N-Hydroxysuccinimide Ester Functionalized Perfluorophenyl Azides

Sep 28, 1993 - photogenerated, highly reactive nitrene intermediate derived from the PFPA. ... new series of photoactivated cross-linking reagents for...
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Bioconjugate Chem. 1994, 5, 151-157

151

N-Hydroxysuccinimide Ester Functionalized Perfluorophenyl Azides as Novel Photoactive Heterobifunctional Cross-Linking Reagents. The Covalent Immobilization of Biomolecules to Polymer Surfaces Mingdi Yan,+ Sui Xiong Cai,+JM. N. Wybourne,§ and John F. W. Keana*s+ Departments of Chemistry and Physics, University of Oregon, Eugene, Oregon 97403. Received September 28, 1993”

The synthesis of N-hydroxysuccinimide (NHS) functionalized perfluorophenyl azides (PFPAs) 2 and 3 is described together with a general method for the covalent modification of polymer surfaces using heterobifunctional, photoactivable cross-linking reagents 1 and 3. The NHS-active ester group becomes covalently attached to the polymer surface via an efficient CH bond insertion reaction of the photogenerated, highly reactive nitrene intermediate derived from the PFPA. The NHS ester is capable of further reaction with a variety of primary amine-containing reagents including biomolecules by way of amide formation. The method is illustrated as follows. Photolysis of polystyrene (PS) and poly(3-octylthiophene) (P30T) thin films spin-coated with NHS PFPA ester 1 or 3 gave films 8 or 9, respectively. Each film was then exposed to an aqueous solution of horseradish peroxidase (HRP), giving films 10 or 11, respectively. The amounts of HRP immobilized on P S and P 3 0 T were calculated from enzyme activity assays to be 0.5 f 0.1 ng/mm2 for loa,1.0 f0.2 ng/mm2 for lla,0.2 f 0.1 ng/mm2 for lob,0.3 f0.1ng/mm2for 1 lb. Using this surface functionalization methodology, biotin-streptavidinbiotin-HRP was constructed on the PS film. The storage stability of HRP thus immobilized through the extended linker, biotin-streptavidin-biotin, was enhanced as compared to that of HRP directly immobilized on the PS surface.

INTRODUCTION

The conjugation of biologicals such as enzymes (I), antibodies (21,DNA (3),and cells (4)to solid supports is of importance in the areas of nucleotide synthesis ( 5 ) , molecular recognition (61,polymeric drugs (3, and biosensors (8). The presence of suitably reactive functional groups on the surface is an essential feature in many of these applications. Thus, we became interested in developing a methodology for surface functionalization. A series of functionalized perfluorophenyl azides (PFPAS)was developed in our laboratory (9,10)and elsewhere (11-13) as a new class of photolabeling reagents with improved CH insertion efficiency over their nonfluorinated analogs. By incorporating a PFPA within a molecule that also contained a protein reactive group, there resulted a new series of photoactivated cross-linking reagents for bioconjugation (14,15). It occurred to us that functionalization of polymer surfaces ought to be possible through the use of a PFPA containing a second reactive functional group such as anN-hydroxysuccinimide (NHS)active ester elsewhere in the molecule. In the process of polymer surface modification, the NHS PFPA ester becomes covalently attached to the polymer surface by way of CH insertion of the highly reactive nitrene intermediate generated by photolysis of the PFPA. The covalentlyattached NHS ester is then allowed to react with NHSreactive groups such as primary amines in proteins or other biomolecules, resulting in the covalent immobilization of proteins or other biomolecules to polymer surfaces. Recently, we communicated the use of NHS-functionalized PFPAs for the surface modification of polymers (16).By

combining this methodology with either deep-UV or electron beam lithography it is possible to generate micrometer-scale patterns of immobilized biomolecules on the polymer surface (17). Herein, we detail the synthesis of NHS PFPA ester 2 as well as 3, which has a six-atom spacer unit between the two reactive groups. We also detail the functionalization of polymer surfaces with 1 (9) and 3. The surface

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R = NHCH2CO-N

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R = OCH,

0

modification process involves a simple spin-coating technique followed by photolysis, providing an attractive alternative to the series of treatments with chemicals as described in other surface functionalization processes (18). We also demonstrate the efficient immobilization of the active enzyme horseradish peroxidase (HRP) to polystyrene (PS) and poly(3-octylthiophene) (P30T) surfaces. EXPERIMENTAL PROCEDURES

+ Department

of Chemistry. Present address: Acea Pharmaceuticals, Inc., 1003 Health Science Road West, Irvine, CA 92715. 6 Department of Physics. Abstract published in Advance ACS Abstracts, January 15, 1994.

