Characterization of Polyelectrolyte−Protein Multilayer Films by Atomic

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Characterization of Polyelectrolyte-Protein Multilayer Films by Atomic Force Microscopy, Scanning Electron Microscopy, and Fourier Transform Infrared Reflection-Absorption Spectroscopy Frank Caruso,*,†,‡ D. Neil Furlong,† Katsuhiko Ariga,§,| Izumi Ichinose,⊥ and Toyoki Kunitake⊥ CSIRO, Molecular Science, Private Bag 10, Clayton South MDC, Victoria 3169, Australia, Supermolecules Project, JRDC, Kurume Research Park, Kurume 839, Japan, and Department of Chemical Science and Technology, Faculty of Engineering, Kyushu University, Fukuoka 812, Japan Received November 24, 1997. In Final Form: February 17, 1998 Protein-containing polyelectrolyte multilayer films of poly(styrenesulfonate) and poly(allylamine hydrochloride), fabricated by the sequential adsorption of polyelectrolyte and anti-immunoglobulin G (anti-IgG) on solid substrates, have been characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared reflection-absorption spectroscopy (FTIRRAS). Visualization of the film structure on the nanometer scale, by AFM and SEM, showed that either layered or disordered films were formed depending on the number of polyelectrolyte layers separating each protein layer. For films where each anti-IgG layer was separated by one polyelectrolyte layer, an open, disordered film structure was observed and significant protein aggregation occurred. In contrast, for films in which the anti-IgG layers were separated by five polyelectrolyte layers, a layered structure with uniform protein layers was formed. Film thicknesses determined by SEM measurements were consistent with those calculated from quartz crystal microbalance measurements. FTIR-RAS confirmed the presence of anti-IgG in the multilayer films, with the amide I and II bands due to anti-IgG clearly visible in the spectra, and provided direct evidence that anti-IgG was not denatured. Both types of films fabricated are interesting for biosensing applications: the first provides ordered, functional protein layers within a polyelectrolyte matrix for sensing investigations, and the second serves as a useful functional film for applications where an increased binding capacity of the film is sought.

Introduction The construction of functional protein-containing films is an important area of research in biotechnology. Over the years, immobilization of proteins onto solid surfaces and in various films and assemblies has been accomplished by a number of methods, including spontaneous adsorption from solution,1-3 covalent binding,4,5 and entrapment within polymer gels, membranes, or surfactant matrixes.6-8 In addition to these more conventional * To whom correspondence should be addressed. Fax: +49 30 6392 3102. E-mail: [email protected]. † CSIRO, Molecular Science. ‡ Present address: Max-Planck-Institute of Colloids and Interfaces, Rudower Chaussee 5, D-12489 Berlin, Germany. § Supermolecules Project, JRDC. | Present address: Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 6300101, Japan. ⊥ Kyushu University. (1) Lin, J. N.; Drake, B.; Lea, A. S.; Hansma, P. K.; Andrade, J. D. Langmuir 1990, 6, 509. (2) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1993, 142, 503. (3) Blomberg, E.; Claesson, P.; Froberg, J.; Tilton, R. Langmuir 1994, 10, 2325. (4) Lin, J. N.; Herron, J.; Andrade, J. D.; Brizgys, M. IEEE Trans Biomed. Eng. 1988, 35, 466. (5) Leggett, G. J.; Roberts, C. J.; Williams, P. M.; Davies, M. C.; Jackson, D. E.; Tendler, S. J. B. Langmuir 1993, 9, 2356. (6) Gunasingham, H.; Teo, P. Y. T.; Lai, Y.-H.; Tang, S. G. Biosensors 1989, 4, 349. (7) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1991, 63, 49.

