Langmuir 1997, 13, 5507-5510
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Study of Mixed Enzyme/Pyrrole Derivative Langmuir-Blodgett Films for Biosensing Application C. A. Nicolae,† S. Cantin-Rivie`re, A. El Abed,* and P. Peretti Groupe de Recherche en Physique et Biophysique, Univeriste´ Rene´ Descartes 45, Rue des Saints-Pe` res, 75270 Paris, France Received February 5, 1997. In Final Form: April 22, 1997
Introduction Chemical sensors are devices that convert a quantity of a solute which has to be detected into an electrical or an optical signal. Among these sensors, biosensors use biological molecules, mainly enzymes, as recognition elements.1,2 It is essential that the used enzymes should be immobilized in a matrix in order to prevent their denaturation. Some immobilization techniques are noncovalent adsorption onto the physical transducer,3 covalent linking,4-6 and polymer matrix immobilization.7 However, it has been shown that the use of the Langmuir-Blodgett (LB) technique for the immobilization of enzymes is a good way to elaborate smart biosensors. During this last decade, different biosensors (glucose, urea, ethanol, etc.) have been produced by immobilizing enzymes into the LB matrix.8-11 The great advantage of LB technology is the close-packed deposition of the sensing molecules in very thin layers. Moreover, the high surface-to-volume ratio of these films implies that any small variation of the physical or chemical properties at the surface of the film induces large changes in the volume of the film. Consequently, this method is interesting for the elaboration of very sensitive biosensors. For alcohol sensing, an alcohol dehydrogenase (ADH) which necessitates a coenzyme, a nicotinamide adenine dinucleotide (β-NAD+ in its oxidized form or NADH in its reduced form), can basically be used. The detection principle is then based on the ADH-catalyzed conversion of the nondetectable ethanol; the detectable element is the reduced form of the co-enzyme NADH:
CH3CH2OH (ethanol) + β-NAD+ f ADH f CH3CHO (aldehyde) + NADH + H+ (1) NADH is then reoxidized into β-NAD+ at the electrode. It has been proven that the ethanol sensor sensibility improves if the charges resulting from the β-NAD+/NADH redox reaction are transmitted to the electrode via a mediator.12 Conductive polymers can play this role, especially the polypyrrole,8 which has a relatively high † Permanent address: Dipartimento di Fisica, Universita degli Studi di Parma, Viale delle Scienze, 43100 Parma, Italy. * Author to whom correspondence should be addressed.
(1) Sharma, A.; Rogers, K. R. Meas. Sci. Technol. 1994, 5, 461. (2) McCurley, M. F. Biosens. Bioelectron. 1994, 9, 527. (3) Clarck, L. C.; Lions, C. Anna. Acad. Sci. 1962, 102, 29. (4) Guilbault, G. G.; Montalvo, J. G. J. Am. Chem. Soc. 1969, 91, 2164. (5) Tran-Minh, C.; Broun, G. Anal. Chem. 1975, 45, 1359. (6) Kobos, R.; Rechnitz, G. A. Anal. Lett. 1977, 10, 751. (7) Eremenko, A.; Kurochkin, I.; Chernov, S.; Barmin, A.; Yaroslavov, A.; Moskvitina, T. Thin Solid Films 1995, 260, 212. (8) Li, J. R.; Cai, M.; Chen, T. F.; Jiang, L. Thin Solid Films 1989, 180, 205. (9) Arisawa, S.; Arise T. Thin Solid Films 1992, 209, 259. (10) Pal, P.; Nandi, D.; Misra, T. N. Thin Solid Films 1994, 239, 138. (11) Turko, I. V.; Lepesheva, G. I.; Chashchin, V. L. Thin Solid Films 1993, 230, 70. (12) Wang, J.; Naser, N. Electroanal. 1995, 4, 362.
