Langmuir 1994,lO, 153-158
153
Cysteine-SpecificSurface Tethering of Genetically Engineered Cytochromes for Fabrication of Metalloprotein Nanostructures Hun-Gi Hong, Min Jiang, Stephen G. Sligar,’ and Paul W. Bohn* Beckman Institute for Advanced Science and Technology and Departments of Chemistry and Biochemistry, University of Illinois at Urbana-Champaign, 405 North Mathews, Urbana, Illinois 61801 Received April 21,1993. In Final Form: November 8,1993@ The preparation of oriented metalloprotein nanostructures through introduction of specific and complementary reactive groups on the solid and protein surfaces is critically dependent on the reaction conditions used to prepare the solid surface. Key problems include the hydrolytic stability of the Si-0 bond, the low reactivity of simple nucleophilic silane reagents, protein physisorption, and identification of conditions for producing monolayer protein coverages. These problems are largely circumvented by utilizing a two-step linker synthesis, in which the surface is first prepared with a monolayer of (3-aminopropy1)silane.(3-APS), and the resulting structure is derivatized with the heterobifunctional reagent N-succinimidyl6-maleimidocaproate(EMCS).The maleimide functionality is then presented to the protein, into which a single unique cysteine residue has been introduced by genetic engineering techniques. Hydrolytic stability is dramatically enhanced by including a postreaction curing step, in which the solid surface temperature is elevated to drive the alkoxysilanecondensation reaction to completion. Finally substituing a gas-phase chemical vapor deposition procedure for the liquid-phase reaction of the 3-APS produces dramatically better control over coverage and quality of the resulting films. Scheme 1
Introduction Heme proteins are excellent candidates for the fabrication of novel biomaterials due to their unique electronic and optical properties. However, before these materials can be effectively implemented in materials or devices, efficient and rapid schemes for preparation of oriented heme protein nanostructures a t a wide variety of surfaces must be developed. In these schemes it is critical that the spatial relationship between the molecular and laboratory coordinate systems be encoded directly within the fabrication chemistry. A straightforward way to accomplish this spatial relationship is to use de novo genetic engineering techniquesto build single-sitemutants with unique reactivity in a fixed and known orientation relative to an optical marker. The specific scheme employed in our laboratories employs a cysteine residue to introduceunique reactivity and the heme prosthetic group as an orientable optical marker. Several schemes have been employed to exploit the presence of a unique cysteine in the solvent accessible region of a protein. Recently, we reported that high surface coverage chemisorbed layers of mutants of cytochrome b5 on Si02 can be obtained by covalent linkage to a unique surface cysteine residue introducedinto the protein surface by genetic engineering techniques.l Linking to dielectric surfaces, such as Si02, has been accomplished either through the use of an amino-terminal silane ((3-aminopropyl)trimethoxysilane, 3-APS) and the heterobifunctional reagent N-succinimidyl 6-maleimidocaproate (EMCS),in a three-step sequence, as shown in Scheme 1la or by coupling through a thiopropylsilane to form a disulfide linkage, in a two-step sequence, as shown in Scheme 2.1b In addition, a pyridine hemochrome assay (PHCA)2 in conjunction with visible absorption measure-
EMCS
Scheme 2
-
O 0\ SI(CH2)3SSCHz Cyt bs
0 ’
ments can be used to determine the surface molar absorptivity for oriented arrays of metalloproteins, in general, and of cytochrome bg mutants in particular.laThis leads directly to the ability to characterize the surface coverage of supermolecular arrays of heme proteins by simple in situ absorption measurements of the Soret resonance. In addition, Blasie, Dutton, and their coworkers have used the naturally occurring cysteine in yeast cytochrome c to bind that protein to surfaces through a disulfide linkage to an undecanethiol silane.3 Nevertheless, optimizing the fabrication of these oriented arrays of metalloproteins requires overcoming some (2)(a) Paul, K.G.; Theorell, H.; Akeson, A. Acta Chem. Scand. 1953,
* Authors to whom correspondence should be addressed. @
Abstract published in Advance ACS Abstracts, December 15,
1993. (1)(a) Hong, H.-G.; Sligar, S. G.; Bohn, P. W. Anal. Chem. 1993,65, 1635. (b) Stayton, P. S.; Olinger, J. M.; Jiang M.; Bohn, P. W.; Sligar, S. G. J. Am. Chem. SOC.1992,114,9298.
