Langmuir 1996, 12, 6721-6723
Selective Solubilization Process Applied to the Study of Enzyme-Containing Langmuir-Blodgett Films Nicolas Dubreuil, Ste´phane Alexandre, Delphine Lair, and Jean-Marc Valleton* Polyme` res, Biopolyme` res, Membranes, URA 500 CNRS, UFR des Sciences, Universite´ de ROUEN, 76821 Mont-Saint-Aignan Cedex, France Received July 17, 1996. In Final Form: October 2, 1996
Introduction The Langmuir-Blodgett (LB) technology allows the elaboration of three-dimensional ordered structures by using amphiphilic long-chain molecules such as fatty acids or lipids, which are constituents of natural membranes.1,2 The incorporation of biomolecules (enzymes, antibodies, etc.) in LB films provided them with biological activity and biospecific recognition properties. The interest of elaborating such structures is twofold. On one hand, such structures may constitute interesting models for biological membranes and may be used to study complex functions with a simplified model. On the other hand, such structures may be used in the field of biomimetics to design artificial systems inspired by biological structures and/or functions; among them biosensors are a major target. The biosensors based on LB technology use an active film consisting of monolayers of an amphiphilic molecule in which the biomolecule is incorporated.3-5 The major interest in this technique is to create an organization of two components at the molecular scale; the very small thickness of such structures leads to biosensors with short response times. In our group, we have developed a glucose biosensor by transferring a mixed film of glucose oxidase (GOx) and behenic acid (BA) on a gold-coated electrode by LB technology.5 However, in spite of the good response time of the bioelectrode and the linearity of the calibration curves obtained, some reproducibility problems were encountered. Therefore, we decided to study in more detail the formation of the mixed film at the air/water interface before the transfer. This study showed the adsorption of the enzyme between the BA molecules and under the film. A model of the film formed at the air/water interface was also proposed.6 In previous papers, we reported IR spectroscopy7 and scanning force microscopy (SFM)8,9 studies of mixed LB films transferred on HOPG (highly oriented pyrolytic graphite). Recently, we studied the structure of comparable films transferred on mica muscovite, which is a hydrophilic substrate, in order to get stronger interactions between the mixed film and the substrate.
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In this paper, we present an original method to obtain chemical-like information by SFM on these mixed systems transferred on muscovite. This method is based on a selective solubilization process by isopropanol, which removes one of the components while preserving the other. Materials and Methods LB films were prepared with a trough from Atemeta (Paris). The dimensions of the trough are 44 cm × 6.5 cm, and the volume of the subphase is 250 cm3. The amphiphilic molecules were compressed at the air/water interface by a mobile barrier, and the pressure was measured with a Wilhelmy balance. The temperature of the subphase was 22 °C, and the pH was 5.6. GOx from Aspergillus niger (Type X-S from Sigma) and BA (Sigma, purity > 99%) were used without further purification. Chloroform (RP Normapur, purity > 99.2% containing 0.6% ethanol as a stabilizer) was purchased from Prolabo, and isopropanol (purity > 99.7%) was purchased from Carlo Erba. All experiments were carried out by using water obtained by a MilliQ system (Millipore). For the preparation of the mixed film at the air/water interface, the subphase was a 3.2 mg/L aqueous solution of GOx. BA was prepared as a 10-3 M solution in chloroform, and an amount of 0.1 mL was spread by a micropipette. After the BA solution was spread, the enzyme molecules were allowed to adsorb at the air/water interface for 45 min onto and between the polar heads of BA molecules. Then the molecules were compressed up to 30 mN/m. The pressure was maintained for 30 min, and the film was transferred by the classical LB technique at a pressure of 30 mN/m. The transfers were performed on CaF2 slides (Sorem, Uzos, France) for the IR spectroscopy study and muscovite sub strates (Metafix, Montdidier, France) for the SFM study. The CaF2 slides were previously cleaned, rinsed, and sonicated in acetone and finally chloroform. Muscovite substrates were cleaved before any transfer and used without any particular processing. The selective solubilization of the mixed LB films was achieved by rinsing the substrates (CaF2 and muscovite) with isopropanol for a few seconds. The transmission IR spectra of the LB films were recorded before and after the solubilization with a Nicolet 510M FTIR spectrophotometer in the spectral region from 4000 to 1200 cm-1. Moreover, a reference IR spectrum of each CaF2 slide was recorded before any experiment. The IR spectra were obtained by collecting and averaging 30 scans at a resolution of 4 cm-1. Spectra subtractions with reference spectra were performed in order to remove the substrate contribution. Thus, the spectra are simple combination of BA and GOx components.7 SFM experiments were performed with a Nanoscope II model from Digital Instruments (Santa Barbara, USA) with a 140 µm scanner in air (before the solubilization) and in isopropanol by using the so-called liquid cell (after the solubilization). The cantilevers used were characterized by a low spring constant of about 0.06 N/m. All the experiments were performed with the feedback loop on (constant force ) 10-9-10-8 N). No particular image processing was used except a flatten operation.
