Enzymatic Hydrolysis of a Chemisorbed Peptide Film Using Beads

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Langmuir 1997, 13, 4855-4860

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Enzymatic Hydrolysis of a Chemisorbed Peptide Film Using Beads Activated with Covalently Bound Chymotrypsin† David C. Turner,* Mary A. Testoff, David W. Conrad,‡ and Bruce P. Gaber Laboratory for Molecular Interfacial Interactions, Code 6930, and Laboratory for Biosensors and Biomaterials Development, Code 6910, Naval Research Laboratory, Washington, DC 20375 Received March 31, 1997. In Final Form: June 24, 1997X Silica and polyacrylamide microspheres were modified with chemisorbed chymotrypsin and used to enzymatically hydrolyze a peptide thin film which was covalently bound to a flat silica surface. Chymotrypsin was covalently cross-linked to 500 nm silica spherical beads and 30-50 µm polyacrylamide spherical beads and shown to be enzymatically active against thin films of a fluorescent peptide, succinyl-ala-ala-phe7-amido-4-methylcoumarin (SAAP-AMC), and an unlabeled peptide, t-BOC-phe. SAAP-AMC and t-BOCphe were covalently coupled to an aminosilane film on silica and aluminum substrates through an amide linkage. Control experiments showed that free chymotrypsin in solution was able to hydrolyze the amide bond between the phenylalanine and the AMC groups of the chemisorbed peptide, resulting in the release of the AMC group into solution. When they were placed into contact with the SAAP-AMC surface, the chymotrypsin-modified beads also hydrolyzed the phe-AMC bond and released AMC into solution, demonstrating that covalently immobilized enzymes can be used to hydrolyze immobilized organic thin films. The hydrolytic activity of the chymotrypsin beads was also confirmed for a second peptide film, t-BOC-phe, by external reflectance IR spectroscopy.

Introduction Immobilized enzymes and catalysts are commonly used for large scale chemical modification of substrate molecules in the food science and fuel industry or as part of enzymelinked immunoassays (ELISA).1 Recently, a number of experiments have shown that enzymes in solution can be used to modify a substrate which is immobilized to a solid surface.2-6 Others have shown that polymeric catalysts of controlled physical size may be used to selectively modify catalyst-accessible regions of a porous polymer bead.7 Using enzymes for chemical surface modification has advantages originating with the high chemical selectivity of the enzymes, the ability to do chemistry at room temperature under aqueous conditions, and the possibility of carrying out some reactions which cannot be done or require a complex, multiple-step synthesis using conventional chemistry. In addition, unlike other reagents, enzymes can be recovered and reused. Despite these advantages, enzymes have not found widespread application in this field due to the problems associated with irreversible nonspecific adsorption of enzyme to the substrate surface.2 We feel that using immobilized enzymes for chemical surface modification may help to † Portions of this work have appeared in preliminary form in: Turner, D.; Testoff, M.; Gaber, B. Direct enzymatic hydrolysis and patterning of a chemisorbed peptide thin film. Proc. SPIE 1997, 2978, 22-30. * To whom correspondence should be addressed. Phone: (202) 404-6021. Fax: (202) 767-9594. E-mail: [email protected]. ‡ Laboratory for Biosensors and Biomaterials Development. X Abstract published in Advance ACS Abstracts, August 15, 1997.

(1) Reen, D. J. Methods Mol. Biol. 1994, 32, 461-466. (2) Turner, D.; Peek, B.; Wertz, T.; Archibald, D.; Geer, R.; Gaber, B. Langmuir 1996, 12, 4411-4416. (3) Wilson, T. E.; Spevak, W.; Charych, D. H.; Bednarski, M. D. Langmuir 1994, 10, 1512-1516. (4) Hagestam, H.; Pinkerton, T. C. J. Chromatog. 1986, 351, 239248. (5) Hagestam, H.; Pinkerton, T. C. Anal. Chem. 1985, 57, 17571763. (6) Whitesell, J. K.; Chang, H. K.; Whitesell, C. S. Angew. Chem., Int. Ed. Engl. 1994, 33, 871-873. (7) Smigol, V.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1994, 66, 21292138.

