Langmuir 1995,11, 2272-2276
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Photochemical Protein Fixation on Polymer Surfaces via Derivatized Phenyl Azido Group Takehisa Matsuda* and Takashi Sugawara Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565, Japan Received July 18, 1994@ This paper addresses a novel surface processing technology aimed at chemical fixation of proteins on substrate surfaces with micron-order precision that is based on the photochemical reactivity of a phenyl azido group derivatizedeither to a protein on a polymer surface. Phenyl azido-derivatizedproteins (albumin and gelatin) and synthetic polymers (poly(3-azidostyrene) and poly(N,N-dimethylacrylamide-co-3azidostyrene) (IV))were prepared. The adsorption of these photoreactive proteins onto polymer surfaces or nonmodified protein onto a poly(3-azidostyrene)-precoatedsurfacefrom their respective aqueous solutions and subsequent U V irradiation were performed. The chemicalfuration of proteins was verified with surface elemental analysis by electron spectroscopy for chemical analysis and by colorimetric staining using corresponding antibodies. Atomic force microscopic observations showed that this photoinduced protein fixation method allows us to photochemicallyimmobilize a protein on a polymer surface with micron-order precision in a given region.
Introduction Recently, a large number of surface modification methods have been reported to provide a designed surface property where the surface property imparts a decisive role in functioning. For instance, in the field of biochemical or medical technology, biologicallyimportant components heparin for nonthromsuch as enzymes for diagn~sis,l-~ b o g e n i ~ i t y ,and ~ , ~ collagen for cell adhesive matrix have been physically coated or chemically immobilized onto the device surface. Since physically adsorbed components gradually desorb from the polymer surfaces in an aqueous environment, chemical fixation of proteins onto the polymer surface via covalent linkage is essential to ensure its durability. In order to achieve covalent bonding between a protein and a polymer surface, the currently available methods use condensation reactions at watedpolymer interfaces. 1-7 In these cases, active hydrogen-containing functional groups such as amino, carboxyl, hydroxyl, or mercapto groups must exist in the outermost layer of polymer surfaces for chemical bonds with proteins. In addition, chemical fixation with micron-order precision at a given region is essentially difficult by these methods. Our previous paperg described a novel protein fixation method based on the surface photocleavage reaction of o-nitrobenzyl ester which generates a carboxyl group upon UV irradiation. Proteins were chemically fixed with the aid of water-soluble condensation reagents by the condensation reaction between the newly generated carboxyl group at the surface and amino groups of proteins. This method allowed us to immobilize proteins only at a given region of the photoirradiated surface. @
Abstract published in Advance ACS Abstracts, January 15,
1995. (1)Zabonsky, O., Ed. Immobilized Enzymes; CRC Press, Cleveland, OH, 1972. (2)Goldstein, L. Biochim. Biophys. Acta 1973,315,1. (3)Briimmer, W.; Hennrich, N.; Klockow, M.; Lang, H.; Orch, H. D. Eur. J . Biochem. 1972,25,129. (4)Patel, A. B.;Pennington, S. N.; Brown, H. D. Biochim. Biophys. Acta 1969,178,626. (5)Minamoto, Y.; Yugari, Y. Biothechnol. Bioeng. 1980,22,1225. (6)Stanley,W. L.; Watters, G. G.; Chan, B.; Mercer, J. M.Biothechnol. Bioeng. 1975,17,315. (7)Boudrant, J.; Chef'tel, C. Biothechnol. Bioeng. 1975,17,827. ( 8 )Tanzawa,T. Trans.Am. Soc.Artif. Intern. Organs 1973,70,2534. (9)Matsuda, T.; Sugawara, T. Langmuir 1995,11,2267,preceding paper in this issue.
UN3 \ProteinAdsorption
UV Irradiation
/
Figure 1. Schemeof the photochemical protein immobilization method developed here.
