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Dynamic organization of mixed Langmuir films of glucose oxidase and stearylamine at the air–water interface
Colloids and Surfaces B: Biointerfaces 45 (2005) 200–208
Dynamic organization of mixed Langmuir films of glucose oxidase and stearylamine at the air–water interface D. Lair, S. Alexandre ∗ , J.M. Valleton UMR 6522, CNRS, Universit´e de Rouen, 76821 Mont-Saint-Aignan, France Received 16 June 2005; received in revised form 28 July 2005; accepted 22 August 2005
Abstract The structure and the dynamic organization of a mixed Langmuir film of glucose oxidase and stearylamine at the air–water interface have been studied. The film has been first characterized at the air–water interface by surface pressure/area isotherms. The dynamics of the mixed film was studied by following the evolution of the film area at a constant pressure and the evolution of the pressure at a constant area. After transfer of the films on solid substrates, the chemical composition of the mixed film has been quantified by UV–vis and IR spectroscopies. These characterizations were carried out in order to study the incorporation of glucose oxidase into the stearylamine film, and its influence on the structural evolution of the film. From these results, the dynamic organization of this mixed film may be described. For short times, glucose oxidase molecules interact with stearylamine molecules in solution or at the interface; these interactions would lead to the formation of a complex between stearylamine and glucose oxidase molecules. For long times (at least 3 h), a homogeneous mixed film constituted essentially of this complex is obtained at the air–water interface. A detailed analysis by atomic force microscopy allowed us to support this model and the existence of the glucose oxidase/stearylamine complex. © 2005 Elsevier B.V. All rights reserved. Keywords: Glucose oxidase; Stearylamine; Langmuir–Blodgett films; Fourier transform infrared spectroscopy; Atomic force microscopy
1. Introduction Langmuir–Blodgett (LB) films have been used for various purposes because of their thickness of molecular dimensions [1,2]. The incorporation of proteins in LB films is a great challenge in the functionalization of such thin films and may provide interesting models for biological membranes [3–6]. The ability of a protein to recognize a specific molecule allows the formation of a mixed LB film with specific molecular recognition properties. Such a specific function is of great interest in the field of biosensors [7–10]. The main advantage of using LB films to build a biosensor is the possibility to decrease dramatically the response time of the biosensor. In our laboratory, we elaborated a glucose biosensor by transferring a mixed film of glucose oxidase (GOx) and behenic acid on a gold-coated electrode by LB technology [8]. However, in spite of the good response time of the bioelectrode and the linearity of the calibration curves obtained, some reproducibility
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problems were encountered due to the electrochemical detection and the film structures. Therefore, we decided to study in a more detailed way the formation of the mixed GOx/behenic acid film at the air–water interface [11,12]. These studies showed the incorporation of the enzyme in the mixed film in contact with the polar heads of the behenic acid. But the electrostatic interactions between GOx and behenic acid molecules are not favorable. In order to improve electrostatic interactions with GOx molecules, we carried out a study on the formation of a mixed film by using a positively charged amphiphilic molecule: stearylamine [13]. In this paper, we present the results of fundamental studies on the influence of the presence of GOx in the subphase on the structure, the stability and the dynamics of a stearylamine film. Surface pressure/area isotherms and dynamic studies of the GOx/stearylamine film as well as the chemical composition determined by spectrophotometric measurements [12,14] has allowed us to propose a theoretical scheme of the formation of the mixed film at the air–water interface involving the formation of a stearylamine–GOx complex. A detailed analysis by atomic force microscopy [15–17] allowed us to support this model; the time evolution of the mixed
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system has been particularly studied. A selective solubilization method allowed us to obtain chemical information in agreement with our model. 2. Materials and methods GOx from Aspergillus niger (type X-S) and stearylamine (purity 99%) were provided from Sigma and used without further purification. Chloroform (RP Normapur, purity > 99.2% containing 0.6% ethanol as a stabilizer) was purchased from Prolabo. Pure water was obtained by a Millipore system (MilliRO et Milli-Q units) involving reverse osmosis, deionization, an active charcoal cartridge and filtration. LB experiments were carried out with a Langmuir trough from Atemeta (Paris, France) designed in order to minimize the volume of the subphase and therefore the amount of GOx used (dimensions 6.5 cm × 44 cm, volume ≈250 cm3 ). The trough and the mobile barrier were made of PTFE and the surface pressure was measured using a Wilhelmy balance. The plunging barrier used with this trough allowed us to prevent leakage of amphiphilic molecules. The temperature throughout the experiments was 21 ± 1 ◦ C and the system operated in the air. CaF2 slides were obtained from Sorem (Uzos, France). CaF2 Slides (3.5 cm × 0.9 cm × 0.2 cm) were cleaned in an ultrasonic bath with acetone and chloroform (twice each) and then dried for several hours under vacuum: the CaF2 slides were finally hydrophilic. The transmission infrared spectra of transferred films were measured with a Nicolet 510 M FTIR spectrophotometer. The IR spectra were obtained in the spectral region from 4000 cm−1 to 1200 cm−1 by collecting and averaging out 30 scans, at a resolution of 4 cm−1 . UV–vis experiments were performed with a Perkin-Elmer Lambda 2 spectrophotometer, at a slow rate of 60 nm min−1 . Spectra subtractions were performed in order to remove the CaF2 contribution. The subphase was constituted of water or GOx aqueous solution with a concentration of 3.2 mg L−1 . The pH of the subphase was 5.6 ± 0.1. In the case of the GOx solution, the surface of the subphase was cleaned by aspiration just before spreading the stearylamine solution in order to remove most of the enzyme molecules from the air–water interface and to start from a reproducible reference state. Hundred microlitres of a 1 mM solution of stearylamine in chloroform were spread on the surface using a glass capillary micropipette (Nichiryo, Model 800, Tokyo, Japan). A pipette holder was designed in order to keep constant the distance between the capillary and the subphase level for all experiments. Surface pressure/area isotherms were made by compressing ˚ 2 molecule−1 the film at a constant barrier speed of 11 × 10−3 A −1 s . The recording of the surface pressure/area isotherms was started after a delay of 15 min corresponding to the time necessary for the evaporation of the chloroform and the relaxation of the film. For the studies of the film area evolution at a constant pressure, first we maintained the delay of 15 min, then the film was compressed until the imposed pressure. When this pressure was
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reached, a feedback loop was used in order to keep the pressure constant and the barrier displacement was recorded. In the case of the surface pressure evolution studies at con˚ 2 molecule−1 s−1 stant area, the barrier speed was 11 × 10−3 A during the compression phase. The barrier movement was stopped when the imposed pressure was reached and the pressure evolution was recorded. The dynamic evolution of the mixed film was followed for different times corresponding to pressures resulting from the adsorption of the GOx molecules. On a GOx subphase, stearylamine molecules were spread and the system was allowed to self organize for different times When a given surface pressure was reached (adsorption pressure), the mixed film was compressed up to 25 mN/m and transferred. These transfers were carried out by using the LB method with a downward speed 10 times greater than the upward speed (transfer at 1 cm min−1 ). Seven mixed layers were deposited onto CaF2 slides. The quality and the number of the deposited layers were controlled by recording the barrier displacement versus the dipping head position. Then, spectrophotometric measurements of each slide were realized in order to determine the chemical composition of the mixed GOx/stearylamine film. The quantities of GOx and stearylamine in the mixed LB films were determined from the IR and UV–vis spectra by combination of spectra using a previously described method [12,14]. The reference spectra of GOx and of stearylamine each multiplied by a factor and then added in order to give the best fit of the mixed film spectrum. These factors allowed us to calculate the amount of GOx molecules in the mixed films and to know the proportion of stearylamine in the mixed film compared to a pure stearylamine film. For the scanning force microscopy, the films were transferred onto muscovite at a pressure of 25 mN/m. Muscovite slides were freshly cleaved just before transfer. For scanning force microscopy measurements, only one layer was transferred. The scanning force microscopy images presented in this paper were obtained with a Nanoscope II from Digital Instruments—Veeco, in the contact mode, with a 150 m piezoelectric scanner for large-scale investigations. The cantilevers used were characterized by a low spring constant (0.06 N/m). A standard tip of silicon nitride was used. The measurement was performed with the feedback loop on (constant force: 10−9 to 10−8 N). All the investigations were performed in the air. The images are presented in height mode (palette of colour for height: dark colours for low zones, light colours for high zones) and are top-view images. For obtaining chemical information, and identifying the nature of the structures observed, a selective solubilization process [18] was used: after imaging a sample resulting from the transfer of the mixed film, the sample placed in a “liquid cell” was rinsed with isopropanol in order to remove stearylamine structures; previous studies with behenic acid and GOx showed [18] that in such a mixed system only amphiphilic structures were eliminated. Preliminary IR measurements showed with the stearylamine/GOx system a similar behavior: stearylamine is almost completely eliminated by isopropanol rinsing while enzyme is conserved. After this selective solubilization, the sample was imaged again by atomic force microscopy.
