Bioconjugate Chem. 1996, 7, 317−321
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A General Method for the Spatially Defined Immobilization of Biomolecules on Glass Surfaces Using “Caged” Biotin Michael C. Pirrung* and Chia-Yu Huang P. M. Gross Chemical Laboratory, Department of Chemistry, Duke University, Durham, North Carolina 27708-0346. Received November 2, 1995X
A method has been developed to spatially define the immobilization of proteins on surfaces using the classic avidin-biotin link, for which a wide variety of biochemical reagents are commercially available. A derivative of biotin bearing a photoremovable nitrobenzyl group (MeNPOC-biotin) has been prepared in a form suitable for simple linkage to biomolecules and surfaces. It has been used to functionalize bovine serum albumin (BSA) to form MeNPOC-biotin-BSA, which can then be coated onto glass. On photolithographic patterning of the surface, biotins are freed in the irradiated areas, permitting avidin to be localized at the irradiated sites. Subsequent addition of a biotinylated molecule permits its site-specific localization. Patterning of a biotinylated antibody and dye-labeled avidins or streptavidin using this reagent has been demonstrated by fluorescence microscopy.
INTRODUCTION
A crucial technology for the production of a new generation of “biochips” (1-6) is the integration of biological and chemical materials on the microscopic scale. Such devices could have uses ranging from biomolecular electronics (7-10) to studies in cell adhesion to micromachines. Because of the power of parallel processing in the patterning of semiconductors, similar methods have been considered for the patterning of biological components. A method that permits the immobilization of multiple proteins in predefined patterns is essential to construct biochips of any significant complexity. The work reported here permits immobilization of multiple proteins at photolithographically determined sites without risk of photochemical damage. We accomplished this by temporally separating the patterning and binding steps using “caged” biotin linked to the glass surface through a conventional serum albumin coating process. This biotin derivative is protected from its normal binding to avidin by a photoremovable nitrobenzyl group. When exposed to light through a mask, this group is lost, the patterned, irradiated sites gain affinity for avidin, and they hereby can immobilize biotinylated substances. BACKGROUND
Several reports of photolithographic protein immobilization have utilized surface-bound photoactivated reagents like nitroarylazides (11, 12). A technique for attaching biomolecules to surfaces using light but without patterning has been reported; it involves attachment of nitrophenylazide photoaffinity reagents to agarose beads and irradiation of the modified solid in the presence of an antibody to immobilize it for immunoassays. The obvious drawbacks of this technique are the low yields known for the nitroarylazide photo-cross-linking process and possible photochemical damage to the protein. Likewise, nitroarylazides have been connected to urokinase, hyaluronic acid, collagen, fibronectin, or serum albumin by polyethylene glycol spacers and the conju* Author to whom correspondence should be addressed. Fax: (919) 660-1591. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, May 1, 1996.
S1043-1802(96)00013-4 CCC: $12.00
gates irradiated in the presence of polyurethane tubing and other plastics to generate biomolecule-functionalized surfaces. This method suffers from the fact that molecules may be photoactivated when distant from the surface and are therefore wasted. The former strategy, involving generation of reactive sites on the surface, makes more efficient use of the reactants. Performing similar nitroarylazide photochemistry in a spatially localized way on the gate of a field effect transistor has been used to form a BIOCHEMFET (13). The direct photochemical cross-linking (14) of benzophenone-derivatized glass surfaces to proteins in solution has been described (15), and a similar use of light-activatable nitroarylazide compounds attached to a surface through an avidin-biotin link has been reported (16). Patterning such substrates with light permits the creation of ∼2 µm