0 Copyright 1995 American Chemical Society
FEBRUARY 1995 VOLUME 11, NUMBER 2
Letters Comparative Study of Langmuir Monolayers of Immunoglobulines G Formed at the Air-Water Interface and Covalently Immobilized on Solid Supports A. Tronin,*?+ T.Dubrovsky,z and C . Nicolini Institute of Biophysics, University of Genoa, uia Giotto 2, Genoa Sestri Ponente 16153, Italy Received April 12, 1994@ Langmuir films of IgG formed at the air-water interface and covalently immobilized on solid supports were studied by means of fluorometry and ellipsometry. Dependencies of monolayer molecular density and thickness on surface pressure were determined. The data obtained make possible a model of the film molecular organization and its dependence on the surface pressure. The films are of monomolecularlayer character within the entire range of achievablepressures on the air-water interface as well as on the solid supports. The molecular orientation changes with the pressure. In the monolayer on the air-water interface the moleculesare positioned with their Fab-Fab-Fc ploane parallel to the surface at pressures below 20 mNIm; when compressed up to 35 mN/m, they turn by 90" to reach perpendicular position with respect to the surface. Within the range 20 mN/m .C n < 35 mN/m the angle of molecular inclination increases monotonically from 0" to 90" with pressure. The pattern of the orientation-pressure behavior for transferred monolayers is almost the same showing that the film molecular structure does not change upon deposition on to the solid activated substrate.
1. Introduction Langmuir films of antibodies are currently gaining interest for their prospective application to biosensing. Immunoglobulin G forms rather dense and stable films on the air-water interfa~el-~ due to pronounced hydrophobic regions on its solvent-exposed surface. These Permanent address: Institute of Crystallography of Academy of Sciences of Russia, Leninsky pr. 59, Moscow 117333, Russia. q e r m a n e n t address: Bakh Institute of Biochemistry ofAcademy of Sciences of Russia, Leninsky pr. 33, Moscow 117071, Russia. e Abstract published in Advance ACS Abstracts, January 15, 1995. (1)Lvov, Yu. M.; Erokhin, V. V.; Zaitsev, S. Yu. Biol.Mem. 1991,4 (9),1477. (2)Erokhin, V. V.; Kayushina, R. L.; Lvov, Yu.M.; Feigin, L. A. Studia Bwphys. 1989,132,97. (3)Ahluvalia, A,; De Rossi, D.; Ristori, C.; Schirone, A.; Serra, G. Bwsens. Bioelectron. 1991, 7, 207. (4) Dubrovsky, T. B.; Demcheva, M. V.; Savitsky, A. P.; Mantrova, E. Yu.;Yaropolov, A. I.; Savransky, V. V.; Belovolova, L. V. Biosens. Bioelectmn. 1993,8, 377. (5) Tronin, A.; Dubrovsky, T.; De Nitti, C.; Gussoni, A.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994,238, 127.
regions, comprising hydrophobic amino acid residues, are located mainly on the Fc fragment.'j Moreover, IgG is most likely not to denature on the water surface because after being transferred from the surface, it still exhibits immunological a ~ t i v i t y . ~High - ~ stability of the IgG molecule results from the large amount of S-S bonds inside its structure. IgG films can be deposited on solid substrates by Langmuir-Schaefer techniques using either a d s o r p t i ~ n ~ ? ~ or covalent b i n d b ~ g . ~Of- ~special interest is the possibility of assembling antibodies into two-dimensional structures. Already reported are the successful attempts of formation of 2-D ordered structures of proteins, such as streptavidin,8p9ferritin,'O and antibodies of different s ~ b c l a s s e s , ~ ~ - ~ ~ (6) Deisenhofer, J. Protein Data Bank 1982, Brookhaven National Laboratory, Ident. Code N 1FC1, 1FC2. (7) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1992,205, 113. (8)Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; BlanRingsdorf, H.; Kornberg, R. D. Biophys. J. kenburg, R.; Ribi, H. 0.; 1991,59, 387. (9) b i t e r , R.; Motschmann, H.; Knoll, W. Langmuir 1993,9,2430.