0743-7463/94/2410-0153$04.50/0
,
7,1284. (b)Berry, E.A.; Trumpower B. L. Anal. Biochem. 1987,161,l. (3)(a) Pachence, J. M.; Blasie, J. K. Biophys. J. 1987,52, 735. (b) Pachence, J. M.; Fischetti, R. F.; Blasie, J. K. Biophys. J. 1989,56,327. (c)Pachence, J. M.; Amador, S. M.; Maniara, G.; Vanderkooi,J.; Dutton, P. L.; Blasie,J. K. Biophys. J. 1990,58,379.(d) Amador, S.M.;Pachence, J. M.; Fischetti, R.; McCauley, J. P., Jr.; Smith, A. B., III; Blasie, J. K. Langmuir 1993,9,812.
0 1994 American Chemical Society
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154 Langmuir, Vol. 10, No. 1, 1994
serious chemical problems related to the use of macromolecules in heterogeneous reactions. Key problems for production of oriented layers a t Si02 surfaces include (a) the hydrolytic stability of the Si-0 bond, (b) the low reactivity of simple nucleophilic silane reagents, (c) protein physisorption, and (d) identification of conditions for producing monolayer protein coverages. The immobilization of proteins on a variety of solid Burfaces has been extensively studied for applications including protein chromatography, biomedical analysis, biomaterials, and biosensors.e Covalent binding of functional protein to surfaces originated with cross-linking reagents which were synthesized specificallyfor the preparation of multisubunit enzymes and protein conjugates in solution7 and for the immobilization of enzymes on solid supports.8 For example, Bhatia and co-workers reported that high surface coverages of IgG were obtained by using thio-terminal silanes and heterobifunctional cross-linkers for immobilization on Si02 surfaces.9 For immobilization of biomolecules at inorganic surfaces, chemical surface modification employing liquid phase silanization has been employed in the vast majority of reported schemes. However, several difficulties, such as nonuniformity of the deposited silane film thickness, codeposition of polymeric silane particles, and hydrolytic removal of the deposited film, are encountered using this method.lOJ1 Even though large protein surface coverages have been obtained, liquid-phase derivatization strategies are not optimum for the preparation of oriented protein layers. The use of a trifunctional (with respect to the surface) silane reagent is necessary to obtain improved hydrolytic stability. Unfortunately, in liquidphase reaction schemes these reagents are also prone to the formation of multilayers, in which the protein orientation relative to the surface plane is lost. Codeposition of oligomeric silane species on the reactive organosilane film is postulated to be responsible for multilayer deposition. There are indications in the literature that these problems can be overcome by resorting to gas-phase reagent delivery, i.e., chemical vapor deposition, in formation of the initial layer.12 For example, Mittal and O'Kane reported chemical vapor deposition on metal surfaces for alkoxysilanes,l%while Jonsson and co-workers reported that, if the temperature is carefully controlled, aqueous-stable silane films can be obtained by gas phase silanization.13 (4) (a) Mosbach, K. Methods in Enzymology; Academic Press: New York, 1988; Vol. 137. (b) Wingard, L. B., Jr.; Katchalski-Katzir, E.; Goldstein, L. Immobilized Enzyme Principles; Academic Press: New York, 1976. (5) (a) Jennissen, H. P. Ber. Bumen-Ges. Phys. Chem. 1989,93,948. (b) Porath, J. Biotechnol. h o g . 1987, 3, 14. (c) Mizutani, T. J. Liq. Chromatogr. 1985,8, 925. (6) (a) Sevastianov,V.I. Crit.Reu.Biocompat. 1988,4,109. (b)Ivarssan, B.; Lundstrom, I. Crit. Rev. Biocompat. 1986, 2, 1. (c) Brash, J. L. Makromol. Chem. Suppl. 1985, 9, 69. (7) (a) D a , M.; Fox, F. C. Annu. Rev. Biophys. Bioeng. 1979,8,165. (b) Freeman, R. B. Trends. Biochem. Sci. 1979,4,193. (c) Kitagawa, T.; Shimozono, T.; Aikiwe, T.; Yoshida, T.; Nishimura, H. Chem. Pharm. Bull. 1981, 29, 1130. (8)Mattiason, B. J. Appl. Biochem. 1981,3, 183. (9) (a) Bhatia, S. K.; H i c k " , J. J.; Ligler, F. S. J. Am. Chem. SOC. 1992,114,4432. (b) Bhatia, S. K.; Shriver-Lake,L. C.;Prior, K. J.; Georger, J. H.; Calvert, J. M.; Bredehorst, R.; Ligler, F. S. Anal. Biochem. 1989, 178, 408. (10) Haller, I. J. Am. Chem. SOC.1978,100, 8050. (11) (e) Baecom, W. D. Macromolecules 1972,5,792. (b) Fischer, A. B.; Wrighton, M. S.; Umana, M.; Murray, R. W. J.Am. Chem. SOC. 1979, 101,3442. (c) Lochmuller, C. H.; Colborn,A. S.;Hunnicutt,M. L.; Harris, J. M. Anal. Chem. 1983,55,1344. (d) Plueddemann,E. P. Silane Coupling Agents; Plenum: New York, 1982. (12) (e) Mittal, K. L.; O'Kane, D. F. J.Adhes. 1976,8,93. (b) Kurth, D. G.; Bein, T. J. Phys. Chem. 1992,96, 6707. (c) Hertl, W. J. Phys. Chem. 1968, 72, 1248. (d) Gorski, D.; Klemm, E.; Fink, P.; H6rhold, H.-H. J. Colloid Interface Sci. 1988, 126, 445.
