Langmuir 1992,8, 1582-1586
1582
Adsorption Kinetics of Proteins onto Polymer Surfaces As Studied by the Multiple Internal Reflection Fluorescence Method? Masanori Hasegawa Department of Polymer Chemistry, Kyoto University, Kyoto 606, Japan
Hiromi Kitano* Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930, Japan Received September 10, 1991. In Final Form: March 6, 1992 Adsorption processes of proteins labeled with fluorescein (human serum albumin (FITC-HSA) and immunoglobulin G (FITC-IgG)) onto various polymer surfaces were followed by the multiple internal reflection fluorescence (MIRF) method. The FITC-proteins were adsorbed strongly onto hydrophobic polymer surfaces (poly(methy1methacrylate) (PMMA) and polystyrene (PSt)),and the proteins adsorbed were scaicely removed by rinsing with a buffer solution. The initial rate of the adsorption of FITCproteins onto various polymer surfaces showed that the proteins were adsorbed more quickly onto hydrophobic surfaces than hydrophilic surfaces (plasma-treated PMMA, for example). Even the initial rate of adsorption onto a PSt surface (the most hydrophobic materials examined here) was, however, still smaller than that calculated from the diffusion-limited rate of arrival of the protein molecule to the surface, probably due to the hydrodynamic effect and the hydration layer around the protein molecule.
Introduction Analysis of protein adsorption onto polymer surfaces is important in biotechnological and biomedical fields.lI2 Although interactions between proteins and solid surfaces have been extensively studied (adsorption isotherms, for example), only a few studies have been carried out concerning kinetics of the adsorption because of ita experimental difficulties. Chromatographic methods, such as frontal analysis3and repetitive injection analysis,4+ are useful for dynamic analyses of protein adsorption onto polymer beads which are packed into columns. These methods, however, are complicated because of many parameters such as void volume, pore size, flow rate, and so on. Therefore, kinetic analysis of adsorption of proteins onto flat surfaces is superior to the analysis by the column method. For a precise kinetic analysis of the adsorption processes, the initial stage of adsorption (within a half or 1 min after onset of the adsorption process) has to be followed. Internal reflection spectroscopy is a useful method to detect fluorescent molecules adsorbed on or located within several tens of nanometers from the ~ u r f a c e .This ~ method adopts the evanescent surface wave which is generated by the total internal reflection to excite fluorophores near an optical interface.
* To whom all correspondence should be addressed. t
Presented at the 40th Annual Meeting of the Society of Polymer
Science, Japan, at Kyoto in May 1991.
(1) Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986, 79, 1. (2)Mosbach, K., Ed. Methods in Enzymology; Academic Press: New York, 1987; Vol. 135. (3) Tashiro, Y .;Kataoka, K.; Sakurai, Y. J.Colloidlnterface Sci. 1990, 66, 140. (4) Kitano, H.; Nakamura, K.; Hirai, Y.; Kaku, T.; he, N. Biotechnol. Bioeng. 1988, 31, 547. ( 5 ) Nakamura, K.; Hirai, Y.; Kitano, H.; Ise, N. Biotechnol. Bioeng. 1987, 30, 216. (6) Hasegawa, M.; Kitano, H. Biotechnol. Bioeng. 1991, 37, 608. (7) Hirschfeld, T. Can. Spectrosc. 1965, 10, 128.
Q743-7463f92/2408-1582$03.00/0
The internal reflection method has been applied to study protein adsorption onto solid surfacess-10and antibodyantigen reaction1' in recent years. In these studies, however, only the adsorption of proteins onto a quartz plate or fused-silica plate was measured. These plates are expensive and not disposable, which makes it relatively more difficult for us to get a sufficient number of results. In addition, a flow cell system was used in the previous internal reflection studies,"l' and the kinetic analysis of protein adsorption was highly complicated. In this study, the initial stage (within several seconds after the onset of the adsorption) of protein adsorption onto a flat surface of poly(methy1 methacrylate) (PMMA) or polystyrene (PSt)was followed by the multiple internal reflection fluorescence (MIRF) method using a non-flow cell system.12 The cuvettes made of these polymers can be prepared easily and with high reproducibility.
