is given in Figure 6B. A maximum in peak current values is observed for the ratio of 0.2. In a second set of experiments, films of AQ-29D polymer and cytochrome c deposited from a premixed (AQ29D polymer + cytochrome c) solution corresponding to the ratio of 0.2 (see above) were assembled on an AQ29D-pretreated gold resonator by alternate adsorption

followed by intermediary drying. QCM monitoring of film assembly shows linear changes of the frequency with the number of adsorption steps (Figure 7), thus demonstrating that the assembly is stable and that the shifts are proportional to the adsorbed mass. For every layer of adsorbed cytochrome c, the decrease in QCM frequency is of -20 Hz. This change corresponds to a film mass increase of 350 ng cm-2 and a 3 nm thickness for each deposited cytochrome c layer. From crystallographic data on the cytochrome c molecule,41 it can be concluded that monomolecular layers are fixed at every step of the assembly process. Polyheme c-Type Cytochromes: DvH and Dn Cytochrome c3. Tetraheme c-type cytochromes are fast electrochemical systems exhibiting different isoelectric points, charge distribution, and hydrophobic/hydrophilic patches. Two of them have been selected in this work to build up multilayer assemblies: cytochrome c3 from DvH (pI ) 10.5) and cytochrome c3 from Dn (pI ) 7). In a previous paper,15 we have shown that DvH cytochrome c3 can be assembled layer-by-layer with AQ-29D polymer or PSS onto gold or silver electrodes, using electrochemistry and QCM techniques. Linearity in the dependence of the QCM frequency shift on cycles of alternate AQ-29D polymer/DvH cytochrome c3 was observed, thus establishing that the shifts are proportional to the adsorbed mass. Because of the flexibility and swelling property of AQ29D films, it may be suggested that the resulting LBL assembly must be characterized by a fuzzy structure. To prepare “mixed” protein films, we have attempted to build up multilayer films of AQ-29D polymer/DvH cytochrome c3/AQ-29D polymer/cytochrome c. Linearity for the dependence of QCM frequency shift on adsorption cycles is observed only for the three first cycles. As soon as cytochrome c is inserted into the multilayer structure, a marked destabilization occurs, yielding a serrated profile of the same type as that observed above (see Figure 3). When cytochrome c, however, was premixed with a small fraction of AQ-29D polymer (at the ratio of 0.2 as described above), we were able to construct a stable cytochrome c/cytochrome c3 mixed multilayer. Assembling LBL organized films by alternate adsorption of charged macromolecules (polypeptide and/or protein) logically entails that the driving force of adsorption must be essentially electrostatic. In this case, it could be expected that species uncharged or bearing the same charge might only poorly associate, for example, neutral Dn cytochrome

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Figure 8. Dependence of the frequency shift on the number of adsorption cycles for alternate AQ-29D polymer/Dn cytochrome c3: (b) cytochrome c3 adsorption steps and (O) AQ-29D polymer adsorption steps. Concentrations: Dn cytochrome c3, 30 µM; AQ-29D, 4.7 g L-1.

c3 (pI ) 7) and AQ-29D polymer. Figure 8 shows the dependence of the QCM frequency shift on cycles of alternate Dn cytochrome c3/AQ-29D polymer adsorption. The shifts are proportional to the adsorbed mass, thus demonstrating that Dn cytochrome c3 associates with the negatively charged polymer. From decreases in frequency after each adsorption step, a thickness of about 5 nm is calculated for each cytochrome c3 layer. Taking into account the available crystallographic data for this cytochrome (4.5 × 3.2 × 2.8),43 it is concluded that virtually monomolecular layers are formed in the assembly process. The existence of immobilized Dn cytochrome c3 has also been confirmed by CV and SWV (results not shown). When alternate AQ-29D and Dn cytochrome c3 layers are built up, electrochemical results show that there is no linear increase in peak current or charge integrated from CV curves with repetitive adsorption steps. Such a behavior could result from the possibility that molecules too far from the electrode surface are not able to participate in the electron exchange process. That Dn cytochrome c3 displays an affinity toward AQ films is not unexpected. In a previous work,20 we have demonstrated that Dn cytochrome c3 could incorporate efficiently into AQ-29D film, even if the process is more sluggish than for cytochrome c or DvH cytochrome c3. It has been proposed that such an incorporation could be partly explained on the basis of hydrophobic effect considerations. More generally, these experimental results are consistent with the contribution of other “secondary” interactions to the multilayer buildup. Discussion It is well confirmed in this paper that the alternate LBL method can be successfully used to prepare assemblies containing AQ-29D polymer and proteins. It has been shown from QCM mass monitoring that each adsorption step provides a new monolayer of immobilized material. AQ polymers exhibit interesting specific properties in that they can form dispersions in water, which display a marked colloidal state. When deposited on an electrode surface and dried, they give films that can be rehydrated in aqueous solution to form water-containing gels. It can be assumed that these gels retain initial colloidal properties, especially their ability to act as cation(43) Pierrot, M.; Haser, R.; Frey, M.; Payan, F.; Astier, J. P. J. Biol. Chem. 1982, 257, 14341.