features, but these methods require the protein to be present during the irradiation, potentially damaging it. Two methods temporally separate the irradiation and immobilization steps. Keana has used the efficient perfluorophenylazide photochemistry (17, 18) to attach NHS active esters to lithographically patterned regions on a polystyrene film. The active esters are then used to immobilize a protein in the patterned areas (19, 20). Ligler has reported a photolithographic method for the production of patterned surfaces that resist protein adsorption in irradiated areas and permit adsorption in masked areas, but this method is limited to the patterning of one protein per surface (21, 22). Whitesides has used self-assembled monolayers on gold to prepare a number of biofunctionalized surfaces, in some cases involving patterning by stamping (23). The use of light to prepare sites on a surface that can react with biomolecules by well-established, conventional chemistry was a guiding principle in this work. We aimed to use the extensively exploited interactions between avidin and biotin (24) in assembling biomolecular aggregates. Other recent reports describe the preparation of a photoactivatable biotin derivative (caged biotin) based on nitrobenzyl photochemistry and its use for covalent derivatization of aminopropylsilanized glass surfaces (25, 26). This work was aimed at the derivatization of bovine serum albumin (BSA), which is commonly coated onto glass surfaces in biological derivati© 1996 American Chemical Society
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Table 1 BSA/0.1 M NaHCO3 (mg/mL)
MeNPOCiotin-NHS/ DMF (µL)
MeNPOCbiotin/BSA
irradiation time (min)
contrast ratio of pattern
4 4 4 4 4 4 4 4
10 25 50 100 150 175 200 300
1.6 2.4 5.4 11 15 19 23 29
20 20 20 20 20 20 20 20
4:1 7:1 6:1 8:1 22:1 25:1 20:1
zation (27), with a novel photoactivatable biotin. This work particularly aimed to apply to the urea ring of biotin the methylnitropiperonyloxycarbonyl (MeNPOC) group that Holmes has developed as a superior reagent for the photolabile protection of alcohols (28). Figure 1. Fluorescence micrograph of a microscope slide coated with MeNPOC-biotin-BSA, exposed (350 nm, Oriel arc lamp, 20 min) through a mask with 350 µm “stripe” features, and stained with 100 µg/mL FITC-streptavidin in phosphatebuffered saline (PBS). The contrast ratio (average fluorescence intensity of a 150 µm × 100 µm light area:intensity of a 150 µm × 100 µm dark area) is 25:1.
RESULTS AND DISCUSSION
Methylnitropiperonyl alcohol (1) was treated with 0.1 equiv of potassium tert-butoxide and carbonyldiimidazole (to presumably generate the carbonate-imidazolide) and then with biotin methyl ester and NaH (1.2 equiv) to provide the N-1′-protected derivative 2. This structure was assigned on the basis of analogy to previous work and consistency between its spectroscopic properties and previously prepared caged biotin derivatives. A straightforward sequence involving standard peptide-coupling reactions was used to add a 6-aminocaproic acid linker terminated in an NHS active ester. The MeNPOCbiotin-aminocaproic-NHS ester (5) was then used in standard condition (29) for functionalization of BSA. The derivatized protein 6 was separated from excess reagents by either gel filtration or ultrafiltration, and the extent of loading was determined by comparison of the unique MeNPOC absorption at 350 nm ( ) 4595 M-1 cm-1) with the protein band at 260 nm. The average number of MeNPOC biotins per BSA could be varied from ∼2 to 28 by modification of reaction conditions (Table 1).
The MeNPOC-biotin-BSA conjugates were used to coat glass microscope slides under standard conditions (30). A derivatized slide was exposed to 350 nm light from an Oriel arc lamp through a mask with 350 µm linear features. After the surface was stained with FITC-streptavidin, confocal fluorescence micrographs
Figure 2. Z-Series fluorescence micrograph (sum of seven confocal planes at 5 µm spacing) of a microscope slide coated with MeNPOC-biotin-BSA, exposed through a 350 µm stripe mask, and treated with streptavidin (100 µg/mL PBS), biotinylated anti-bovine IgG (50 µg/mL PBS), and then bovine IgGFITC (180 µg/mL PBS). The contrast ratio (300 µm × 200 µm light area:150 µm × 100 µm dark area) is 10:1.