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alleviate the problem of nonspecific adsorption without losing the specificity of enzyme chemistry. In addition, immobilized enzymes may also have applications for the chemical patterning of surfaces. We demonstrate here that an enzyme which has been covalently immobilized to the surface of a spherical bead can chemically modify a chemisorbed thin film substrate on a planar surface. The chemistry occurred without nonspecific adsorption of the enzyme to the substrate surface, and the enzyme-coated beads were routinely recovered for subsequent use. As a model system, the enzyme R-chymotrypsin was used to modify two peptide thin film substrates: the fluorescent peptide succinylala-ala-phe-7-amido-4-methylcoumarin (SAAP-AMC) and the unlabeled peptide t-BOC-phe. Chymotrypsin hydrolyzes the phe-AMC bond in the SAAP-AMC peptide, releasing the AMC group from the peptide and leaving a carboxylic acid as the terminal group on the phenylalanine. When it is conjugated to the phenylalanine, AMC fluoresces at 395 nm. After hydrolysis, the AMC is released into solution and exhibits a red-shifted fluorescence emission at 445 nm. Therefore, a change in the AMC fluorescence spectrum is a simple indication of enzymatic activity on the peptide. Both peptides were covalently coupled to an aminosilane film via amide bond formation. Spherical beads activated with covalently attached R-chymotrypsin were used to hydrolyze the phe-X amide bond in both peptide films. Fluorescence spectroscopy, water contact angle goniometry, and external reflectance infrared spectroscopy were used to follow the changes in the peptide films due to enzymatic hydrolysis. Material and Methods Materials. The fluorescent peptide, succinyl-ala-ala-phe-7amido-4-methylcoumarin (SAAP-AMC, MW 564.6), was obtained from Novabiochem-Calbiochem (La Jolla, CA). t-BOC-phe (Nt-BOC-L-phenylalanine, MW 265.3) and R-chymotrypsin were purchased from Sigma (St. Louis, MO). (4-aminobutyl)dimethylmethoxysilane (BAMM) and (3-aminopropyl)trimethoxysilane (APS) were obtained from United Chemical Technologies, Inc. (Bristol, PA). Fused silica slides were purchased from NSG

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Published 1997 by the American Chemical Society

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Scheme 1. Coupling of SAAP-AMC peptide to a BAMM silane film on a planar silica surface.

Precision Cells (Farmington, NJ) and 500 nm silica beads (monospher 500) were purchased from E. Merck (Darmstadt, Germany). Polished silicon n〈100〉 phosphorus-doped wafers were obtained from WaferNet (San Jose, CA). 3M Emphaze biosupport medium (AB1 activated chromatography beads), EDC (1-ethyl3-(3-(dimethylamino)propyl)carbodiimide hydrochloride), EGS (ethylene glycolbis(succinimidyl succinate)), and NHS (N-hydroxysuccinimide) were purchased from Pierce Chemical (Rockford, IL). Phosphate buffer was 0.1 M sodium phosphate at pH 7.8 unless otherwise noted. Anhydrous methanol was obtained from Aldrich Chemical Company (Milwaukee, WI). All other solvents were of reagent grade, and water was deionized to 18 MΩ/cm resistivity. Immobilization of Peptide to Fused Silica and Aluminum Slides. Fused silica slides and polished silicon wafers were cleaned with HCl/methanol (1:1, v/v) followed by concentrated H2SO4 for 30 min each and rinsed three times in deionized water. To form the aluminum surfaces required for IR spectroscopy, aluminum was deposited on the clean silicon wafers by electron beam evaporation at a pressure of 1.1 × 10-7 Torr and a deposition rate of 15 Å/s to a final thickness of 5000 Å. Aluminum surfaces were treated for 5 min with an oxygen plasma (200 mTorr O2, 100 W RF) before further treatment. Just prior to silane treatment silica and aluminum surfaces were boiled in deionized water for 15 min and dried with a stream of nitrogen. They were then reacted with a solution of 1% (v/v) BAMM or APS in methanol containing 1 mM acetic acid and 5% (v/v) deionized water for 12 h. Following silanization, the slides were rinsed several times with methanol, dried with a nitrogen stream, and baked at 120 °C for 5 min.4 Sessile drop water contact angles were measured for each of the films. SAAP-AMC or t-BOC-phe peptide was coupled to the aminosilane films via peptide bond formation between the carboxylic acid group on the peptide and the primary amine on the silane (Scheme 1). SAAP-AMC couples via the carboxylic acid group on the succinic acid residue while t-BOC-phe couples via the carboxylic acid at the C-terminus of the peptide. A peptidecoupling solution was made by adding 30 mL of a 0.0833 mg/mL peptide solution in methanol to an equimolar solution of EDC (3.4 mg) and NHS (4.6 mg) dissolved in 10 mL of deionized H2O