In this paper, our interest in the microchemical fixation of proteins was extended to utilize photoreactivity of the phenyl azido group which is incorporated into either the protein or polymer surface. This functional group is easily photolyzed and generates a highly reactive intermediate, phenyl nitrene,lo-l2 which can react with neighboring atoms to form a covalent bond. Therefore, introduction of the phenyl azido group onto either proteins or polymer surfaces is expected to lead to the photochemical immobilization of proteins, as schematically shown in Figure 1. In fact, we reported that the phenyl azido-derivatized polymers can be photochemically fixed on material surfaces upon coating and subsequent UV irradiation.13-19 In addition, these methods will permit regional dimensional control of the protein-fixed region with micronorder precision, in principle, because the photochemical reaction occurs only at a UV-irradiated region. Experimental Section Materials. All solventsand reagents were of the highest grade
available. Tetrahydrofuran(THF),isopropyl alcohol, isopropyl ether, NJV-dimethylformamide (DMF),and chloroform were (10)Abramobitch, R. A.;
Davis, B. A. Chem. Rev. 1964,64,149. (11)L'Abbe, G.Chem. Rev. 1969,69,345. (12)Patai, S.,Ed. The Chemistry of Azido Group; Interscience Publishers: London, 1971. (13)Matsuda, T.; Inoue, K.; Sugawara, T. Trans. Am. SOC.Artif. Intern. Organs 1990,36(31,M559-563. (14)Matsuda, T.;Inoue, K.; Ozeki,E.; Akutsu, T.Artif. Organs 1990, 14 (31,74. (15)Matsuda, T.; Inoue, K. Trans. Am. SOC.Artif. Intern. Organs 1990,36,161. (16)Matsuda, T.; Inoue, K.; Akutsu, T. Jpn. J . Artif. Organs 1990, 19 (31,1177. (17)Matsuda, T.; Inoue, IC;Akutsu, T. Jpn. J . Artif. 1990, . Organs 19,1143. (18)Matsuda, T.; Sugawara, T. ASAIO J . 1992,38,M243. (19)Sugawara, T.; Matsuda, T. Jpn. J . Artif. Organs 1992,21(11, 186.
0743-7463/95/2411-2272$09.00/0 1995 American Chemical Society
Protein Fixation on Polymer Surfaces purchased from Wako Pure Chemical Industry Ltd. (Kyoto, Japan). These were used without further purification except for treatment with molecular sieves. Poly(viny1alcohol)(PVA)film was donated by Nitigo Film Ltd. (Osaka, Japan). Poly(ethy1ene terephthalate) (PET) was obtained from Bellco Glass Inc. 2,2'-Azobis(isobutyronitrile) (AIBN), NJV-dimethylacrylamide (DMAAm), 4-azidobenzoic acid, and dicyclohexylcarbodiimide (DCC)were purchased from Tokyo Kasei Organic Chemicals Co. (Tokyo,Japan). N-Hydroxysuccinimidewas a product ofProtein Institute Inc. (Minoh, Japan). Seamless cellulose tubes for dialysis (cutoff MW of ca. 12 000) were purchased from Viskase Sales Corp. Bovine serum albumin (fraction V) was obtained from Sigma Chemical Co. (St.Louis, MO) and IgGfractionrabbit anti-bovine albumin was obtained from Organon Teknika Co. Vectastain ABC-AP Kit was obtained from Vector Laboratories, Inc. Bovine bone gelatin was obtained from Wako Pure Chemical Industry Ltd. Preparation of Photoreactive Compounds and Polymers. All treatments were carried out in the dark. Preparation of N-((4-Azidobenzoyl)oxy)succinimide (I):A solution of DCC (13.3 g, 64.6 mmol) in THF (50 mL) was added dropwise into a solution of N-hydroxysuccinimide (7.43 g, 64.6 mmol) and 4-azidobenzoic acid (9.57 g, 58.7 mmol) in THF (150 mL) cooled in an ice bath with stirring. After 3 h, the reaction mixture was warmed slowly to room temperature and stirring was continued for one night. A white solid was formed and was filtered o f fand the solvent was removed under reduced pressure. The yellow residue obtained was crystallized from isopropyl alcohoVisopropy1 ether: yield, 5.66 g (41%);270-MHz 'H-NMR [(CD&SO; ppm from (CH3)4Sil 6 7-8 (4H, m, phenyl), and 2.7 (4H,t, CH2). Compound I was used for the preparation of phenyl azide-derivatized proteins. Preparation of Photoreactive Polymers. Preparation of 3-azidostyrene (11)and poly(3-azidostyrene) (111)is described in our previous paper.g Copolymer IV,poly(NJV-dimethylacrylamideco-3-azidostyrene), was obtained from the radical copolymerization of NJV-dimethylacrylamide (450 mg, 4.5 "01) with 3-azidostyrene (50 mg, 0.5 mmol) in the presence of AIBN (7.4 mol) as an initiator in DMF. After 20-h mg, 4.5 x polymerization, the solvent was removed under reduced pressure. Deionized water was added to the residue and the solution was dialyzed for 3 days. The product was then freeze-dried. A white solid