like that shown in Figure 1 could be obtained. This experiment was conducted with BSAs bearing a range of caged biotin loadings (Table 1) to optimize the contrast ratio between the illuminated and unilluminated areas. The optimum ratio was obtained with an average of 24 biotins per BSA. Likewise, using a patterned irradiation step followed by streptavidin, a biotinylated antibody, and fluoresceinated antigen, the fluorescence micrograph in Figure 2 was obtained. The three-dimensional appearance of this figure is derived from the summation of multiple confocal planes and shows that the protein-coated surface is somewhat rough but relatively uniformly derivatized by the absorption step. It also shows that the thickness of the protein coating is ∼30 µm. The potential of caged biotin-BSA for the patterning of different biomolecules was demonstrated by another experiment in which a first patterning step was used to immobilize avidin-Texas red and a second step was used to immobilize avidin-fluorescein. The two-color micrograph shown in Figure 3 demonstrates little cross-talk
Caged Biotin
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ecules, which are available by either chemical derivatization or recombinant DNA techniques (31). The general interest of the method lies in its potential to construct complex patterns of proteins on the micrometer scale without risk of photochemical damage and its convergence with reagents widely used for the linking of biomolecules, namely bovine serum albumin, avidin, and biotin. The avidin-biotin link and the absorption of BSA to glass used in this method are quite robust, though denaturants are still capable of disrupting these interactions. Possible applications of patterned proteinaceous surfaces prepared using this method include mimicking of cell surfaces and the development of multianalyte fluoroimmunosensors. MATERIALS AND METHODS Figure 3. Two-color fluorescence micrograph after exposure of a microscope slide coated with MeNPOC-biotin-BSA through a 500 µm stripe mask, treatment with avidin-Texas Red, followed by another exposure through a 500 µm stripe mask rotated 90° to the former pattern, and treatment with avidinfluorescein. The signal:noise ratio is 30:1 for the fluorescein image (300 µm × 200 µm light area:150 µm × 100 µm dark area) and 30:1 for the Texas Red image (200 µm × 133 µm light area: 200 µm × 133 µm dark area).
Figure 4. Fluorescence micrograph of a MeNPOC-biotin-BSAcoated microscope slide after exposure (350 nm, Oriel arc lamp, 20 min) through a resolution target and staining with 100 µg/ mL FITC-streptavidin.
(s:n > 30:1) between sites and demonstrates that essentially all of the biotins are released in the first irradition step, since no fluorescein-avidin is immobilized in the Texas Red region. When avidin-fluorescein is used as the first stain and avidin-Texas Red is used as the second stain, green fluorescence is seen in the area of intersection (data not shown). This control demonstrates that the red fluorescence in the area of intersection in the micrograph shown in Figure 3 is not due to energy transfer from fluorescein to Texas Red. Treatment of the first slide with avidin-Texas Red overnight ensured saturation of free biotin groups on the surface. The control shows some cross-talk between green and red because the slide was treated only for 1 h with avidinfluorescein before the second patterning step. More complex features of even smaller dimensions were written by exposure through a resolution target as a mask (Figure 4). Significance. This paper reports a novel and general technique for localization of biomolecules at microscopic locations on glass surfaces utilizing biotinylated biomol-