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utilizing different approachesto produce such structures: specific binding of streptavidinor antibodies to preformed layers of modified phosph01ipids;~~~J~ nonspecific adsorption of proteins to hydrophobic substrates;13flow-mediated formation of protein monolayers on mercury surfaces.1° While the same is true for Langmuir films of antibodies formed on the air-water interface, until now there are no data on their structure. It is not obvious a priori that these IgG films are two-dimensionally ordered. Since all factors governing the structure (such as shape of the molecule, nature of interactive forces, distributions of charge, and hydrophobic and hydrophilic regions) for protein molecules are very complicated, the structure of the superficial array is likely to be more diverse. Aggregates of proteins floating on the surface and multilayer formation are possible. Moreover, due to the same reasons, the array structure may depend significantly on external conditions, such as surface pressure, subphase pH, and surface residence time. It is obvious that the problem of orientation of protein molecules in films and possible ways of the manipulation of this orientation are of great importance for practical applications. In case of immunosensing elements, for example,the orientation of IgG molecules should be such that the Fab fragments be directed outward with respect to the film support. Protein molecules spred on water surface are likely to be subject to reorientation as a response to external factors. Salesse et al.14have shown that orientation of rhodopsin molecules on water surface depends on surface pressure. However, the possibility to orient the molecules on water surface is not enough for manufacturing films with desired molecular disposition. The films should be deposited on some solid support and therefore another problem arises as to whether or not the molecular orientation remains unchanged upon deposition. To address these two problems, we studied IgG films formed at the air-water interface and deposited on substrates. We applied ellipsometry to measure film thickness and fluorescence measurements to determine film molecular density. Ellipsometry provides a unique tool for studying the films directly on the water surface since it can be applied in situ, which has been efficiently used by many inve~tigators.~J~-'~ In the case of highly nonspherical molecules with significant differences in dimensions in various directions (as in the case of I@), thickness of the monolayer depends markedly on orientation of the molecules, and thus ellipsometry, being very sensitive to the film thickness, can provide information about molecular orientation. This possibility to determine molecular orientation was used in a paper by Salesse et al.14 cited above for the films of rhodopsin, the molecule of which is approximated by a cylinder with height differing from the diameter. 2. Materials and Methods 2.1. Materials. For protein deposition we used mouse monoclonal antibodies generated against anti(2,4-dichlorophenoxylacetic acid (MAb 063BF2, purified from ascites fluid by (10)Yamaki, M.; Matsubara, K.; Nagayama, K. Langmuir 1993,9, 3154. (11)Uzgiris, E. E.; Kornberg, R. D. Nature 1993,301,125. (12)Uzgiris, E. E. Biochem. J. 1987,242,293. (13)Barraud, A.;Perrot, H.; Billard, V.; Martelet, C.; Therasse, J. Biosens. Bioelectron. lWS,8, 39. (14)Salesse, C.;Ducharme, D.; Leblanc, R. M.; Boucher, F. Biochemistry 1990,29,4567. (15)Engelsen, D.; Konig, B. J. Chem. Soc.,Faraday Trans. 1974,70, 1603. (16)Ducharme, D.; Salesse, C.; Leblanc, R. M.; Meller, P.; Mertesdorf, C . ; Ringsdorf, H. Langmuir 1993,9, 2145.
Letters protein A chromatography), obtained from Dr. M. Dovis, University of Brescia, Italy. (3-Glycidoxypropyl)trimethoxysilane (GOPTS)was purchased from Aldrich. 2.2. Preparation of the Surfaces. Undoped silicon wafers were used as supports. The supports were treated with boiling chloroform, rinsed on a glass filter, and dried under nitrogen. These supports were silanized with (3-glycidoxypropy1)trimethoxysilane in accordance with the technique proposed by Ma1mq~ist.l~The density of the adsorbed organosiloxane polymer was controlled by the microgravimetric method and its average value was 4-8 nmol/cm2.18 Prepared in this way, the surface of the substrate has many exposed active epoxy groups which react readily with protein amino groups. This surface preparation method has been used successfully for previous immobilization of LB films of antibodies on quartz surface^.^ 2.3. Deposition of the Films. Antibody monolayers were formed in a Langmuir trough (KSV 5000 LB, Finland). Decimolar carbonate buffer (pH 8.6) was used as the subphase. It was prepared using distilled and deionized (Millipore, 18.2 MQ cm) water. A 100-pL portion of IgG solution in Tris buffer (100 mM, pH 7.3) with concentration of 1 mg/mL was spread on the subphase by Hamilton rheodyne syringe. The transfer of LB antibody films from the subphase surface onto the reactive supports was performed by "touching" the silanated support in parallel mode to the subphase surface (analogous t o the Langmuir-Schaefer method). After film deposition,the samples were dried in nitrogen, incubated for 1h under ambient conditions to be sure that all epoxy-amino group reactions are completed, washed with deionized water, and dried again under nitrogen. 