This paper addresses the synthetic problems associated with the formation of oriented metalloprotein layers. We report that gas phase silanization, coupled with the use of the heterobifunctional linker chemistry shown in Scheme 1, shows significant advantages over the less efficient chemistry shown in Scheme 2 and over liquid-phase reactions for the preparation of monolayer-coverage cytochrome ba covalently bound to SiO2. Gas-phase silanization permits control of silane delivery rate to the surface and of the temperature of the surface-silane reaction, leading to stable and reproducible monolayer silane films. In addition, the selection of the highly and specifically reactive heterobifunctional cross-linker,EMCS, allows self-assembly of proteins with solvent-accessible cysteines to obtain monolayer coverage of protein on reactive maleimido-terminated surfaces. The quantities of covalently bound heme protein on Si02 are easily determined by using measured surface molar absorptivities for oriented mutants, which are calibrated by PHCA determinations of adsorbed protein.
Experimental Section Reagents and Materials. The usual surface for the coating of heme proteins was a 24 mm X 60 mm X 150 pm TkZn glass substrate (Corning Glass). Silicon wafers, 3.2 in. diameter, 0.16 mm thick, double-polished, boron doped to 16 Q cm and in the (100) orientation were supplied by Monsanto. (3-Mercaptopropy1)trimethoxysilane (MPS), (3-aminopropy1)trimethoxysilane (3-APS),(3-iodopropy1)trimethoxysilane(IPS),(3-bromopropyl)trimethoxysilane (BPS), (3-acryloxypropyl)trimethoxysilane (AcPS) and n-propyltrimethoxysilane (NPS) were purchased from Petrarch System (Huls America Inc.). N-Succinimidyl 6-maleimidocaproate (EMCS) was purchased from Fluka. Toluene and chloroform were dried over CaHa before use. Deionized (DI) water (18.2 MQ cm) was obtained from a Milli-Q system (Millipore, Bedford MA) with an Organex-Q final stage. A 20 mM phosphate buffer solution(pH = 8.0) was used for preparation of all cytochrome bh solutions. Highly purified and concentrated wild type and mutant genotypes of cytochrome b b (T8C, T65C) were prepared as described previously.1b Liquid-Phase Silanization. Glass surfaces were cleaned by immersion in a hot 1:4 mixture of concentrated HlOz and NH,OH for 10 min followed by rinsing several times with deionized water. Next, the substrates were consecutivelytreated with hot, concentrated sulfuric acid for 30 min twice, followed by rinsing and sonication in DI water. Finally, the substrates were dried with forced Nz. For the liquid phase chemical surface modification, the substrates were placed in a 2% (w:v) solution of the appropriate trimethoxysilane (MPS, BPS, IPS, AcPS, or NPS) in dry CHCls and refluxed for 3-4 h. After silanization, the substrates were washed thoroughly in consecutive portions of CHCl3, ethanol, and DI water before deposition of protein. Gas-Phase Silanization. Borondoped silicon wefers were used to investigate the optimum conditions for the preparation of monolayers of 3-APS. Prior to uBe, wafers were cut into 1cm X 1cm squares and were cleaned by immersion for 5 min each in a series of oxidative basic (NH,OH/HaO?/H20 = 1:1:5) and acidic (HCl/HzO1/HzO = 1:1:5) etches. The silicon slides were then rinsed thoroughly in DI water and dried in Nz. Clean glass cover slips were prepared as described above. Silane reagents were purified by vacuum distillation once prior to use. The apparatus for gas phase silanization was adapted from an earlier version reported in the literature.'g The solid substrates (either silicon single crystal or glass cover slips) were transferred to the apparatus shown in Figure 1. The temperature of the oven surrounding the gas-phase reactor was elevated to 90 5 "C, and vacuum distillation of the reactive silane was started with evacuation of the apparatus to 0.03 Torr. The silane was slowly vacuum distilled for 10-20 h. The silane flask was kept at room temperature during distillation. After vacuum distillation of the ~~
(13) Jonsson,U.; Olofsson,G.; Malmqviat, M.; Ronnberg, I. Thin. Solid Films 1985, 124, 117.
