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Characterization of Flexibility of Ultrathin Protein Films by Optical Sensing Eduard Brynda,*,† Milan Houska,† Andreas Wikerstal,‡ Zbynek Pientka,† Jan E. Dyr,§ and Albrecht Brandenburg‡ Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 162 06 Prague 6, Czech Republic, The Fraunhofer Institute of Physical Measurement Techniques, Freiburg, Germany, and Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague 2, Czech Republic Received December 2, 1999. In Final Form: January 31, 2000 Films consisting of three cross-linked molecular layers of human serum albumin (HSA) or three HSA layers alternating with three heparin layers were immobilized on surfaces of grating coupler sensors and hydrophobized glass. Optical responses of the coated sensors to pH changes were observed. A decrease in the effective refractive index, ∆Neff, measured by a grating coupler was observed when the pH of buffers contacting the films was increased. The process was quite reversible, which indicated that the mass of the film remained constant when the buffers were repeatedly exchanged. Theoretical treatment of the grating coupler optics related the decrease in ∆Neff to expansion of the films. The interpretation of sensor measurements was confirmed by atomic force microscopy which revealed reversible changes in the thickness of HSA/heparin film induced by pH changes of the buffers.
Introduction Tailor-made films of biological macromolecules immobilized on solid surfaces are of great interest both for the modeling of biological processes and for various practical applications in which a specific function of biomolecules can be utilized. Affinity surfaces with immobilized antibodies, antigens, and other ligands are currently used for isolation and purification of biologically active compounds, for immunoanalytical assays, and for biosensors. Immobilized enzymes are used in heterogeneous catalysis and enzymatic sensors. An improvement of biological compatibility of medical devices has been achieved by coating their surfaces with various types of molecules like, e.g., anticoagulants, cell adhesive proteins, and antibodies for cell seeding media. Besides common ways of preparation of immobilized monolayers by physical adsorption or covalent binding to a chemically active surface, more sophisticated methods have been developed allowing preparation of three-dimensional molecular assemblies suitable for specific applications. Immobilization of ligands in a thin hydrogel matrix attached to the surface of optical transducers, e.g. (carboxymethyl)dextran gel, has been used for fabrication of affinity biosensors.1,2 Alternating protein-polyelectrolyte multilayers have been prepared by successive adsorption of proteins or nucleic acids and oppositely charged polyelectrolytes.2,3,4,5 Multilayer assemblies consisting of polyelectrolytes and various enzyme layers6,7 capable of sequential catalysis7
and applicable for enzymatic sensors8,9 or of polyelectrolytes and immunoglobulins applicable for immunosensing10 have been prepared in this way. Blood-compatible coatings of medical devices sufficiently stable under physiological conditions have been fabricated by covalent cross-linking alternating multilayers of human serum albumin (HSA) and anticoagulant heparin polyanions.11,12 Assemblies consisting only of proteins have been obtained by washing chemically nonreactive polyelectrolytes out of alternating multilayer films after covalent cross-linking the proteins.13 In this way immunosensors have been prepared by immobilization of multilayers of various monoclonal antibodies on the surfaces optical transducers.14 In addition to biological properties of individual molecules in the assembly (e.g. receptor activity), physical properties of the thin film are important for its function. For example, a flexible film of loosely cross-linked receptor molecules14 makes binding of analytes more efficient due to easy diffusion of analytes to the receptors inside the film and the freedom for the receptors and analytes to adopt an optimum mutual orientation. Mobile HSA molecules in the upper region of a loosely cross-linked film can create a compatible interface between the solid surface and blood, in which HSA is the most abundant protein.12 Optical methods employing the evanescent light wave penetrating from the surface of an optical element into the adjacent medium, such as Fourier transform infrared
†
Academy of Sciences of the Czech Republic. The Fraunhofer Institute of Physical Measurement Techniques. § Institute of Hematology and Blood Transfusion. ‡
(1) Lo¨fås, S.; Johnsson, B. J. Chem. Soc., Chem. Commun. 1990, 21, 1526. (2) Karlson, R.; Fa¨lt, A. J. Immunol. Methods 1997, 200, 121. (3) Decher, G.; Hong, J.-D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831. (4) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26, 5396. (5) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 797, 284. (6) Lvov, Y., In Protein Architecture, Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald H., Eds.; Marcel Dekker: New York, Basel, 1999; p 125.