for a total volume of 40 mL. The aminosilane-treated surfaces were immersed in the coupling solution and treated for at least 12 h at 25 °C. Following treatment the slides were rinsed several times with 0.5% (v/v) TWEEN 20 in sodium phosphate buffer followed by methanol (to remove uncoupled adsorbed peptide) and dried with nitrogen. The SAAP-AMC-modified surfaces of the slides were characterized by surface wettability and fluorescence spectroscopy (SLM Instruments, Inc., Urbana, IL). The fluorescent intensity of immobilized SAAP-AMC was measured using an excitation wavelength of 325 nm and emission wavelength of 395 nm.8 t-BOC-phe was coupled only to APS-modified aluminum substrates and was characterized using surface contact angle and external reflectance IR spectroscopy. Immobilization of r-Chymotrypsin to Silica Beads. Colloidal silica beads (500 nm) were activated with R-chymotrypsin following the procedure of Jadaud and co-workers.9,10 Monodisperse beads (500 nm diameter) were rinsed three times with deionized water by pelleting in a centrifuge at 3300 rpm for 10 min. The beads were cleaned with HCl/methanol (1:1, v/v) for 30 min, rinsed three times with deionized water, treated with concentrated sulfuric acid for 30 min and rinsed three times with deionized water. Beads were treated with a 1% (v/v) BAMM solution in methanol containing 1 mM acetic acid and 5% (v/v) deionized water for 12 h. Following BAMM treatment, the beads were rinsed three times with methanol and baked at 120 °C for 1 h.4 Next the beads were immersed in a 1% glutaraldehyde solution in phosphate buffer and the solution was gently shaken for 2 h.9,10 After the samples were centrifuged, they were rinsed three times times with methanol followed by one time with phosphate buffer. The beads were then resuspended in 0.1 M sodium borate solution at pH 7.8 (1 g of beads/2.5 mL) containing R-chymotrypsin (60 mg/1 g of beads). The suspension was gently shaken at 4 °C for 18 h and then rinsed three times with phosphate buffer. To further stabilize the R-chymotrypsin and minimize (8) Zimmerman, M.; Quigley, J. P.; Ashe, B.; Dorn, C.; Goldfarb, R.; Troll, W. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 750-753. (9) Jadaud, P.; Wainer, I. W. J. Chromatogr. 1989, 476, 165-174. (10) Jadaud, P.; Thelohan, S.; Schonbaum, G. R.; Wainer, I. W. Chirality 1989, 1, 38-44.