S0743-7463(97)00117-0 CCC: $14.00
Figure 1. Schematic view of the alcohol biosensor: alcohol dehydrogenase (ADH) immobilized in a LB matrix of poly(hexadecylpyrrole) (P-3HDP); the long chains are hexadecyl (CH3(CH2)14CH2-).
electrical conductivity (10-2-10-1 S/cm) due to orbital overlapping and cation doping. Using LB technology, Pal et al. realized recently an ethanol biosensor by incorporating ADH in a LB film of stearic acid.10 Our aim is to elaborate an ethanol biosensor with a very high sensitivity. We think that we could obtain better sensitivity of the existing biosensors by incorporating the enzyme into a conductive polymer matrix using LB technology, in order to increase the electron-transfer rate. Consequently, the pyrrole monomer must be amphiphilic to allow it to spread at the air-water interface. This monomer is a pyrrole derivative which is rendered amphiphilic by grafting chemically on it a long hydrophobic alkyl chain. We chose hexadecylpyrrole (3HDP) for which the alkyl chain is CH3(CH2)16-. This compound, if polymerized, forms poly(3-hexadecylpyrrole) (P3HDP). In a previous work, monolayers of this compound have been studied by one of us.13 We study in this work the interactions between the 3HDP monomer and the ADH enzyme, in order to obtain homogeneous mixed (3HDP/ ADH) LB films and to optimize the distribution of the enzyme within the 3HDP film (Figure 1). In the next step, the polymerization of the pyrrole will be electrically performed by applying an electric potential on the working electrode. This process should lead to a better immobilization and distribution of the enzyme inside the P3HDP polymer matrix than if the enzyme was incorporated into the polymer after polymerization. Thus, the same molecule acts either for the enzyme immobilization and for the electron-transport mediation. This paper presents, on one hand, some new results obtained on the pure 3HDP monolayers at the air-water interface (Langmuir films) and, on the other hand, preliminary results of the mixed (3HDP/ADH) Langmuir and Langmuir-Blodgett films. We used fluorescence14 and Brewster angle15,16 microscopies set on Langmuir balances to study the Langmuir films and atomic force microscopy for the LB films observations.17 Experimental Section A Kru¨ss balance (one barrier, floating balance) was employed for the monolayers studied. The trough was filled with ultrapure water from an Elga system. The compression speed was 3 × 10-4 (nm2/molecule)/s. ADH from yeast (EC 1.1.1.1), β-NAD+ from yeast, and Tris buffer were purchased from Sigma-Aldrich. 3-Hexadecylpyrrole (3HDP) was synthesized by Prof. G. Ruggeri (13) Nicolae, C. A.; Fontana, M. P.; Capelletti, R.; Paradiso, R.; Bonfilio, A.; Parodi, M. T.; Ciardelli, F.; Rugerri, G. Molec. Cryst. Liq. Cryst. 1995, 266, 277. (14) Lo¨sche, M.; Mo¨hwald, H. Rev. Sci. Instrum. 1984, 55, 1968. (15) He´non, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936. (16) Riviere, S.; He´non, S.; Meunier, J. Phys. Rev. E 1994, 49, 1375. (17) Binning, G.; Quate, C. F.; Gerber, Ch. Phys. Rev. Lett. 1986, 56, 930.
© 1997 American Chemical Society
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Figure 2. Surface pressure (π) vs molecular area (A) isotherms; subphase, purified water (pH ) 5.7); temperature, 20 °C; and nitrogen atmosphere. (a) Pure 3HDP film; (b) mixed 3HDP/ ADH film. at the Department of Chemistry and Industrial Chemistry of Pisa, Italy. The enzyme solution was prepared by diluting 2.5 mg ADH of and 10 mg of β-NAD+ in 1 mL of Tris buffer solution (75 U for every 100 µL). The mixed ADH/3HDP Langmuir films as well as the pure 3HDP ones were observed with both fluorescence and Brewster angle microscopies. The Brewster angle microscopy experiments were performed at the Laboratoire de Physique Statistique, Ecole Normale Superieure.15 This technique, using the properties of reflectivity of an interface illuminated at the Brewster angle with light polarized in the plane of incidence, allows for a direct observation of monolayers or multilayers at the air-water interface. First-order phase transitions, i.e., coexistence between two phases, have been studied in this way.16 The fluorescence microscopy observations were made by means of an Olympus-BX30 microscope, set on a Riegler&Kirstein Langmuir trough. An AIS (MXRi2) video camera with an image intensifier which allows for a very high sensitivity (10-6 Lux) enables us to visualize the film and record images on a video tape. The Langmuir films were observed either using solutions containing 1% (by weight) NBD-stearic acid or without any fluorescent dye. The NBD group has an absorption band in the blue range (470 nm) and a fluorescence band in the yellow range (530 nm). Hence, using dichroic filters, the wavelength of the incident light was set in the 0.45-0.48 µm range, and the observation was allowed for wavelengths greater than 500 nm. A Nanoscope III of Digital Instruments (University of Parma, Italy) was used in order to get images of multilayers transferred onto freshly cleaved mica substrates.