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3. Results 3.1. Surface pressure/area isotherms The surface pressure/area isotherm of stearylamine on pure water (as a reference) and the surface pressure/area isotherm of the mixed film GOx/stearylamine are shown in Fig. 1. We present also in this figure a surface pressure/area isotherm of GOx obtained in the same conditions as other isotherms (compression after a 15-min delay). When the pressure is higher than 2 mN/m, the shape of the surface pressure/area isotherm of the stearylamine film on water subphase confirms that this film is in a liquid expanded phase. ˚ 2. The limiting molecular area of the stearylamine is (17 ± 1) A This value is slightly lower than the molecular area at basic pH ˚ 2 ) [19,20]. Since, the stearylamine molecules are (about 20 A positively charged at pH 5.6, we can expect a partial dissolution of the amphiphilic molecules in the aqueous subphase [21–23]. For the mixed GOx/stearylamine film, an apparent stearylamine molecular area was defined as the ratio of the film area by the number of stearylamine molecules spread at the air/subphase interface. This notion is introduced to indicate that the corresponding measurement refers to stearylamine but takes into account the possible presence of GOx molecules at the air–water interface. The surface pressure/area isotherm of the mixed film crossed the isotherm of the stearylamine at about 38 mN/m. Below this pressure, the apparent stearylamine molecular area is greater when GOx is present in the subphase. This observation implies that molecules of GOx are inserted between stearylamine molecules at the air–water interface. When the pressure increases (below 38 mN/m), the distance between the two isotherms decreases. The intersection observed at 38 mN/m corresponds mainly to a decrease of the amount of stearylamine molecules at the air–water interface. When GOx is present, the
Fig. 1. Surface pressure/area isotherms of a stearylamine film (a) and mixed film obtained with GOx concentration of 3.2 mg/l in the subphase. The recording was done after a relaxation time of 15 min. The compression speed ˚ 2 molecule−1 s−1 : (a) [GOx] = 0 mg/l; (b) [GOx] = 3.2 mg/l; was 11 × 10−3 A (c) reference isotherm obtained for a pure (no stearylamine) GOx subphase [GOx] = 3.2 mg/l.
dissolution of the stearylamine is more important during the compression of the mixed film: the presence of GOx in the subphase facilitates the dissolution of the stearylamine molecules in the subphase. For a surface pressure higher than 48 mN/m, we observe a change of the slope. This may be due to an expulsion of GOx molecules from the interface. A similar phenomenon was observed by Subirade et al. in the case of another mixed film [24]. They studied the interactions between a wheat lipid transfer protein and phospholipids. The results showed that the surface pressure/area isotherms of the mixed films were shifted toward a lower molecular area when the protein was introduced in the subphase. This shift toward a lower molecular area is due to the presence of specific interactions between the protein and the phospholipid: the protein drags some lipid molecules in the subphase. Therefore, the analogy between our results and the results obtained by Subirade et al. allow us to consider the existence of interactions between stearylamine and GOx. 3.2. Dynamics of the mixed films The area evolution of a pure monolayer of stearylamine obtained without GOx in the subphase is shown as a reference for various imposed surface pressure (Fig. 2a –c ). When the pressure increases, we observe a faster decrease of the film area. This decrease is due to the dissolution of stearylamine molecules in the subphase. A similar study was done using a GOx solution as the subphase. For high pressures (40 mN/m Fig. 2c) the decrease of the area is slower when GOx is present in the subphase. For lower pressures, the kinetics are more complex (Fig. 2a and b): in a first time, the area decreases. This decrease is slower in the case of the mixed film in comparison with the stearylamine alone. After the decrease, the area increases. The shape of these curves may be explained by a competition between the partial dissolution of the stearylamine in the subphase and the adsorption of the GOx
Fig. 2. Evolution of the film area for various imposed surface pressures. For the stearylamine film, A/A0 is the ratio of the molecular area to the initial molecular area: (a ) π = 10 mN/m; (b ) π = 25 mN/m; (c ) π = 40 mN/m. For the study of the mixed film, the subphase is constituted of a GOx solution with a concentration of 3.2 mg/l. A/A0 is the ratio of the mixed film area to the initial mixed film area. All curves were obtained by using an adsorption time of 15 min: (a) π = 10 mN/m; (b) π = 25 mN/m; (c) π = 40 mN/m.
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3.3. Evolution of the chemical composition of the mixed film
Fig. 3. Evolution of the surface pressure at a constant area for a mixed film prepared with a GOx concentration of 3.2 mg/l: (a) without stearylamine molecules at the air–water interface; (b) πinit = 0.5 mN/m; (c) πinit = 10 mN/m; (d) πinit = 25 mN/m; (e) πinit = 40 mN/m.
molecules at the air–water interface. The initial decrease of the film area is mainly due to the partial solubilization of the stearylamine molecules in the subphase. Simultaneously, molecules of GOx adsorb between stearylamine molecules at the air–water interface. Another study of the dynamics of the film was performed at constant area in order to have an other insight in the changes of the structure of the Langmuir films as a function of time. After spreading the stearylamine solution on the GOx subphase and evaporation of the chloroform, the mixed film is compressed with a constant barrier speed until a given pressure. When this pressure is reached, the barrier is stopped. The evolution of the surface pressure is followed at constant area. Whatever the imposed area, the surface pressure reaches a value of about 25 mN m−1 (Fig. 3). The shapes of the kinetics obtained at different pressures seem to be the result of the competition between the two phenomena: partial solubilization of stearylamine molecules and adsorption of GOx molecules. For higher pressures (Fig. 3c–e), in a first time, the surface pressure of the mixed film decreases (stearylamine solubilization); this initial decrease is followed by a progressive increase of the pressure (GOx adsorption). For low pressures (Fig. 3b), we observed a fast increase of the surface pressure of the film with the stearylamine in comparison to the one of the GOx solution without the amphiphile (Fig. 3a): the surface pressure of a subphase of GOx increases to 7 mN m−1 whereas the pressure of the film in presence of stearylamine tends toward 25 mN m−1 for the same time (15 h). This increase is explained by the adsorption of the GOx molecules at the air–water interface: the kinetic adsorption is faster in presence of the stearylamine molecules at the air–water interface. The adsorption curve of the mixed film obtained for a low initial pressure (Fig. 3b) shows an inflexion at about 12 mN m−1 : this inflexion may correspond to an evolution of the structure of this film at the air–water interface. In order to understand the complexity of this adsorption process (Fig. 3b), we studied the evolution of the chemical composition and structure of the mixed film for various adsorption times.