was obtained: yield, 274 mg (55%);IR 2113 cm-l (v(N3)). The composition of this copolymer,NJV-dimethylacrylamide/3azidostyrene = 0.93/0.07 by molar ratio, was determined by elemental analysis: Found: C, 56.76; H, 8.77; N, 13.24. The sample of this copolymer contains 43 mol % water. Preparation of Photoreactive Proteins. To a solution of bovine serum albumin (200 mg) in phosphate-buffered saline (PBS; pH 7.4, 100 mL) was added compound I (450 mg, 1.5mmol) under stirringwith coolingin an ice bath. The solution was continuously stirred overnight at 4 "C. After filtration, the reaction mixture after filtration was subjected to dialysis usinga seamless cellulose tube in deionized water for 3 days. After it was freeze-dried under vacuum, a while solid was obtained: yield, 54 mg. The derivatized BSA showed a characteristic IR band at 2132 cm-l ascribed t o the azido group. Preparation of phenyl azidoderivatized gelatin was carried out according to the same method as described above. The derivatization of gelatin was confirmed by IR spectra with the characteristic band a t 2127 cm-l ascribed to the azido group. Photochemical Fixation. (I) Method via Photoreactive Protein. An aqueous solution of phenyl azido-derivatizedalbumin (5 wt %, 1mL) was spread on a PVA film (dimensions, 20 x 20 mm; thickness, 25 pm). After 1h of incubation, the unadsorbed proteins were sucked off with a Pasteur pipet. After air-drying, the film was UV irradiated for 40 s using a Toshiba UV lamp (H-400P; 400 W) at a distance of 30 cm (intensity, 2.2 mW/cm2). The treated surface was washed several times with 1.0 M NaCl aqueous solution and PBS. The samples for electron spectroscopy for chemical analysis (ESCA)were further washed with deionized water. A micron-order protein array was prepared as follows. Copolymer (IV)was applied in a thin layer and photochemically fixed on the surface of a Corning tissue culture dish. An aqueous solution of phenyl azido-derivatized albumin (5 wt %, 1mL) was spread on the surface. Subsequently, the unadsorbed protein
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was sucked off with a Pasteur pipet. The photomask, which has a pattern consisting of an array of slits 0.50 mm and 0.35 mm in width, was then placed on the surface. The surface was U V irradiated for 40 s, and unfured protein was thoroughly removed with 1.0 M NaCl aqueous solution and PBS. Microfuration of photoreactive BSA was verified with staining of the fixed proteins using avectastain ABC-APkit, which made it possible to visualize the protein-fmed area under a light microscope. The staining was performed in accordance with the manufacturer's instructions. Briefly, the first step is treatment with a buffer solution containing mouse monoclonal antibody. After a biotinylated secondary antibody containing buffer solution is applied, avidin DH and biotinylated alkaline phosphatase H-mixed solutions were subsequently added to the surface. Alkaline phosphatase substrate was applied. The colorimetric enzyme-substrate reaction made it possible to visualize the protein-fixed region. Photoreactive gelatin was treated in the same manner as the photoreactive BSA described above. Van Gieson (dye; a mixture of picric acid and acid fuchsine) staining was used for photochemically fmed gelatin. (ZI) Method via Photoreactive Surface. A chloroform solution of photoreactive polymer (111)(1.0 wt %) was cast on a PET film (20 x 20 mm; thickness, 50 pm). An aqueous solution of BSA (5 wt %) was cast on the surface. Subsequently, after air-drying, the sample was W irradiated as in the method described above. Physical Measurments. FT-IR spectra were obtained on Nicolet 5DX. lH-NMR spectra were recorded with a JEOL GX270 (Tokyo, Japan) [in (CD3)zSO a t 30 "C; ppm from (CH3)4Sil instrument. ESCA spectra were recorded on Shimadzu ESCA 750 (Kyoto, Japan) instrument. The Cls spectra were deconvoluted into subpeaks by computer-aided processing. Atomic force microscope (AFM)images were obtained on NanoScope I1 (Digital Instruments Co. Ltd.; Santa Barbara, CA) using 200pm cantilevers (narrow-leg type) with a calculated force constant of 0.06 N/m, operating a t -1.0 V setpoint voltage and scanning rate of 1.34 Hz. All the data manipulations and image processing were carried out with Digital Instruments software. Microscopic observation was carried out on an Olympus Vanox-S system (Tokyo, Japan).