Surface Hydrolysis Using Immobilized Chymotrypsin leaching, the beads were immersed in 10 mM EGS in phosphate buffer for 2 h. Finally, the bead suspension was rinsed with 0.5% (v/v) TWEEN 20 in phosphate buffer, followed by three phosphate buffer rinses to ensure removal of any noncovalently bound R-chymotrypsin. Immobilization of r-Chymotrypsin to 3M Polymer Microspheres. 3M Emphaze polymer beads (30-50 µm diameter, bis-acrylamide/azlactone copolymer, 10 mg) were treated with R-chymotrypsin (10 mg) in 1 mL of 0.6 M sodium citrate buffer containing 0.1 M MOPS, pH 7.5, for 2 h at 25 °C with slow shaking. After the mixture was centrifuged at 2400 rpm for 5 min, the supernatant was removed and the beads were subjected to 1 mL of 1.0 M Tris, pH 8.0, for 2 h at 25 °C with slow shaking to quench the enzymatic attachment. After centrifuging, the beads were washed by vortexing for at least 15 min in 50 mM sodium phosphate buffer, pH 7.2, followed by a second wash in 1 M sodium chloride. After centrifuging, the beads were washed twice in 50 mM sodium phosphate buffer and pelleted. Standard Curve. A standard curve of free AMC fluorescence versus concentration was prepared to measure the activity of the R-chymotrypsin-modified beads. Stock solutions of SAAPAMC peptide varying in concentration from 0.83 ng/mL to 0.83 µg/mL in 1 mL aliquots were incubated with 20 µg of R-chymotrypsin for 24 h at 25 °C to completely hydrolyze the AMC from all of the SAAP-AMC peptide. The fluorescence spectrum of each sample was measured using an SLM/AMINCO 8000 fluorimeter (SLM Instruments, Inc., Urbana, IL) at an emission wavelength of 445 nm (345 nm excitation) to determine the total fluorescence from free AMC. The AMC fluorescence was then plotted against concentration for each of the samples and fitted via linear least-squares to determine the slope and intercept. Assay of Immobilized r-Chymotrypsin. One milliliter of an 8.3 × 10-5 mg/mL solution of SAAP-AMC in phosphate buffer was subjected to approximately 6 mg of immobilized R-chymotrypsin-activated silica beads. At time points of 10 min, 1 h, and 2 h the suspension was pelleted by centrifugation at 3300 rpm for 3 min at 25 °C and the supernatant was removed. In solution, R-chymotrypsin is known to cleave the fluorescent AMC group from SAAP-AMC so the enzymatic hydrolysis can be followed using fluorescence spectroscopy.8 When it is cleaved from the peptide, the fluorescence from the AMC is red-shifted so the relative fractions of hydrolyzed and intact peptide can be determined. The fluorescence spectrum of SAAP-AMC in solution was measured at an emission wavelength of 395 nm (325 nm excitation). The fluorescence spectrum of free AMC in solution was measured at an emission wavelength of 445 nm (345 nm excitation). The activity of the R-chymotrypsin-modified beads was determined by measuring the intensity of the free AMC fluorescence in the supernatant as a function of time and then calculating the amount of hydrolyzed SAAP-AMC using the standard curve. To demonstrate that the observed hydrolysis was due only to the immobilized R-chymotrypsin, the bead supernatant was checked for enzymatic activity. The supernatant was collected by centrifugation at 3300 rpm for 3 min at 25 °C and filtered through a 0.2 µm membrane filter. The activity of the supernatant against SAAP-AMC was measured as described above. Activity of Solution r-Chymotrypsin against the SAAPAMC Surface. One milliliter of a 0.2 mg/mL R-chymotrypsin solution was placed on the surface of a SAAP-AMC wafer. At time points of 2 and 4 h the enzyme solution was pipeted from the surface and the substrate fluorescence at 395 nm (325 nm ex) was measured. The substrate fluorescence measures the amount of complete SAAP-AMC remaining on the surface. Following the fluorescence measurements the enzyme solution was placed back onto the surface of the slide for further time points. Assay of the Activity of r-Chymotrypsin Beads against Peptide Surfaces. The SAAP-AMC-modified fused silica surface was subjected to 1 mL of a 6 mg/mL suspension of R-chymotrypsin-modified silica beads for times of 10 min, 1 h, 2 h, 4 h, and 12 h. At each time point, the bead suspension was pipeted off the substrate surface and the supernatant was collected by centrifugation and filtration through a 0.2 µm membrane. The silica substrate was rinsed with deionized water and dried with nitrogen. No free AMC was observed remaining

Langmuir, Vol. 13, No. 18, 1997 4857 on the silica substrate surface or in the rinse, so all released AMC was presumed to be in the bead supernatant. Substrate fluorescence at 395 nm was measured to determine the fraction of unhydrolyzed peptide remaining on the surface. AMC release to the supernatant due to enzyme hydrolysis was determined by measuring the fluorescence of the bead supernatant at 445 nm. Following the fluorescence measurement, the R-chymotrypsin beads were redispersed into the supernatant, and the suspension was placed back onto the peptide-modified surface for further time points. t-BOC-L-phe-modified aluminum substrates were subjected to a suspension of R-chymotrypsin-modified 3M Emphaze beads for 2 h at 25 °C. The bead suspension was pipeted off the surface, and the aluminum substrates were rinsed several times with deionized water followed by methanol. Substrates were dried with nitrogen and then characterized by contact angle and external reflectance IR spectroscopy. Contact Angle Measurements. The water contact angle of each fused silica slide was measured by the static sessile drop method described by Zisman.11 The average of three measurements on each substrate surface was recorded. Contact angles were recorded for the bare silica or aluminum surface, the aminosilane surface, the peptide surface, and the peptide surface following R-chymotrypsin treatment. External Reflectance Infrared Spectroscopy. Aluminum substrates were analyzed with an FT IR spectrometer (Nicolet, Magna-IR 750, Series II), equipped with a liquid nitrogen-cooled MCT detector. The sample chamber and the interferometer were purged with dry nitrogen. 1000 scans were collected at a resolution of 2 cm-1.