Langmuir, Vol. 10, No.1, 1994 155
Surface Tethering of Cytochromes Pressure gauge Valve
4
4
Silane flask
I '
-'
n
Table 1. Evaluation of Silane Linker Monolayers silane film reactive group contact angle (deg) 55.4 2.0 APS -NH2 53.9 1.7 MPS -SH IPS -I 55.7 1.9 BPS -Br 51.5 2.6 AcPS -O-CO-CH=CH2 59.8 i 0.9 NPS -CH, 54.1 1.8
** *
I
Substrate Oven
Figure 1. Silanization apparatus used for chemical vapor deposition of silane. silane, the oven temperature was elevated to 155f 5 OC and kept at this temperature for 7-8 h under vacuum (below 0.01 Torr) to drive the silanization surface reaction to completion. The system was then allowed to cool to room temperature and was slowly back filled with dust-free dry Nz. The silanized surfaces were stored in absolute ethanol at room temperature until use. All transfer lines, except the silane flask and cold trap, were heatedduring gas transfer to prevent condensation of the reactive silane. Treatment with Heterobifunctional Cross-Linker. Following preparation of the aminosilane layer at the Si02 surface, either by liquid-phase or gas-phase reagent deliverysystems, the silanizedglass surfaceswere treatedwith 2mM EMCSin absolute ethanol for 2-8 h under adry Nz atmoephere. The Si02 substrates were then removed, rinsed with fresh absolute ethanol, and stored in ethanol until use. Immobilization of Cytochrome b. Wild type and the T8C mutant of cytochrome ba, concentrated (3.1 mM) in phosphate buffer, were diluted to 35-50 pM with 20 mM phosphate buffer solution and used directly for the immobilization of cytochrome ba to the surface. One hundred microliters of concentrated (1.6 mM) solution of the T65M mutant was diluted in 1 mL of phosphate buffer and treated with 1mg of dithiothreitol (DTT). Next, the T65Csolutionwas loaded onto aP4 gel filtration column (Biorad),whichwas equilibratedwithphosphate buffer, including 1 mM EDTA, prior to use, to separate T65C monomer from DTT. A red band from the column was isolated and diluted to 40 pM with phosphate buffer. This solution was used for the immobilization of the T65C mutant. Glass substrates which had previouslybeen prepared with thiol-reactiveimmobilizedreagents and stored in ethanol were washed with 20 mM phosphate buffer (pH = 8.0) solution thoroughly. The lower half of each glass substrate was immersed in the phosphate buffer solutions of cytochrome b~at room temperature for the allotted time. Typical immersed area was 14.4 cm2. The incubation time was varied from a few hours to 2.5 days. After the incubation, the surfaces were rinsed with phosphate buffer extensively. After the surfaces were dried with nitrogen,visibleabsorption spectrawere obtained to determine the density of surface-bound protein. Instrumentation. Visible absorption spectra were acquired using a computer-interfaced double beam grating UV-visible spectrophotometer (Cary 3, Varian Instruments). In all measurementsthe spectral resolution was 2 nm, and the data interval was 1.5 nm. Each spectrum was obtained by signal averaging 100scans to obtain acceptable signal-to-noiseratio (p-p noise = 2 X lo-' absorbance unit). The total time required for the acquisition of each spectrum was ca. 4 min. All absorption measurementswere carried out at normal incidenceto the surface. UV spectra of surfaces silanized with APS and EMCS, before adsorption of cytochrome bs, were used as blank spectra. The ellipsometer used was a thin film ellipsometer (43603-200F, Rudolph Research). The refractive index for the silane film was assumed to be 1.46.1° Ellipsometric measurements were performed with 546.1-nm radiation at 70° angle of incidence only on silicon substrates silanized by chemical vapor deposition and were made at severaldifferent locationson each surface. Contact angle meaaurements were performed using an in-house constructed contact anglegoniometerand were made on sessiledrops by measuring the tangent to the drop at its intersection with the surface. Relative humidity in the chamber was maintained at 100% ' by f i i g wells in the sample chamber with deionized water. The volume of the drop used was 3 pL. All reported values are
the average of at least six measurements taken at different locationson the film surface. Measurements of angle were taken within 10 s after formation of the sessile drop. Pyridine Hemochrome Assay. Two milliliters of 20 mM potassium phosphate buffer (pH = 8.0), 0.5 mL of pyridine, and 0.25 mL of NaOH (1M)were mixed. This mixture was repeatedly pipetted onto the portion of the surface immobilized with cytochrome bb. Two milliliters of this rinse solution was divided into two 1-mL quartz cuvettes, which were placed in the sample and referencepaths of the double beam spectrophotometer. After baseline measurement, a small quantity of dithionite was added to the sample cuvette, and the visible absorption difference spectrum of the reduced minus oxidized pyridine hemochrome was obtained.