*

@

General. Melting points were taken on a Mel-Temp melting point apparatus and are uncorrected. Preparative TLC was performed on Analtech GF precoated silica gel (1000 pm) glass-backed plates (20 X 20 cm). ‘H NMR spectra were recorded on a 300-MHz General Electric QE-

1043-1802/94/2905-0151$04.50/00 1994 American Chemical Society

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Yan et at.

Bioconjugate Chem., Vol. 5, No. 2, 1994

300. Chemical shifts are reported in 6 units referenced to residual proton signals of the deuterated solvents. Infrared spectra were obtained on a Nicolet 5DXB FT-IR spectrometer. UV-vis spectra were obtained on a Perkin Elmer Lambda 6 UV/vis spectrophotometer. Mass spectra were recorded on a VG ZAB-2-HF mass spectrometer with a VG-11-250 data system in the electron ionization mode (70 eV). Microanalyses were performed by Desert Analytics of Tucson, AZ. Spin-coating was performed with a Model l-EClOlD-R485 photoresist spinner (Headway Research Inc.) with the spin speed set at 1000rpm. Baking was accomplished in an oven (Model 1410,VWR Scientific) preheated to 60 "C in air for 20 min. Photolysis was carried out at ambient temperature in a Rayonet photoreactor with 254 nm lamps (400 W) for 5 min. Materials. Circular micro cover glasses (radius 10 mm) were purchased from VWR Scientific, Inc. Triethylamine, dicyclohexylcarbodiimide (DCC),N-hydroxysuccinimide (NHS), glycine ethyl ester hydrochloride, and 5-aminopentanoic acid were used as received from Aldrich. Polystyrene (PS) beads were used as received (MW 125 000-250 000, Polysciences Inc.). Poly(3-octylthiophene) (P30T) was prepared following a known procedure (19). Horseradish peroxidase (HRP) was purchased as a salt-free powder (type IV-A, activity about 1,000 units/mg) from Sigma and used without further purification. Bovine serum albumin (BSA) (fraction V powder) was used as received from Sigma. 2,2'-Azinobis(3-ethylbenzthiazoline-6-sulfonicacid) diammonium salt (ABTS) was purchased from Sigma and was used without further purification. N-(5-Aminopentyl)biotinamide, streptavidin, and biotin-HRP conjugate were used as received from Molecular Probes, Inc. (Eugene, OR). Hydrogen peroxide (30%) was used as received from J. T. Baker (20). NaHC03 buffer (0.1 M) was prepared by dissolving 8.4 g of NaHC03 in 1.0 L of doubly distilled water. Phosphate buffer (0.1 M, pH 7.0) was prepared from K H ~ P O ~ / K Z H in P Odoubly ~ distilled water. Tetrahydrofuran (THF)was dried over sodium/benzophenone ketyl. Dimethylformamide (DMF) was dried over molecular sieves. Nitromethane and chloroform (CHC13)were dried over CaClz pellets (4-8 mesh). N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)glycineEthyl Ester (5). A mixture of glycine ethyl ester hydrochloride (217 mg, 1.55 mmol) and triethylamine (158 mg, 1.56 mmol) in 7.0 mL of T H F was stirred for 20 min. 4-Azido-2,3,5,6-tetrafluorobenzoic acid (9) (369 mg, 1.57 mmol) and DCC (324 mg, 1.57 mmol) were added, and the mixture was stirred overnight and filtered. The filtrate was evaporated, and the residue was treated with 20 mL of ethyl acetate. The mixture was filtered, and the filtrate was washed with 0.1 N HCl(2 X 10 mL). The solution was dried and evaporated to leave a solid which was purified by preparative TLC with 1:2 CHCl3-hexanes as the ' ) of 5 as a colorless developing solvent to give 160 mg (32% powder, mp 85-86 OC: lH NMR (CDC13) 6 1.32 (t, J = 7.1 Hz, 3 H), 4.24 (d, J = 4.8 Hz, 2 H), 4.27 (9, J = 7.1 Hz, 2 H), 6.54 (m, 1 H); FTIR 2128, 1744, 1686, 1649, 1523, 1488, 1225, 1001 cm-'. Anal. Calcd for CllHgF4N403 C: 41.26;H,2.52;N,17.50. Found: C,41.46;H,2.37;N,17.66. N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)glycine(6). To a solution of 5 (60 mg, 0.19 mmol) in 0.5 mL of methanol was added 1 N NaOH (0.25 mL, 0.25 mmol), and the solution was stirred for 1h. The solution was acidified by addition of 2 N HC1 to pH < 1, and the precipitate was filtered and dried to leave 23 mg of 6 as a white powder. The filtrate was extracted with THF/CHC13 (l:l,3 x 3 mL), and the extracts were combined and evaporated to

