Abstract

USING THE SELF-ASSEMBLY TECHNIQUE FOR THE FABRICATION OF ULTRA-THIN FILMS OF A PROTEIN

C.E. Borato¹, P.S.P. Herrmann ^1,2, L.A. Colnago ^1,2, O.N. Oliveira Jr.³ and

L. H. C. Mattoso^1,2,* * To whom correspondence should be addressed.

¹Centro Nacional de Pesquisa e Desenvolvimento de Instrumentação Agropecuária,

CNPDIA/EMBRAPA, São Carlos, C.P. 741, 13560-970, SP, Brazil,

Fax: 55-16-2725958, Phone: 55-16-2742477

² Instituto de Química de São Carlos, Universidade de São Paulo, USP, SP, Brazil, 13560-970

³ Instituto de Física de São Carlos, Universidade de São Paulo, USP, SP, Brazil,

C. P. 369, 13560-970

(Received: June 11, 1997; Accepted: October 30, 1997)

INTRODUCTION

The fabrication of materials whose properties can be controlled at the molecular level has been the subject of a number of research projects in the last few years (Oliveira et al., in press), mainly because of their possible application in sensors and microelectronic devices. In addition to replacing commercially available materials in conventional applications, there is the challenge of fabricating novel materials to be used as circuit elements in molecular electronics (Carter, 1987). The principal requirement for such applications is nanometric control over the structure, such as in the ability to form ultra-thin films. The first option for producing such structures is the Langmuir-Blodgett (LB) method (Roberts, 1990), in which the material of interest is dissolved in a volatile organic solvent, spread and ordered on an aqueous subphase and then transferred to a solid substrate. Repeated immersions and withdrawals of the same substrate lead to the formation of multilayer structures. The interesting advantages of these films are that their thickness is on the order of only a few nm and they show surface uniformity, a high degree of orientational order and possibility of controlling the molecular architecture.

Recently, a much simpler technique was developed which is an advantageous alternative for fabrication of ultra-thin films. It is the self-assembly (SA) method, based on the spontaneous adsorption of polymeric materials by electrostatic interaction (Decher et al., 1992; Ferreira et al., 1994). In contrast to the LB method, the SA technique allows the fabrication of organized ultra-thin films without requiring sophisticated and expensive equipment. In the SA technique, alternating layers of polycations and polyanions are deposited in a uniform and controlled manner. The SA films are extremely promising for a number of applications, including light-emitting diodes (LEDs) whose feasibility has already been demonstrated (Onitsuka et al., 1996). The self-assembly technique is also interesting for immobilizing proteins in a controlled fashion, as has been previously demonstrated (Lvov and Decher, 1994; Lvov et al., 1995; Kayushina et al., 1996). However, the adsorption kinetics and the influence of important parameters on the formation of the self-assembled film have not yet been investigated. The self-assembly technique of proteins allows, in principle, a greater control over the resulting structure which is potentially important for applications in biotechnology and molecular electronics.

In this work, self-assembled films are produced from lysozyme alternated with poly(styrene sulfonate). The adsorption kinetics of lysozyme layer formation, especially the influence of experimental conditions such as immersion time, drying method, solution pH and ionic strength, is investigated in detail using UV-spectroscopy.

EXPERIMENTAL

Lysozyme from Sigma and poly(styrene sulfonate) - PSS - from Aldrich are used as received. Quartz slides are used as substrates after a hydrophilization treatment described elsewhere (Ferreira, 1994). A schematic representation of the self-assembly method involves the following steps: (Figure 1).

i)preparation of stock solutions of a given pH, concentration and ionic strength;

ii)immersion of the hydrophilic substrate in the polycation solution for a certain period of time;

iii)washing of the substrate to eliminate residual, nonadsorbed material and drying of the sample;

iv) detection of the adsorbed material by measuring UV absorbance;

v)immersion of the hydrophilic substrate in the polyanion solution for a certain period of time;

vi)repetition of steps ii) to v) for the formation of alternating bilayers.

For kinetic studies only steps i) to iv) are carried out and the immersion time is taken as the cumulative period of time from several immersions. UV absorbance at a given wavelength is then plotted against time. Aqueous solutions are prepared using ultrapure water supplied by a Millipore system comprising reverse osmosis followed by ion exchange and filtration steps (Milli-Q, Millipore GmbH). Ultraviolet (UV) absorption spectra are obtained in a Shimadzu spectrophotometer model UV-1601PC.