Experimental Section Materials. Human serum albumin (HSA, fatty acid free, A3782) and human immunoglobulin G (IgG, 14506) were purchased from Sigma (St. Louis, MO). Fluorescein isothiocyanate (FITC,Art24546, Aldrich, Milwaukee, WI) was used to label the proteins. Labeling was carried out in pH 9.5 carbonate buffer (0.05 M) for 2 h at 5 O C . I 3 Free FITC was separated from fluorescein bound to the protein by gel filtration using a Sephades G-25 column (2 cm i.d. x 30 cm) with water as a mobile phase. The labeled protein was fractionated with the number of fluorescein groups per protein molecule (F/P ratio) by using (8)Lok, B. K.; Cheng, Y.; Robertson, C. R. J. Colloid Interface Sci. i9s3,91,a7. (9) Hlady, V.; Reinecke, D. R.; Andrade, J. D. J. ColloidInterface Sci. 1986,111,555. (10)Beissinger, R. L.; Leonard, E. F., ASAIO. J . 1980, 3, 160. (11)Sutherland, R. M.;Dihne, C.; Place, J. F.; Ringrose, A. S. Clin. Chem. 1984, 30, 1533. (12)Harrick, N. J.; Loeb, G. I. Anal. Chem. 1973, 45,687.
0 1992 American Chemical Society
Adsorption Kinetics of Proteins onto Polymer Surfaces
Figure 1. Schematic of the MIRF method 0,FITC-protein, --,fluorescence, A, optical wave guide; B, dichroic mirror; C, cutoff filter. ion exchange chromatography (DEAE-cellulose, 2 cm i.d. X 30 cm).l3 HSA of F/P = 1and IgG of F/P = 2.4 were used in this work. Apparatus for MIRF. The MIRF apparatus is illustrated in Figure 1. The light source was a 10-mWargon laser (LAJ2223, Toshiba Electronics, Tokyo, Japan) operated at 488.0 nm. The excitation beam is introduced into the wave guide through the prism. The light beam strikes the interface between the wave guide and sample solution at an angle (8) of 80° and is reflected at the interface. When the light is reflected,an evanescent surface wave is generated and penetrates into the sample solutionbeyond the interface. The penetration depth (d,) of the evanescent wave calculated from eq 114is 1500 A
where nl and n2 are the refractive indices of the two media, 8 is the angle of the incident light beam, and X is the wavelength of the incident light. The excitation beam is emitted inside of the wave guide with multiple reflections. When a FITC-protein is adsorbed onto the surface of the wave guide, fluorescein is excited by the evanescent wave and emits fluorescence. The fluorescence moves back to the opposite direction of the excitation beam and comes out the wave guide through the prism. The fluorescence is separated from the excitation beam using a dichroic mirror and a cutoff filter (R515, Schott) and is finally detected by a photomultiplier (R-1547, Hamamatsu Photonics, Hamamatsu, Japan). A cuvette for the MIRF method consists of two parts, a wave guide and a cover. The wave guide with prisms is made of poly(methyl methacrylate) (PMMA)or polystyrene (St). These parts are wed only once. The wave guides have an intrinsic background signal due to the surface properties of the wave guide.16 The relative fluorescence intensity corresponding to the background signal of the wave guides used here was 15 f 1, and we could assume that surface properties of the wave guides were constant. The surface area of the wave guide with which the sample solution can be in contact is 4 cm2(1X 4 cm). The cover part with porta for injection of the sample solutions is made of stainless steel. A silicone rubber gasket is attached to a stainless steel support to seal two parts. The totalthickness of the cell is about 0.5 mm, and the total cell volume including the injection ports is about 0.25 mL. The path of a fluorescence signal through the cutoff filter is detected by the photomultiplier and the fluorescence intensity was integrated for 1s. A monochromatorreference is alsodetected and the difference between the intensities of the referencesignal and the fluorescence signal is calculated with a microcomputer (5-3100 GT, Toshiba Electronics, Tokyo, Japan). Assay Procedure. The wave guide of the MIRF was washed with a soak solution, rinsed with deionized water, and treated with a halogenated hydrocarbon, Daiflon 5-3 (Daikin Industries, Osaka, Japan), before use. The cover part was washed several times with deionized water using an ultrasonicater (UT-204, Sharp, Tokyo, Japan). The sample solutions were prepared with a 0.01 M acetate buffer (pH 3.8, 4.5, and L O ) , 0.01 M phosphate buffer (pH 5.5 (13) Maeda, H.; Ishida, N.; Kawauchi, H.; Tuzimura, K. J . Biochem. 1969,135,777.
(14) Born, M.; Wolf, E. Principles of Optics, 5th ed.; Pergamon: New York, 1975. (15) Rockhold, S. A.; Quinn, R. D.; Van Wagenen, R. A,; Andrade, J. D.; Reichert, M. J . Electroanal. Chem. 1983, 150, 261.