General. Ether and THF were distilled from a deep blue solution resulting from benzophenone and sodium. Dichloromethane and chloroform were refluxed and distilled from calcium hydride. DMF was stirred with calcium sulfate, filtered, and distilled from calcium hydride. BSA, streptavidin, streptavidin-fluorescein, bovine IgG-FITC, and anti-bovine IgG-biotin were purchased from Sigma. Bodipy avidin-Texas Red, Bodipy avidin-fluorescein, and biotin-FITC were purchased from Molecular Probes, Inc. All washings were accomplished by dipping the slide in the specified solution. 3′,4-(Methylenedioxy)-6′-nitroacetophenone. To a solution of 3′,4′-(methylenedioxy)acetophenone (Aldrich, 5 g, 30.4 mmol) being stirred in 50 mL of TFA was added sodium nitrite (6.3 g, 91.3 mmol) in one portion. A redbrown gas was generated. The mixture was stirred overnight. Water was added, and the solution was extracted with CH2Cl2 (100 mL × 3). The organic layer was washed with saturated NaHCO3 and water and dried (MgSO4). Solvent was removed under reduced pressure. Purification by flash column chromatography (hexane: EtOAc ) 2:1) afforded 3.75 g of yellow solid (60%). 1H NMR (CDCl3, 300 MHz): δ 7.53 (s, 1 H), 6.75 (s, 1 H), 6.19 (s, 1 H), 2.49 (s, 3 H). 13C{1H} NMR (CDCl3, 75 MHz): δ 199.2, 152.6, 148.7, 139.9, 134.9, 106.0, 104.7, 103.6, 30.1. This compound is known (32, 33). Methylnitropiperonyl Alcohol (1). The above ketone (3.1 g, 14.8 mmol) was dissolved in a mixture of dry THF and absolute EtOH (50 mL each). To this solution was added 560 mg (14.8 mmol) of NaBH4, and it was stirred at room temperature overnight. The reaction was quenched with 1 N HCl, and solvent was removed under reduced pressure. The residue was partitioned between CH2Cl2 and water. The organic layer was dried (MgSO4) and solvent removed under reduced pressure. Purification by flash chromatography (hexane:EtOAc ) 3:1) afforded 3.53 g of yellow solid (96%). 1H NMR (CDCl3, 300 MHz): δ 7.43 (s, 1 H), 7.25 (s, 1 H), 6.11 (dd, J ) 3.3 Hz, J ) 1.2 Hz, 2 H), 5.43 (q, J ) 6.3 Hz, 1 H), 2.50 (br, 1 H), 1.51 (d, J ) 6.3 Hz, 3 H). 13C{1H} NMR (CDCl3, 75 MHz): δ 152.3, 146.8, 141.3, 139.0, 106.3, 105.1, 102.9, 65.6, 24.1. This compound is known (28, 34). MeNPOC-biotin-OMe (2). Four reactions of the same scale were set up simultaneously. To methylnitropiperonyl alcohol (1) (715 mg, 3.39 mmol) in 5 mL of dry THF was added KOtBu (43 mg, 0.39 mmol). After 10 min, carbonyldiimidazole (829 mg, 5.08 mmol) was added. The mixture was stirred at room temperature for 3 h. THF was removed under reduced pressure and the residue triturated with CCl4. The precipitate was filtered through Celite, and the filtrate was evaporated. The residue was redissolved in 15 mL of dry CHCl3 (amylenestabilized). Biotin-OMe (500 mg, 1.94 mmol) and NaH
320 Bioconjugate Chem., Vol. 7, No. 3, 1996