Results and Discussion Scheme 2 summarizes the surface reactions in the immobilization procedure used previously, in which the surface is derivatized with the linker molecule in a single step, and covalent attachment of cytochrome bg to the silane surface is achieved through disulfide (as shown in Scheme 2) or thioether (e.g. by use of a halopropylsilane reactive linker) linkages. To prepare a reactive organic surface for protein binding, several silane films were evaluated as summarized in Table 1. Contact angles of the films were used to evaluate film quality and reproducibility. All of these films were prepared by liquid phase silanization reactions. The water contact angles from Table 1clearly show that the wetting properties of these silane films are indistinguishable with respect to the identities of the reactive groups. This fact is supported by the contact angle (54.1') of the nonreactive methyl terminal of NPS. The low measured contact angles (51.559.8") of these silane films can be directly attributed to the short (C3) chain length and consequent ready access of the solvent to exposed surface sites, of the silanes used, since the native hydrophobicity of all terminal groups, except those in APS and MPS,is quite high. Bain and co-workers have reported that wettability strongly depends on the alkyl chain length and the polarity of the tail group of a-derivatized alkanethi01s.l~The silane films exhibited contact angles with low standard deviations for water drops, indicating that the silane films are macroscopically homogeneous. Surface coverages of cytochrome bs bound using the single-step reaction scheme were determined by using visible absorption spectroscopy in conjunctionwith PHCA, as described previously.la The absolute number of molecules present on the surface is measured in the PHCA, and the geometric surface area is known for a given sample. Thus, I?, the surface coverage can be calculated and the corresponding absorbance of the surface before hemochrome treatment used to calculate the absorption crosssection. Surface coverages can then be calculated in fractions of a monolayer by comparing to the full tightpacked monolayer density. rsat,the saturated monolayer surface density of 1.1X 1013cm-2, is obtained by inverting the 900 A2 surface footprint (the crystallographic dimen(14)Bain, C.D.;Evall, J.; Whitesides, G.M.J. Am. Chem. SOC.1989, 111,7155.
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156 Langmuir, Vol. 10, No. 1, 1994
Table 2. Cytochrome Reactivity on Mixed Functionalized Surfaces surface compositionn 0.00
rm* 0.00
0.00
0.17 0.50
0.25 0.22 0.31
0.17 0.17 0.15
1.00
-0-
acryloxy . cured
r*c
Surface composition expressed as the fraction of mercaptopropylsilane. The remainder of the mixed surface is composed of n-propylsilane. Surface coverage for T8C cytochrome b5 mutant. e Surface coverage for wild-type cytochrome ba.
+C15Br 0 7
-+C158r - cured
(16) Mathews, F. S.;Levine, M.; Argoe, P. J. Mol. B i d . 1972,64,449. (16) von Bdman, F.S.;Schuler, M. A.;Jollie, D. R.; Sligar, S.G . h o c . Natl. Acad. Scr. U.S.A. 1986,83, 9443.