Langmuir, Vol. 8, No.6,1992 1583 and 6.0), 0.01 M HEPES (N-(2-hydroxyethyl)piperazine-N’-2ethanesulfonic acid) buffer (pH 7.0 and 8.0), and a CHES (2(cyc1ohexylamino)ethanesulfonic acid) buffer (pH 9.0). The ionic strength of the buffer solutions was adjusted to 0.01 with NaCl. The sample solution (0.25 mL) was injected into the cell (without pretreatment) by an autopipetter (Microelectropette, Matrix Technologies, Lowell, MA) to keep the injection speed constant. Calibration Experiment. For calibration experiments, the surface concentration of proteins on the MIRF cell was determined as follows: The protein solution of various concentrations (1-20 pg/mL) was injected into the MIRF cell. The protein concentration of the solution injected and that recovered from the cell 600 s after the injection was immediately estimated from fluorescence intensity at 520 nm (excitation 480 nm) using a fluorescence spectorophotometer (F-410,Hitachi, Tokyo,Japan). The surfaceconcentration of the protein (C,) could be determined from the difference of the concentrations before and after the injection. Plasma Treatment. The PMMA cuvettes for the MIRF method were exposed to oxidative plasma using a glow discharge reactor (LCVD 19,Shimadzu, Kyoto, Japan).16 The pressure in the reaction chamber was reduced to lW3 Torr, followed by introduction of Ar into the chamber. The pressure in the chamber was kept at about 0.1 Torr afterward. Plasma was generated at a power of 30 W and the cuvettes were exposed to plasma for a predetermined period of time. Contact Angle Measurement. Static contact angles, OH*, of water on the surface of cuvettes were measured at 23 OC and 75% relative humidity by the sessile drop method (CA-D, Kyowa Kagaku, Tokyo,Japan).lo The 8H$J was determined 10times to obtain a reliable average value. An uncertainty of the contact angles was *3O. ATR-FTIR Measurement. In order to obtain information on the surface of the cuvettes exposed to plasma, attenuated totalreflection Fourier transform IR (ATR-FTIR)was measured by using a Perkin-Elmer FT-IR spectrometer, Model 1760X. Thallium bromide was used as the prism of the ATR-FTIR. Results a n d Discussion A. Reliability of Fluorescence Signals. At first, we examined the contribution of fluorescence of the bulk solution to the total MIRF signal. The total MIRF fluorescence intensity may involve the fluorescence from adsorbed proteins and the background signals from the bulk protein s o l ~ t i o n .The ~ background signal has two sources: the one occurring from the protein molecules above the cuvette excited by the evanescent wave, and the other due to the fluorophore excited by the light scattered a t the interface between the wave guide and sample s o l ~ t i o n .To ~ evaluate the actual surface concentration of the proteins, subtraction of the contribution of the background signals from the total MIRF signal has to be carried out. The contribution of the background signals to the total MIRF signal can be distinguished from an immediate decrease of fluorescence intensity after the replacement of the protein solution in the cell with the buffer s01ution.l~ Curve A in Figure 2 shows the typical profile of the MIRF signal on the injection of the FITC-HSA into the PMMA cell. The MIRF signal increased immediately after injection of the sample solution. Curves B and C in Figure 2 show the effect of replacement of the protein solution in the cell by either buffer solution or nonlabeled protein solution ca. 200 s after the injection. The sample solutions in the cell were removed a t point a in Figure 2B,C by an aspirator. Then 3 mL of buffer solution was allowed to flow into the cell, and the cell was fully aspirated again. At the point b in Figure 2B,C, 0.25 mL of buffer or nonlabeled protein solution was injected into the cell. No (16) Suzuki,M.; Kishida,A.;Iwata, H.; Ikada, Y. Macromolecules 1986, 19, 1804.
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Figure 3. Time dependence of the fluorescence intensity: [FITC-HSA]o = 30 pg/mL, pH 7.0,O.Ol M HEPES buffer (I= 0.01 M). (A) The same data as Figure 2A. (B)The cuvette had been incubated with nonlabeled HSA solution for 10 min beforehand.