(7) Ariga, K.; Kunitake, T. Ibid., p 169. (8) Hodak, J.; Etchenique, R.; Calvo, E.; Singhal, K.; Barlett, P. Langmuir 1998, 13, 2708. (9) Zhang, Xi; Sun, Y.; Shen, J. In Protein Architecture, Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald H., Eds.; Marcel Dekker: New York, Basel, 1999; p 229. (10) Caruso, F. Ibid., p 193. (11) Brynda, E.; Houska, M. J. Colloid Interface Sci. 1996, 183, 18. (12) Brynda, E.; Houska, M.; Jirousˇkova´, M.; Dyr, J. E. J. Biomed Mater. Res., in press. (13) Brynda, E.; Houska, M. Macromol. Rapid Commun. 1998, 19, 173. (14) Brynda, E.; Houska, M.; Brandenburg, A.; Wikerstal, A.; Sˇ kvor, J. Biosens. Bioelectron. 1999, 14, 363.
10.1021/la991558r CCC: $19.00 © 2000 American Chemical Society Published on Web 04/06/2000
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multiinternal reflection spectroscopy (FTIR MIRS),11,13 surface plasmon resonance,15 optical waveguide light mode spectroscopy using grating coupler,16 resonant mirror,17 and various types of interferometry,18 are particularly suitable for real-time observation of processes taking place in thin films immobilized on the optical surface. The intensity of evanescent light decays exponentially with the distance from the surface, the penetration depth increasing with increasing light wavelength. Thus, only a medium in the close vicinity of the surface is effectively detected by the optical device. In biosensors, a specific optical response is caused by adding a mass into the light penetration depth due to the binding of analytes to relevant bioreceptors immobilized at the optical surface. In our previous work14 we have observed reversible grating coupler responses of immobilized antibody films to repeated changes between phosphate-buffered physiological saline, pH 7.4 (PBS), and citrate buffer, pH 4 (CB). In this work, optical responses of the sensors with immobilized HSA or HSA/heparin multilayer films to changes in pH of surrounding buffers were measured. Theoretical treatment of the sensor optics was provided suggesting that reversible responses were due to changes in the film thickness, t, and refractive index, n. A relation between t and n during the changes was determined by a constant mass of the film, because n is proportional to the film density. This explanation was supported by the direct observation of the HSA/heparin film expansion and compression with an atomic force microscope (AFM). The method can be used for characterization of flexibility of biomolecular assemblies as well as for the study of interactions between the immobilized molecules.
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Figure 1. Formation of HSA/heparin three-layer film by successive adsorption on a grating coupler surface. The arrows denote the exchange of solutions at the surface: pH 7.4, PBS; pH 4, CB; HSA 1.5 mg/mL CB; heparin 1.5 mg/mL CB.