The chemical composition at different stages of the GOx adsorption at the air–water interface in presence of stearylamine was quantified. For an adsorption pressure given, the mixed film was compressed up to 25 mN m−1 and transferred onto a CaF2 slide (seven layers). The transfer quality is different according to the times of adsorption: for lower times (leading to surface pressures below 12.5 mN m−1 ) the transfers are irregular and transfer ratios are greater than 1, which corresponds to the existence of complex structures. For greater times of adsorption (leading to surface pressures above 12.5 mN m−1 ) the transfers are quite regular and the transfer ratios are close to 1. This result indicates a change in the system at this key surface pressure of 12.5 mN m−1 . The chemical composition of the corresponding mixed LB films was analyzed by UV–vis and IR spectroscopies. The IR reference spectra of stearylamine and GOx in KBr pastille are represented in Fig. 4A: the main vibrations for stearylamine and GOx are presented in Table 1. The IR spectra of the mixed GOx/stearylamine samples transferred for different adsorption pressures of 2.5, 10 and 25 mN m−1 are presented in Fig. 4B. We observed that the characteristic sharp adsorption bands for stearylamine decrease continuously from 2.5 mN m−1 to 25 mN m−1 . The intensity of the broad absorption bands for GOx increases up to an adsorption pressure of 10 mN m−1 and remains stable up to 25 mN m−1 . The amount of stearylamine and GOx molecules per monolayer and surface unit is reported as a function of the adsorption pressure in Fig. 5A and B. The quantification of GOx from the IR and UV–vis measurements (Fig. 5B) shows that the GOx quantity increases with the adsorption pressure. The quantity of GOx per monolayer for an adsorption pressure of 2.5 mN m−1 can be evaluated to (0.7 ± 0.2) × 1010 molecules mm−2 . For an Table 1 The main vibrations of stearylamine and glucose oxidase in KBr pastille Wavenumber (cm−1 ) 3330 3301 2962 2955 2915 2848 1657 1625 1565 1543 1473 1463 1424 1382 1244
Characteristic vibration Stearylamine
Glucose oxidase
NH2 asymmetrical stretching NH asymmetrical stretching CH2 asymmetrical stretching CH3 asymmetrical stretching CH2 asymmetrical stretching CH2 symmetrical stretching C O stretching from amide NH2 deformation (amide) NH2 deformation NH deformation amide II CH2 deformation CH2 scissoring C N stretching CH3 deformation OH deformation
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Fig. 4. (A) IR reference spectra of stearylamine and glucose oxidase in KBr pastille: (a) stearylamine, (b) glucose oxidase; (B) IR spectra of mixed glucose oxidase/stearylamine films, transferred on CaF2 slides (seven layers) for different adsorption pressures: (a) π = 2.5 mN/m, (b) π = 10 mN/m, (c) π = 25 mN/m.
adsorption pressure of 20 mN m−1 and above, the quantity is (2.5 ± 0.2) × 1010 molecules mm−2 . Simultaneously, the quantity of stearylamine (Fig. 5A) decreases quickly to tend toward a low amount from an adsorption pressure of 12.5 mN m−1 . This decrease of the quantity of the stearylamine can be explained by a decrease of the amount of the stearylamine molecules at the air–water interface. The quantification from the IR and UV–vis spectra shows that the mixed film, after an adsorption pressure of 12.5 mN m−1 , is constituted mainly of the GOx molecules adsorbed. In previous works, a GOx film was studied at the air–water interface [25]. These studies showed that this film could not be maintained stable at a constant pressure. Consequently, the transfer of a GOx film was impossible at a pressure of 25 mN m−1 ; this is not the case of the transferred film in presence of the stearylamine. 3.4. Evolution of the structures observed by atomic force microscopy
Fig. 5. Composition of the mixed films for various adsorption times. Quantity of stearylamine (A) per monolayer in the mixed films: (䊉), calculation from the IR spectra. Quantification of glucose oxidase (B) per monolayer in the mixed films: (ν), calculation from the IR spectra; (), calculation from the UV–vis spectra.
The mixed system was allowed to evolve for different times (as in Fig. 3b) until given surface pressures are reached (correspondence time/pressure). Then the resulting mixed films were transferred at a surface pressure of 25 mN/m on muscovite slides. The samples were then analyzed by scanning force microscopy. The images presented in Fig. 6 correspond to samples obtained for six different times: (A) 15 min (π = 2.5 mN/m);
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Fig. 6. Evolution of the structure of a mixed film of glucose oxidase and stearylamine observed by atomic force microscopy during the adsorption of GOx under a stearylamine monolayer initially in the gas/liquid phase transition. GOx concentration in the subphase: 3.2 mg/l: (A) π = 2.5 mN/m (15 min); (B) π = 10 mN/m (120 min); (C) π = 20 mN/m (320 min); (D) π = 25 mN/m (1200 min). Scan size 10 m × 10 m. The disappearance of separate structures is observed as a function of time.
(B) 120 min (π = 10 mN/m); (C) 320 min (π = 20 mN/m); (D) 1200 min (π = 25 mN/m). In every case, the scan size is 10 m × 10 m. From this study, the main conclusion is the disappearance, as a function of time, of structures at the micron scale. For low pressures (Fig. 6A, 2.5 mN/m; 15 min), well-defined structures are observed, characterized by domains of two different heights constituted of globules of diameters ranging from 40 nm to 50 nm as observed on the image of Fig. 7. Progressively, the structures disappear above 12.5 mN/m: in Fig. 6C, only “ghosts” of these structures can be observed. Within the same time, (Fig. 6C), small holes appear probably due to the transfer, which is a sign of a change in the nature of the layer. In Fig. 6D (π = 25 mN/m; 1200 min) holes are no longer visible, which is the sign of the end of changes in the layer. In this last image, the layer appears uniform with a few defects. The change in the nature of the layer as a function of time, is clear. However, the identification of the nature of the structures observed is not obvious. The selective solubilization process was used at two different pressures: 10 mN/m and 12.5 mN/m; the first value corresponds to a system in which the initial structures are still clearly visible; the second value corresponds to the key step during which the
Fig. 7. Details of a small domain observed on the image of Fig. 1 Scan size 10 m × 10 m; the small domain and the surrounding zone are constituted of globules of diameters ranging from 40 nm to 50 nm.
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Fig. 8. Identification of the structures thanks to a selective solubilization process involving isopropanol washing. GOx concentration in the subphase: 3.2 mg/l; π = 10 mN/m (120 min) (A and B); (A) before isopropanol washing (B) after isopropanol washing; π = 12.5 mN/m (120 min) (C and D); (C) before isopropanol washing (D) after isopropanol washing. Scan size 10 m × 10 m.
system changes dramatically. For both values of the surface pressure, a sample, initially placed in the liquid cell, was first imaged in the air, and then imaged after rinsing it with isopropanol in order to compare in each case the same zone. For the first sample (10 mN/m) (Fig. 8A and B), it appears that some structures are removed by the action of the isopropanol; the structures removed being stearylamine molecules, it seems that two types of structures (which perhaps may not be clearly distinguished) are removed: a stearylamine film covering the larger GOx structures, and a great number of small structures of steraylamine between these large GOx structures. For the second sample (12.5 mN/m) (Fig. 8C and D), isopropanol seems to have no action. The two images obtained before (Fig. 8C) and after rinsing the sample with isopropanol (Fig. 8D) are quite identical. This means that stearylamine is no more present in the sample under an isolated form. This is in agreement with IR spectra which showed a very low quantity of stearylamine at this pressure. 4. Discussion We have studied the influence of the presence of GOx in the subphase on a stearylamine film. From the surface pressure/area
isotherms and the film evolution studies, we found that stearylamine molecules dissolved partially in the subphase and GOx molecules were present at the air–water interface and under the stearylamine film. The nature of the interactions between the GOx molecules and the polar heads of the stearylamine molecules is probably electrostatic. All the experiments were performed with a pH of 5.6. The pKa of stearylamine is 10.6 in solution [24] and the isoelectric point of GOx is 4.2 [26]. Therefore, all the stearylamine molecules are positively charged and the GOx molecules in the subphase are globally negatively charged. This is a priori favorable to electrostatic interactions between GOx and stearylamine molecules. Moreover, we observe that the adsorption kinetic of the GOx is faster in presence of stearylamine. Next, the dynamic organization of the mixed Langmuir film of GOx and stearylamine has been studied by spectrophotometry and atomic force microscopy. We observe from the spectroscopic measurements that the GOx quantity present in the mixed film increases with time to reach a maximum value for an adsorption time corresponding to a surface pressure of 12.5 mN/m. In the same time, the stearylamine quantity decreases to tend toward a slight amount. The atomic force microscopy study shows the disappearance, as a function of time, of the structures observed
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at the micron scale for the lower times of GOx adsorption. For high adsorption pressures (above 12.5 mN/m), it was found from both studies that stearylamine is no more present in the sample under an isolated form. These studies show that the structure of the mixed film evolves fundamentally: this evolution can be due to a partial hydrophobization of the GOx which interacts with stearylamine
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molecules. These interactions between GOx molecules and stearylamine molecules in solution has led us to assume the formation of a stearylamine–GOx complex stabilized by electrostatic (and eventually local hydrophobic) interactions. This stearylamine–GOx complex may be constituted of one (or several) GOx molecule(s) to which a little number of stearylamine molecules are bound.