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Figure 4. Plots of surface concentration of FITC-HSA VB fluorescence intensity observed at 23 O C and pH 7.0, 0.01 M HEPES buffer (I= 0.01 M). 0
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Figure 2. Time dependence of the fluorescence intensity: [FITC-HSA]o = 30 pg/mL, pH 7.0,O.Ol M HEPES buffer (I= 0.01 M). (A) At t = 10s,a FITC-HSA solution was injected. (B) At arrow a, a FITC-HSA solution was removed and at arrow b a buffer solution was injected. (C) At arrow a, a FITC-HSA solution was removed and at arrow b a solution of HSA without FITC was injected. significant decrease of the intensity was observed after replacement of the FITC-HSA solution. These results show that the contribution of the bulk protein solution to the total MIRF signal is negligibly small and the desorption and exchange of the protein adsorbed on the PMMA cell surface are negligible. In addition, we measured the MIRF signal from the cell surface which had been precoated with the nonlabeled proteins to reconfirm the absence of the contribution of fluorescence of the bulk solution to the total MIRF signal. Curve B in Figure 3 shows the intensity change after injection of the FITC-HSA solution into the cell which had been incubated with the nonlabeled protein solution (250 pg/mL) for 10 min just before the injection. The signals obtained here were negligibly small in comparison with the signal obtained from the cell without precoating (curve A). No more proteins could be adsorbed onto the cell which had been fully covered with the nonlabeled proteins, and the total MIRF fluorescence intensity involved only the fluorescence from the protein molecules adsorbed onto the cell surface. In this study, therefore, the total MIRF signals were not corrected by the background signal. B. Adsorption of FITC-HSA. Figure 4 shows the relationship between the surface concentration (C,)of FITC-HSA and the total MIRF intensity. The total MIRF
t
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W A I (MW Figure 5. Initial adsorption rate (dCJdt) of FITC-HSA onto PMMA surface at 23 "C and pH 7.0, 0.01 M HEPES buffer (I = 0.01 MI. intensity was linearly related to the surface concentration of the protein. From the initial slope of the increase in fluorescence as exemplified in Figure 2A, we could evaluate the initial adsorption rate of proteins onto the polymer surface by using the linear relationship in Figure 4. Figure 5 shows the dependence of the initial adsorption rate on the initial solution concentration (CO) for FITCHSA. The initial adsorption rate (dC,/dt) increased linearly with an increase in CO. When the adsorption is 'diffusion-limited" we could calculate the theoretical adsorption rate (dC,/dtth,,,) from eq 217 dC,/dttheo, = C0(D/7rt)'.' where D is the diffusion coefficient of HSA calculated . ' ~ 6shows from Perrin's relation (6.8 X lo-' ~ m ~ / s ) Figure a linear relationship between dCddt and (evaluated from the data in Figure 21, which suggests that the protein adsorption in this study is 'diffusion-limited". The ratio of (dC,/dt)/(dC,/dttheo,) ( = R ) reflects the probability of (17)MacRitchie, F. Adu. Protein Chem. 1978, 32,283. (18) Wallevik, K. Biochim. Biophys. Acta 1973, 322,75. (19) Perrin, F. J . Phys. Radium 1936, 7 , 1.
Adsorption Kinetics of Proteins onto Polymer Surfaces
Langmuir, Vol. 8, No. 6, 1992 1585 Table I. Effects of Plasma Exposure Time on Contact Angle and R Value
plasma exposure time," s
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Figure 6. Plots of adsorption rate (dCJdt) of FITC-HSA vs l/t0.6. The same data as Figure 2A.
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Figure 7. pH dependence of the value of R for FITC-HSA at 23 OC: cuvette, PMMA; [FITC-HSA]0,30 &mL. Buffer: pH 3.8, 4.5, and 5.0,O.Ol M acetate buffer; pH 5.5 and 6.0, 0.01 M phosphate buffer; pH 7.0 and 8.0,O.Ol M HEPES buffer; pH 9.0, 0.01 M CHES buffer. Ionic strength of each buffer waa adjusted to 0.01 by NaCl. cu E
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Under 0.1 Torr Ar at 30 W. * Static contact angle of HzO. At pH 7.0, 23 O C , [FITC-HSAIo = 30 pg/mL. 1.0
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Figure 8. Effect of ionic strength on the value of dCJdt for FITC-HSA at 23 OC and pH 7.0 cuvette, PMMA; [FITC-HSA'J0, 30 kg/mL; buffer, pH 7.0,O.Ol M HEPES buffer. Ionic strength was adjusted by NaCl. irreversible binding by a collision of the protein molecule to the polymer surface. Figure 7 shows the plots of the value of R vs pH. The R value had a maximum near the PI of HSA (4.&5.3),8 where HSA is the most hydrophobic. The R value obtained here, however, was always smaller than unity. A similar tendency (the experimental adsorption rate is smaller than that for theoretical "diffusion-limited" adsorption) has been reported frequently.20 The small R value has been attributed to some barrier which the protein molecules have to break before they are adsorbed onto the surface.21 The barrier has been considered to be caused by electrostatic repulsion between the surface and the proteins. The PMMA surface used here may be slightly negatively charged because some of the PMMA residues are usually hydrolyzed to methacrylic acid. The R value, however, decreased with a decrease in pH below the PI, where HSA is positively charged, which suggests that the contribution of the electrostatic repulsion on the barrier is not so large under the present experimental conditions. Figure 8 shows the effect of ionic strength (0.01-0.1 M) on the adsorption rate a t pH 7.0. No significant change (20) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988,125,246. (21) van Dulm, P.; Norde, W. J. Colloid Interface Sci. 1983, 91,248.