methods, sol-gel entrapment,9,10 Langmuir-Blodgett deposition,11-16 and electropolymerization17 have also been extensively used. Recently, a new versatile technique based on the consecutive alternate adsorption of anionic and cationic polyelectrolytes18-23 has been utilized to (8) Hall, E. A. H.; Hall, C. E.; Marttens, N.; Mustan, M. N.; Datta, D. In Uses of Immobilized Biological Compounds; Guilbault, G. G., Ed.; Kluwer Academic Press: Dordrecht, 1993; pp 11-21. (9) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (10) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120 A. (11) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1991, 205, 113. (12) Ahluwalia, A.; De Rossi, D.; Monici, M.; Schirone, A. Biosens. Biolectron. 1991, 6, 133. (13) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1992, 210/211, 710. (14) Dubrovsky, T. B.; Demcheva, M. V.; Savitsky, A. P.; Mantrova, E. Yu.; Yaropolov, A. I.; Savransky, V. V.; Belovolova, L. V. Biosens. Bioelectron. 1993, 8, 377. (15) Dubrovsky, T.; Vakula, S.; Nicolini, C. Sens. Actuators, B 1994, 22, 69. (16) Tronin, A.; Dubrovsky, T.; De Nitti, C.; Gussoni, A.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 238, 127. (17) Scouten, W. H.; Luong, J. H. T.; Brown, S. Tibtech 1995, 13, 178 and references therein. (18) Decher, G.; Hong, J. D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (19) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831. (20) Decher, G.; Schmitt, J. Prog. Colloid Polym. Sci. 1992, 89, 160. (21) Lvov, Y.; Decher, G.; Mo¨hwald, H. Langmuir 1993, 9, 481. (22) Decher, G.; Lvov, Y.; Schmitt, J. Thin Solid Films 1994, 244, 772. (23) Sukhorukov, G. B.; Schmitt, J.; Decher, G. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 948.

S0743-7463(97)01288-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/15/1998

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construct multilayer protein-containing films by the adsorption of oppositely charged polyelectrolytes and proteins.24-27 This method has also been extended to fabricate multilayer films of nucleic acids,28-30 bilayer membranes,31 and inorganic colloids.32-35 In a previous publication26 it was demonstrated that alternating multilayer films of polyelectrolytes [poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH)] and anti-immunoglobulin G (anti-IgG) could be successfully constructed to contain at least five protein layers. The method described for fabricating the polyelectrolyte and protein multilayer films was shown to have the advantages of allowing the number of protein layers, film thickness, and film structure to be controlled. The capacity of the protein-containing multilayer films to bind antigens in solution was found to be dependent on the number of polyelectrolyte interlayers separating the protein layers. The binding capacity of the film was linearly dependent on the number of anti-IgG layers in the multilayer film when anti-IgG layers were separated by one PSS layer, whereas the films where anti-IgG layers were separated by five polyelectrolyte (PSS(PAH/PSS)2) layers displayed only the outer anti-IgG layer to be immunologically active. The difference in activity was attributed to the structure of the multilayer films. The data obtained from quartz crystal microbalance (QCM) measurements indicated that the multilayer film, with PSS(PAH/PSS)2 separating the protein layers, was a dense, layered film through which antibody permeation was restricted. For the film where PSS was the interlayer, the data suggested that protein aggregation occurred and the film structure was less ordered. Hence, it was shown that the capacity of the polyelectrolyte/protein films for antibody-antigen recognition could be increased by reducing the number of polyelectrolyte interlayers between the immunoglobulin layers. In this article, we examine the structure of the protein/ polyelectrolyte multilayer films, and the protein distribution and orientation in the films, using atomic force microscopy (AFM) and scanning electron microscopy (SEM). In addition, Fourier transform infrared reflection-absorption spectroscopy (FTIR-RAS) is employed to investigate the nature of the protein in the films. The results obtained from the different techniques will also be compared. Experimental Section Materials. Cationic poly(allylamine hydrochloride) (PAH), Mr 50000-65000, and anionic poly(sodium 4-styrenesulfonate) (PSS), Mr 70000, were purchased from Aldrich Chemical Co. 3-Mercaptopropionic acid (MPA) was obtained from Sigma. The (24) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (25) Lvov, Y.; Ariga, K.; Kunitake, T. Chem. Lett. 1994, 2323. (26) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir, 1997, 13, 3427. (27) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotech. Bioeng. 1996, 51, 163. (28) Sukhorukov, G. B.; Mo¨hwald, H.; Decher, G.; Lvov, Y. M. Thin Solid Films 1996, 284/285, 220. (29) Sukhorukov, G. B.; Montrel, M. M.; Petrov, A. I.; Shabarchina, L. I.; Sukhorukov, B. I. Biosens. Bioelectron. 1996, 11, 913. (30) Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043. (31) Ichinose, I.; Fujiyoshi, K.; Mizuki, S.; Lvov, Y.; Kunitake, T. Chem. Lett. 1996, 257. (32) Fendler, J. H.; Meldrum, F. C. Adv. Mater. 1995, 7, 607. (33) Keller, S. W.; Kim, H.-N.; Mallouk, T. E. J. Am. Chem. Soc. 1994, 116, 8817. (34) Ichinose, I.; Senzu, H.; Kunitake, T. Chem. Lett. 1996, 831. (35) Schmitt, J.; Decher, G.; Dressick, W. J.; Brandow, S. L.; Geer, R. E.; Shashidhar, R.; Calvert, J. M. Adv. Mater. 1997, 9, 61.