Fig. 9. Schematic representation of the dynamic organization of the mixed glucose film oxidase/stearylamine at the air–water interface for different adsorption times.
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The consequence of this hypothesis is that the evolution of the system for long times leads to the formation of a film constituted essentially of this complex explaining the disappearance of stearylamine in an isolated form. From the results at the air–water interface and after transfer, the dynamic organization of the mixed film GOx/stearylamine during the adsorption of the GOx molecules may be described in the following model (Fig. 9): after the spreading of the stearylamine solution on the GOx aqueous subphase, for an adsorption pressure of 2.5 mN m−1 , the stearylamine positively charged molecules dissolve partially in the subphase. In the same time, the GOx molecules are being incorporated at the air–water interface in the stearylamine film and interact with the stearylamine film leading to an adsorption of the GOx molecules under the stearylamine film. As we showed previously, the quantity of stearylamine molecules determined from the IR spectroscopy decreases by 30% for an adsorption time of 120 min (πads = 10 mN m−1 ). The ionization of its polar head decreases its hydrophobic character and allows its dissolution. For short adsorption times (tads ≤ 120 min: πads ≤ 10 mN m−1 ), the GOx interacts with the stearylamine molecules at the air–water interface or in solution to form a stearylamine–GOx complex more hydrophobic than GOx. From an adsorption time of 160 min (πads ≥ 12.5 mN m−1 ), the complex formed in the subphase adsorbs between the remaining stearylamine molecules. This is in good agreement with the IR and UV–vis spectroscopies because the quantity of GOx in the mixed film increases with the adsorption pressure. When the film is compressed to 25 mN m−1 , the compression phenomenon has an influence on the dissolution of the stearylamine film at the air–water interface and in interaction with the enzyme molecules in the subphase. We have considered a mechanism which leads probably to the formation of a mixed GOx/stearylamine miscellous structure: these structures progressively formed should be expelled by the compression from the air–water interface considering it hydrophilic character. At the same time with this formation, the free space is taken up quickly by the stearylamine–GOx complex formed in the subphase and by some GOx molecules. This mechanism takes into account the results obtained by spectroscopies: from an adsorption pressure of 12.5 mN m−1 , the quantity of the GOx molecules is maximum whereas the quantity of the stearylamine decreases by 90%. The GOx molecules without stearylamine are probably diluted in the complexed film or expelled from the interface. For an adsorption time superior to 200 min (πads ≥ 15 mN m−1 ), and after the compression to 25 mN m−1 , the film at the air–water interface is a film constituted of these complexes. The quantity of GOx per monolayer is just as it would characterize the presence of a mixed layer constituted of a complex stearylamine–GOx at the air–water interface. Therefore, it is necessary to wait at least 160 min corresponding to an adsorption pressure of 12.5 mN m−1 in order to obtain
a mixed film with a maximum amount of GOx. The organization and the stabilization of this mixed film is a relatively fast process compared to the dynamic organization of the mixed GOx/behenic acid [12]. In this paper, we focused on the organization and the dynamics of the mixed GOx/stearylamine LB film. Some experiments were carried out in order to test the activity of the GOx. The preliminary results demonstrate that GOx in such structures constituted of the stearylamine–GOx complex still has good activity. An implication of these results is that, when waiting at least 3 h, the mixed GOx/stearylamine system tends to form a homogeneous film constituted of the stearylamine–GOx complex. This result is a priori potentially interesting in the field of biosensors because of the homogeneity of the films formed and because of the hydrophobization of GOx (the stearylamine–GOx complex) which might be favorable to prevent GOx leakage from the sensing film. References [1] G. Roberts, Langmuir–Blodgett Films, Plenum Ed., 1990. [2] M.C. Petty, Thin Solid Films 210/211 (1992) 417. [3] M.C. Wilkinson, B.N. Zaba, D.M. Taylor, D.L. Laidman, T.J. Lewis, Biochim. Biophy. Acta 857 (1986) 189. [4] I. Vikholm, O. Teleman, J. Colloid Interface Sci. 168 (1994) 125. [5] M.A. Bos, T. Nylander, Langmuir 12 (1996) 2791. [6] V. Rosilio, M.M. Boissonade, J. Zhang, L. Jiang, A. Baszkin, Langmuir 13 (1997) 4669. [7] S. Arisawa, T. Arise, R. Yamamoto, Thin Solid Films 209 (1992) 259. [8] C. Fiol, J.M. Valleton, N. Delpire, G. Barbey, A. Barraud, A. RuaudelTeixier, Thin Solid Films 210/211 (1992) 489. [9] S.U. Zaitsev, Colloids Surf. A: Physicochem. Eng. Asp. 75 (1993) 211. [10] A.F. Collings, F. Caruso, Rep. Prog. Phys. 60 (1997) 1397. [11] N. Dubreuil, S. Alexandre, C. Fiol, J.M. Valleton, J. Colloid Interface Sci. 181 (1996) 393. [12] S. Alexandre, N. Dubreuil, C. Fiol, D. Lair, F. Sommer, Tran Minh Duc, J.M. Valleton, Thin Solid Films 293 (1997) 295. [13] D. Lair, Ph.D. thesis, Universit´e de Rouen, France, 1998. [14] C. Fiol, S. Alexandre, N. Dubreuil, J.M. Valleton, Thin Solid Films 261 (1995) 287. [15] G. Binnig, C.F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930. [16] S.W. Hui, R. Viswanathan, J.A. Zasadzinski, J.N. Israelachvili, Biophys. J. 68 (1995) 171. [17] F. Sommer, S. Alexandre, N. Dubreuil, D. Lair, Tran Minh Duc, J.M. Valleton, Langmuir 13 (1997) 791. [18] N. Dubreuil, S. Alexandre, D. Lair, J.M. Valleton, Langmuir 12 (1996) 6721. [19] V.L. Shapovalov, Russ. Chem. Bull. 45 (1996) 1611. [20] P. Ganguly, D.V. Paranjape, F. Rondelez, Langmuir 13 (1997) 5433. [21] C.G. Lyons, E.K. Rideal, Proc. R. Soc. Ser. A 124 (1929) 333. [22] G.L. Gaines, Nature 298 (1982) 544. [23] B.P. Binks, Adv. Colloid Interface Sci. 34 (1991) 343. [24] M. Subirade, C. Salesse, D. Marion, M. Pezolet, Biophys. J. 69 (1995) 974. [25] N. Dubreuil, Ph.D. thesis, Universit´e de Rouen, France, 1995. [26] R. Wilson, A.P.F. Turner, Biosens. Bioelectron. 7 (1992) 165.