Figure 9. pH dependence of the value of R for FITC-IgG at 23 OC: cuvette, PMMA; [FITC-IgG10,10 rglmL. Other conditions were the same as given in Figure 7.
was observed a t a higher ionic strength (0.1 M) where the electrostatic repulsion may be shielded, which supports the negligible role of electrostatic repulsion described above. Another possibility of the barrier of the protein adsorption is a hydrodynamic effect between polymer surfaces and protein molecules, that is, the resistance of bulk water against approaching protein molecules to the polymer surface.22 In addition, protein molecules are solvated by many water molecules, and for the irreversible adsorption of the protein molecules onto hydrophobic polymer surfaces, water molecules around the protein molecules have to be moved away, which might consume energy. C. Importance of Hydrophobic Interaction on Adsorption of Proteins. We followed the adsorption of FITC-HSA onto a more hydrophobic surface than PMMA (8&0 = 72O). The R evaluated from the adsorption experiment of the FITC-HSA ontoPSt (8&0 = 93') surface was lager than that onto PMMA surface and relatively closer to unity ( R = 0.55 at pH 7.0 and 23 OC, under the same conditions the value of R for a PMMA cuvette was 0.41). Furthermore, the adsorption of FITC-HSA onto a more hydrophilic surface, plasma-treated PMMA, occurred much more slowly than that onto the nontreated PMMA surface (Table I). The ATR-FTIR data showed that free carboxyl groups on the PMMA surfaces appeared with the plasma treatment. It is well-known that methyl ester groups on the PMMA surface are hydrolyzed to carboxyl groups by plasma treatment under Ar gas,23which largely decreases the hydrophobicityof the PMMA surface. These results suggest the importance of hydrophobic interaction for the adsorption of proteins to polymer surfaces. Figure 9 shows the R value estimated for IgG at various pH values. The absence of a maximum in the pH dependence of the R value for IgG (PI = E~8-7.3:~which is (22) Adamczyk, Z.; Adamczyk, M.; van de Ven, T. G. M. J. Colloid Interface Sci. 1983, 96,204. (23) Salvati, L., Jr.; Hook, T. J.; Gardella, J. A,, Jr. Polym. Eng. Sci. 1987, 27, 939. (24) Schultze, H. F.; Hermans, J. F. Molecular Biology of Human
Proteins with Special Reference to Plasma Proteins I; Elsevier: Amsterdam, 1966.
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more hydrophobic than HSA, might be due to a selfaggregation of IgG molecules near PI (by the complete dimerization, for example, the R value would decrease to 0.89 of that for a monomer system). The R value, therefore, would slightly decrease near PI. adsorption In literature citations of the batch isotherms of protein molecules to polymer surfaces very frequently showed that hydrophobic interaction between protein molecules and polymer surfaces is influential to the amount of proteins; however, this has not been estimated precisely due to experimental difficulties. The findings obtained in this work indicated that the hydrophobicity of the polymer surfaces onto which proteins are adsorbed strongly affects the initial adsorption rate of the protein molecules. (25)Brash, J. L.;Uniyal, S.J. Polym. Sci., Polym. Symp. 1979,66,377.
Hasegawa and Kitano
Acknowledgment. We thank Professor Norio Ise, Department of Polymer Chemistry, Kyoto University, for his encouragement and helpful suggestions throughout this work. We are indebted to Drs. T. Matsuda, H. Iwata, and A. Kishida a t the Research Institute of National Cardiovascular Center, Suita, Osaka, Japan, for allowing us to carry out the plasma treatment and contact angle measurements. We are grateful to Daikin Industries, Osaka, Japan, for their support to carry out the evanescent wave experiments. This work was supported by a Grantin-Aid (03236225) from the Ministry of Education, Science and Culture, Japan.
Registry No. PMMA, 9011-14-7; PSt, 9003-53-6.