give another portion (32 mg) of 6 as a white powder (combined yield 55 mg, 99%), mp 147-148 OC: lH NMR (CDC13 + DMSO-&) 6 4.34 (d, J = 4.8 Hz, 2 H), 6.53 (m, 1H); MS 292 (2, M+),264 (20, M+-N2), 190 (20, NC6F4c o ) , 162 (100, NC6F4). N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)glycine NSuccinimidyl Ester ( 2 ) . A solution of 6 (39.3 mg, 0.134 mmol), DCC (29.3 mg, 0.142 mmol), and NHS (16.6 mg, 0.144 mmol) in 0.5 mL of T H F was stirred at 25 "C overnight. The resulting mixture was filtered, and the filtrate was evaporated to leave a solid which was redissolved in 1.0 mL of CHzCl2 and filtered. The filtrate was evaporated to leave 42 mg (80%)of 2 as a white powder, mp 145-146 OC: lH NMR (CDC13) 6 2.88 (s, 4 H), 4.64 (d, J = 5.4 Hz, 2 H), 6.55 (m, 1 H); FTIR 2129,1792, 1748, 1718,1699,1649,1520,1489,1204 cm-l; MS 389 (8, M+), 275 (60, M+ - NHS), 247 (27, M+ - NHS - Nz), 218 (65, M+ - CONHS - N2 - H), 190 (45, NCGF~CO), 162 (100, NC6F4); high-resolution MS calcd for C13H~F4N505 389.0382, found 389.0405. 5-(4-Azido-2,3,5,6-tetrafluobenzamido)pentanoic Acid (7). To a solution of 5-aminopentanoic acid (238 mg, 2.03 mmol) in 5 mL of 1N NaOH was added 4-azido2,3,5,6-tetrafluorobenzoylchloride (9) (239 mg, 0.942 mmol). The mixture was stirred a t 25 "C for 20 min and acidified with 2 N HCl to pH < 1. The white precipitate was filtered, washed with 0.1 N HCl(5 X 3 mL) and water (4 X 3 mL), and dried under reduced pressure to give 273 mg (87%) of 7 as a white powder, mp 160-161 OC: lH NMR (CDC13 D M s 0 - d ~ 6) 1.44 (m, 4 H), 2.08 (t, J = 6.9 Hz, 2 H), 3.15 (9, J = 6.0 Hz, 2 H), 7.87 (s, 1 H); MS 334 (5, M+),317 (4, M+ -OH), 306 (40, M+ - Nz), 190 (15, NCsF&O), 162 (100, NCsF4); high-resolution MS calcd for C ~ ~ H I O F ~ 334.0687, N ~ O ~found 334.0710. N-Succinimidyl5-(4-Azido-2,3,5,6-tetrafluorobenzamido)pentanoate (3). NHS PFPA ester 3 was prepared from 100 mg of acid 7 in a manner similar to 2 and was obtained as a white power (yield 112 mg, go%), mp 93-95 OC: lH NMR (CDCl3) 6 1.77 (m, 2 H), 1.85 (m, 2 H), 2.69 (t, J = 6.6 Hz, 2 H), 2.84 (s, 4 H), 3.51 (9, J = 6.2 Hz, 2 H), 6.22 (m, 1H); FTIR 2127,1817,1786,1742,1681, 1649,1602,1526,1487,1260,1209,1069 cm-'; MS 431 (5, M+), 403 (3, M+ - Nz), 317 (22, M+ - NHS), 289 (8, M+ - NHS - Nz), 162 (100, NC6F4); high-resolution MS calcd for C16H13F4N505 431.0850, found 431.0866. Surface Modification of PS with 1 and 3 To Give Films 8a and 9a. PS beads (50.0 mg) were dissolved in 1.0 mL of xylenes to form a colorless solution. Two drops of the solution was placed on the surface of a circular micro cover glass, and the glass was spun a t 1000 rpm for 2 min to give a smooth PS thin film. A solution of 2.5 mg of NHS PFPA ester 1 (9) or 3 in 0.5 mL of nitromethane was prepared. About 40 pL of the solution was placed on top of the PS film, and the glass was spun at 1000 rpm for 1min. The resulting film was baked for 20 min in an oven preheated to 60 OC and was photolyzed with 254-nm lamps for 5 min to give film 8a or 9a. The decomposition of azide upon photolysis was monitored by FTIR using a NaCl disk as the support. The FTIR spectrum showed strong absorption a t 2128 cm-' due to the azide group after the solution of 1 or 3 was spin-coated on the PS film. This absorption completely disappeared after the film was irradiated with 254-nm lamps for 5 min. Surface Modification of P 3 0 T with 1 and 3 T o Give Films 8b and 9b. P30T (20.0 mg) was dissolved in 1.0 mL of CHC13 to form a red solution. Four drops of the solution was placed on a circular micro cover glass and