Results The R-chymotrypsin-modified silica beads showed enzymatic activity against SAAP-AMC in solution as shown in Figure 1. The fluorescence due to unhydrolyzed SAAPAMC decreases to background level after 10 min (Figure 1a) while the fluorescence due to hydrolyzed AMC increased concomitantly to a maximum value after 10 min (Figure 1b). The beads retained their activity for several months in storage at 4 °C and also through dozens of rinses with 0.5% Tween 20 in phosphate buffer. Also, no activity was observed in filtered bead supernatant, indicating that no leaching of the covalently bound R-chymotrypsin from the surface of the beads was occurring. Similar results were observed with the R-chymotrypsin activated 3M Emphaze beads. Sessile drop water contact angle and fluorescence spectroscopy was used to follow the formation of the peptide thin films on the planar silica surface. The BAMM aminosilane surface had an average contact angle of 59 ( 2° which changed to 53 ( 2° following the coupling of the SAAP-AMC. These contact angles are consistent with a relatively hydrophobic silane and peptide being coupled to a hydrophilic silica surface and may indicate low overall coverage of peptide on the silica surface. Fluorescence spectroscopy, however, clearly shows the presence of SAAP-AMC peptide on the surface (Figure 2). The silica surface-bound SAAP-AMC was hydrolyzed readily by solution phase R-chymotrypsin as shown in Figure 2. The SAAP-AMC fluorescence is seen to reduce to background within approximately 2 h, indicating the release of the AMC fluorophore from the surface due to hydrolysis. A standard curve relating free AMC fluorescence and AMC concentration was determined to measure the activity of the R-chymotrypsin microspheres as well as an approximate density of the hydrolyzable SAAP-AMC peptide attached to the silica surfaces. A linear leastsquares fit to the standard curve gave a slope of m ) 8.07 × 108 FI/(µmol/mL) AMC, with an intercept of 919 FI (FI (11) Zisman, W. in Contact Angles, Wettability and Adhesion; Vol. 43 of Advances in Chemistry; Fowkes, F., Ed.; American Chemical Society: Washington, DC, 1964; pp 1-51.

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Figure 1. Activity of R-chymotrypsin-modified silica microspheres against SAAP-AMC in solutions. Fluorescence spectra of (a) SAAP-AMC in solution (325 nm ex) and (b) hydrolyzed AMC in solution (345 nm ex) at several time points. Note that an isoemissive point is present in both spectra, compatible with a two-state population of the AMC fluorophore. Legend: t ) 0, filled circle; t ) 10 min, open triangle; t ) 1 h, filled triangle; t ) 2 h, open circle.

Figure 3. Activity of R-chymotrypsin-modified microspheres against a covalently attached SAAP-AMC surface film. (a) Fluorescence spectra of SAAP-AMC bound to a silica surface (325 nm ex) and (b) fluorescence spectra of hydrolyzed AMC released into solution (345 nm ex) at several time points. Legend: t ) 0, diamond; t ) 10 min, hexagon; t ) 1 h, circle; t ) 2 h, square; t ) 4 h, up triangle; t ) 12 h, down triangle.

Figure 2. Activity of solution phase R-chymotrypsin against an SAAP-AMC surface film. Fluorescence spectra of SAAPAMC bound to a silica surface (325 nm ex) at several time points. Legend: t ) 0, circle; t ) 2 h, square; t ) 12 h, triangle.

) measured fluorescence intensity value). By following the enzymatic degradation of 1 mL of an 8.3 × 10-5 mg/ mL SAAP-AMC solution with approximately 6 mg of R-chymotrypsin silica beads, we were able to determine an activity of 1.7 × 10-6 µmol/min of SAAP-AMC hydrolyzed per milligram of activated beads. This compares to an activity of free R-chymotrypsin of 40-60 µmol/min per milligram of R-chymotrypsin, indicating the equivalent of approximately 4 × 10-5 mg of active R-chymotrypsin per milligram of silica. This corresponds to approximately 168 equiv of fully active R-chymotrypsin molecules per bead. The activity of the R-chymotrypsin 3M Emphaze beads was comparable to the activity of the activated silica beads. The packing density of hydrolyzable SAAP-AMC on a 1 in. × 1 in. planar silica surface was determined by allowing free R-chymotrypsin to fully hydrolyze the peptide film until the substrate fluorescence decreased to back-