Results and Discussion Pure 3HDP Monolayers in Ambient Atmosphere. The 3HDP monomer has been shown to form homogeneous and well-packed monolayers at the air-water interface, showing a steep surface pressure (π) vs molecular area (A) isotherm.13 This isotherm shows a well-condensed phase where the area occupied by each molecule is about 0.23 nm2 (Figure 2a). We studied the pure 3HDP Langmuir monolayers with fluorescence and Brewster angle microscopies. Both techniques show the same features. During the gasliquid condensed-phase transition, we observe large and rigid condensed domains (whose surface is about 1 mm2) with sharp boundaries coexisting with the gaseous phase (Figure 3). At molecular areas lower than 0.23 nm2 (in the condensed state), the film appears homogeneous and rigid. We noticed that the film can be compressed to high surface pressures, up to 50 mN/m, without undergoing any collapse process. At higher surface pressures, collapse occurs and small 3D crystals (whose surfaces are a few squared micrometers) are observed on the microscopy images. Their number increases vs surface pressure. It is interesting to note that they melt on decompressing the
Notes
Figure 3. Domains observed during the gas-condensed phase transition of the pure 3HDP using Brewster angle microscopy (BAM). The bright region corresponds to the condensed phase and the dark region corresponds to the gas phase.
Figure 4. Surface pressure (π) increase vs time (t) of the mixed 3HDP/ADH film after an injection of a fresh ADH solution. The 3HDP monolayer was compressed earlier until its surface pressure reached a value of about 3 mN/m.
film. Then, the film displays the same feature as for compression. Although it is known that the pyrrole moiety oxidates easily in the presence of oxygen and light, π-A isotherms of the 3HDP monolayer at the air-water are not sensitive to oxidation. For this reason and since our aim is, also, to build the alcohol biosensor as simply as possible, we first decided to study the mixed ADH/3HDP film in ambient atmosphere. In fact, as will be shown below, it appeared that the control of the atmosphere is very important to prevent the oxidation of the 3HDP monomers which would inhibit pyrrole polymerization. Mixed 3HDP/ADH Langmuir Films in Ambient Atmosphere. Let us first describe the experimental procedure we use to build the mixed ADH/3HDP Langmuir films. First, the 3HDP monolayer is compressed to a few micronewtons/meter, corresponding to its uniform twodimensional condensed state, as can be observed with Brewster angle microscopy or fluorescence microscopy. Then a few drops of ADH solution are carefully injected just below the 3HDP monolayer. The surface pressure is then recorded as a function of time (Figure 4). One can observe that a few minutes after having injected the enzyme, the surface pressure starts to increase slowly and reaches saturation after a time of 20-30 min. The
Notes
variation ∆π of surface pressure is a few micrometers/ meter, indicating that an adsorption of the enzyme in the 3HDP monolayer occurs. We notice that ∆π increases with the injected enzyme solution volume, ∆v. For a given ∆v, the value of ∆π is very sensitive to the enzyme solution age. Fresh solution gives greater ∆π. Consequently, we prepared a fresh ADH solution for each experiment. Once the enzyme is adsorbed at the air-water interface within the 3HDP film, we expand the mixed film completely and recompress it again. The surface pressure π is then recorded as a function of the 3HDP molecular area A. The π-A isotherms of the mixed films appear to be shifted to large areas with respect to the pure 3HDP ones. This result confirms that (i) the enzyme is adsorbed at the air-water interface and (ii) the complete expansion of the film after the enzyme is initially adsorbed does not induce its desorption. Unfortunately, we have no direct evidence that the enzyme is adsorbed in its native form. This will be confirmed once the alcohol biosensor is built and tested. Effect of the Atmospheric Oxygen on the Mixed 3HDP/ADH Langmuir Films. We noticed that neither the π-A isotherm shapes nor the shifts in molecular area were reproducible, even when injecting only fresh enzyme solutions. After having varied many experimental parameters, such as the subphase buffering, the enzyme injection technique, etc., we found that only the control of the atmosphere (by using pure nitrogen atmosphere) above the film can give reproducible π-A isotherm diagrams. In nitrogen atmosphere, the features of the mixed ADH/ 3HDP film are as follows: the isotherm is