(91 mg, 60% dispersion in mineral oil) were added. The mixture was heated to reflux under argon for 72 h. The reaction was quenched with a few drops of acetic acid. The four reaction mixtures were combined, and the solvent was removed under reduced pressure. Purification by flash chromatography on silica (3% acetone, 3% MeOH in CHCl3) afforded 1.4 g (37%) of a yellow foam. Sp: 79-80 °C. IR (film): 3284, 1779, 1730 cm-1. 1H NMR (CDCl3, 300 MHz): δ 7.51 (d, J ) 3.9 Hz, 1 H), 7.38 (d, J ) 9.0 Hz, 1 H), 4.51 (q, J ) 6.3 Hz, 1 H), 6.11 (s, 2 H), 5.93 (d, J ) 12.3 Hz, 1 H), 4.84 (m, 1 H), 4.21 (m, 1 H), 3.66 (s, 3 H), 3.21 (m, 1 H), 3.10 (m, 1 H), 2.97 (m, 1 H), 2.33 (t, J ) 7.5 Hz, 2 H), 1.67 (m, 7 H), 1.47 (m, 2 H). 13C{1H} NMR (CDCl3, 75 MHz): δ 173.8, 155.4, 155.3, 152.5, 147.2, 141.1, 135.2, 106.8, 105.0, 103.0, 71.1, 62.6, 57.6, 55.1, 51.5, 38.6, 33.3, 28.1, 27.8, 24.5, 22.4. MS-FAB: m/z 494 (M- - H). Anal. Calcd for C21H25N3O9S: C, 50.91; H, 5.05; N, 8.48. Found: C, 50.99; H, 5.12; N, 8.52. MeNPOC-biotin-OH (3). MeNPOC-biotin-OMe (1.21 g, 2.45 mmol) was dissolved in 15 mL of THF and 15 mL of 1 N HCl. The mixture was heated at reflux for 24 h, and the solvent was removed under reduced pressure. Purification by flash chromatography (10% MeOH in CHCl3) afforded 897 mg (76%) of a yellow foam. Sp: 9192 °C. IR (film): 3280, 1775, 1723 cm-1. 1H NMR (CDCl3/CD3OD, 300 MHz): δ 7.50 (d, J ) 3.3 Hz, 1 H), 7.31 (d, J ) 5.1 Hz, 1 H), 6.49 (q, J ) 3 Hz, 1 H), 6.14 (s, 2 H), 4.81 (m, 1 H), 4.22 (m, 1 H), 3.20 (m, 1 H), 3.03 (m, 2 H), 2.33 (t, J ) 7.2 Hz, 2 H), 1.68 (m, 7 H), 1.49 (m, 2 H). 13C{1H} NMR (CDCl3, 75 MHz): δ 177.7, 157.2, 157.1, 152.6, 147.2, 141.2, 134.9, 106.6, 105.1, 103.0, 71.3, 62.8, 57.8, 54.9, 38.6, 33.5, 28.3, 28.1, 24.9, 22.3. MSFAB: m/z 480 (M- - H). Anal. Calcd for C20H23N3O9S: C, 49.89; H, 4.78; N, 8.73. Found: C, 49.66; H, 4.87; N, 8.54. MeNPOC-biotin-AC-OH (4). To a stirring solution of MeNPOC-biotin-OH (400 mg, 0.83 mmol) and Nhydroxysuccinimide (105 mg, 0.92 mmol) in 5 mL of anhydrous dimethoxyethane and 3 mL of anhydrous DMF was added 1,3-dicyclohexylcarbodiimide (171 mg, 0.83 mmol). The mixture was stirred under argon overnight and filtered through Celite. The filtrate was transferred to a round bottom flask, and 6-aminocaproic acid (109 mg, 0.83 mmol) was added. The mixture was stirred under Ar overnight and filtered through Celite. The filtrate was evaporated and placed in a high vacuum to remove DMF. Purification by flash chromatography (10% MeOH in CHCl3 to 20% MeOH in CHCl3) afforded 349 mg (70%) of a brown syrup. IR (film): 3419, 1768, 1648 cm-1. 1H NMR (CD3OD, 300 MHz): δ 7.97 (t, J ) 5.4 Hz, 1 H), 7.48 (s, 1 H), 7.24 (s, 1 H), 6.37 (q, J ) 6.3 Hz, 1 H), 6.14 (s, 2 H), 4.88 (m, 1 H), 4.26 (dd, J ) 7.8 Hz, J ) 3.6 Hz, 1 H), 3.29 (t, J ) 1.5 Hz, 2 H), 3.16 (m, 2 H), 3.06 (m, 1 H), 2.25 (t, J ) 7.5 Hz, 2 H), 2.16 (t, J ) 7.5 Hz, 2 H), 1.67 (m, 11 H), 1.49 (m, 2 H), 1.36 (m, 2 H). 13C{1H} NMR (CDCl , 75 MHz): δ 176.8, 173.8, 156.2, 3 152.5, 150.5, 147.2, 140.9, 134.7, 106.4, 104.8, 103.0, 70.9, 62.5, 57.6, 54.9, 50.1, 38.9, 35.5, 33.6, 29.4, 28.6, 27.8, 27.6, 25.9, 24.1, 22.0. MS-FAB: m/z 593 (M- - H). Anal. Calcd for C26H34N4O10S‚2H2O: C, 49.52; H, 5.40; N, 8.89. Found: C, 49.53; H, 5.66; N, 8.83. MeNPOC-biotin-AC-NHS (5). MeNPOC-biotin-ACOH (113 mg, 0.19 mmol) and N-hydroxysuccinimide (24 mg, 0.21 mmol) were dissolved in 5 mL of dry DMF. Dicyclohexylcarbodiimide (39 mg, 0.19 mmol) were added with stirring under Ar. The mixture was stirred at room temperature overnight and filtered through Celite, washing with 2 mL of dry DMF. The filtrate was brought to