ground level and all hydrolyzable AMC had been released into solution. By measuring the fluorescence of the free AMC in solution and using the standard curve, we found a density of approximately 1.4 × 1012 SAAP-AMC molecules/cm2 or 7.14 × 103 Å2/SAAP-AMC. It should be emphasized that this is the density of hydrolyzable peptide on the surface, and that may not be equal to the absolute density of the peptide on the surface. Once the R-chymotrypsin-modified silica, 3M Emphaze beads, and SAAP-AMC surface had been characterized, we demonstrated that the activated beads could be used to enzymatically modify the immobilized peptide film. A suspension of the beads was placed upon the SAAP-AMC surface as described in the Materials and Methods section. The beads settled out of suspension and came to rest on the surface of the silica plate. The plate was periodically agitated to move the beads to untouched regions of the surface. At several time intervals the bead suspension was removed from the surface of the plate and the fluorescence from the peptide film on the plate was measured as well as the fluorescence from free AMC in the suspension. Figure 3a shows the observed decrease in SAAP-AMC substrate fluorescence, and Figure 3b shows the concomitant increase of released AMC fluo-

Surface Hydrolysis Using Immobilized Chymotrypsin

rescence in the silica bead suspension. Complete hydrolysis of the film typically was achieved after approximately 12 h. The water contact angle of the hydrolyzed peptide film was found to be 41 ( 2°. This is a ∼12° decrease in contact angle from that of the unmodified SAAP-AMC film. This increase in hydrophilicity is an expected result of the removal of the hydrophobic AMC group and the presence of the free carboxylic acid left after the hydrolysis. Using the density of 1.4 × 1012 SAAPAMC molecules/cm2 from above, and knowing that 1 mg of R-chymotrypsin beads hydrolyzed all of the SAAP-AMC in a 6.4 cm2 area, we can estimate the activity of the immobilized R-chymotrypsin against the immobilized SAAP-AMC surface to be ∼2 × 10-7 µmol/min of bound SAAP-AMC hydrolyzed per milligram of activated beads. This is approximately a factor of 10 smaller than the activity of the activated beads against a solution SAAPAMC substrate. Control experiments with inactive silica microspheres were carried out to prove that the removal of the AMC group from the SAAP-AMC film was due to enzymatic hydrolysis. A suspension of untreated silica beads was placed onto a SAAP-AMC surface, and fluorescence experiments were carried out as described above. No evidence of AMC release from the surface was observed. A second set of control experiments using heat-inactivated R-chymotrypsin-modified beads was also carried out. The R-chymotrypsin beads were heat shocked at 100 °C for 1 h. No hydrolytic activity was observed against SAAPAMC peptide in solution. When the heat-shocked R-chymotrypsin beads were used to treat to the SAAP-AMC surface, no evidence of AMC release was observed. Hydrolysis experiments were also carried out against the t-BOC-phe films on aluminum. The R-chymotrypsin 3M beads were placed on the peptide-modified aluminum for 2 h and then removed. Water contact angles were 26 ( 3° for the APS silane film, 18 ( 3° for the t-BOC-phe film, and 23 ( 3° for the hydrolyzed peptide surface. The R-chymotrypsin should remove the entire t-BOC-phe peptide from the surface, leaving the APS silane film as it was before peptide attachment. Within error, the water contact angles of the APS film and the hydrolyzed peptide film agree, in support of this conclusion. Further evidence for hydrolysis was obtained using external reflectance IR spectroscopy. Figure 4 shows the change in the IR spectra for the t-BOC-phe film before and after treatment with R-chymotrypsin beads. Following hydrolysis the amide I/carbonyl region of the spectrum (1650-1750 cm-1) is nearly eliminated and the C-H stretch region (28003000 cm-1) is reduced but not entirely eliminated. In addition, the peak at 1500-1600 cm-1, which may correspond to the signal from the phenyl ring in the peptide, is reduced after treatment with the R-chymotrypsin beads. These changes are consistent with the removal of the entire peptide film from the surface, leaving the aminosilane film behind. Discussion The fluorescence data shown in Figure 3 strongly suggest that the covalently immobilized R-chymotrypsin is in fact hydrolyzing the phe-AMC amide bond in the immobilized SAAP-AMC film. The decrease in fluorescence of the SAAP-AMC substrate (Figure 3a), combined with the observed increase of the fluorescence due to solution phase AMC (Figure 3b), indicates at a minimum that the fluorescent AMC group has been removed from the substrate surface into solution and also has been hydrolyzed and released from the peptide. We have verified that this was not due to R-chymotrypsin leaching from the surface of the beads into solution by looking for