Experimental Section Materials. Human serum albumin (HSA), 99% (by agarose electrophoresis), globulin free, and dextran sulfate (DS) sodium salt prepared from dextran of Mw 5000 were from Sigma. Unfractionated heparin, from porcine intestinal mucose, sodium salt, weight-average molecular weight of 13 500-15 000, was from Calbiochem. Activated heparin (hereinafter heparin) was prepared by partial depolymerization of unfractionated heparin with nitrous acid.19 The procedure cleaves heparin molecules into shorter chains each with one end aldehyde group. Heparin preparation used in this work was a mixture of heparin chains12 of molecular weight in the range of 2000-7000. Human fibrinogen (Fbg), grade TF, free of plasminogen, fibronectin, and factor XIII was a generous gift from B. Blomba¨ck (IMCO Stockholm, Sweden). Glutaraldehyde was freshly distilled at 20 kPa under nitrogen into water to form a stock solution containing 20% glutaraldehyde. The solutions were prepared in citrate buffer, 0.1 M, pH 4 (CB), phosphate-buffered physiological saline, pH 7.4 (PBS), and 50 mM TRIS, 0.1 M NaCl, pH 7.4 (TRIS). Refractive indexes of PBS and CB were adjusted to be the same. Buffers within this range of pH were prepared by mixing CB with PBS. Grating Coupler Chips. The sensor chips ASI-3 200 with a Ta2O5 waveguide and a grating period of 0.75 µm were purchased from Artificial Sensing Instruments, Zurich. Grating Coupler Sensors. The chips were measured in a reflected TE mode (electrical field vector perpendicular to the plane of incidence) using a grating coupler instrument as described by Brandenburg et al.20 Changes in the coupling angle were recorded, from which the changes in the effective refractive (15) Lundstro¨m, I. Biosens. Bioelectron. 1994, 9, 725. (16) Lukosz, W. Biosens. Bioelectron. 1992, 6, 261. (17) Cush, R.; Cronin J. M.; Steward W. J.; Maule, C. H.; Molloy, J.; Goddard, N. J. Biosens. Bioelectron. 1993, 8, 347. (18) Fattinger, C.; Koller, H.; Schlatter, D.; Wehrly, P. Biosens. Bioelectron. 1993, 8, 99. (19) Larm, O.; Larsson, R.; Olsson, P. Biomater. Med. Devices Artif. Organs 1983, 11, 161. (20) Brandenburg, A.; Gombert, A. Sens. Actuators, B 1993, 17, 35.
Figure 2. Grating coupler response of a cross-linked HSA/ heparin three-layer film to the exchange of buffers. The arrows denote the exchange of solutions at the surface: pH 7.4, PBS; pH 4, CB; pH 6.15, pH 5.5, and pH 4.9 mixtures of CB and PBS. index, ∆Neff, were calculated using the relation
Neff ) a0 sin R0 + kλ/Λ
(1)
where n0 is the refractive index of air, R0 the air-coupling angle, k the diffraction order, λ the wavelength (632.8 nm), and Λ the grating period. Multilayer films were prepared on the chip surface in a flow cell located in the instrument, in which solutions were exchanged. The formation of the film (Figure 1) and its optical response to contacting solutions (Figure 2) were observed in real time. Atomic Force Microscopy (AFM). AFM observations were performed on a Multimode AFM Nanoscope IIIa (Digital Instruments) using a SiN microcantilever OMCL TR400 (Olympus) 100 µm long with a spring constant 0.08 N/m. The samples were studied immersed in appropriate buffer using the tapping mode at driving frequency about 8.8 kHz. To avoid the sample damage the energy of tapping was kept as low as possible. Free-oscillation amplitude was about 1 V, and the setpoint was adjusted just below the amplitude when cantilever pulls off the surface. Scan rates were 4 and 1 µm/s for 10 µm and 350 nm scans, respectively. AFM Sample Preparation. Microscopic glass slides were soaked in a solution of 10 µL of dichlorodimethylsilane in 75 mL of toluene for 30 min at room temperature, washed with toluene, acetone, and water, and baked at 130 °C for 2 h. The values of advancing and receding contact angles of 89.4 ( 0.2 and 64 ( 0.4°, respectively, were determined by the Wilhelmy plate method.
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The slide was placed in a flow cell, in which adsorption solutions were successively replaced. In this way multilayer protein films were prepared on the surface area in contact with the solutions. A line was scratched over the film with a sharp knife, and the sample was transferred into the AFM instrument. A drop of CB coated the film area all the time during the manipulation. Buffers at the sample surface were exchanged in situ in the AFM instrument. Grating Coupler Theoretical Calculations. To make an estimation of the “expansion” and “compression” of the crosslinked films, the theoretical effective refractive index, Neff, was calculated using an approach presented by Vassel21 for multilayer systems. The film was approximated with a single layer of a uniform thickness and refractive index. The effective refractive index, Neff ) 1.927 315, for TE light (λ ) 632.8 nm) mode guided in an ASI-3 200 chip in water was calculated using the following parameters: refractive index of the supporting glass nsubstrate ) 1.5315; thickness of Ta2O5 waveguide 156 nm; refractive index of the waveguide nguide ) 2.222; refractive index of water ncover ) 1.334. The compression and expansion of the film were calculated taking into consideration a constant mass of the film
Γfilm ) tfilmFfilm ) constant
(2)
where Γfilm is the mass, tfilm is the film thickness, and Ffilm is the film density. By the increase of the film thickness, the density and hence the refractive index of the film are decreased.