Caruso et al. water used in all experiments was prepared in a three-stage “Milli-Q” purification system to a conductivity of less than 1 µS cm-1. Protein donkey anti-sheep IgG (anti-IgG) was supplied by Sigma and used as received. 2-(N-morpholino)ethanesulfonic acid (MES) was obtained from Aldrich. Protein solutions were made by diluting the stock solution of anti-IgG to the desired concentration with 0.05 M MES (containing 0.082 M NaCl, pH adjusted to 6.0 using NaOH). Substrate Preparation. Details on the preparation of the QCM electrodes (AT-cut, gold-coated electrodes, Kyushu Dentsu, Japan) for SEM measurements have been described elsewhere.36 Glass microscope slides were used as the substrates for AFM and FTIR-RAS measurements. These surfaces were cleaned by ultrasonication in acetone for 10 min, followed by rinsing with ethanol and drying with nitrogen. For the AFM experiments, a 10 nm chromium adhesion layer was sputter-coated onto the glass slides, followed by a gold layer of 100 nm. Glass slides for FTIR-RAS experiments were coated using the same procedure to give a 30 nm chromium layer and a 250 nm gold layer. Polyelectrolyte and Protein Multilayer Film Formation. The precursor film (PAH/PSS)2 was prepared on MPA-modified gold substrates by repeating two alternate adsorption cycles of PAH and PSS. A thicker precursor film of (PAH/PSS)12 (also assembled on gold substrates pretreated with MPA) was used for SEM experiments so as to enable determination of film thicknesses; this allows comparison of the film thicknesses from SEM with those determined from QCM measurements. Details on the preparation of these precursor films has been described in a previous publication.37 The surfaces were then alternately immersed in solutions of anti-IgG in MES buffer for ca. 45 min and PSS for 1 min. The surfaces were rinsed with pure water (2 × 1 min) and dried with nitrogen between each deposition. The anti-IgG/PSS deposition cycle was repeated until the desired number of anti-IgG layers was achieved.26 The concentration of the anti-IgG solution used in the AFM and SEM experiments was 20 µg mL-1. In the FTIR-RAS experiments the concentration was increased to 100 µg mL-1. These concentrations produce saturation coverage of the surfaces.38,39 Atomic Force Microscopy (AFM) Measurements. AFM imaging was performed in air using a Digital Instruments Nanoscope IIIa AFM (Santa Barbara, CA) in tapping mode (TM). Less damage occurs to biological specimens such as proteins when using tapping mode.40 Further details on the TM principle can be found elsewhere.40 Raw AFM data obtained were processed by flattening and plane fitting. Scanning Electron Microscopy (SEM) Measurements. For the SEM measurements, QCM electrodes covered with thin multilayer films were cut and then sputter-coated with a 2 nm thick platinum layer using an ion-coater (Hitachi E-1030 ion sputter) at a current of 15 mA and pressure of 10 Pa. SEM micrographs were obtained with a Hitachi S-900 (Japan) scanning electron microscope at an acceleration voltage of 25 kV. Fourier Transform Infrared Reflection-Absorption Spectroscopy (FTIR-RAS) Measurements. FTIR-RAS spectra were recorded using a Nicolet 710 spectrometer equipped with a MCT detector. The FTIR-RAS spectrum of a bare gold plate was used as the reference. Cast films of PAH and PSS were prepared by depositing an aliquot of 3 mg mL-1 PAH or PSS solution onto a gold substrate and allowing it to dry overnight. All spectra were collected at a spectral resolution of 4 cm-1. Quartz Crystal Microbalance (QCM) Measurements. Details on the QCM measurement system can be found in earlier publications.36,38 QCM experiments were performed by immersing a QCM electrode in a polyelectrolyte or protein solution (36) Caruso, F.; Serizawa, T.; Furlong, D. N.; Okahata, Y. Langmuir 1995, 11, 1546. (37) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422. (38) Caruso, F.; Rodda, E.; Furlong, D. N. J. Colloid Interface Sci. 1996, 178, 104. (39) Geddes, N. J.; Martin, A. S.; Caruso, F.; Urquhart, R. S.; Furlong, D. N.; Sambles, J. R.; Than, K. A.; Edgar, J. A. J. Immunol. Methods 1994, 175, 149. (40) Fritz, M.; Radmacher, M.; Cleveland, J. P.; Allersma, M. W.; Stewart, R. J.; Gieselmann, R.; Janmey, P.; Schmidt, F. C.; Hansma, P. K. Langmuir 1995, 11, 3529 and references therein.