completely from the surface, solvent accesses and hydrolyzes the unreacted Si-OR bond, resulting in a more hydrophilic surface, or solvent hydrolyzes the terminal functionality to generate a hydrophilic group hydroxyl group a t the distalend of the linker. However, if the latter explanation were correct, then we should observe a difference between the iodo-terminated (hydrolyzable)and methyl-terminated (non-hydrolyzable) species. Since no difference between these species (uncured) is observed, we conclude that hydrolysis a t the surface end of the linker molecule is the more important factor in determining the stability of the short-chain linkers. Since each linker molecule is trifunctional a t the silane end, the high temperature annealing drives the surface derivatization reaction further to completion, resulting in alarger average number of Si-0 bonds being formed than in the uncured materials. As a result, the cured films are more resistant to hydrolysis. Similar effects have been observed previously. Haller reported that a substantial removal of APS group due to hydrolysis was observed upon immersion in water or in aqueous solutions for times longer than a few minutes,1° and the lack of long-term hydrolytic stability of APS-derivatization in an aqueous environment has been observed on porous glass substrates." The other approach involves limiting access of the solvent to the solvolytically unstable bond. The effect of increasing the length of the alkyl linker to c16 from C3 is also readily apparent. The absolute magnitude of the contact angle is significantly increased, although these are probably still not wellordered close-packed films. In addition the stability of the linker to solvolysis is also enhanced as demonstrated by the fact there is little change over 70 h of immersion in buffer. The next problem to be addressed in protein reactivity for specific orientation of macromolecules a t surfaces concerns the effects of physisorption on the course of the protein immobilization reaction. These effects were investigated by comparing the adsorption of wild-type (no cysteine residues) and the T8C mutant (cysteine specifically introduced a t amino acid residue 8)on surfaces with varying fractions of thiol-reactive groups. Table 2 shows the coverages of wild type and T8C cytochrome b5 measured from the Soret absorbances,l* using appropriate absorption cross sections for randomly and nonrandomly oriented species determined previously.lg Coverageswere measured after reaching steady state in 24-36 h. The coverages of T8C and wild-type were not significantly (17) (a) Weetall, H. H.; Filbert,A. M. In Methods of Enzymology; Academic Press: New York, 1974; Vol. 34, pp 59-72. (b) Weetall, H. H. In Methods of Enzymology; Academic Press: New York, 1976; Vol. 44, pp 134-148. (18) (a) Chottard, G.;Michelon, M.; Herve, G.Biochim. Biophys. Acta 1987,916,402. (b) Smith,D.W.; Williams, R. J. P. Struct.Bonding 1970, 7, 1. (19) Optical waveguide linear dichroism shows the orientation of the heme plane to be 59.5 1.3O for T8C resulting in an absorption c r w section of 6.78 X 10-'6 cm*for TSC, while the randomly-oriented wildtype species has u.b = 4.98 X 10-16 cm*.
*
Surface Tethering of Cytochromes altered by the composition of the reactive silane surface, indicating that the wild type of cytochrome bg, which does not have a reactive cysteine residue, is easily physically adsorbed on the mercaptopropyl/n-propyl mixture surfaces. This observation indicates that there is nonspecific interaction between the nonreactive cytochrome b6 and thiol-reactive organic surfaces. This nonspecific adsorption component is quite resistant to removal by repeated washings in buffer, and it is likely to be present as a part of specifically adsorbed protein population. In addition the coverages of T8C do not vary significantly with surface composition, indicating that the problem lies with the protein not the surface reactive groups. In fact for MPS the activity of the surface thiol group remained quite stable during the protein adsorption process, as was confirmed by reactivity testing of the silane films with Ellman’s reagent beforeand after protein a d s ~ r p t i o n .The ~ surface coverages of protein determined from heme absorbance is ca. 30% on the pure thiol base. From these results, we conclude that the adsorbed T8C population is composed of two components: a nonspecifically-bound physisorbed fraction, and a covalently bound chemisorbed fraction. Several experiments were implemented to examine the use of sonication to remove the physisorbed component at regular time intervals during the protein linking reaction. In a typical experiment, one reactive cycle was composed of static immersion for 5 h and sonication for 20 min, and the coverage reached a plateau of 0.37rmtafter 10reaction cycles. However, since this represents only a small improvement over the coverages obtained without sonication, this strategy was not pursued further. Taken together these results point to the fact that the derivatization strategies illustrated in Scheme 2 are illsuited to the preparation of full monolayer coverages of oriented metalloprotein layers. To increase the surface coverage of covalently bound protein, it is necessary to increase reactivity of the linker molecule, the accessibility of the linker molecule, or both. Bhatia and co-workers reported that high surface coverages of IgG were obtained by using thiol-terminated silanes and heterobifunctional cross-linkers for immobilization of antibodies on silicasg The surface reactions in the immobilization step, which are summarized in Scheme 1,utilize a 3-APS treatment in liquid phase to provide free primary amine groups. Subsequently, the succinimide group of EMCS reacts to form an amide linkage. In this preparation, the aminoterminated surface was normally incubated in 2 mM EMCS solution in ethanol for 7-8 h. The reactive cysteine adds across the double bond to generate a thioether linkage, as shown in Scheme 1. The stability of the maleimide group is quite good. Gregory20 reported t h a t t h e rate of hydrolysis of maleimide to the maleamic acid is negligible below pH 7.5. In common with other alkylating reagents, the maleimide functionality is quite reactive to the thiolate anion in weak basic buffer.21 Thus, the combination of an amino-terminal silane and a heterobifunctional crosslinker should offer a convenient method for covalent attachment of functional proteins to silica surfaces a t high concentration. Figure 3 shows the time course of protein adsorption obtained for samples in which the 3-APS layer was prepared by liquid-phase immersion. While the nonspecific adsorption of nonreactive wild-type cytochrome b5 increases initially and then remains roughly constant over the remainder of the reaction time, the coverage of the (20)Gregory, J. D.J . Am. Chem. SOC.1965,77,3922. (21)Partis, M.D.;Griffiths, D. G.; Robert, G. C.; Beechey, R. B. J. Protein Chem. 1983, 2, 263.