Figure 1: Schematic diagram of the steps followed in producing alternating ultra-thin films by the self-assembly technique (Ferreira, 1994).

RESULTS AND DISCUSSION

The self-assembly technique is based on the spontaneous adsorption of charged molecules by electrostatic interaction. Although it is a relatively new method for building ultra-thin films (Decher et al., 1992; Ferreira et al., 1994), its has been growing very rapidly due to its possible application for a wide variety of compounds (Lvov and Decher, 1994; Lvov et al., 1995; Kayushina et al., 1996). However, the mechanisms involved in the adsorption process are far from being fully understood. The aim of the present paper is thus to investigate the kinetics of adsorption of a single layer of lysozyme on quartz substrates, also comparing the data with results obtained for conducting polymers, and to produce multilayer structures from lysozyme alternated with a polyelectrolyte.

Lysozyme absorption increases with substrate immersion time in the ultraviolet region, as shown in the spectra of Figure 2, and therefore UV spectroscopy is a suitable tool for studying the kinetics of adsorption. Figure 3 shows the UV absorbance at 219 and 285 nm for a layer of lysozyme as a function of immersion time. In this case, the aqueous solution containing lysozyme has a pH = 6.4 at which lysozyme behaves as a polycation (isoelectric point of 11), thereby being electrostatically attracted by the hydrophilic substrate. Absorbance increases rapidly as immersion time increases in the initial stage, indicating the fast adsorption of lysozyme molecules onto the substrate. Even though complete adsorption may take much longer, a reasonable amount of lysozyme has already been adsorbed within 10 min of immersion. Together with the ease of fabrication, this makes the SA method a very convenient way of immobilizing the protein on a solid substrate.

With regard to the kinetics of adsorption, UV absorbance increases exponentially at least in the first few minutes of immersion which is indicative of a first-order kinetics process. The data are not sufficient to indicate whether there is a small plateau or kink after this initial process, before proceeding to a steady state corresponding to complete adsorption. The possibility of a two-step process arises because it has actually been observed for poly(o-methoxyaniline) (POMA) - a conducting polymer (Raposo et al., 1996). The kinetics for POMA adsorbed on glass comprise an initial, fast step characteristic of a first-order kinetics process, followed by a slower second step represented by a Johnson-Mehl-Avrami function with tⁿ in the exponential. The main differences between the lysozyme and POMA adsorption processes lie in the time scales. While for POMA the initial rise has a characteristic time of only a few seconds (5-10s) (Raposo et al., 1996, submitted), for lysozyme this characteristic time is on the order of 8-10 minutes. Furthermore, complete adsorption is achieved with POMA deposited on glass within 5-10 minutes, as the characteristic time of the second step is on the order of hundreds of seconds. For lysozyme, on the other hand, complete adsorption only takes place after several hours and depends on the conditions used.

Wavelength (nm)

Figure 2: UV spectra of lysozyme (10^-4M, pH=6.4) absorbed on the substrate with an increase in absorption occurring as the immersion time of the substrate increases, as plotted in Figure 3 for two selected wavelengths.

That the kinetics may depend on the type of material investigated is illustrated by comparing the data mentioned above for POMA (Raposo et al., 1996) and those by Ferreira and Rubner (1995) for poly(thiophene-3-acetic-acid) (PTAA). The latter author suggested that adsorption of PTAA on glass could be diffusion controlled as in a Langmuir-Schaefer type adsorption, at least for the first three minutes of immersion. The calculated effective diffusion coefficient for various polymer concentrations varies over four orders of magnitude; however, Ferreira and Rubner (1995) then concluded that the Langmuir-Schaefer relationship is not valid for PTAA in the time regime accessible by UV experiments. The results for POMA, on the other hand, cannot be explained by a t^1/2 dependence - indicative of a diffusion controlled process - even for the first minute of immersion. Taken together these results for POMA (Raposo et al., 1996) and PTAA (Ferreira and Rubner, 1995) indicate that mechanisms involved in the kinetics of adsorption appear to vary, even when the two examined materials are conducting polymers. For the protein lysozyme, the time scale also appears to be different.