Polyelectrolyte-Protein Multilayer Films

Figure 1. TM AFM image of a (PAH/PSS)2 film on a gold substrate pretreated with MPA.

Figure 2. SEM micrograph of a (PAH/PSS)12 film on a MPAtreated gold QCM electrode. for a given time, rinsing the surface with pure water, nitrogen drying, and recording the in air frequency changes due to adsorption.26

Results and Discussion AFM and SEM Data. Figure 1 shows the AFM image of a thin (PAH/PSS)2 precursor film on a gold substrate pretreated with MPA. The root-mean-square (RMS) roughness of the film is 0.8 nm. This value agrees with that obtained for PAH/PSS multilayer films by smallangle X-ray reflectivity measurements (0.8 ( 0.2 nm).23 A scanning electron micrograph of a (PAH/PSS)12 film assembled on a gold QCM electrode pretreated with MPA is shown in Figure 2. (A thicker polyelectrolyte precursor film was used in the SEM experiments, compared to that used in AFM, to allow determination of the film thickness

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Figure 3. TM AFM image of anti-IgG immobilized on a precursor film of (PAH/PSS)2 on a gold substrate pretreated with MPA.

by SEM. The RMS surface roughness is similar for PAH/ PSS multilayers of different thicknesses.23) The SEM micrograph shows the film is smooth with no distinct features on the surface. The AFM image for anti-IgG immobilized on a precursor film of (PAH/PSS)2 on a MPA-treated gold substrate is shown in Figure 3. The 1 × 1 µm2 area is well covered with anti-IgG and the film has a RMS roughness of 1.7 nm. Most of the anti-IgG on the surface have a diameter in the range of 40-80 nm. From Figure 3 the mean (( standard deviation, SD) diameter for anti-IgG in this size range is 65 ( 11 nm. Some larger anti-IgG structures (> 100 nm diameter) are also present in the image. The diameters observed are significantly larger than that of individual anti-IgG molecules; the dimensions of IgG determined from X-ray crystallography data are approximately 10 × 14 × 5 nm.41-43 This discrepancy in size may be due to tip convolution effects that enhance the x- and y-dimensions of imaged proteins in AFM, giving rise to an observed diameter larger than the actual protein diameter.40,44 For example, a diameter of 20 nm has been measured for lysozyme adsorbed on mica using AFM,40 which is ca. 5 times larger than that determined from X-ray diffraction. Hence, it is conceivable that some of the structures observed in Figure 3 are individual anti-IgG molecules while some of the larger structures (>100 nm) are aggregates, as confirmed by the SEM data below. Analysis of the heights of imaged anti-IgG of ∼65 nm size in Figure 3 gives a mean (( SD) height of 4.5 ( 1.5 nm. The larger anti-IgG structures seen have heights of ∼11 nm. AFM height measurements can be taken to be the true vertical dimensions of the anti-IgG molecules, assuming flattening of the proteins is negligible. The mean height observed suggests that most of the anti-IgG lies flat on the polyelectrolyte precursor film. This value is close to the smallest dimension of 5 nm for anti-IgG. Wa¨livaara et al.45 have recently reported similar heights (41) Silverton, E. W.; Navia, M. A.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5140. (42) Marquart, M.; Deisenhofer, J. Immunol. Today 1982, 3, 160. (43) Amit, A. G.; Mariuzza, R. A.; Phillips, S. E.; Poljak, R. J. Science 1986, 233, 747. (44) Davies, J.; Roberts, C. J.; Dawkes, A. C.; Sefton, J.; Edwardes, J. C.; Glasbey, T. O.; Haymes, A. G.; Davies, M. C.; Jackson, D. E.; Lomas, M.; Shakesheff, K. M.; Tendler, S. J. B.; Wilkins, M. J.; Williams, P. M. Langmuir 1994, 10, 2654. (45) Wa¨livaara, B.; Warkentin, P.; Lundstro¨m, I.; Tengvall, P. J. Colloid Interface Sci. 1995, 174, 53.