Langmuir, Vol. 10, No. 1, 1994 157 2.0
-
1
-D- TBC wild type
+ 1.5-
0
10
20
30
40
50
Immersion Time (h)
Figure 3. Time course of protein surface derivatkationon3-APS substrates prepared by the liquid-phase immersion technique: open symbol, T8C mutant; filled symbol, wild-type cytochrome bg. The measurement uncertainty is ea 0.1 monolayer. thiol-reactive mutant T8C monotonically increases over the entire time period. This higher coverage (ca. 1.7 monlayers) of T8C might be due to the three-dimensional polymerization of (3-aminopropy1)trimethoxysilaneduring the liquid phase silanization step. This polymerization of the silane reagent could introduce open, thick polymeric regions containing many more reactive primary amine groups per unit area than would be present in a uniform monolayer, thus resulting in a highly nonuniform surface presented to the heterobifunctional linker, and ultimately to the protein. Chemical surface modifications involving silanes in the liquid phase have previously been observed t o result in several difficulties, such as nonuniformity of the deposited silane film thickness, codeposition of polymeric silane particles, and hydrolytic instability of the deposited film? For preparation of reproducible, oriented, and well-controlled structures, it is critical to have single monolayer coverage of the aminosilane as a base for treatment with the heterobifunctonal linker. Given the naturally higher propensity for oligomerization of the silane reagent in the liquid-phase, a straightforward method to overcome the above difficulties is to implement a gasphase (i.e. chemical vapor deposition) reagent delivery system. In order to identify optimum conditions for monolayer deposition of aminosilane, precleaned silicon wafers were used as substrates to facilitate the study of the depoeited films by ellipsometry. The large refractive index difference between silicon and typical organic films allows estimates of film thickness to be made directly from the ellipsometric parameters, i.e., without full inversion of the ellipsometry equations, for films less than 5 nm thick. For thin organic films on silicon, the recovered thickness is relatively insensitive to changes in $, but highly sensitive to changes in A. Therefore, simple measurements of A can provide good estimates of film thickness in the monolayer regime. For example, Lee and co-workersreported that a lo change in A corresponds to a 10 f 1.5A change in total thickness of a zirconium 1,lO-decanebis(phosph0nate)multilayer on silicon.22 This estimate is in good agreement with measurements made from inverting the ellipsometry equations and is relatively independent of the identity of the organic film. The thickness of silane filmsis dependent on control variables such as pressure used during the silane distillation, oven temperature, silanization/annealing time, and the boiling point of the silane used. Line pressure (22)Lee,H.;Kepley, L. J.; Hong,H.-G.;Mallouk,T. E. J. Am. Chem. SOC.1988,110, 618.