Dynamic organization of mixed Langmuir films of glucose oxidase and stearylamine at the air–water interface D. Lair, S. Alexandre ∗ , J.M. Valleton UMR 6522, CNRS, Universit´e de Rouen, 76821 Mont-Saint-Aignan, France Received 16 June 2005; received in revised form 28 July 2005; accepted 22 August 2005
Abstract The structure and the dynamic organization of a mixed Langmuir film of glucose oxidase and stearylamine at the air–water interface have been studied. The film has been first characterized at the air–water interface by surface pressure/area isotherms. The dynamics of the mixed film was studied by following the evolution of the film area at a constant pressure and the evolution of the pressure at a constant area. After transfer of the films on solid substrates, the chemical composition of the mixed film has been quantified by UV–vis and IR spectroscopies. These characterizations were carried out in order to study the incorporation of glucose oxidase into the stearylamine film, and its influence on the structural evolution of the film. From these results, the dynamic organization of this mixed film may be described. For short times, glucose oxidase molecules interact with stearylamine molecules in solution or at the interface; these interactions would lead to the formation of a complex between stearylamine and glucose oxidase molecules. For long times (at least 3 h), a homogeneous mixed film constituted essentially of this complex is obtained at the air–water interface. A detailed analysis by atomic force microscopy allowed us to support this model and the existence of the glucose oxidase/stearylamine complex. © 2005 Elsevier B.V. All rights reserved. Keywords: Glucose oxidase; Stearylamine; Langmuir–Blodgett films; Fourier transform infrared spectroscopy; Atomic force microscopy
1. Introduction Langmuir–Blodgett (LB) films have been used for various purposes because of their thickness of molecular dimensions [1,2]. The incorporation of proteins in LB films is a great challenge in the functionalization of such thin films and may provide interesting models for biological membranes [3–6]. The ability of a protein to recognize a specific molecule allows the formation of a mixed LB film with specific molecular recognition properties. Such a specific function is of great interest in the field of biosensors [7–10]. The main advantage of using LB films to build a biosensor is the possibility to decrease dramatically the response time of the biosensor. In our laboratory, we elaborated a glucose biosensor by transferring a mixed film of glucose oxidase (GOx) and behenic acid on a gold-coated electrode by LB technology [8]. However, in spite of the good response time of the bioelectrode and the linearity of the calibration curves obtained, some reproducibility
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problems were encountered due to the electrochemical detection and the film structures. Therefore, we decided to study in a more detailed way the formation of the mixed GOx/behenic acid film at the air–water interface [11,12]. These studies showed the incorporation of the enzyme in the mixed film in contact with the polar heads of the behenic acid. But the electrostatic interactions between GOx and behenic acid molecules are not favorable. In order to improve electrostatic interactions with GOx molecules, we carried out a study on the formation of a mixed film by using a positively charged amphiphilic molecule: stearylamine [13]. In this paper, we present the results of fundamental studies on the influence of the presence of GOx in the subphase on the structure, the stability and the dynamics of a stearylamine film. Surface pressure/area isotherms and dynamic studies of the GOx/stearylamine film as well as the chemical composition determined by spectrophotometric measurements [12,14] has allowed us to propose a theoretical scheme of the formation of the mixed film at the air–water interface involving the formation of a stearylamine–GOx complex. A detailed analysis by atomic force microscopy [15–17] allowed us to support this model; the time evolution of the mixed
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system has been particularly studied. A selective solubilization method allowed us to obtain chemical information in agreement with our model. 2. Materials and methods GOx from Aspergillus niger (type X-S) and stearylamine (purity 99%) were provided from Sigma and used without further purification. Chloroform (RP Normapur, purity > 99.2% containing 0.6% ethanol as a stabilizer) was purchased from Prolabo. Pure water was obtained by a Millipore system (MilliRO et Milli-Q units) involving reverse osmosis, deionization, an active charcoal cartridge and filtration. LB experiments were carried out with a Langmuir trough from Atemeta (Paris, France) designed in order to minimize the volume of the subphase and therefore the amount of GOx used (dimensions 6.5 cm × 44 cm, volume ≈250 cm3 ). The trough and the mobile barrier were made of PTFE and the surface pressure was measured using a Wilhelmy balance. The plunging barrier used with this trough allowed us to prevent leakage of amphiphilic molecules. The temperature throughout the experiments was 21 ± 1 ◦ C and the system operated in the air. CaF2 slides were obtained from Sorem (Uzos, France). CaF2 Slides (3.5 cm × 0.9 cm × 0.2 cm) were cleaned in an ultrasonic bath with acetone and chloroform (twice each) and then dried for several hours under vacuum: the CaF2 slides were finally hydrophilic. The transmission infrared spectra of transferred films were measured with a Nicolet 510 M FTIR spectrophotometer. The IR spectra were obtained in the spectral region from 4000 cm−1 to 1200 cm−1 by collecting and averaging out 30 scans, at a resolution of 4 cm−1 . UV–vis experiments were performed with a Perkin-Elmer Lambda 2 spectrophotometer, at a slow rate of 60 nm min−1 . Spectra subtractions were performed in order to remove the CaF2 contribution. The subphase was constituted of water or GOx aqueous solution with a concentration of 3.2 mg L−1 . The pH of the subphase was 5.6 ± 0.1. In the case of the GOx solution, the surface of the subphase was cleaned by aspiration just before spreading the stearylamine solution in order to remove most of the enzyme molecules from the air–water interface and to start from a reproducible reference state. Hundred microlitres of a 1 mM solution of stearylamine in chloroform were spread on the surface using a glass capillary micropipette (Nichiryo, Model 800, Tokyo, Japan). A pipette holder was designed in order to keep constant the distance between the capillary and the subphase level for all experiments. Surface pressure/area isotherms were made by compressing ˚ 2 molecule−1 the film at a constant barrier speed of 11 × 10−3 A −1 s . The recording of the surface pressure/area isotherms was started after a delay of 15 min corresponding to the time necessary for the evaporation of the chloroform and the relaxation of the film. For the studies of the film area evolution at a constant pressure, first we maintained the delay of 15 min, then the film was compressed until the imposed pressure. When this pressure was
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reached, a feedback loop was used in order to keep the pressure constant and the barrier displacement was recorded. In the case of the surface pressure evolution studies at con˚ 2 molecule−1 s−1 stant area, the barrier speed was 11 × 10−3 A during the compression phase. The barrier movement was stopped when the imposed pressure was reached and the pressure evolution was recorded. The dynamic evolution of the mixed film was followed for different times corresponding to pressures resulting from the adsorption of the GOx molecules. On a GOx subphase, stearylamine molecules were spread and the system was allowed to self organize for different times When a given surface pressure was reached (adsorption pressure), the mixed film was compressed up to 25 mN/m and transferred. These transfers were carried out by using the LB method with a downward speed 10 times greater than the upward speed (transfer at 1 cm min−1 ). Seven mixed layers were deposited onto CaF2 slides. The quality and the number of the deposited layers were controlled by recording the barrier displacement versus the dipping head position. Then, spectrophotometric measurements of each slide were realized in order to determine the chemical composition of the mixed GOx/stearylamine film. The quantities of GOx and stearylamine in the mixed LB films were determined from the IR and UV–vis spectra by combination of spectra using a previously described method [12,14]. The reference spectra of GOx and of stearylamine each multiplied by a factor and then added in order to give the best fit of the mixed film spectrum. These factors allowed us to calculate the amount of GOx molecules in the mixed films and to know the proportion of stearylamine in the mixed film compared to a pure stearylamine film. For the scanning force microscopy, the films were transferred onto muscovite at a pressure of 25 mN/m. Muscovite slides were freshly cleaved just before transfer. For scanning force microscopy measurements, only one layer was transferred. The scanning force microscopy images presented in this paper were obtained with a Nanoscope II from Digital Instruments—Veeco, in the contact mode, with a 150 m piezoelectric scanner for large-scale investigations. The cantilevers used were characterized by a low spring constant (0.06 N/m). A standard tip of silicon nitride was used. The measurement was performed with the feedback loop on (constant force: 10−9 to 10−8 N). All the investigations were performed in the air. The images are presented in height mode (palette of colour for height: dark colours for low zones, light colours for high zones) and are top-view images. For obtaining chemical information, and identifying the nature of the structures observed, a selective solubilization process [18] was used: after imaging a sample resulting from the transfer of the mixed film, the sample placed in a “liquid cell” was rinsed with isopropanol in order to remove stearylamine structures; previous studies with behenic acid and GOx showed [18] that in such a mixed system only amphiphilic structures were eliminated. Preliminary IR measurements showed with the stearylamine/GOx system a similar behavior: stearylamine is almost completely eliminated by isopropanol rinsing while enzyme is conserved. After this selective solubilization, the sample was imaged again by atomic force microscopy.