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Figure 5. TM AFM image of a (PAH/PSS)2/(anti-IgG/PSS)4/ anti-IgG multilayer film on a gold substrate pretreated with MPA. The interlayer between anti-IgG layers is PSS, and antiIgG forms the outermost layer.

Figure 4. SEM micrograph of a (PAH/PSS)12/anti-IgG film on a MPA-treated gold QCM electrode.

(∼4 nm) for individual human IgG molecules adsorbed on methylated silica surfaces using AFM in tapping mode. Figure 4 shows the scanning electron micrograph of a (PAH/PSS)12/anti-IgG film on a MPA-treated gold QCM electrode. The micrograph shows many isolated anti-IgG molecules of 10-20 nm size covering most of the surface. Unlike AFM measurements, where the anti-IgG dimensions are exaggerated by tip convolution effects, the antiIgG dimensions from SEM accurately reflect the size of the protein. Figure 4 suggests that most of the anti-IgG seen in Figure 3 represent individual anti-IgG molecules. There are, however, also some larger structures (size ca. 80-150 nm) in this micrograph that correspond to antiIgG aggregates. This is also consistent with what is observed in the corresponding AFM image (Figure 3). Although the precursor film used for film assembly in the SEM measurements ((PAH/PSS)12) is thicker than that used in the AFM measurements ((PAH/PSS)2), QCM data reveal the amount of anti-IgG adsorbed onto these surfaces is the same (ca. 4.0 ( 0.2 mg m-2), indicating that essentially no diffusion of anti-IgG into the films occurs. This surface coverage (Γ) is in agreement with that previously found for anti-IgG on (PAH/PSS)5 films26 and suggests that on average a monolayer of anti-IgG forms on the precursor films (PAH/PSS)n where n ) 2, 5, or 12. The QCM results indicate an end-on orientation of anti-IgG on the polyelectrolyte films with repelling F(ab) fragments (Γ ) 3.7 mg m-2).46 For this anti-IgG orientation (indicated solely by QCM data), a height of ca. 10 nm would be expected for individual anti-IgG molecules.46,47 QCM results, however, give a mean surface coverage value (and height of anti-IgG) for the entire surface. Hence, taking into account the AFM and SEM results above, the (46) Buijs, J.; Lichtenbelt, J. W. Th.; Norde, W.; Lyklema, J. Colloids Surf. B: Biointer. 1995, 5, 11. (47) Grabbe, E. S. Langmuir 1993, 9, 1574.

QCM surface coverage data can be explained by most of the polyelectrolyte surface being covered with individual anti-IgG molecules (in a flat orientation), as well as some anti-IgG aggregates. AFM images and SEM micrographs of multilayer films, where the protein layers are separated by five polyelectrolyte layers (PSS(PAH/PSS)2), and where anti-IgG forms the outermost layer, are similar to those shown in Figures 3 and 4, respectively. The interlayer PSS(PAH/PSS)2 provides a surface for anti-IgG immobilization that is similar to the precursor (PAH/PSS)2 and (PAH/PSS)12 films; the amount and distribution of anti-IgG immobilized on these films are the same. This is also consistent with QCM measurements, which yield a linear relationship of frequency decrease (corresponding to the mass of antiIgG on the surface) with the number of anti-IgG layers for polyelectrolyte/anti-IgG multilayer films when the interlayer is PSS(PAH/PSS)2.26 Figure 5 shows the AFM image of a polyelectrolyte/ anti-IgG multilayer film on a gold substrate precoated with (PAH/PSS)2 when the interlayer between anti-IgG layers is PSS, and anti-IgG forms the outermost layer. In contrast to the multilayer film where the interlayer was PSS(PAH/PSS)2 (see above), anti-IgG aggregation is clearly evident in this image. The surface is considerably rougher than those shown in Figures 1 and 3; the RMS roughness for this surface over the 1 × 1 µm2 area is 4.7 nm. Aggregates ranging in size from 150 to 230 nm are observed. The mean (( SD) diameter for these aggregates is 200 ( 25 nm, and the mean height is 20 ( 3 nm. Some smaller structures of diameter ca. 100 nm and height ca. 7 nm are also seen in the image; these too are aggregates of anti-IgG. Thus, in this film, individual anti-IgG molecules are not imaged. A noticeable feature of this image is the nonuniform coverage of the surface by antiIgG. The film structure is clearly open with areas as large as 100 × 50 nm2 unfilled in the upper layers of the film. This open, disordered structure can allow IgG to penetrate into the film and interact with immobilized anti-IgG. This type of structure is consistent with the QCM immunosensing results which indicated that antigen (IgG) in solution penetrates such films to bind with immobilized anti-IgG.26 The scanning electron micrograph of the film (PAH/ PSS)12/(anti-IgG/PSS)4/anti-IgG on a gold QCM electrode pretreated with MPA (Figure 6) clearly shows significant