shifted to large molecular areas with respect to the pure 3HDP one (Figure 2b), which again indicates the adsorption of the enzyme into or below the pyrrole monolayer, the slope is lower than in the case of the pure monolayer which probably results from a continuous expulsion of the ADH molecules from the 3HDP monolayer during the compression. This lower slope may also be due to a less condensed structure of the 3HDP monolayer in the presence of the enzyme. At the end of the compression, the 3HDP molecular area (A ∼ 0.23 nm2) indicates that the film consists of pure 3HDP molecules. As we pointed out earlier, the pure 3HDP monolayer isotherms show very good reproducibility in ambient atmosphere. We considered then that the problems of reproducibility of our mixed film isotherms could be due to pyrrole oxidation by the oxygen in the atmosphere. Consequently, one should wonder whether the nonreproducibility of the mixed film in the ambient atmosphere arises from the effect of the oxygen on the pyrrole moiety, which would be enhanced by the enzyme presence, or whether this nonreproducibility is due to the presence of the enzyme itself. The only effect of the enzyme need not be considered since the instability of the mixed film disappears when the atmosphere is oxygen free. Hence, as we will see in the following, we can attribute this instability to the oxidation of the 3HDP which is revealed in the presence of the enzyme. Indeed, it is known that the oxygen reacts with pyrrole. This reaction leads to many different oxidation products during time. Until now, all these compounds are not well known.18 Consequently, it appears obvious that, in ambient atmosphere, the enzyme interacts differently with the different oxidation products. It follows that the ADH enzyme is more or less adsorbed at the air-water interface. In ambient atmosphere and in the case of the pure 3HDP monolayer, the oxidation products are probably also (18) Bazin, M. Private communications, Laboratoire de Photobiologie, Muse´um National d’Histoire Naturelle, Paris.
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Figure 5. Fluorescent domains observed during the gascondensed phase transition of the pure 3HDP monolayer using fluorescence microscopy. No additive fluorescent dye was used. The image was recorded a few minutes after the pure 3HDP monolayer was spread at the air-water interface in ambient atmosphere. The dark region corresponds to the gas phase, the weak fluorescent region corresponds to the condensed phase of the pure 3HDP, and the stronger fluorescent region corresponds to oxidation products of the pure 3HDP.
present but nondetected on the π-A isotherms. Their formation does not change significantly the molecular area. To check this assumption and since we taught that some of the 3HDP oxidation products could be small poly-3HDP chains which should be fluorescent, we have carried out fluorescence microscopy experiments on the pure 3HDP monolayer without adding any fluorescent probe. We observe that, during the gas-liquid condensed-phase transition, slightly homogeneously fluorescent domains are present. The fluorescence intensity of these domains becomes less and less homogeneous during time (Figure 5). This confirms that different oxidation products appear as a function of time. In nitrogen atmosphere, the pure 3HDP monolayers also show weak but homogeneous fluorescent domains. Upon compression of the monolayer, the surface occupied by these domains increases until a single homogeneous fluorescent phase is obtained. This occurs when the surface pressure starts to increase. Consequently, the fluorescent regions of the film, at zero surface pressure, correspond to the condensed state. Contrary to the experiments performed in ambient atmosphere, the intensity of the fluorescence of the condensed phase remains constant vs time. To explain this result, one should note that the 3HDP monomer does not absorb in the wavelength range we used (450-480 nm) but the poly-3HDP may absorb at this wavelength range. Consequently, we first wondered if this unexpected fluorescence is due to an eventual polymerization of the 3HDP monomer when it is spread at the air-water interface. The answer is negative for two reasons: (i) After the first cycle of compression and decompression, we still observe, using fluorescence microscopy, the coexistence between the condensed domains and the gaseous phase. This coexistence would not be retained if a polymerization had occurred. (ii) The 3HDP LB film built in the above conditions is colorless, whereas the poly3HDP Langmuir-Blodgett film is brown.13 Actually, this fluorescence observed only in the condensed phase can be explained by very strong intermolecular interactions in this phase. Indeed, the strong interactions between the 3HDP molecules may induce a shift in the absorption band of the pyrrole moiety. This is confirmed by the pink color of the pyrrole compound in the 3D crystalline phase18 even when oxidation is prevented. Hence, as a conclusion, one necessarily has to control the atmosphere above the film