2.4. Measurements of Protein Film Surface Density. Films of antibodies with a fluorescent Eu label were deposited onto quartz substrates with strictly controlled surface area. W-p-Isothiocyanatobenzyl derivative of diethylenetriamineWPJV3,iPtetraacetic acid, which chelates the Eu3+ atom (Wallac, Finland) was used for IgG labeling. The degree of antibody labeling with europium chelate was found to be of the ratio of 1:l. The labeled protein as such is practically nonfluorescent. After the deposition samples were immersed in Enhancement Solution (Wallac), in which europium is efficiently dissociated from the labeled compound within a few minutes. Free Eu3+ rapidly forms a new, highly fluorescent and stable chelate inside a protective micelle with the components of the Enhancement Solution and total amount of Eu atoms in solution was measured according to DELFIA time-resolved fluorescence technique and thus the surface density of initial IgG monolayer was determined. Fluorescent signal was measured usinga 1232 DELFIA research fluorometer (Wallac,Finland). The sensitivity of this technique is very high. Due to amplification by a factor of lo6ofthe fluorescence ofthe europium chelate in Enhancement Solution and time resolution, the sensitivity of Eu volume concentration determination is better than lO-13moYL. It means, that in a volume of 0.2 mL, which was normaly used, an amount of 1O-l' mol of Eu can be detected. Taking into account the area of the sample of about 1 cm2 from which Eu was extracted, we obtain sensitivity for surface density detection of mol/cm2. For a general description of the DELFIA method see ref 19, and for the application to IgG LB films see ref 4. 2.5. Ellipsometric measurements were performed using a PCSAnull ellipsometer LEPh-2 (SpecialDesign and Production Bureau for Scientific Devices of the Siberian branch ofthe Russian Academy of Sciences, Novosibirsk) using a He-Ne laser (wavelength 632.8 nm). The accuracy ofthe device is 0.02"with respect to Y and A. Measurement procedures and data treatment were different for deposited films and for films formed on the water surface. For deposited films an angle of incidence of 70" was chosen. A two-zone technique was used for measurements. A model of two isotropic layers was used for data treatment. The upper layer represents the protein layer and the lower one accounts for superficial imperfections of the substrate. The technique is described in more detail elsewhere.6 In the case of measurements of the films directly on the water surface we used an angle of incidence of 56", one-zone measurements, and a one-
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(17)Malmqvist, M.; Olofsson, G. U.S. Patent 4,833,093, 1989. (18)Dubrovsky, T.B.; Erokhin, V. V.; Kayushina, R. L. Biol. Membr. 1992,6 (l),130. (19)Hemmila, I.; Dakubu, S.; Mukkala, V.; Siitari, H.; Lovgren, T. Anal. Biochem. 1984,137,335.
Letters looo
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Incidence angle, deg Figure 1. Dependencies of ellipsometricparameters Y (crosses) and A (squares) on angle of incidence for pure water: lines, calculation for ideal boundary between media with nupper = 1 and nlower = 1.334; points, measurements of thin water layer (0.2-0.3 mm) covering rough glass plate. layer model. The accuracy of measurements on water surface was lower than that of the solid samples mainly due to two reasons: (i) device imperfections effecting one-zone measurements and (ii)residual capillar waves of the surface resulting in some increase of signal noise. The systematic errors were estimated by comparison with two-zone measurements and found to be less than 10’ in Y and A. The measurements were carried out on solid samples with ellipsometric parameters chosen in the same range as for the measurements a t the air-water interface. The influence of the second factor led to errors of 2% in Y? and 10%in A. A home-built trough was used for ellipsometric observations of the monolayers at the air-water interface. This all-Teflon trough (surface dimensions 164 cm2 and depth 0.4 cm) was installed at the ellipsometer. A Wilhelmy system was used to measure the surface pressure. Vibration of the water surface was dampened by a glass plate, immersed into the water. The plate was adjusted so as to provide a water depth of about 0.20.3 mm coveringthe glass plate. The plate surface was roughened to avoid interference in the water layer. To be sure that interference phenomenon is absent and thus that the one-layer model is feasible, we measured ellipsometricparameters of pure water at various angles of incidence (Figure 1). Two-zone averaging was used for these measurements. Shown by the lines are corresponding theoretical dependencies for ideal air-water interface ( n w a k r = 1.334for 632.8 nm). Good agreementbetween theoretical and experimental data proves that interference effects can be neglected.