Polyelectrolyte-Protein Multilayer Films

Figure 6. SEM micrograph of a (PAH/PSS)12/(anti-IgG/PSS)4/ anti-IgG film on a MPA-treated gold QCM electrode. The interlayer between anti-IgG layers is PSS, and anti-IgG forms the outermost layer.

aggregation of anti-IgG. The film structure is disordered and numerous holes exist in the film. This SEM micrograph, like the AFM image for the corresponding film, is also consistent with the notion that antigen can diffuse into the film to interact with anti-IgG immobilized in the multilayer films. Figure 7 shows a cross-sectional view (SEM micrograph) of the (PAH/PSS)12/anti-IgG film displayed in Figure 4. The film follows the profile of the gold QCM electrode and has a thickness of 90 ( 10 nm. (The SEM micrograph for the precursor (PAH/PSS)12 film yielded a thickness of 100 ( 10 nm.) The errors represent variations over different areas of the SEM micrographs. The film thicknesses from QCM data can be calculated using d (nm) ) -0.018 ∆F (Hz),37 assuming a density of 1.2 × 106 g m-3 for both the polyelectrolyte and protein layers.48 Using the QCM frequency shift (∆F) of -5400 Hz for the (PAH/PSS)12/ anti-IgG film, a thickness of 97 ((10) nm is calculated, which is in agreement with that determined from SEM. A cross-sectional view of the (PAH/PSS)12/(anti-IgG/PSS)4/ anti-IgG multilayer film is shown in the SEM micrograph in Figure 8 (same multilayer film as that shown in Figure 6). A thickness of 150 ( 15 nm is observed for this film. This value is consistent with the 155 ((15 nm) nm determined from QCM data (∆F ) -8600 Hz) for this polyelectrolyte multilayer film. The excellent agreement between the thicknesses determined from SEM and QCM measurements validates the use of 1.2 × 106 g m-3 for the density of both the polyelectrolyte and protein layers. FTIR-RAS Spectra. FTIR-RAS spectra (Figures 9 and 10) were recorded for MPA-treated gold (spectrum a), the polyelectrolyte precursor film (PAH/PSS)2 (spectrum b), (48) Polymer Handbook; Brandrup, J., Immergut, E., Eds.; John Wiley and Sons: New York, Chichester, Brisbane, Toronto, 1975; Part 5.

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Figure 7. SEM micrograph (cross-sectional view) of the (PAH/ PSS)12/anti-IgG film on a MPA-treated gold QCM electrode displayed in Figure 4.

Figure 8. SEM micrograph (cross-sectional view) of the (PAH/ PSS)12/(anti-IgG/PSS)4/anti-IgG multilayer film on a MPAtreated gold QCM electrode displayed in Figure 6.

and protein/polyelectrolyte multilayer films of the form (PAH/PSS)2/(anti-IgG/PSS)m/anti-IgG where m ) 0, 2, 4

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Figure 9. FTIR-RAS spectra of polyelectrolyte/anti-IgG multilayer films on a gold substrate in the region 3600-2400 cm-1: (a) MPA-treated gold; (b) precursor film (PAH/PSS)2; (c), (d), and (e) protein/polyelectrolyte multilayer films of the form (PAH/ PSS)2/(anti-IgG/PSS)m/anti-IgG, where m ) 0, 2, 4, respectively.

Figure 10. FTIR-RAS spectra of polyelectrolyte/anti-IgG multilayer films on a gold substrate in the region 1800-950 cm-1: (a) MPA-treated gold; (b) precursor film (PAH/PSS)2; (c), (d), and (e) protein/polyelectrolyte multilayer films of the form (PAH/PSS)2/(anti-IgG/PSS)m/anti-IgG, where m ) 0, 2, 4, respectively.