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3. Results 3.1. Surface pressure/area isotherms The surface pressure/area isotherm of stearylamine on pure water (as a reference) and the surface pressure/area isotherm of the mixed film GOx/stearylamine are shown in Fig. 1. We present also in this figure a surface pressure/area isotherm of GOx obtained in the same conditions as other isotherms (compression after a 15-min delay). When the pressure is higher than 2 mN/m, the shape of the surface pressure/area isotherm of the stearylamine film on water subphase confirms that this film is in a liquid expanded phase. ˚ 2. The limiting molecular area of the stearylamine is (17 ± 1) A This value is slightly lower than the molecular area at basic pH ˚ 2 ) [19,20]. Since, the stearylamine molecules are (about 20 A positively charged at pH 5.6, we can expect a partial dissolution of the amphiphilic molecules in the aqueous subphase [21–23]. For the mixed GOx/stearylamine film, an apparent stearylamine molecular area was defined as the ratio of the film area by the number of stearylamine molecules spread at the air/subphase interface. This notion is introduced to indicate that the corresponding measurement refers to stearylamine but takes into account the possible presence of GOx molecules at the air–water interface. The surface pressure/area isotherm of the mixed film crossed the isotherm of the stearylamine at about 38 mN/m. Below this pressure, the apparent stearylamine molecular area is greater when GOx is present in the subphase. This observation implies that molecules of GOx are inserted between stearylamine molecules at the air–water interface. When the pressure increases (below 38 mN/m), the distance between the two isotherms decreases. The intersection observed at 38 mN/m corresponds mainly to a decrease of the amount of stearylamine molecules at the air–water interface. When GOx is present, the
Fig. 1. Surface pressure/area isotherms of a stearylamine film (a) and mixed film obtained with GOx concentration of 3.2 mg/l in the subphase. The recording was done after a relaxation time of 15 min. The compression speed ˚ 2 molecule−1 s−1 : (a) [GOx] = 0 mg/l; (b) [GOx] = 3.2 mg/l; was 11 × 10−3 A (c) reference isotherm obtained for a pure (no stearylamine) GOx subphase [GOx] = 3.2 mg/l.
dissolution of the stearylamine is more important during the compression of the mixed film: the presence of GOx in the subphase facilitates the dissolution of the stearylamine molecules in the subphase. For a surface pressure higher than 48 mN/m, we observe a change of the slope. This may be due to an expulsion of GOx molecules from the interface. A similar phenomenon was observed by Subirade et al. in the case of another mixed film [24]. They studied the interactions between a wheat lipid transfer protein and phospholipids. The results showed that the surface pressure/area isotherms of the mixed films were shifted toward a lower molecular area when the protein was introduced in the subphase. This shift toward a lower molecular area is due to the presence of specific interactions between the protein and the phospholipid: the protein drags some lipid molecules in the subphase. Therefore, the analogy between our results and the results obtained by Subirade et al. allow us to consider the existence of interactions between stearylamine and GOx. 3.2. Dynamics of the mixed films The area evolution of a pure monolayer of stearylamine obtained without GOx in the subphase is shown as a reference for various imposed surface pressure (Fig. 2a –c ). When the pressure increases, we observe a faster decrease of the film area. This decrease is due to the dissolution of stearylamine molecules in the subphase. A similar study was done using a GOx solution as the subphase. For high pressures (40 mN/m Fig. 2c) the decrease of the area is slower when GOx is present in the subphase. For lower pressures, the kinetics are more complex (Fig. 2a and b): in a first time, the area decreases. This decrease is slower in the case of the mixed film in comparison with the stearylamine alone. After the decrease, the area increases. The shape of these curves may be explained by a competition between the partial dissolution of the stearylamine in the subphase and the adsorption of the GOx
Fig. 2. Evolution of the film area for various imposed surface pressures. For the stearylamine film, A/A0 is the ratio of the molecular area to the initial molecular area: (a ) π = 10 mN/m; (b ) π = 25 mN/m; (c ) π = 40 mN/m. For the study of the mixed film, the subphase is constituted of a GOx solution with a concentration of 3.2 mg/l. A/A0 is the ratio of the mixed film area to the initial mixed film area. All curves were obtained by using an adsorption time of 15 min: (a) π = 10 mN/m; (b) π = 25 mN/m; (c) π = 40 mN/m.
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3.3. Evolution of the chemical composition of the mixed film
Fig. 3. Evolution of the surface pressure at a constant area for a mixed film prepared with a GOx concentration of 3.2 mg/l: (a) without stearylamine molecules at the air–water interface; (b) πinit = 0.5 mN/m; (c) πinit = 10 mN/m; (d) πinit = 25 mN/m; (e) πinit = 40 mN/m.
molecules at the air–water interface. The initial decrease of the film area is mainly due to the partial solubilization of the stearylamine molecules in the subphase. Simultaneously, molecules of GOx adsorb between stearylamine molecules at the air–water interface. Another study of the dynamics of the film was performed at constant area in order to have an other insight in the changes of the structure of the Langmuir films as a function of time. After spreading the stearylamine solution on the GOx subphase and evaporation of the chloroform, the mixed film is compressed with a constant barrier speed until a given pressure. When this pressure is reached, the barrier is stopped. The evolution of the surface pressure is followed at constant area. Whatever the imposed area, the surface pressure reaches a value of about 25 mN m−1 (Fig. 3). The shapes of the kinetics obtained at different pressures seem to be the result of the competition between the two phenomena: partial solubilization of stearylamine molecules and adsorption of GOx molecules. For higher pressures (Fig. 3c–e), in a first time, the surface pressure of the mixed film decreases (stearylamine solubilization); this initial decrease is followed by a progressive increase of the pressure (GOx adsorption). For low pressures (Fig. 3b), we observed a fast increase of the surface pressure of the film with the stearylamine in comparison to the one of the GOx solution without the amphiphile (Fig. 3a): the surface pressure of a subphase of GOx increases to 7 mN m−1 whereas the pressure of the film in presence of stearylamine tends toward 25 mN m−1 for the same time (15 h). This increase is explained by the adsorption of the GOx molecules at the air–water interface: the kinetic adsorption is faster in presence of the stearylamine molecules at the air–water interface. The adsorption curve of the mixed film obtained for a low initial pressure (Fig. 3b) shows an inflexion at about 12 mN m−1 : this inflexion may correspond to an evolution of the structure of this film at the air–water interface. In order to understand the complexity of this adsorption process (Fig. 3b), we studied the evolution of the chemical composition and structure of the mixed film for various adsorption times.