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Figure 4. External reflectance IR spectra of t-BOC-phe peptide on aluminum (solid line) and the same surface after 2 h of hydrolysis using R-chymotrypsin 3M Emphaze microspheres (dotted line). The amide I/carbonyl region is 1650-1750 cm-1, the C-H stretch is 2800-3000 cm-1, and the phenyl region is 1500-1600 cm-1. The spectra are acquired as difference spectra against a clean aluminum surface. The negative absorbances are probably due to a low level of water contamination being subtracted out in the difference spectrum.

the activity in the supernatant of the bead suspension as a function of time. Supernatant was collected from a pelleted suspension of beads or through a 0.2 µm filter. No leaching was observed over many days of incubation of the EGS cross-linked beads. It is worth noting that R-chymotrypsin beads cross-linked with only glutaraldehyde did show substantial leaching, and that was the reason we chose to carry out the subsequent EGS step. At least two possible scenarios could explain the hydrolysis data shown in Figure 3: (1) the enzyme is specifically hydrolyzing the phe-AMC bond in the peptide film and releasing the AMC into solution or (2) the whole peptide film or the peptide and silane films are detaching from the solid surface into solution and then the AMC group is cleaved by the R-chymotrypsin microspheres while the peptide or a fragment of the peptide is in solution. The second scenario may occur as a result of some nonspecific physical interaction of the beads with the peptide thin film. Scenario 1 is the expected explanation and is entirely consistent with all of our data. Scenario 2 is incompatible with our control experiments. With heat-inactivated R-chymotrypsin beads, as well as bare silica beads, no removal of the SAAP-AMC from the substrate surface was observed by fluorescence; i.e., the fluorescence signal from the surface remained constant over the time scale of the experiment. Therefore, the beads alone are not causing the peptide (or the AMC group) to be released from the surface and any modification of the surface must be the result of the direct enzymatic activity of the activated beads against the covalently bound peptide, in support of scenario 1. The change in water contact angle is also in agreement with this conclusion because R-chymotrypsin bead treatment of the surface results in an increase in hydrophilicity of the surface consistent with the appearance of hydrophilic carboxylic acid groups on the surface due to enzyme-mediated hydrolysis. Experiments with t-BOC-phe peptide films on aluminum substrates are also in agreement with the conclusion that the R-chymotrypsin beads are directly hydrolyzing the covalently immobilized peptide film. The IR spectra in Figure 4 show nearly complete elimination of the amide I/carbonyl region of the IR spectra following enzymatic treatment with the R-chymotrypsin beads. In addition, the C-H stretch region and the region we have attributed

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to the phenyl ring show a substantial reduction or complete elimination of signal. These results are consistent with the (expected) complete removal of the t-BOC-phe peptide from the surface, leaving the APS silane film behind. Once again water contact angle measurements help to confirm this conclusion. The final contact angle after R-chymotrypsin bead treatment (23°) increased from the 19° of the t-BOC-phe surface toward the value observed on unmodified APS (26°), as would be expected if the surface was to become more like APS and less like t-BOC-phe on APS. The nonspecific adsorption of proteins onto solid surfaces is a major difficulty encountered with all systems that rely upon specific interactions of proteins with solid surfaces. To date, this problem has been observed most often in the biosensor and bioassay community12-14 and has been studied in detail by several groups.15,16 In previous work using enzymes to modify surface chemistry we found nonspecific enzyme adsorption to be a serious impediment to surface chemical modification.2 By covalently immobilizing the enzyme itself to a solid surface, the problem of nonspecific enzyme adsorption to the substrate surface virtually disappears. The work described in this paper showed no evidence from spectroscopy or contact angle measurements that enzyme was adsorbing to the peptide surface during or after microsphere treatment. Therefore the chemical modification of the peptide film has been carried out without contamination of the surface. The resulting surface is clean and can be subjected to further chemical processing steps. The system we have chosen to work with, however, still has several weaknessessprimarily the low activity of the immobilized R-chymotrypsin and the low surface coverage of hydrolyzable SAAP-AMC. Using steric considerations alone, a close-packed film of R-chymotrypsin on the surface of a 500 nm silica bead would require approximately 105 copies of the enzyme per bead. This would imply an activity for each bead equivalent to 105 R-chymotrypsin molecules, assuming no loss of activity upon immobilization. We measured an activity per bead equivalent to approximately 168 R-chymotrypsin molecules, indicating a 1000-fold loss in activity from the ideal case. This loss in activity could be due to either a low surface packing density of the enzyme, an unfavorable orientation of the active site, or direct loss of activity due to the covalent conjugation. We have not identified which effect is primarily responsible for the loss of R-chymotrypsin activity in our system. The coverage density of hydrolyzable SAAP-AMC peptide was approximately 100 times lower than the ideal close-packed density. The rate at which the activated beads modified the immobilized SAAPAMC was a factor of 10 slower than the rate of hydrolysis against free SAAP-AMC. The factors controlling the rate and extent to which the enzyme can hydrolyze this surface include the local molecular packing of the peptide film, the length of the molecular tether to the surface, and masstransport limitations of the reagents. Whitesell and coworkers have shown that highly ordered substrate films will restrict access of the enzyme to an individual substrate molecule as a result of steric hindrance.6 Also, an insufficient tethering distance of the substrate molecule and enzyme from their respective surfaces will, at a minimum, limit the number of molecular orientations and (12) Flounders, A. W.; Brandon, D. L.; Bates, A. H. Appl. Biochem. Biotechnol. 1995, 50, 265-284. (13) Morgan, H.; Pritchard, D. J.; Cooper, D. M. Biosens. Bioelectron. 1995, 10, 841-846. (14) Bhatia, S. K.; Hickman, J. J.; Ligler, F. S. J. Am. Chem. Soc. 1992, 114, 4432-4433. (15) Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstrom, I. J. Colloid. Interface Sci. 1987, 119, 203-210. (16) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-340.