[ ] [] [ ] n1 1/t1 1 ) n0 + kt0 n2 1/t2 1
(3)
where the subscripts 1 and 2 represent two different density states and [n0, t0] represents the initial film with the refractive index n0 and thickness t0 (in our case n0 ) solution index and t0 ) 0; i.e., the thickness is really arbitrary because a film with the same refractive index as the ambient solution has the density of the solution independent of its thickness). From the expression, for example the refractive index of state two, n2, can be derived knowing [n1, t1] and t2.
t1 n2 ) n0 + (n1 - n0) t2
(4)
The thickness of the film measured by AFM in CB was used as t1 for the calculations. The corresponding refractive index, n1, of the film in CB was calculated from the thickness measured by AFM and the change in the effective refractive index, ∆Neff, measured by the grating coupler instrument, when the film was built on the waveguide surface. Using the starting values [n1, t1], theoretical dependence of ∆Neff on the film thickness was calculated.
Results and Discussion Figures 1 and 2 illustrate the typical grating coupler sensor experiment. Figure 1 shows the preparation of an assembly consisting of alternating HSA and heparin molecular layers. The first molecular layer was obtained by adsorption of HSA from CB on Ta2O5 surface. The adsorbed HSA monolayer was stable in CB; however, about 38% of adsorbed HSA could be washed from the surface in PBS. The irreversible adsorption is assumed to be due to hydrophobic interaction of HSA molecules with the surface. The partial desorption in PBS might be due to some negative surface charge on Ta2O5 attracting HSA molecules (isoelectric point 4.6) positively charged in CB at pH 4 and repulsing them when their neat charge reversed to a negative one in PBS at pH 7.4. Upon replacement of HSA solution with CB and then with heparin solution, heparin polyanions adsorbed on the HSA layer. The alternating HSA/heparin multilayer film was formed by repeating the successive adsorption of HSA (21) Vassel, M. O. JOSA 1974, 64, 166.
and heparin. The adsorption is driven by electrostatic attraction between the molecules in solution and oppositely charged molecular layer previously adsorbed on top of the assembly, because at pH 4 HSA is positively charged and heparin is charged negatively.11,22 The multilayer assembly in heparin solution was heated to 50 °C for 3 h in order to conjugate heparin molecules with HSA by forming a covalent bond between the aldehyde end of activated heparin19 and HSA amino group. The final treatment with 0.2% glutaraldehyde in CB solution was necessary to stabilize the film by covalent cross-linking of the assembly via some HSA amino groups. Without the cross-linking the film would dissolve in PBS (except for the first HSA layer adsorbed directly on Ta2O5). Optical responses of the cross-linked HSA/heparin multilayer to buffers of various pH are shown in Figure 2. The first replacement of CB by PBS removed from the surface molecules, that were not covalently fixed in the cross-linked assembly. The decrease in the mass attached to the sensor surface is reflected by the lower ∆Neff measured in CB after the first soaking with PBS (Figure 2). In an analogous experiment on a germanium reflection element, we detected by FTIR MIRS8 that about 30% of heparin was released in PBS. Comparing the decrease in ∆Neff of -4.5 × 10-4 (Figure 2) with the increase of 5.1 × 10-4 due to adsorption of the three heparin layers (Figure 1), we can assume that some heparin and also HSA molecules were removed from the film. Only an insignificant decrease in the ∆Neff was observed in CB if the exchange of CB and PBS buffers was repeated (not shown in Figure 2). As Figure 2 shows further, a gradual stepwise decrease in pH (in mixed PBS/CB buffers) increases ∆Neff. The difference in ∆Neff between the final value in CB (pH 4.0) and starting value in PBS (pH 7.4) is the same as the difference observed after direct exchange of PBS for CB. When PBS was replaced by water, ∆Neff decreased at first because the refractive index of solution within the penetration depth of the evanescent light decreased and, after a while, it started to increase again above its value measured in PBS. The changes in ∆Neff observed at various pH are probably due to some changes in the film. It is to be emphasized that no changes in ∆Neff were detected if the above buffers were exchanged at the bare (uncoated) sensor surface. Multilayer films consisting