3. Results and Discussion In the case of partially soluble,surface-activesubstances such as amphiphilic proteins, it is impossible to determine the area occupied by one molecule on the surface simply by dividing the number of the molecules in the trough by the trough area since the molecules pass constantly from the surface to the volume and vice versa. The coefficient of surface/volume distribution depends, above all, on surface ~ r e s s u r e .The ~ use of the fluorescence europium labeling makes it possible to determine the molecular density of the film. The dependence of both surfacedensity and corresponding area per molecule on the surface pressure are presented in Figure 2. Both curves have pronounced inflections in the range of 20-30 mN/m. In general, bends in n-A isotherms are associated with phase transitions in the film. Although the given n-A curve is not properly an isotherm since the total amount of molecules at the surface is not constant, we propose that some changes in film structure occur. In order to consider molecular packing of the monolayer let us assume that IgG molecule is approximated by an
,
Langmuir, Vol. 11,No. 2, 1995 387 2
Area per IgG molecule, nm2
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01 0
Surface density, pmol/cm, 2,5
1
1
I
10
20
30
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Surface pressure, mN/m
Figure 2. Dependencies of surfacedensity (triangles) and area per molecule (squares) on surface pressure in IgG monolayers on the water surface. IgG surfaceconcentration was determined with using fluorescent europium label.
El
Figure 3. Model of IgG molecular orientation in the monolayer at the water-air interface: a, characteristic dimensions of an IgG molecule; b, the plane of the IgG molecule larger crosssection is parallel to the interface plane (low pressures); c, the film remains in 2-Dform and molecules begin to tilt; d, the plane of the IgG molecule larger cross section is perpendicular t o the interface plane (high pressures).
ellipsoid of rotation with diameter of about 14 nm and small axis of about 4 nm (Figure 3a). This assumption is rather natural as a first approximation since the junction of Fab fragments is flexible and they can rotate by 120150”in the plane of the molecule around its center. The largest cross section of this ellipsoid is of 160 nm2, the smallest one is of 60nm2. The bend in “area per moleculesurface pressure” curve occurs at 200-150 nm2, which
Letters
388 Langmuir, Vol. 11, No. 2, 1995 14
Thickness, nm
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"i
01 10
I 30
20
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Surface pressure, mN/m Figure 4. Dependence of the IgG film thickness at the waterair interface on the surface pressure. Ellipsometric measurements. coincides with the larger cross section. After this bend, the film becomes more elastic, small decrease of area causes large increase of pressure, and it is natural to suppose that upon the bend we observe a transition into close packed state. It means that up to a surface pressure of 25-30 mN1m the IgG molecules are oriented parallel to the water surface(Figure 3b),since any other orientation would result in smaller cross section. With increase of surface pressure there are two possible ways of changes of molecular packing: 1. Being compressed to the dense packing state (area per molecule 200-150 nm2) at 30 mN/m, the molecules aggregate and form various complexes and multilayers. 2. The film remains in 2-D form, and starting from 30 mNlm, molecules begin to tilt (Figure 312). At a pressure near 40 mNIm, the IgG molecules achieve the densest packing, area per molecule coincides with the smallest cross section (70 nm2), which means that the molecules are perpendicular to the surface (Figure 3d). The second pattern of behavior seems to be more likely. In fact, a sharp decrease of the film elasticity most likely should be observed in the first case (like in the case of monolayer collapse), while in the second one the film remains more or less elastic and it is impossible to achieve area per molecule less than 70 nm2. Interesting enough is the coincidence of value of the area per molecule at 40 mNlm with that obtained by Uzgiris and Kornbergll (also 70 nm2)for close packed (crystallized) IgG by means of electron diffraction. To check these models, we measured the thickness of the film by ellipsometry. We suppose,that the dependence of film thickness versus surface pressure should be quite different for these cases. For the first one it should be unlimited growth of thickness (at least in order of several molecular dimensions); in the second one the thickness should not exceed the largest molecular dimension. The dependence of monolayer thickness on surface pressure is shown in Figure 4. As it can be seen, the curve has two plateaus: the first at pressures below 20 d l m with a value of 5 nm, the other at pressures above 35 mN/m with a value of 13 nm. Remarkable is the coincidence of these experimental thicknesses with the model characteristic dimensions of the IgG molecule