Pirrung and Huang
a total volume of 8 mL with dry DMF. This solution was kept in a dessicator at 4 °C for direct derivatization of BSA. MeNPOC-biotin-AC-BSA (6). MeNPOC-biotin-ACNHS (200 mL of a 23.75 mM solution in DMF) was mixed with 4 mg of BSA in 2 mL of 0.1 M NaHCO3/0.2 M NaCl solution; the reaction mixture was brought to a total volume of 3 mL with PBS, and it was allowed to stand at 4 °C overnight. The product was purified by ultrafiltration (Centricon-30 microconcentrator at 4 °C, 6000 rpm). Alternatively, a gel filtration column (Sephadex G-50, fine) could be used to purify the product. The concentrated product was diluted in PBS to about 2 mg of BSA/mL and kept at 4 °C for further use. The ratio of MeNPOC-biotin-AC to BSA can be varied by using different amounts of the active ester (Table 1). The loading was determined by comparison of the unique MeNPOC absorption at 346 nm ( ) 4595 M-1 cm-1) with the protein band at 280 nm. Immobilization of MeNPOC-biotin-AC-BSA on a Solid Support. A microscopic slide (precleaned with absolute EtOH) was treated with 1 mL of 30 µM MeNPOC-biotin-AC-BSA in phosphate-buffered saline (PBS) solution (pH 7.4) for 2 h, washed with PBS for 5 min, and dried. Spatially Addressable Immobilization of Fluorescein-Streptavidin on a Solid Support. A MeNPOC-biotin-AC-BSA-derivatized slide was exposed to UV light at 350 nm (Oriel arc lamp, 4 mW/cm2) through a mask (stripe mask or resolution target) for 20 min in PBS. The slide was washed in PBS for 5 min and treated with 1 mL of 100 µg/mL FITC-streptavidin in PBS for 1 h. The slide was then washed in PBS for 10 min, washed with deionized water for 1 min, and dried. Table 1 shows the different contrast ratios of the fluorescence images which were collected when MeNPOC-biotin-ACBSA conjugates with different biotin:BSA ratios were used. Spatially Addressable Immobilization of Fluorescein-Biotin on a Solid Support. A MeNPOCbiotin-AC-BSA-coated slide was exposed through a 350 µm stripe mask, treated with streptavidin (200 µg/mL PBS) for 1 h, washed in PBS for 10 min, and dried. The slide was then treated with biotinylated fluorescein (100 µg/mL PBS) for 2 h and washed in PBS for 10 min and deionized water for 1 min. Spatially Addressable Immobilization of Fluorescein-Antibody on a Solid Support. A MeNPOCbiotin-AC-BSA-coated slide was exposed through a 350 µm stripe mask, treated with streptavidin (100 µg/mL PBS) for 3 h, washed in PBS for 10 min, and dried. The slide was then treated with biotinylated anti-bovine IgG (50 µg/mL in PBS) for 2 h and washed in PBS for 10 min. The slide was stained with bovine IgG-FITC (180 µg/ mL PBS) for 1 h and washed in PBS for 10 min and deionized water for 1 min. Two-Color Experiments: Immobilization of Fluorescein-Streptavidin and Fluorescein-Texas Red on a Solid Support. A MeNPOC-biotin-AC-BSAderivatized slide was exposed to UV light at 350 nm through a mask for 20 min in contact with PBS. The slide was washed in PBS for 5 min and treated with 1 mL of 100 µg/mL avidin-Texas Red in PBS overnight. The slide was then washed in PBS for 10 min and exposed again at 350 nm through a mask with a stripe 90° to the first mask. The slide was washed in PBS for 5 min and treated with 1 mL of 100 µg/mL avidinfluorescein in PBS for 1 h. A similar experiment with green stain first and red stain second gave a similar outcome.