Results The fundamental concept of the newly devised surface protein immobilization method, which can permit the regional control of the protein-fxed site on a twodimensional surface, is based on photochemical reactivity of the phenyl azido group. A salient feature of the photochemistry of the phenyl azido group is the facile elimination of molecular nitrogen from the azido group upon UV irradiation and the formation of phenyl nitrene.lgT21 The photochemically generated phenyl nitrene, which is a highly reactive species in a triplet state, undergoes formation of a covalent bond with neighboring atoms. If such a reaction effectively takes place a t a proteidsurface interface, a protein can be chemically fixed only at a photoirradiated region. In order to fix proteins only a t a given part of the surface, two approaches, both utilizing the photoreactivity of the phenyl azido group, were developed, as shown in Figure 1. One is v i a photoreactive group-derivatized proteins. The other is surface modification with a photoreactive group. Irrespective of the approach, proteins, which are preadsorbed on a surface, are chemically fixed upon W irradiation. The photochemical fixation of proteins was verified by ESCA spectra and a colorimetric staining method as follows. Preparation of Photoreactive Proteins and Polymers. Phenyl azido derivatization of bovine serum albumin and bovine bone gelatin was carried out by the (20) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Arbrecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. K. Science 1969,243,1586. (21) Lin, J. N.; Drake, B. A.; Lee, S.; Hansma, P. K.; Andrade, J. D. Langmuir 1990,6,509.
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Bindii Energy (eV)
Figure 2. ESCA spectra of (a) nontreated PVA film surface and (b) phenyl azido-derivatized BSA-fixed surface.
condensation reaction of amino groups of proteins with the active ester-containing photoreactive compound (I)in a buffered solution a t low temperature (4 "C). The derivatization of two proteins was confirmed by the characteristic IR absorption peak of azido group a t around 2130 cm-l. Poly(3-azidostyrene) (111) and poly(NJVdimethylacrylamide-co-3-azidostyrene)(IV)were prepared by radical polymerization or copolymerization of 3-azidostyrene with NJV-dimethylacrylamide, respectively. The latter photoreactive copolymer contains 7 mol % of the phenyl azido group per molecule. Protein Fixation via Photoreactive Proteins. Photochemical fixation of photoreactive BSA onto the poly(vinyl alcohol) (PVA) surface was demonstrated. First, BSA was adsorbed on the PVA film by casting and airdrying of an aqueous solution of photoreactive BSA. After W irradiation, the treated surface was extensively washed with a highly concentrated salt aqueous solution (1.0 M NaC1) and then with deionized water. ESCA spectra were recorded to verify the photochemical fixation of phenyl azido-derivatized BSA onto the PVA surface. Figure 2 shows ESCA spectra of nontreated PVA and photochemically treated surfaces. Marked differences in the surface elemental ratio and subpopulations of C l s spectra between these spectra were observed. The Nls/ C l s peak area ratio (corrected for the total relative sensitivity) of the photoreactive BSA-treated surface was 0.12,whereas that of the nontreated PVA surface was