solely of HSA (without heparin) were prepared by the successive adsorption of HSA and dextran sulfate polyanions in a way analogous as the HSA/heparin films described above. Dextran sulfate does not contain reactive groups which would be involved in the glutaraldehyde cross-linking so that after crosslinking of HSA with glutaraldehyde, dextran sulfate molecules were removed from the protein film by washing with PBS. The complete expulsion of dextran sulfate polyanions from the cross-linked HSA films by reverting the positive protein charge in CB to a negative one in PBS was confirmed earlier using FTIR MIRS.13 The optical responses of a cross-linked film composed of three HSA layers to the replacement of the buffers are shown in Figure 3. The film behavior is similar to that observed with the HSA/heparin film. The initial decrease in ∆Neff after the first wash with PBS was apparently due mainly to the removal of dextran sulfate. A corresponding increase was observed when dextran sulfate was readsorbed again into the film from dextran sulfate solution in CB (DS in Figure 3). The full reversibility of the changes in ∆Neff associated with the following repeated changes (22) Houska, M.; Brynda, E. J. Colloid Interface Sci. 1997, 188, 243.
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Figure 3. Grating coupler response of a cross-linked HSA three-layer film to the exchange of buffers. The arrows denote the exchange of solutions at the surface: pH 7.4, PBS; pH 4, CB; DS, dextran sulfate 1 mg/mL CB.
between PBS and CB indicates that no HSA was further washed out of the film. The preservation of adsorbed HSA mass over the changes of buffers have been confirmed also by measuring FTIR MIRS spectra of similar HSA multilayers prepared on germanium reflective elements. To interpret changes in ∆Neff of the films studied, we considered two explanations. First, it can be assumed that although the mass of the film itself is constant, it can reversibly change due to adsorption and desorption of citrate ions in CB and PBS, respectively. The increase in ∆Neff induced by CB might be caused either by an increase in the film mass due to the reversible adsorption of citrate ions or by a change in the film structure. The former explanation does not seem plausible because the increase was observed also when PBS was replaced by water (Figures 2 and 3) or when 0.1 M NaCl solution, pH 6, in contact the film was replaced by a 0.15 M NaCl/HCL, pH 4 (not shown). Evidently, structural changes in the film accompanied with changes in the film density and thickness were responsible for the optical response. Relative changes in ∆Neff measured on HSA/heparin and HSA films with decreasing pH of the buffers are shown in Figure 4. (The relative change is expressed as ∆Neff/∆Neff0, where ∆Neff is a value in a buffer and ∆Neff0 is a value in PBS.) The most extensive relative decrease accompanied the transfer of the HSA/heparin film from CB to PBS. In this case, HSA molecules reversed their charge to a negative side and the electrostatic attraction between heparin and HSA changed to repulsion. The repulsion should expand the cross-linked film. Similarly, the repulsion would have dissolved the film if it had not been cross-linked. To avoid optical changes due to different refractive indexes of the buffer background, the dependence was measured in mixed CB/PBS buffers of a constant refractive index. As the ionic strength, i.s., of CB (pH 4) and PBS were of 0.11 and 0.15, respectively, an effect of ionic strength took part in the film response. A decrease in ∆Neff observed when CB (pH 4, i.s. 0.11) was replaced with CB/NaCl solution (pH 4, i.s. 0.15) was about 10% of that induced by the replacement CB with PBS (Figure 4). Also in this case, the decrease in ∆Neff could be related to an expansion of the film due to weakening the electrostatic attraction at higher ionic strength of buffer. Another example of the grating coupler response to structural changes of immobilized protein layers is shown in Figure 5. Fibrinogen was adsorbed on the grating
Figure 4. Dependence of the relative change in ∆Neff on pH of CB/PBS buffers measured by GC chips coated with crosslinked films composed of three layers of HSA/heparin (triangles) and HSA (squares). The open square is a value observed with the HSA film when CB was replaced with mixture of CB and 0.2 M NaCl of pH 3.96 and ionic strength 0.15. ∆Neff is the value in a buffer; ∆Neff0 is the value in PBS.