described above. Transition between the two plateaus takes place at a pressure near 30 d l m , corresponding with the observed bend in JG-Acurve. The second plateau shows that it is impossible to make IgG films thicker than the largest dimension of the molecule. This is a direct indication of the two-dimensionalstructure of the protein film. In general, ellipsometric measurements conform to the second model of the molecular packing of the IgG film. To appreciate the reliability of these data, let us consider the accuracy of thickness determination. Ellipsometric parameters at certain surface pressures are given in Table 1. Since for very thin films parameter Y depends almost exclusively on the parameters of the substrate (which in this case is water), it is impossible to resolve the thickness and the refraction index of the protein film. We calculated the film thickness assuming an index of refraction to be equal to 1.5. This value was obtained for deposited fivelayer thick films, the thickness of which (60nm) was large enough to resolve both parameters. The forth column of the table presents thicknesses of the layer with n = 1.5 that fit best to measured Y and A; corresponding calculated parameters are shown in columns 5 and 6. Descrepancy between measured and calculated data is less than experimentalerrors. Variation of \Ymeaaured with film thickness can be attributed to the systematic error of one-zone measurements. (It should be noted that systematic error in this case is not a constant shift but a complicate function of optical system under investigation, thus shift in Y dependson A). Descrepancy of A is less than random error. Since in case of thin films, thickness is mainly determined by A, so does its accuracy 6d = (ad1aA)dA; the derivative adlaA for our system was determined to be 0.5 d d e g , the corresponding accuracy of thickness determination is given in the seventh column. It is evident that difference in the film thickness at 20 and 40 mN1m exceeds by far the experimental error. The other important problem is that of film stability. As it was mentioned,the molecules leave the surface when the film is compressed. If the total area of the film is kept constant after the compression, the pressure decreases, and the higher the starting pressure the faster ita decrease. Besides the pressure, the rate of this process depends on many factors, such as temperature, trough geometry, etc. In our case it varied from 0 at 20-25 mNlm (film was almost stable) to 1-3 mN1m per second at 40 mN1m. We did our measurements either fast enough to refer them to definite pressure or in the regime of constant pressure. We never observed any increase of either molecular density or thickness of the film with time a t high pressure (35-40 mN/m), which means that molecules expelled from the layer do not accumulatejust below the monolayer in forms of any aggregates but dissolve in the subphase. In other words, it means that the film is stable in the sense that external conditions (pressure) being fixed, the internal parameters (density and thickness) remain constant. Fortunately, the relative variation of protein bulk concentration is negligible and does not change the subphase parameters. In order to check whether the molecular packing of the film formed on the water surface is maintained upon deposition or not, we measured the same dependence of thickness on surface pressure for the deposited protein films. The corresponding curve is given in Figure 5. It
Table 1 pressure, mN/m 20 30 40
A measured -1O"OO'
-19"50' -26"05'
Y measured 4"37' 4"31' 4"25'
thickness, nm 5.1 11 13.2
A calculated
Y calculated
- lO"09' -21'47' -26"04'
4"37' 4"35' 4"35'
accuracy, nm k0.5 hl.0 zk1.3
Langmuir, Vol. 11, No.2, 1995 389
Letters Thickness, n m
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orientation may be stable, but when touched by the substrate, the molecules tend to lay parallel on the surface to minimize the interaction energywith the former. Some part of the molecules is expelled from the surface to leave space for the remainder. Only when the angle is rather high and so is the molecular overlap in the film is the exclusion less energetically favorable than the planeparallel alignment and the thickness of the film begins to grow. Except for this difference, the covalent immobilization on the substrate preserves the general long-range molecular packing of the monolayer formed on the water surface.
4. Conclusions
45 2
S u r f a c e p r e s s u r e , mN/m
Figure 5. Dependence of the deposited I&
film thickness on the surface pressure of deposition, ellipsometric measurements.
can be noticed that the curves for films on the water surface as well as on solid substrate have almostthe same pattern; this is considered to be evidence for the preservation of molecular packing in the film during deposition. Minor differencesin thickness may be due to many factors, such as difference in ellipsometry data treatment, substrate roughness or minor changes in conformation of IgG molecules (since Fab-Fc bonds are rather flexible). It is noteworthy that for deposited films the transition between the two plateaus is steeper and starts at higher pressures. We presumethat the shift in the beginning of the thickness growth as well as the steeper character of the curve are due to interactions between IgG molecules and the substrate. The GOPTS-activated substrate binds lysine amino groups which are more or less uniformly distributed over the surface of the IgG molecule? When the plane of molecules begins to incline, the angle between their planes and the surface is small. On the water surface such
Consideringthe questions formulated in the introduction to this paper, one can see that experimental data are most probably in line with interpretation that IgG molecules form 2-D monolayers on the water surface. Orientation of the protein molecules changes with surface pressure. At surface pressures below 20 mNlm the molecules are positioned with their Fab-Fab-Fc plane parallel to the surface; when compressed up to 35 mNIm they turn by 90" to reach perpendicular position with respect t o the surface. Molecular orientation remains unchanged upon transfer to the solid activated substrate. The possibility of manipulating the orientation of protein molecules in monolayer on water surface and its preservation upon deposition can be regarded as an approach to fabrication of films with desired molecular orientation.
Acknowledgment. This work has been partially supported by the EL.B.A. Foundation (Portoferraio (LI), Italy). LA940320U