Results (1) Roberts, G. Langmuir-Blodgett films: Plenum: New York, 1991. (2) Barraud, A. J. Chim. Phys. 1985, 82, 683-685. (3) Moriizumi, T. Thin Solid Films 1988, 160, 413-429. (4) Arisawa, S.; Arise, T.; Yamamoto, R. Thin Solid Films 1992, 209, 259-263. (5) Fiol, C.; Valleton, J. M.; Delpire, N.; Barbey, G.; Ruaudel-Texier, A. Thin Solid Films 1992, 210/211, 489-491. (6) Dubreuil, N.; Alexandre, S.; Fiol C.; Valleton, J. M. J. Colloid Interface Sci. 1996, 181, 393-398. (7) Fiol, C.; Alexandre, S.; Dubreuil N.; Valleton, J. M. Thin Solid Films 1995, 261, 287-295. (8) Alexandre, S.; Dubreuil, N.; Fiol C.; Valleton, J. M. Microsc. Microanal. Microstruct. 1994, 5, 359-371. (9) Dubreuil, N.; Alexandre, S.; Fiol C.; Valleton, J. M. Langmuir 1995, 11, 2098-2102.
S0743-7463(96)00681-6 CCC: $12.00
1. Evidence of the Selective Solubilization Process. The selective solubilization of BA in the mixed LB films was demonstrated by using IR spectroscopy. The spectra of eight layers of mixed BA/GOx films transferred on CaF2 slides (Figure 1a) are characterized by peaks at 2956 (γasCH3), 2916 (γasCH2), 2850 (γsCH2), 1702 (γCdO), and 1463 (γCH2) cm-1 characteristic of the presence of BA molecules. The stretching bands (γCH2) are narrow and strong (with an intensity of 4 × 10-2 and 6 × 10-2 absorbance units) and have the same wavenumber and shape as in a BA/KBr pastille.7 © 1996 American Chemical Society
6722 Langmuir, Vol. 12, No. 26, 1996
Notes
Figure 1. IR transmission spectra of eight layers of BA/GOx transferred on CaF2 slides in the spectral regions 3800-2500 and 2000-1200 cm-1: (a) before the solubilization by isopropanol; (b) after the solubilization by isopropanol in which the peaks characteristic of BA have disappeared and the peaks characteristic of GOx are still present with the same intensity.
The spectra are also characterized by a broad peak from 3000 to 3600 cm-1 (γasNH and γOH) and peaks at 1657 (γCdO amide I) and 1540 cm-1 (γNH amide II) of the amide group characteristic of the presence of the enzyme. These peaks have a weak intensity because of the amount of enzyme and have the same wavenumber as in a GOx/ KBr pastille.7 After the action of isopropanol, we observed in the spectra the quasi-elimination of BA peaks while the GOx peaks still have the same intensity (Figure 1b). This result suggested a complete removal of BA molecules, which were present in the LB film while the enzyme molecules were preserved. A similar study was achieved with muscovite, since it is the substrate used for SFM study. This study has been successful, since isopropanol, like on CaF2, eliminated BA molecules in the mixed film when the film was deposited on muscovite. The next step has been to apply this selective solubilization of BA in the mixed LB films deposited on muscovite to identify the chemical nature of the structures observed with SFM. 2. Selective Solubilization Applied to SFM. The structure of mixed BA/GOx films transferred on muscovite has been first observed by SFM at the microscopic scale in the air with the contact mode. The image (Figure 2) presents quasi-circular structures. The area of these structures is about 0.1 µm2; some of them may reach 2 µm2. In order to appreciate the height of the circular structures, a section profile of the image was used. The heights of 2.9 and 2.5 nm measured on this profile may be related to the length of the BA molecule or to
the difference between the size of the GOx molecule10 (7.0 nm × 5.5 nm × 8.0 nm) and the length of the BA molecule.11 The circular structures might be BA bilayer or enzyme monolayer domains. However, because a bilayer of BA on muscovite is, a priori, not stable in air because of the polar heads in contact with air, the hypothesis of the enzyme monolayer appears more probable. After the observation in the air, the sample was rinsed with isopropanol. The observation of the sample in the liquid cell in isopropanol revealed similar circular structures (Figure 3). The study of a profile showed that the heights of the circular structures are about 6.3, 6.4, and 6.5 nm and may be related to the dimensions of the GOx molecule. On the basis of IR spectra, we were able to assume that isopropanol eliminated BA molecules in the structure and preserved the enzymatic structures. This result demonstrates that the circular structures correspond to enzymatic structures that were surrounded by the BA monolayer. The selective solubilization by isopropanol allowed us to carry out a chemical identification of the components present in the structure. Conclusion In this paper, we have demonstrated the possibility of a selective solubilization of one component of a mixed LB film consisting of behenic acid and glucose oxidase: (10) Hecht, H. J.; Schomburg, D.; Kalisz H. M.; Schmid, R. D. Biosensors Bioelectronics 1993, 8, 197-203. (11) A. Dhatathreyan, Colloids Surf. 1988, 29, 425.
Notes
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Figure 2. SFM image, obtained with the contact mode in air, of a mixed layer consisting of BA and GOx transferred on muscovite. The heights measured in this profile (2.9 and 2.5 nm) may be related to the length of the BA molecule11 or to the difference between the size of the GOx molecule10 and the length of the BA molecule.
Figure 3. SFM image, obtained in the “liquid cell” in isopropanol after the solubilization, of a mixed layer consisting of BA and GOx transferred on muscovite. The circular structures are clearly visible. The heights of these circular structures (6.3, 6.4, and 6.5 nm) may be related to the dimensions of the GOx molecule.
isopropanol is able to solubilize behenic acid while the enzymatic structures are preserved. This result has been applied to SFM analysis: it is possible to conclude if the structure observed consists of behenic acid or glucose oxidase. It might be also used for chemical quantification by IR. It should be possible, after the elimination of behenic acid,
to quantify the enzyme in the mixed film with a better precision than before the solubilization. It would be interesting to extend this concept of selective solubilization in mixed thin films, which provides SFM with chemical information potentialities, to other mixed systems. LA9606815