Lojou et al

exchangers. It has been shown in this work that an AQ ionomer such as AQ-29D is able to adsorb on a gold surface to give relatively stable cast films. The same conclusion has been gained in the case of PSS and Nafion (results not shown). Considering that AQ-29D polymer adsorbs more extensively on a crude gold surface than on gold made more hydrophilic through the covalent fixation of a thiol (aldrithiol), it is suggested that favored adsorption could partly result from hydrophobic interactions between the AQ film and the gold surface. Very similar conclusions have been proposed for other polymers such as poly(vinyl alcohol).6 A drawback in the case of simply adsorbed films is that they can be more easily released from the electrode surface, especially when submitted to strong interactions from species present in the solution in contact with the modified electrode. This point is well highlighted in the case of gold/AQ-29D film in the presence of solutions containing polyelectrolytes such as PEI or PLL or cytochrome c. No stable films can be constructed with these molecules. However, stabilization of deposited polymer multilayers can be observed upon the addition of a salt (NaCl). It has been proposed that salt ions incorporated in a polyelectrolyte multilayer assembly are involved in an exchange equilibrium, where salt counterions participate in charge neutralization.36 Mixing cytochrome c with negatively charged polymer, for example, AQ-29D, results in a decrease in the global positive charge of the protein and thus tends to diminish the attraction between positively charged cytochrome c and negatively charged polymer. Leaching of polyelectrolyte bilayers from the substrate has been observed in several cases including poly(acrylic acid) and poly(styrenesulfonate).44 It has been explained on the basis of modifications occurring when the electrostatic complex between both polymers is formed, thus resulting in the disruption of polymer/surface bonds. The release process is avoided when polymer is bound to the electrode surface via covalent bonding (e.g., when using MPS modifier). For multiply charged polypeptides and polymers, strong adsorption of an electrostatic nature is expected. This is the case for negatively charged AQ-29D polymer and positively charged PLL or PEI. In contrast, the interaction between AQ-29D and the gold surface seems to be governed more by other factors, presumably the hydrophobic effect. Previous works19,20 have established that basic proteins are able to incorporate in AQ films. Despite interactions within the films, it has been suggested from spectroscopic UV-vis data45 that proteins retain a near-native conformation at neutral pH. This assumption is supported by the fact that cytochromes retain their metal-reductase activity when incorporated in AQ films.46 On this basis, multilayer assemblies have been performed using AQ29D acting as a negatively charged polyionic species toward positively charged proteins such as lysozyme, cytochrome c, and DvH cytochrome c3. Lysozyme is a rather compact protein with an ellipsoid shape.47 The lysozyme molecule carries a net charge of +9 at pH 5.6, with most of the positive charge located around the active site cleft which in turn is situated on a side parallel to the long axis of the ellipsoid. On the side opposite the active cleft is a relatively large hydrophobic patch that may be involved in the dimerization of lysozyme, known to take place in concentrated solutions.48 Because the positive charges are (44) Hoogeven, N. G.; Cohen Stuart, M. A.; Fleer, G. J. Langmuir 1996, 12, 3675. (45) Hu, N.; Rusling, J. F. Langmuir 1997, 13, 4119. (46) Lojou, E Ä .; Bianco, P. Electrochim. Acta 2002, 47, 4069. (47) Imoto, T.; Johnson, L. N.; North, A. C. T.; Phillips, D. C.; Rupley, J. A. In The Enzymes, 3rd ed.; Boyer, P., Ed.; Academic Press: New York, 1972; Vol. 7.