Matsuda and Sugawara negligibly small (below 0.01). The relative fraction of the C l s subpeak in the binding energy region of 287-288 eV, which was assignable to the carbonyl carbon, was 0.03 for the nontreated PVA surface and 0.08 for the photoreactive BSA-treated surface. Even upon vigorous washing with PBS and deionized water, little appreciable difference in NIC ratio and C l s subfractions was observed for the photochemically treated surface. On the other hand, physically adsorbed photoreactive BSA on the PVA surface was scarcely left by washing, which was evidenced by ESCA spectra. These results indicate that photoreactive BSA is chemically fmed onto the PVA surface. Since photochemical reaction of phenyl azido-derivatized proteins occurs only a t W-irradiated parts, regionally restricted surface fmation of proteins and the formation of a proteinaceous molecular array can be easily carried out as follows. After protein adsorption, a photomask with a multiple-stripe pattern (width of split, 0.50 and 0.35 mm) was placed on the surface. After W irradiation, unfixed protein was extensively washed with a highly concentrated salt aqueous solution and PBS. The photochemically fixed protein was stained with the enzyme-labeled antibody technique using a chromogenic substrate. Figure 3 shows the micrographsof the stained surface. The linewidths of the stained part were 0.50 and 0.35mm, respectively, which were identical with those of the slits of the photomask. Regionally restricted fmation of photoreactive gelatin was carried out similarly to the method used for photoreactive BSA. Very fine glass photomasks with a multiple metal-coated circular pattern (internal diameter (a) 120 pm, (b) 60pm, and (c) 20 pm) were used. Micrographs of the treated surface stained by the Van Gieson method are shown in Figure 4. Only the outer part of the circle was colored red, indicating that a photoreactive gelatin-based proteinaceous pattern was obtained with micron-order precision. Protein Fixation uia Photoreactive Surface. An alternative method of protein immobilization is the surface derivatization of the photoreactive center, followed by protein adsorption and subsequent W irradiation, as mentioned above. Poly(3-azidostyrene)(111)was applied in a thin layer to the PET surface. After BSA adsorption, W irradiation was carried out. ESCA analysis indicates that nonreactive BSA was chemically fmed on the surface.
Figure 3. Micrographs of (a) photomask and (b) chemically albuminated micropattern on poly(NJV-dimethylacrylamide-co-3azidostyrene)(IV)photochemically prefured surface. The photoreactive BSA was stained by enzyme-labeled antibody techniques using a Vectastain ABC-AP kit, which was obtained from Vektor Laboratories, Inc.
Protein Fixation on Polymer Surfaces
Langmuir, Vol. 11, No. 6, 1995 2275
-
Nlnoscopr I 1 Par d m t r r s :
1m.o Am P4.1 b l U
Safroles
XU/tcen
Figure 6. 1 x 1pea AFM image showing the boundary region of the irradiated part (chemically gelatinated surface) and nonirradiated part (poly(N,ZV-dimethylacrylamide-co-3-azidostyrene) (nr)photochemically fixed surface). The maximum height of the protein-fixed layer is ca. 3 pm.
_-
_ _ _ _ _ _ _ _ _
~
Figure4. Micrographs of chemically gelatinated micropattern (internal diameter, (a) 120 pm, (b) 60 pm, and (c) 20 pm)on poly(NJV-dimethylacrylamide-co-3-azidostyrene)(TV) photochemicallyfixed surface. The photoreactive gelatin was stained by Van Gieson method.