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158 Langmuir, Vol. 10,No.1, 1994
z'ol3 I
I
-La
0.0
t 18-8-8 -0-
-m-
10-8-8 5-8-8
-A- 5-4-8
4 0
20
40
60
80
Immersion Time (h)
Figure 4. Coverage of T8C mutant as a function of immersion time (h) in the protein solution for surfaces prepared using different chemical vapor deposition conditions. Each curve is labeled by three numbers which give vacuum distillation time, silanization time, EMCS reaction time. The measurement uncertainty is 0.1 monolayer.
was maintained a t 0.01 Torr and 3-APS was used in this experiment, and distillation and annealing times were varied to optimize the reaction conditions. Excellent quality films were obtained for vacuum distillation times of 10-12 h and annealing times of 7-8 h, the resulting films being 14 f 3 A thick. Water contact angles measured on these filmswere similar to those of 3-APS films prepared by liquid phase silanization (cf. Table 1). During deposition of these high-quality films, the silane flask was not heated. Heating of the silane (70 OC) resulted in deterioration of sample quality and introduced large changes in A (20-30O). When the distillation time was raised to 18h or more, multilayer (t 15 nm) deposition was observed. Since the thickness of 3-APS fiims obtained from liquid phase silanization is around 5-8 nm, gas-phase and liquidphase reaction conditions produce very similar results under these conditions. Finally we note that although the reactivity of the glass surfaces is expected to be somewhat different than that of adventitious Si02 on Si, these measurements and conditions can act as a guide for the preparation of monolayers of APS on glass as well. Reasoning that the ellipsometry experiments with 3-APS films on silicon had produced reasonable estimates of the conditions for preparing good-quality films on glass, we proceeded to evaluate the glass derivatization reactions by the saturation coverage of T8C mutant protein which could be bound to the surface. Figure 4 shows the coverage of T8C mutant immobilized with EMCS on 3-APS films prepared by vapor phase silanization. Each curve is labeled by three numbers which give vacuum distillation time, silanization time, and EMCS reaction time, where the vacuum distillation time refers to the length of time in which the silane reagent is delivered to the solid surface, the silanization time refers to the time for annealing at high temperature and driving the surface hydrolysis reactions to completion, and the EMCS reaction time refers to the length of time used for the immobilization of the
heterobifunctional linker. The figure clearly shows that the protein surface coverage critically depends on the preparation conditions for the underlying silane film. The preparation of the initial 3-APS layer is composed of three steps: vacuum distillation delivery of aminosilane to the solid surface; covalent attachment of the silane reagent to the glass surface; and finally EMCS immobilization through amide bond formation. Coverages larger than a single monolayer are clearly obtained for 18 h reagent delivery times, consistent with the observation from ellipsometry that this condition resulted in formation of very thick (2' > 5 nm) 3-APS layers. For 10 h reagent delivery time, nearly complete (ca. 85% ) monolayer coverage of protein is obtained, again consistent with the monolayer 3-APS coverage deduced from the ellipsometry experiment. Shorter reagent delivery time (curve 5-84) or shorter curing times (curve 5-4-8) result in films with smaller limiting coverages; ca 27-33% for the 5-8-8 film, and little, if any, bound protein for the 5-4-8 film. The 5-8-8 film seems to have produced a stable 3-APS film of less than monolayer coverage, while the 5-4-8 film, with its shorter curing time, has probably been entirelyremoved from the surface during immersion in the protein buffer solution. It is worthwhile to note that the coverage of covalently bound protein is unchanged after 36 h, suggesting that multilayer adsorption, for example by physisorption to the underlying protein monolayer, is not obtained. Thus, the silane reagent delivery time and curing time are both seen to be crucial to the formation of monolayer thick 3-APS layers, which can then be used as substrates for the immobilization of oriented23metalloprotein nanostructures.
Conclusions Key problems in the preparation of metalloprotein monolayers have been addressed. These include the hydrolytic stability of the Si-0 bond, the low reactivity of simple nucleophilic silane reagents, protein physisorption, and identification of conditions for producing monolayer protein coverages. These problems are largely circumvented by substituting a two-step linker synthesis for the simple single-step synthesis used previously. In addition the use of the gas-phase introduction (chemical vapor deposition) of 3-APS reagent allows much enhanced control over the coverage of the resulting protein films and the stability of the linker species used to anchor them to the surface. Acknowledgments. The authors wish to acknowledge D. Seielstad for generously making the pentadecylbromosilane available to us and J. A. Katzenellenbogen for numerous insightful discussions. This work was supported by the Biotechnology Research and Development Corporation and by the Department of Energy through Grant DE FG02 88ER13949. (23) Orientations of the samples prepared from the 10-8-8 set of conditions were determined by optical waveguide measurements and determined to be 60.1 f 1.7', in excellent agreement with the results obtained from liquid phase reactions producing submonolayer protein coverages. Measured orientation of the T65C mutant was 48.5 h 3.8O.