It is found that the drying process also affects the amount of adsorbed material, as observed in Figure 4. Since lysozyme does not unfold during dehydration (Prestrelski et al., 1993), this can be understood in terms of the moisture dependence of the packing capability of the molecules. Intense drying may reduce the lysozyme molecular volume (Yip and Ward, 1996), leaving further free space for other molecules to adsorb, when compared with immersions carried out with films containing larger amounts of entrained water. The effect of solution pH, in the range from 4 to 8, on the adsorption kinetics is also investigated. The results are displayed in Figure 5, from which one can observe that the shape of the absorbance versus time curve is not significantly affected, but absorbance is considerably higher for lower pHs. This occurs because as the proton concentration in the solution increases, the positive charge density within the protein molecules also increases, which facilitates the electrostatic attraction to the substrate. A similar behavior has been obtained for poly(o-ethoxyaniline) adsorbed on glass using the self-assembly technique (Mattoso et al., 1996).

In order to distinguish between the effects of proton concentration (pH) and those of ionic strength, the absorption kinetics of solutions with a constant pH (6.4) but different ionic strengths (by salt addition) are investigated (Figure 6). In contrast to the pH effect, by increasing the ionic strength the absorption intensity tends to decrease. The only exception is in the initial stage of deposition for 10^-4M NaCl concentration. It seems that salt ions present in the lysozyme solution screen the positive charge of the protein, decreasing the driving force of electrostatic attraction.

Figure 3: UV absorbance for lysozyme at 219 nm (--o--) and 285 nm (--n --) (extracted from Figure 2) versus immersion time of the substrate. The absorbance intensity at 219 nm was divided by a factor of 8 in order to fit in this figure.

Figure 4: Adsorption kinetics of lysozyme adsorbed onto quartz after being dried by various drying methods: a) forced drying by blowing hot air, b) spontaneous drying in ambient air and c) no drying.

Figure 5: Adsorption kinetics for lysozyme (10^-4 M) assembled from solutions at different pHs (4.0, 6.4, 8.0).

Figure 6: Adsorption kinetics of lysozyme assembled from solutions (10^-4 M, pH = 6.4) prepared with various NaCl concentrations (10^-3 M, 10^-4 M and no salt).

Figure 7: Multilayer formation of alternating lysozyme/PSS bilayers using the self-assembly technique. Conditions: pH=4.0, polyion concentration of 10^-4M.

As can be seen in Figure 7, this self-assembly technique can also be used to produce alternating ultra-thin films of lysozyme and a polyanion (PSS), under selected conditions. The multilayer formation confirms the suggestion by Haggerty et al. (1991, Haggerty and Lenhoff, 1993), that lysozyme adsorption be governed by electrostatic interaction. It is observed that absorbance at 285nm increases linearly, within experimental error, with the number of bilayers (lysozyme/PSS), indicating that a constant amount of material is being adsorbed in each immersion process. In previous work Lvov and Decher (1994) initially failed to obtain a linear behavior on the multilayer growth of lysozyme/PSS film due to protein coagulation. The authors confirm that the choice of appropriate solution and self-assembling conditions are of key importance for obtaining a uniform growth. Further studies (Lvov and Decher, 1994; Lvov et al., 1995; Martinez, 1997) have shown the possibility of using the self-assembly technique for several other systems, including conducting polymers, polyacids, proteins and viruses forming multicomponent structures. However, the precise conformational arrangement of the molecules in the films and its dependence on the self-assembling parameters are still unknown. On-going atomic force microscopy studies (Martinez, 1997) are being conducted in our laboratories to investigate the effect of film preparation conditions on the structuring of the protein layers, as well as on the structure of the lysozyme molecule itself.

In summary, the possibility of using the self-assembly technique for producing ultra-thin films of a protein has been demonstrated. The influence of several self-assembly parameters on adsorption kinetics has also been elucidated. These results are highly promising as they illustrate that multilayered, ultra-thin films of proteins can be produced if alternated with charged polyions, which might be of great interest for biotechnology and molecular electronics applications. This is important insofar as protein properties may be tailored by the adequate choice of experimental conditions for protein adsorption and also the polyion to be used for the alternating multilayers. While the fabrication of SA films from a number of conducting polymers has been well established, the same is not true of SA films made from proteins. In this context, the results presented here may pave the way for a number of exciting possibilities, such as the fabrication of 3-D crystals from proteins.

ACKNOWLEDGMENTS

The financial support given by CNPq, FAPESP and EMBRAPA is gratefully acknowledged.

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