Figure 5. Contraction of fibrinogen layer adsorbed on grating coupler sensor in water. An arrow indicates an exchange of solutions at the sensor surface: Fbg, fibrinogen in TRIS, curve 1, adsorption from 5 µg/mL Fbg in TRIS; curve 2, adsorption from 200 µg/mL Fbg in TRIS.
coupler from TRIS at concentrations 5 µg/mL (curve 1) and 200 µg/mL (curve 2). Two different arrangements of Fbg molecules on the surface obtained at these concentrations were observed earlier by scanning and transmission electron microscopy.23 In a monolayer adsorbed at low concentration of fibrinogen in solution, both central and end domains of fibrinogen molecules were accessible for binding of the specific antibodies indicating fibrinogen adsorption side on the surface; in a monolayer adsorbed at high solution concentration, only antibodies specific to the end domain of fibrinogen could bind to the monolayer indicating that fibrinogen molecules were densely packed (23) Brynda, E.; Houska, M.; Lednicky´, F. J. Colloid Interface Sci. 1986, 113, 164.
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Figure 7. Three-dimensional AFM height image of a crosslinked HSA three-layer film prepared on hydrophobized glass washed with PBS and measured in CB. The scan size is 350 nm.
(a)
Figure 6. Theoretical dependence of ∆Neff on the thickness of HSA/heparin and HSA films immobilized on a grating coupler chip. The thickness in CB measured by AFM and the refractive index of the film in CB, n0, calculated from the ∆Neff values determined by grating coupler measurements were used as starting values for calculation of the curves.
end-on the surface (standing on the surface).24 When TRIS was replaced by water at the grating coupler surface occupied by a low concentration of Fbg (Figure 5, curve 1), a decrease in ∆Neff was observed due to the refractive index of water being lower than that of TRIS. Similarly, the increase in ∆Neff was observed at the beginning of the experiment when water was exchanged for TRIS at the bare grating coupler surface. When TRIS was replaced with water at the surface occupied with a high concentration of Fbg (curve 2), first ∆Neff decreased due to decrease in the refractive index of solution within the penetration depth of the evanescent wave but then it increased due to shrinking of the adsorbed Fbg layer. In solution, Fbg molecules precipitate when TRIS is diluted with water. A theoretical dependence of ∆Neff on the film thickness is shown in Figure 6. The curves were calculated by considering the relation between the film thickness and refractive index determined by the constant film mass (see the theory above) and the film thickness measured in CB by AFM. The AFM thickness of HSA/heparin and HSA three-layer films in CB was 20.5 and 24.5 nm, respectively. Refractive indexes of these films in CB of 1.49 and 1.47, respectively, were calculated from the AFM thickness and ∆Neff obtained from grating coupler measurements. A higher refractive index fits with a higher density of the HSA/heparin film contracted in CB due to the electrostatic attraction. One can see ∆Neff decreasing with the increasing film thickness. The thicknesses of 43.1 and 37 nm were calculated from grating coupler data for HSA/heparin and HSA films in PBS, respectively. The three-dimensional AFM height image of the crosslinked HSA three-layer film prepared on the hydrophobized glass washed with PBS and measured in CB is shown in Figure 7. An average thickness of the film was about (24) Dyr, J. E., Tichy´, I.; Jirousˇkova´, M.; Tobisˇka, P.; Slavı´k, R.; Homola, J.; Brynda, E.; Houska, M.; Suttnar, J. Sens. Actuators, B 1998, 51, 268.