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eventually could restrict the substrate from reaching the enzyme active site. Although it is unlikely that high packing density is a problem for our system, it is quite likely that the relatively short tethers we have used for immobilizing the substrate may cause the low hydrolyzable density of the film. In any case, the reaction between the immobilized R-chymotrypsin and the immobilized SAAP-AMC is obviously mass-transport limited under these circumstances. The low activity may be improved by optimizing the enzyme and substrate immobilization conditions to increase packing density, orient the enzyme active site away from the surface, and provide for increased dynamics using alternative tethering chemistries. For R-chymotrypsin, glutaraldehyde coupling was chosen for simplicity but arguably is a rather crude choice when compared with many alternative approaches.12-14 It certainly doesn’t provide a long tether for the enzyme and was not implemented in a way which orients the enzyme with respect to the surface. In this system, and other enzymesubstrate systems in general, optimal activity will be achieved when the active enzyme can freely interact with the appropriate moieties in the substrate film. Longer covalent tethers and methods for controlling molecular packing and orientation will help immensely in this regard. Finally, while we have demonstrated enzymatic hydrolysis of a substrate on a surface, it would also be desirable to demonstrate immobilized-enzyme-mediated catalytic addition to the surface. Obviously, this would be a much more powerful tool than simple chemical hydrolysis and would provide the opportunity for doing true in situ synthesis on the surface. This will require optimization of the catalytic activity of the enzyme as well as a host of other reaction conditions such as solution reactant concentration, pH, ionic strength, and examination of nonaqueous solvent systems.17 In summary, the use of a covalently immobilized enzyme alleviates many of the problems encountered when using solution phase enzymes for chemical modification of organic thin films by eliminating enzyme fouling of the surface and enabling recovery of the enzyme. Successive modification of the same thin film could allow several steps of complex chemistry to be carried out with the high chemical specificity characteristic of enzymes. In addition, since the enzyme is immobilized to a solid support, it is plausible that with positional control the immobilized enzyme systems could be used for chemical patterning of substrate thin films. Thus, localized patterning of an immobilized substrate surface could also be carried out with the exceptional chemical specificity of enzymatic chemistry. The ultimate patterning resolution would be dependent on the size and shape of the surface to which the enzyme is immobilized as well as the ability to bring that surface into contact with a substrate surface with precision. For example, the use of closed loop piezoelectric positioners such as those used in scanning probe microscopes to control the enzyme surface position could result in near molecular scale resolution. Our laboratory is investigating some of these possibilities for further study. Acknowledgment. We thank Pete Isaacson for preparing the aluminum films and Charles Patterson for assistance with the IR experiments. We thank Dr. William Lacy, Dr. Brett Martin, and Dr. Michael Markowitz for scientific discussions. This work was supported by NRL core funding from the Office of Naval Research. LA970324+ (17) Khmelnitsky, Y. L.; Welch, S. H.; Clark, D. S.; Dordick, J. S. J. Am. Chem. Soc. 1994, 116, 2647-2648.