(spectra c-e). It has been demonstrated that these films show desirable immunosensing characteristics.26 The spectra for the regions 3600 to 2400 cm-1 and 1800 to 950 cm-1 are shown in Figures 9 and 10, respectively. Between 2400 and 1800 cm-1 the spectra show no significant features. The only noticeable peak in the spectrum of MPA-modified gold (a) is that at 1410 cm-1, which is assigned to a combination of the symmetric COOstretching band of the carboxylate anion and the R-CH2 scissors deformation of the carboxylic acid.49,50,51 The very weak, broad peak around 1700 cm-1 may be assigned to the carboxylic acid CdO stretch.49,50 No bands due to the C-OH stretching of the carboxylic acid groups (υs(COH), 1200 cm-1) or the asymmetric COO- stretching band of the carboxylate anion (υas(COO-), 1610 cm-1) are identifiable in the spectrum. Further, no bands due to the asymmetric (υas(CH2), 2920 cm-1) and symmetric (υs(CH2), 2850 cm-1) methylene stretching bands are ob(49) Smith, E. L.; Alves, C. A.; Anderegg, J. W.; Porter, M. D.; Siperko, L. M. Langmuir 1992, 8, 2707. (50) Jordan, C. E.; Frey, B. L.; Korngruth, S.; Corn, R. M. Langmuir 1994, 10, 3642. (51) Sun, L.; Crooks, R. M.; Ricco, A. J. Langmuir 1993, 9, 1775.

Caruso et al.

served. The presence of these two bands and an intense υs(CH2) band are indicative of a highly organized monolayer.52 The absence of these bands in the measured spectrum is not surprising given that MPA does not form a well-ordered self-assembled monolayer on the gold surface.53 MPA, however, is useful in that it renders the surface sufficiently negative to aid the subsequent adsorption of positively charged polyelectrolyte. After deposition of (PAH/PSS)2 onto the MPA-modified gold surface a number of new bands appear (spectrum b). The bands at 3056, 2921, and 2854 cm-1 are all due to the presence of PAH in the film. A cast film of PAH onto gold displayed peaks at 3323 (weak), 3100 (broad), 2925 (strong), and 2858 (shoulder) cm-1. In the region 36002400 cm-1 a cast film of PSS on gold showed weak bands at 2925 and 2848 cm-1. In the region 1800-950 cm-1, the new bands that appear at 1610 and 1525 cm-1 are due to PAH, and the bands in the 1500-950 cm-1 region are due to PSS; these bands are present in the cast films of PAH and PSS, respectively. Since we are interested in the nature of the protein in the films, we will concentrate on the assignment of the anti-IgG IR bands. The changes in the FTIR-RAS spectra after exposure of the gold surface with a precursor (PAH/PSS)2 film to an anti-IgG solution show that anti-IgG is adsorbed onto the polyelectrolyte surface (spectrum c, m ) 0). Three new dominant bands appear at 3290, 1645, and 1530 cm-1. The band at 3290 cm-1 is assigned to N-H stretching vibrations (υN-H) and is known to occur from a combination of the amide and amine N-H frequencies.50 The bands at 1645 and 1530 cm-1 are assigned to the amide I and amide II bands of anti-IgG. The amide I band originates predominantly from the CdO stretching vibrations of the peptide bond groups and the amide II band arises from N-H in-plane bending and C-N stretching modes of the polypeptide chains.54,55 There is also a slight enhancement in the absorbance of peaks at 3060, 2930, 1450, and 1410 cm-1 with deposition of anti-IgG. Anti-IgG layers alternating with PSS to give a total of 3 (m ) 2) and 5 (m ) 4) anti-IgG layers in the multilayer film were subsequently deposited onto the existing thin film (m ) 0). The measured FTIR-RAS spectra for these films are also shown in Figures 9 and 10 (spectra d and e, m ) 2 and 4, respectively). It can be clearly seen that the υN-H, amide I and amide II bands all increase in absorbance with an increasing number of protein layers in the film. The frequency at which the peak maxima occurs for these bands remains unchanged with the addition of protein layers. The existing peaks at 3060, 2930 (split into two peaks, 2955 and 2930 cm-1), 1450, and 1410 cm-1 also increase in absorbance, and the shoulder at 2870 cm-1 (υs(CH2)) becomes more distinct for the multilayer films where m ) 2 and 4. The appearance of the amide I and II bands, and their increasing absorbance with increasing number of antiIgG deposition cycles, confirms the incorporation of antiIgG into the multilayer films with each successive adsorption cycle. The absorbance of the υN-H (3290 cm-1), amide I (1645 cm-1), and amide II (1530 cm-1) bands varies linearly with the number of layers. At these frequencies only anti-IgG bands contribute to the absorbance. Figure (52) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (53) Sondag-Huethorst, J. A. M.; Scho¨nenberger, C.; Fokkink, L. G. J. J. Phys. Chem. 1994, 98, 6826. (54) Jackson, M.; Haris, P.; Chapman, D. J. Mol. Struct. 1989, 214, 329. (55) Walton, A. G.; Blackwell, J. Biopolymers; Academic Press: New York, 1973; Chapter 5.