The chemical composition at different stages of the GOx adsorption at the air–water interface in presence of stearylamine was quantified. For an adsorption pressure given, the mixed film was compressed up to 25 mN m−1 and transferred onto a CaF2 slide (seven layers). The transfer quality is different according to the times of adsorption: for lower times (leading to surface pressures below 12.5 mN m−1 ) the transfers are irregular and transfer ratios are greater than 1, which corresponds to the existence of complex structures. For greater times of adsorption (leading to surface pressures above 12.5 mN m−1 ) the transfers are quite regular and the transfer ratios are close to 1. This result indicates a change in the system at this key surface pressure of 12.5 mN m−1 . The chemical composition of the corresponding mixed LB films was analyzed by UV–vis and IR spectroscopies. The IR reference spectra of stearylamine and GOx in KBr pastille are represented in Fig. 4A: the main vibrations for stearylamine and GOx are presented in Table 1. The IR spectra of the mixed GOx/stearylamine samples transferred for different adsorption pressures of 2.5, 10 and 25 mN m−1 are presented in Fig. 4B. We observed that the characteristic sharp adsorption bands for stearylamine decrease continuously from 2.5 mN m−1 to 25 mN m−1 . The intensity of the broad absorption bands for GOx increases up to an adsorption pressure of 10 mN m−1 and remains stable up to 25 mN m−1 . The amount of stearylamine and GOx molecules per monolayer and surface unit is reported as a function of the adsorption pressure in Fig. 5A and B. The quantification of GOx from the IR and UV–vis measurements (Fig. 5B) shows that the GOx quantity increases with the adsorption pressure. The quantity of GOx per monolayer for an adsorption pressure of 2.5 mN m−1 can be evaluated to (0.7 ± 0.2) × 1010 molecules mm−2 . For an Table 1 The main vibrations of stearylamine and glucose oxidase in KBr pastille Wavenumber (cm−1 ) 3330 3301 2962 2955 2915 2848 1657 1625 1565 1543 1473 1463 1424 1382 1244
Characteristic vibration Stearylamine
Glucose oxidase
NH2 asymmetrical stretching NH asymmetrical stretching CH2 asymmetrical stretching CH3 asymmetrical stretching CH2 asymmetrical stretching CH2 symmetrical stretching C O stretching from amide NH2 deformation (amide) NH2 deformation NH deformation amide II CH2 deformation CH2 scissoring C N stretching CH3 deformation OH deformation
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Fig. 4. (A) IR reference spectra of stearylamine and glucose oxidase in KBr pastille: (a) stearylamine, (b) glucose oxidase; (B) IR spectra of mixed glucose oxidase/stearylamine films, transferred on CaF2 slides (seven layers) for different adsorption pressures: (a) π = 2.5 mN/m, (b) π = 10 mN/m, (c) π = 25 mN/m.
adsorption pressure of 20 mN m−1 and above, the quantity is (2.5 ± 0.2) × 1010 molecules mm−2 . Simultaneously, the quantity of stearylamine (Fig. 5A) decreases quickly to tend toward a low amount from an adsorption pressure of 12.5 mN m−1 . This decrease of the quantity of the stearylamine can be explained by a decrease of the amount of the stearylamine molecules at the air–water interface. The quantification from the IR and UV–vis spectra shows that the mixed film, after an adsorption pressure of 12.5 mN m−1 , is constituted mainly of the GOx molecules adsorbed. In previous works, a GOx film was studied at the air–water interface [25]. These studies showed that this film could not be maintained stable at a constant pressure. Consequently, the transfer of a GOx film was impossible at a pressure of 25 mN m−1 ; this is not the case of the transferred film in presence of the stearylamine. 3.4. Evolution of the structures observed by atomic force microscopy
Fig. 5. Composition of the mixed films for various adsorption times. Quantity of stearylamine (A) per monolayer in the mixed films: (䊉), calculation from the IR spectra. Quantification of glucose oxidase (B) per monolayer in the mixed films: (ν), calculation from the IR spectra; (), calculation from the UV–vis spectra.
The mixed system was allowed to evolve for different times (as in Fig. 3b) until given surface pressures are reached (correspondence time/pressure). Then the resulting mixed films were transferred at a surface pressure of 25 mN/m on muscovite slides. The samples were then analyzed by scanning force microscopy. The images presented in Fig. 6 correspond to samples obtained for six different times: (A) 15 min (π = 2.5 mN/m);
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Fig. 6. Evolution of the structure of a mixed film of glucose oxidase and stearylamine observed by atomic force microscopy during the adsorption of GOx under a stearylamine monolayer initially in the gas/liquid phase transition. GOx concentration in the subphase: 3.2 mg/l: (A) π = 2.5 mN/m (15 min); (B) π = 10 mN/m (120 min); (C) π = 20 mN/m (320 min); (D) π = 25 mN/m (1200 min). Scan size 10 m × 10 m. The disappearance of separate structures is observed as a function of time.
(B) 120 min (π = 10 mN/m); (C) 320 min (π = 20 mN/m); (D) 1200 min (π = 25 mN/m). In every case, the scan size is 10 m × 10 m. From this study, the main conclusion is the disappearance, as a function of time, of structures at the micron scale. For low pressures (Fig. 6A, 2.5 mN/m; 15 min), well-defined structures are observed, characterized by domains of two different heights constituted of globules of diameters ranging from 40 nm to 50 nm as observed on the image of Fig. 7. Progressively, the structures disappear above 12.5 mN/m: in Fig. 6C, only “ghosts” of these structures can be observed. Within the same time, (Fig. 6C), small holes appear probably due to the transfer, which is a sign of a change in the nature of the layer. In Fig. 6D (π = 25 mN/m; 1200 min) holes are no longer visible, which is the sign of the end of changes in the layer. In this last image, the layer appears uniform with a few defects. The change in the nature of the layer as a function of time, is clear. However, the identification of the nature of the structures observed is not obvious. The selective solubilization process was used at two different pressures: 10 mN/m and 12.5 mN/m; the first value corresponds to a system in which the initial structures are still clearly visible; the second value corresponds to the key step during which the
Fig. 7. Details of a small domain observed on the image of Fig. 1 Scan size 10 m × 10 m; the small domain and the surrounding zone are constituted of globules of diameters ranging from 40 nm to 50 nm.
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Fig. 8. Identification of the structures thanks to a selective solubilization process involving isopropanol washing. GOx concentration in the subphase: 3.2 mg/l; π = 10 mN/m (120 min) (A and B); (A) before isopropanol washing (B) after isopropanol washing; π = 12.5 mN/m (120 min) (C and D); (C) before isopropanol washing (D) after isopropanol washing. Scan size 10 m × 10 m.
system changes dramatically. For both values of the surface pressure, a sample, initially placed in the liquid cell, was first imaged in the air, and then imaged after rinsing it with isopropanol in order to compare in each case the same zone. For the first sample (10 mN/m) (Fig. 8A and B), it appears that some structures are removed by the action of the isopropanol; the structures removed being stearylamine molecules, it seems that two types of structures (which perhaps may not be clearly distinguished) are removed: a stearylamine film covering the larger GOx structures, and a great number of small structures of steraylamine between these large GOx structures. For the second sample (12.5 mN/m) (Fig. 8C and D), isopropanol seems to have no action. The two images obtained before (Fig. 8C) and after rinsing the sample with isopropanol (Fig. 8D) are quite identical. This means that stearylamine is no more present in the sample under an isolated form. This is in agreement with IR spectra which showed a very low quantity of stearylamine at this pressure. 4. Discussion We have studied the influence of the presence of GOx in the subphase on a stearylamine film. From the surface pressure/area
isotherms and the film evolution studies, we found that stearylamine molecules dissolved partially in the subphase and GOx molecules were present at the air–water interface and under the stearylamine film. The nature of the interactions between the GOx molecules and the polar heads of the stearylamine molecules is probably electrostatic. All the experiments were performed with a pH of 5.6. The pKa of stearylamine is 10.6 in solution [24] and the isoelectric point of GOx is 4.2 [26]. Therefore, all the stearylamine molecules are positively charged and the GOx molecules in the subphase are globally negatively charged. This is a priori favorable to electrostatic interactions between GOx and stearylamine molecules. Moreover, we observe that the adsorption kinetic of the GOx is faster in presence of stearylamine. Next, the dynamic organization of the mixed Langmuir film of GOx and stearylamine has been studied by spectrophotometry and atomic force microscopy. We observe from the spectroscopic measurements that the GOx quantity present in the mixed film increases with time to reach a maximum value for an adsorption time corresponding to a surface pressure of 12.5 mN/m. In the same time, the stearylamine quantity decreases to tend toward a slight amount. The atomic force microscopy study shows the disappearance, as a function of time, of the structures observed
D. Lair et al. / Colloids and Surfaces B: Biointerfaces 45 (2005) 200–208
at the micron scale for the lower times of GOx adsorption. For high adsorption pressures (above 12.5 mN/m), it was found from both studies that stearylamine is no more present in the sample under an isolated form. These studies show that the structure of the mixed film evolves fundamentally: this evolution can be due to a partial hydrophobization of the GOx which interacts with stearylamine
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molecules. These interactions between GOx molecules and stearylamine molecules in solution has led us to assume the formation of a stearylamine–GOx complex stabilized by electrostatic (and eventually local hydrophobic) interactions. This stearylamine–GOx complex may be constituted of one (or several) GOx molecule(s) to which a little number of stearylamine molecules are bound.