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when studying pyrrole derivative monolayers at the airwater interface. Moreover, we investigated the mixed films by means of fluorescence microscopy (also without adding any fluorescent probe), in nitrogen atmosphere. As we noticed earlier, the pure 3HDP monolayer contains only two phases, a gaseous one which is progressively transformed into a condensed phase. The addition of ADH solution into the subphase, during the coexistence between the gaseous phase and the condensed phase of the 3HDP monolayer, leads to an increase of the area covered by the condensed domains. This indicates that the adsorption of the enzyme occurs onto the polar heads of the 3HDP molecules packed into the condensed domains. Following the insertion of the enzyme, the density of the 3HDP molecules in the condensed domains decreases. Consequently, their fluorescence intensity should normally decrease. However, since this fluorescence is already very weak, we think that its decrease is not detectable by our yet very sensitive camera (10-6 Lux). We would like to mention that the film becomes very rigid a few minutes after the ADH is added. Compressing the mixed film to non-zero surface pressures gives a very stable, homogeneous, and rigid film. When the mixed film was observed by means of fluorescence microscopy using the NBD dye in controlled atmosphere, the film shows the same features as when observed without dye. The only difference is the expulsion of the dye from the condensed domains to their boundaries. Transfer of the Mixed Film onto Solid Substrates. In a previous work,13 X multilayers of pure 3HDP have been transferred onto solid substrates in normal conditions and with relatively high speeds. Lowering the speed of the downstroke, we succeeded in transferring good quality Y multilayers of 3HDP and also of mixed ADH/3HDP film onto mica substrates. The transfer ratios were 1 ( 0.2 at target surface pressures of 25-30 mN/m and with dipping speeds of 10-170 µm/s. In order to know whether the enzyme is simultaneously transferred with the 3HDP molecule, we performed atomic force microscopy experiments on samples consisting of three layers. At the micrometric scale, the LB film of the pure 3HDP compound shows a uniform pattern (Figure 6a), while the mixed film shows a granular homogeneous structure (Figure 6b), indicating that, indeed, the mixed film is totally transferred. Conclusion Despite of the similarity between the isotherms of pure Langmuir monolayers of hexadecylpyrrole obtained in ambient atmosphere and in nitrogen atmosphere, we have shown that it is necessary to work in oxygen-free atmosphere. This has been achieved by studying the mixed film of 3HDP and alcohol dehydrogenase enzyme at the air-water interface. We have shown that the oxygen of the ambient atmosphere induces the formation of oxidation products of the 3HDP molecules as a function
Notes
Figure 6. AFM images of trilayers of Langmuir-Blodgett films transferred onto mica substrates: (a) pure 3HDP LB film; (b) mixed ADH/3HDP LB film.
of time. These oxidation products are revealed in the presence of the enzyme which interacts strongly with the 3HDP molecules. Then, we studied, in nitrogen atmosphere, pure Langmuir monolayers of 3HDP by means of fluorescence and Brewster angle microscopies. We observed the coexistence between a gaseous phase and large domains of a condensed phase, even without adding any fluorescent probe for fluorescence microscopy experiments. Lastly, we have successfully transferred Y LB layers of the pure 3HDP and of the mixed ADH/3HDP films. We confirmed this by atomic force microscopy by comparing the pure 3HDP LB films and the mixed LB films. Acknowledgment. We are grateful to G. Ruggeri from the Department of Chemistry and Industrial Chemistry of Pisa for supplying the pyrrole derivative, to J. Meunier from the Laboratoire de Physique Statistique de l’Ecole Normale Supe´rieure of Paris for allowing us to carry out the Brewster angle microscopy experiments, and M. Bazin for helpful discussions. LA970117+