Polyelectrolyte-Protein Multilayer Films

Figure 11. Amide I band absorbance and QCM anti-IgG layer frequency changes plotted as a function of the number of antiIgG layers for multilayer films of the form (PAH/PSS)2/(antiIgG/PSS)m/anti-IgG (m ) 0-4) on a gold substrate pretreated with MPA.

11 shows the absorbance values for the amide I band plotted as a function of the number of anti-IgG layers. The QCM anti-IgG layer frequency changes for the same multilayer films are also shown in Figure 11.26 There is good correlation between the FTIR-RAS and QCM results, with the exception of the data for the first anti-IgG layer. It should be pointed out that the FTIR-RAS measurements are sensitive only to the vibrational modes that are perpendicular to the substrate, and hence the absorbance could possibly display a dependency on the protein orientation with respect to the number of anti-IgG layers. Suffice it to say, the FTIR-RAS spectral data clearly confirm that anti-IgG is incorporated in the films. Both the AFM and SEM data in this work are consistent with the QCM results; the first anti-IgG layer on the precursor polyelectrolyte film forms essentially a uniform layer, and subsequent anti-IgG layers, which are separated by one PSS layer, form aggregated multilayer structures. The frequencies at which the υN-H, amide I, and amide II bands occur for the polyelectrolyte/anti-IgG multilayer films compare well with those reported for IgG in solution (υN-H 3295 cm-1, amide I 1644 cm-1, amide II 1550 cm-1).56 The amide I and II peak positions are, however, slightly lower than those previously reported for the surface binding of IgG onto an activated gold surface using FTIR

Langmuir, Vol. 14, No. 16, 1998 4565

(amide I 1665 cm-1, amide II 1545 cm-1).57 FTIR studies on IgG in solution have shown that the maximum of the amide I band shifts to 1625-1630 cm-1 (from 1644 cm-1) upon IgG denaturation.56,58 In addition, FTIR spectra for denatured proteins are generally characterized by bands at around 1625 and 1520 cm-1.59-62 The absence of such bands in the FTIR-RAS spectra for anti-IgG in the multilayer films presented in this work suggests that no significant denaturation of anti-IgG occurs. This finding is consistent with the conclusion drawn from earlier work concerning the immunosensing behavior of such multilayer films.26 In that work it was found that anti-IgG retains its activity in the films and is able to interact with IgG in solution. Functional protein layers are necessary for antibody-antigen based biosensing films, as the ability for an immobilized antibody receptor layer to detect antigens is crucially dependent on the extent of denaturation of the immobilized antibodies. Conclusions The AFM, SEM, and FTIR-RAS data reveal that polyelectrolyte and protein multilayer films can be constructed with regularly ordered protein layers or with open, permeable structures, in both cases containing biologically active protein. The structure of the film is determined by the polyelectrolyte interlayer separation between the protein layers for the polyelectrolytes studied. The layer-by-layer self-assembly method provides a general means for fabricating multilayer protein-containing films with different structural architectures. This should allow its exploitation in the preparation of functional thin films for biosensing devices. Acknowledgment. Frank Caruso acknowledges the Department of Industry, Science and Tourism for financial assistance. LA971288H (56) Abaturov, L. V.; Nezlin, R. S.; Vengerova, T. I.; Varshavsky, J. M. Biochim. Biophys. Acta 1969, 194, 386. (57) Geddes, N. J.; Paschinger, E. M.; Furlong, D. N.; Caruso, F.; Hoffmann C. L.; Rabolt, J. F. Thin Solid Films 1995, 260, 192. (58) Abaturov, L. V.; Varshavsky, J. M. Stud. Biophys. 1969, 13, 47. (59) Lenormant, H. Trans. Faraday Soc. 1956, 52, 549. (60) Lenormant, H. Bull. Soc. Chim, 1953, 214. (61) Bamford, C. H.; Hanby, W. E.; Happey, F. Proc. R. Soc. London, Ser. A 1951, 205, 30; 1951, 206, 407. (62) Ambrose, E. J.; Elliot, A. Proc. R. Soc. London, Ser. A 1951, 205, 47; 1951, 206, 206; 1951, 208, 75.