Fig. 9. Schematic representation of the dynamic organization of the mixed glucose film oxidase/stearylamine at the air–water interface for different adsorption times.
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The consequence of this hypothesis is that the evolution of the system for long times leads to the formation of a film constituted essentially of this complex explaining the disappearance of stearylamine in an isolated form. From the results at the air–water interface and after transfer, the dynamic organization of the mixed film GOx/stearylamine during the adsorption of the GOx molecules may be described in the following model (Fig. 9): after the spreading of the stearylamine solution on the GOx aqueous subphase, for an adsorption pressure of 2.5 mN m−1 , the stearylamine positively charged molecules dissolve partially in the subphase. In the same time, the GOx molecules are being incorporated at the air–water interface in the stearylamine film and interact with the stearylamine film leading to an adsorption of the GOx molecules under the stearylamine film. As we showed previously, the quantity of stearylamine molecules determined from the IR spectroscopy decreases by 30% for an adsorption time of 120 min (πads = 10 mN m−1 ). The ionization of its polar head decreases its hydrophobic character and allows its dissolution. For short adsorption times (tads ≤ 120 min: πads ≤ 10 mN m−1 ), the GOx interacts with the stearylamine molecules at the air–water interface or in solution to form a stearylamine–GOx complex more hydrophobic than GOx. From an adsorption time of 160 min (πads ≥ 12.5 mN m−1 ), the complex formed in the subphase adsorbs between the remaining stearylamine molecules. This is in good agreement with the IR and UV–vis spectroscopies because the quantity of GOx in the mixed film increases with the adsorption pressure. When the film is compressed to 25 mN m−1 , the compression phenomenon has an influence on the dissolution of the stearylamine film at the air–water interface and in interaction with the enzyme molecules in the subphase. We have considered a mechanism which leads probably to the formation of a mixed GOx/stearylamine miscellous structure: these structures progressively formed should be expelled by the compression from the air–water interface considering it hydrophilic character. At the same time with this formation, the free space is taken up quickly by the stearylamine–GOx complex formed in the subphase and by some GOx molecules. This mechanism takes into account the results obtained by spectroscopies: from an adsorption pressure of 12.5 mN m−1 , the quantity of the GOx molecules is maximum whereas the quantity of the stearylamine decreases by 90%. The GOx molecules without stearylamine are probably diluted in the complexed film or expelled from the interface. For an adsorption time superior to 200 min (πads ≥ 15 mN m−1 ), and after the compression to 25 mN m−1 , the film at the air–water interface is a film constituted of these complexes. The quantity of GOx per monolayer is just as it would characterize the presence of a mixed layer constituted of a complex stearylamine–GOx at the air–water interface. Therefore, it is necessary to wait at least 160 min corresponding to an adsorption pressure of 12.5 mN m−1 in order to obtain
a mixed film with a maximum amount of GOx. The organization and the stabilization of this mixed film is a relatively fast process compared to the dynamic organization of the mixed GOx/behenic acid [12]. In this paper, we focused on the organization and the dynamics of the mixed GOx/stearylamine LB film. Some experiments were carried out in order to test the activity of the GOx. The preliminary results demonstrate that GOx in such structures constituted of the stearylamine–GOx complex still has good activity. An implication of these results is that, when waiting at least 3 h, the mixed GOx/stearylamine system tends to form a homogeneous film constituted of the stearylamine–GOx complex. This result is a priori potentially interesting in the field of biosensors because of the homogeneity of the films formed and because of the hydrophobization of GOx (the stearylamine–GOx complex) which might be favorable to prevent GOx leakage from the sensing film. References [1] G. Roberts, Langmuir–Blodgett Films, Plenum Ed., 1990. [2] M.C. Petty, Thin Solid Films 210/211 (1992) 417. [3] M.C. Wilkinson, B.N. Zaba, D.M. Taylor, D.L. Laidman, T.J. Lewis, Biochim. Biophy. Acta 857 (1986) 189. [4] I. Vikholm, O. Teleman, J. Colloid Interface Sci. 168 (1994) 125. [5] M.A. Bos, T. Nylander, Langmuir 12 (1996) 2791. [6] V. Rosilio, M.M. Boissonade, J. Zhang, L. Jiang, A. Baszkin, Langmuir 13 (1997) 4669. [7] S. Arisawa, T. Arise, R. Yamamoto, Thin Solid Films 209 (1992) 259. [8] C. Fiol, J.M. Valleton, N. Delpire, G. Barbey, A. Barraud, A. RuaudelTeixier, Thin Solid Films 210/211 (1992) 489. [9] S.U. Zaitsev, Colloids Surf. A: Physicochem. Eng. Asp. 75 (1993) 211. [10] A.F. Collings, F. Caruso, Rep. Prog. Phys. 60 (1997) 1397. [11] N. Dubreuil, S. Alexandre, C. Fiol, J.M. Valleton, J. Colloid Interface Sci. 181 (1996) 393. [12] S. Alexandre, N. Dubreuil, C. Fiol, D. Lair, F. Sommer, Tran Minh Duc, J.M. Valleton, Thin Solid Films 293 (1997) 295. [13] D. Lair, Ph.D. thesis, Universit´e de Rouen, France, 1998. [14] C. Fiol, S. Alexandre, N. Dubreuil, J.M. Valleton, Thin Solid Films 261 (1995) 287. [15] G. Binnig, C.F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930. [16] S.W. Hui, R. Viswanathan, J.A. Zasadzinski, J.N. Israelachvili, Biophys. J. 68 (1995) 171. [17] F. Sommer, S. Alexandre, N. Dubreuil, D. Lair, Tran Minh Duc, J.M. Valleton, Langmuir 13 (1997) 791. [18] N. Dubreuil, S. Alexandre, D. Lair, J.M. Valleton, Langmuir 12 (1996) 6721. [19] V.L. Shapovalov, Russ. Chem. Bull. 45 (1996) 1611. [20] P. Ganguly, D.V. Paranjape, F. Rondelez, Langmuir 13 (1997) 5433. [21] C.G. Lyons, E.K. Rideal, Proc. R. Soc. Ser. A 124 (1929) 333. [22] G.L. Gaines, Nature 298 (1982) 544. [23] B.P. Binks, Adv. Colloid Interface Sci. 34 (1991) 343. [24] M. Subirade, C. Salesse, D. Marion, M. Pezolet, Biophys. J. 69 (1995) 974. [25] N. Dubreuil, Ph.D. thesis, Universit´e de Rouen, France, 1995. [26] R. Wilson, A.P.F. Turner, Biosens. Bioelectron. 7 (1992) 165.