Figure 6. Micrographs of BSA chemically fixed micropattern
Regional fixation of BSA on the photoreactive surface was carried out by using a photomask that had a parallel array of hexagonal patterns (line width, 130 pm) before UV irradiation. The patterns of photochemically fixed BSA, stained with the enzyme-labeled antibodytechnique, are shown in Figure 5. Due to the hydrophobic nature of the substrate surface, nonspecific absorption cannot be completely circumvented. Atomic Force Microscopic Image. It is of interest how precisely the method developed here permits regional protein fixation in terms of dimension. The atomic force microscope (AFM) is available for analyzing the surface topographyin angstroms to nanometer order and has been used to obtain atomic-resolution images of synthetic and biological polymers such as polyalanine and fibrin.22*23
AFM images were taken of photoreactive gelatin which was photochemically fixed on a poly(NJV-dimethylacrylamide-co-3-azidostyrene) (rv)precoated PET surface, which significantly reduced nonspecific adsorption of protein due to its hydrophilic surface nature. The AFM images, after the low path filtering treatment with the NanoScope I1 software, are shown in Figure 6. The nonirradiated surface was quite flat; roughness was within several tenths of a nanometer. The borderline between nonirradiated and irradiated regions was relatively linear. The maximum thickness of the photoreactive gelatin-fixed layer was approximately 3 pm.
(22) Yeung, C. W. T.; Moule, M. L.; Yip, C. C. Biochemistry 1980,19, 2196.
(23)Synder, U. IC;Thompson, J. F.; Landy, A Nutum 1989,341, 255.
on poly(3-azidostyrene)-precoatedsurface.
Discussion Avariety of methods have been developed to immobilize biologically important components such as enzymes, peptides, and polysaccharides onto polymer surfaces. One of the most conventional methods is to utilize the condensation reactions between functional groups of a
2276 Langmuir, Vol. 11, No. 6, 1995
biosubstance and a polymer surface in aqueous solutions. However, these methods have considerable limitations in terms of availability of the surface functional group and dimensional precision. In this paper, a photoinduced protein-fixation method utilizing a photoreactive phenyl azido group was developed to chemically fur proteins onto polymer surfaces; it is essentially free from the limitations mentioned above. The alkyl or aryl azido group is easily photolyzed to generate nitrene upon W irradiation. The photogenerated nitrene is highly reactive and is capable of forming a covalent bond with neighboring atoms.1°-12 Aryl azides including phenyl azide are more thermally and chemically stable than alkyl or acyl azide. By virtue of its photoreactivity and stability, ((azidobenzoy1)oxy)succinimide and related heterobifunctional compounds have been used for photoaffinity labeling of proteins, by which active sites in ligand-receptor, enzyme-substrate, and antigenantibody couples have been explored. For example, photoreactive aryl azide derivatives of insulin were prepared and were used to label an insulin receptor protein in the cellular membrane.22 We utilized this heterobifunctional compound to derivatize the phenyl azido group into a protein and developed a photoinduced protein surface-fixationmethod which permits micron-order regional precision. Upon mixing with a protein, an activated ester group can specifically react with the primary amino group ofproteins, resulting in derivatization with the phenyl azido group. As shown in Figure 2, the chemical fixation of photoreactive BSA was evidenced by ESCA measurements. Regionally precise microfixation of the photoreactive BSA
Matsuda and Sugawara and the photoreactive gelatin, which is attained by an W-irradiated photomask, was visualized with subsequent staining and microscopic observation (Figures 3 and 4). In both cases, the regional control of the protein-fured area was within micron-order precision. The AFM observation clearly determined the spatial configuration of immobilized gelatin atX-, Y-, and Z-axes, where the XY-plane is the surface and the Z-axis is the thickness or height. As demonstrated in the proteinaceous micropattern with its quite linear edge (Figure 5), micronorder precision at them-plane was achieved, as expected. In this particular case, the maximum thickness of the immobilized layer was around 3 pm. The alternative method is via surface derivatization of photoreactive groups. Phenyl azido-derivatized polymer (111)and copolymer (IV),both of which were prepared for this purpose, were coated on polymer surfaces. Chemical furation of proteins was achieved as in the method using photoreactive protein. The microfixation of BSA, as clearly seen in Figure 6 , was attained.
Conclusions A novel protein-furation method utilizing phenyl azido groups was established. The photoinduced protein fixation method allows us to fix proteins on a given substrate with dimensional micron-order precision at a given portion without any difficulty. Photochemical protein fixation technologies developed here could be applied to artificial organs, biosensors, and cellular engineering where proteins play a key role in functionalization. LA940568J