Buildup of Polyelectrolyte-Protein Multilayers

mainly located on the C-terminal lobe of the lysozyme molecule, it can be speculated49 that direct contact with the negative charges of AQ-29D occurs via this terminal lobe. The lateral electrostatic interaction in the interfacial region would determine the orientation of the molecule. At neutral pH, it can be expected that lysozyme molecules adsorb sideways onto the AQ-29D polymer surface. After adsorption, the lysozyme layer should exhibit the hydrophobic patch opposite the positively charged active cleft, thus more favorable to the adsorption of a further hydrophobic AQ-29D layer. This alternate electrostatic/ hydrophobic facing could partly contribute to the buildup of the multilayer architecture. From the results presented in this report, it has been concluded that one monolayer of lysozyme and one monolayer of AQ-29D adsorb alternately at each AQ-29D/lysozyme step. It is interesting now to speculate on the possible structure of these polyelectrolyte/protein layers. AQ films are known to contain large amounts of water and have a wide-mesh structure that enables them to incorporate protein molecules. It is thus likely that lysozyme can enter the AQ29D monolayer or, more precisely, give a mixed lysozyme/ AQ-29D layer. Somewhat fuzzy structures have been postulated for superlattice architecture,3 and it is suggested that the “patched” nature of a protein surface charge can favor polyion anchoring.4 As a consequence, it appears that flexible polyions may creep into protein molecules, thus acting as a glue. Flexible polyelectrolytes produce more compact complexes with their partners, displaying higher degrees of contact. This must be the case for the AQ-29D polymer and its protein partners. The interactions between polyelectrolyte multilayers and proteins are governed by several parameters not exclusively of an electrostatic nature. This is of relevance in the case of Dn cytochrome c3. For this cytochrome, allowance must be made for electrostatic effects but also for nonelectrostatic factors including hydrophobic effects and surface topography. Cytochromes of the c3-type family are characterized by the presence of four low redox potentials inserted in a relatively short polypeptide chain of about 115 residues. A three-dimensional model of Dn cytochrome c3 has been established on the basis of the primary structure and of X-ray data at 2.5 Å resolution.43 By generation of a three-dimensional model using interactive computer graphic methods, it has been proposed that heme 4 (sequential numbering) is the interacting site.50 The electrostatic potential surface of Dn cytochrome c3 clearly shows51 that the region surrounding the specific exposed heme 4, which is supposed to participate in intermolecular electron exchange, is lined with a number (48) Deonier, R. C.; Williams, J. W. Biochemistry 1970, 9, 4260. (49) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438. (50) Cambillau, C.; Frey, M.; Mosse´, J.; Guerlesquin, F.; Bruschi, M. Proteins: Struct., Funct., Genet. 1988, 4, 63. (51) Florens, L.; Ivanova, M.; Dolla, A.; Czjzek, M.; Haser, R.; Verger, R.; Bruschi, M. Biochemistry 1995, 34, 11327.

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of positively charged lysine residues. These residues would interact with complementary domains of the partner’s surface. It can be inferred that Dn cytochrome c3 has a marked dipolar character unlike DvH cytochrome c3 which displays a more regular distribution of compensating positive and negative charges, except in the region around heme 4.51 Thus Dn cytochrome c3 is also able to associate with AQ-29D films via electrostatic interaction involving the positive region of the protein molecule. Nevertheless, in this situation, the external layer after adsorption would bear a high density of negative charges capable of preventing further adsorption of the AQ-29D layer. This is not observed from QCM and electrochemical data. It has been established52 that the ion-exchange selectivity of ionomer films is dependent upon the chemical/ ionic interactions of the ions in the film. This could explain the selectivity of the AQ-29D film for the large hydrophobic ions in aqueous solutions. When exchanging species have different hydrophobic properties, the interaction between a charged species and the hydrophobic region(s) of the exchanging film can be the driving force for the thermodynamics of the association process. Based on such considerations, it not unexpected that the AQ-29D polymer is able to associate with Dn cytochrome c3. Relatively large amounts of Dn cytochrome c3 are able to enter AQ films, as demonstrated in our previous work.20 Further support for the idea that hydrophobic interactions may play an important part in the adsorption is provided by the ability to build up stable assemblies of alternate AQ-29D/Dn cytochrome c3 layers. An important correlation has been found between the structure of the polyion, specifically the hydrophobic or hydrophilic nature of the polymer backbone, and the surface regions of the partner macromolecule.21 This might be the case, especially for Dn cytochrome c3. It has been demonstrated in a previous work51 that this cytochrome develops a marked ability to penetrate several lipid films. A reason would be that hydrophobic contacts occur via a particular surface loop of this cytochrome c3. This domain, in which hydrophobic and charged residues were found to alternate, has been proposed to be the site of interaction with lipids. It has been suggested that in addition to electrostatic interaction, a partial penetration of the extending loop into the lipid layer occurs, forming hydrophobic contacts. Another argument in favor of the occurrence of hydrophobic interaction can be found in the specific behavior exhibited by Dn cytochrome c3 incorporated into layers of phosphatidylcholine cast onto a carbon electrode as established in a previous work.53 It can be concluded that the interaction between proteins, especially cytochromes, and the AQ-29D polymer must result from a compromise between several factors not exclusively of an electrostatic nature. LA030286W (52) Szentirmay, M. N.; Martin, C. R. Anal. Chem. 1984, 56, 1898. (53) Bianco, P.; Haladjian, J. Electrochim. Acta 1994, 39, 911.