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Caged Biotin ACKNOWLEDGMENT
Financial support was provided by NIH GM-46720 and the ONR (N00014-94-1-0364). Consultation with Prof. W. M. Reichert and Amy Blawas of the Duke Biochemical Engineering Department is acknowledged. LITERATURE CITED (1) Wangermann, G. (1989) Topical aspects of bioelectronics. Stud. Biophys. 132. 946. (2) Hamann, C. (1987) Bioelectronics - perspectives and reality. Naturwiss. Reihe 51, 95-102. (3) Gil’manshin, R. I., and Lazarev, D. I. (1987) Biotechnology as a source of materials for electronics. Biotekhnologiya 3, 421-32. (4) Drexler, K. E. (1986) Engines of Creation, Anchor/Doubleday, New York. (5) Molecular Electronic Devices (1987) Vol. I, II, MarcelDekker, New York. (6) Wrighton, M. S. (1985) Prospects for a new kind of synthesis: assembly of molecular components fo achieve functions. Comments Inorg. Chem. 4, 269-94. (7) Stayton, P. S., Olinger, J. M., Wollman, S. T., Bohn, P. W., and Sligar, S. G. (1994) Engineering proteins for electrooptical biomaterials. Adv. Chem. Ser. 240, 475-90. (8) Hong, H. G., Jiang, M., Sligar, S. G., and Bohn, P. W. (1994) Cysteine-specific surface tethering of genetically engineered cytochromes for fabrication of metalloprotein nanostructures. Langmuir 10, 153-8. (9) Bohn, P. W., Sligar, S. G., Hong, H. G., Jiang, M., Thurman, E. M., and Cong, Y. (1993) Oriented monolayers of genetically engineered cytochrome b5. Implications for molecular devices. Proc.-Electrochem. Soc. 93-7, 845-8. (10) Stayton, P. S., Olinger, J. M., Jiang, M., Bohn, P. W., and Sligar, S. G. (1992) Genetic engineering of surface attachment sites yields oriented protein monolayers. J. Am. Chem. Soc. 114, 9298-9. (11) Swanson, M. J., Dunkirk, S. G., Pietig, J. A., and Guire, P. E. (1992) Use photochemistry to modify membranes. CHEMTACH 22, 624-6. (12) Dunkirk, S. G., Gregg, S. L., Duran, L. W., Monfils, J. D., Haapala, J. E., Marcy, J. A., Clapper, D. L., Amos, R. A., Guire, P. E. (1991) Photochemical coatings for the prevention of bacterial colonization. J. Biomater. Appl. 6, 131-56. (13) Lowe, C. R., and Earley, F. G. P. (1985) Diagnostic device incorporating a biochemical ligand. U.S. Patent 4,562,157. (14) Sigrist, H., Gao, H., and Wegmu¨ller, B. (1992) Lightdependent, covalent immobilization of biomolecules ‘inert’ surfaces. Bio/Technology 10, 1026-8. (15) Rozsnyai, L. F., Benson, D. R., Fodor, S. P. A., and Schultz, P. G. (1992) Photolithographic immobilization of biopolymers on solid supports. Angew. Chem., Int. Ed. Engl. 31, 759. (16) Pritchard, D. J., Morgan, H., and Cooper, J. M. (1995) Micron-scale patterning of biological molecules. Angew. Chem., Int. Ed. Engl. 34, 91-93. (17) Young, M. J. T., and Platz, M. S. (1989) Polyfluorinated aryl azides as photoaffinity labeling reagents; the roomtemperature carbon-hydrogen insertion reactions of singlet pentafluorophenyl nitrene with alkanes. Tetrahedron Lett. 30, 2199-202. (18) Leyva, E., Munoz, D., and Platz, M. S. (1989) Photochemistry of fluorinated aryl azides in toluene solution and in frozen polycrystals. J. Org. Chem. 54, 5938-45. (19) Yan, M., Cai, S. X., Wybourne, M. N., and Keana, J. F. W. (1993) Photochemical functionalization of polymer surfaces
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