(b)
Figure 8. (a) AFM height image of a cross-linked HSA/heparin three-layer film immobilized on hydrophobized glass and measured in PBS. The scan size is 10 µm; the contrast covers 50 nm. The black band on the left-hand side is a scratch made in the film with a knife. (b) Surface profile of the sample in PBS obtained by averaging line profiles over a stripe between 3 and 6 nm of vertical scale in (a). The dashed line shows the sample measured in CB.
24.5 nm. The height of hills on the film surface of about 4 and 8 nm coincides with HSA molecular dimensions. Unlike the z-dimension, the displayed x,y-dimensions are much larger than the molecular ones due to a shape of AFM tip of a radius between 10 and 20 nm. The AFM height image of a cross-linked HSA/heparin three-layer film immobilized on hydrophobized glass and measured in PBS is shown in Figure 8a. A black band is a scratch made in the film with a knife. The surface profile of the sample in Figure 8b was obtained by averaging the
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Table 1. Thickness of HSA/Heparin Three-layer Film Measured by AFM after Repeated Exchanges of Buffers order of buffer replacement fresh film 1st 2nd 3rd 4th 5th
buffer CB PBS CB PBS CB PBS
film thickness (nm) 17.5 25.8 ( 0.3 20.3 ( 0.7 25.0 ( 2.0 20.3 ( 0.8 26.1 ( 0.3
horizontal position of AFM tip over the 3 nm width of a stripe perpendicular to the scratch edge (between 3 and 6 nm of vertical scale in Figure 8a). One can see a contraction of the film as the PBS (full line) was replaced by CB (dashed line). Table 1 shows the repetitive expansion and contraction of the HSA/heparin film measured in PBS and CB, respectively, after exchanging the buffers. The increase in the film thickness from 17.5 to 20.3 nm measured in CB after the first soaking of the freshly prepared film with PBS might be caused by weakening the electrostatic attraction in the film due to partial washing out heparin molecules. The film behavior corresponds qualitatively with that predicted by the grating coupler theory. A smaller film expansion observed by AFM in comparison with that calculated from the grating coupler response might be due to oversimplification of the grating coupler theory, different properties of the film prepared on hydrophobized glass and on more hydrophilic charged grating coupler chips, or an inaccurate AFM measurement of the thickness of very flexible protein films. Within an experimental error we were not able to observe changes in the thickness of the HSA film. Unlike the expanded HSA/heparin film controlled by strong electrostatic repulsion, the expanded HSA film might be too soft even for the light tapping of AFM used in these experiments. Figures 7 and 8 show HSA and HSA/heparin multilayers as homogeneous films of rather uniform
average thickness, which was an assumption of the grating coupler theoretical treatment used above. Conclusions Cross-linked films composed of three molecular layers of HSA/heparin or HSA were immobilized on the surface of grating coupler sensors. The decrease in ∆Neff was observed when pH of buffers in contact with the films increased (from pH 4 to pH 7.4). The reversibility of the process indicated that the mass of the film remained constant during the exchange of buffers. Theoretical treatment of the grating coupler sensor optics related the decrease in ∆Neff to expansion of the films. The theoretical description of the phenomenon was supported by a direct AFM observation of the repetitive expansion and compression of HSA/heparin film in PBS, pH 7.4, and CB, pH 4, respectively. The work provides a basis for understanding our earlier grating coupler experiments on cross-linked multilayer films of monoclonal antibodies.14 We observed that more loosely cross-linked films exhibited larger pH-induced changes in ∆Neff as well as better binding of antigens. According to the conclusions derived in the present work, a larger decrease in ∆Neff associated with the film transfer to PBS can be related to a more extensive expansion of the film facilitating the penetration of antigens to active centers located inside the film. In general, the described optical method can be used for characterization of flexibility of biomolecular assemblies as well as for the study of interactions between the immobilized molecules. Acknowledgment. This research was supported by the Grant Agency of the Czech Republic under Contract 102/99/0549 and by the Ministry of Education, Youth, and Sports of the Czech Republic under Contracts ME 082 (1997) and IGA MZ 4642-3. LA991558R