+

Immobilizatlon of Biomolecules to Polymer Surfaces

was spun for 2 min to give a red thin film. About 40 p L of a solution of 2.5 mg of 1 or 3 in 0.5 mL of methanol was placed on top of the P 3 0 T film and was spun for 1 min. The resulting film was baked at 60 "C and photolyzed with 254-nm lamps for 5 min to give film 8b or 9b. A control experiment using a NaCl disk as the support and FTIR to monitor the decomposition of the azido group was performed in a similar manner as described above. The IR spectrum showed strong azide absorption at 2128 cm-' after the solution of 1 or 3 was spin-coated on the P30T film and complete disappearance of this absorption when the film was irradiated with 254-nm lamps for 5 min. Immobilization of HRP To Give Films 10 and 11. HRP (2.0 mg) was dissolved in 1.0 mL of 0.1 M pH 8.2 NaHCO, buffer containing 10.0 mg of BSA to form a light brown solution (50 pM). The film obtained above (8 or 9) was incubated under this solution at 25 "C for 3 h and was washed thoroughly with NaHC03 buffer followed by phosphate buffer to give film 10 or 11. Two control experiments using PS as the polymer substrate were conducted. In the first experiment, a circular micro cover glass was spin-coated with 5% PS solution in xylenes for 2 min (for details see the spincoating procedure above). The film was baked, photolyzed, incubated in 50 pM HRP solution at 25 OC for 3 h and rinsed thoroughly with NaHC03 buffer followed by phosphate buffer. In the second control experiment, a circular micro cover glass was spin-coated with 5% PS solution in xylenes, then with a solution of 0.5 w t % methyl 4-azido-2,3,5,6-tetrafluorobenzoate (4) in nitromethane on top of the PS film. The film was baked, photolyzed, incubated in 50 pM HRP solution at 25 "C for 3 h, and rinsed thoroughly with NaHC03 buffer followed by phosphate buffer. A control experiment was performed using P30T as the support. A circular micro cover glass was spin-coated with 2% P30T solution in chloroform for 2 min, baked, photolyzed, incubated in 50 pM HRP solution at 25 "C for 3 h, and rinsed thoroughly with NaHC03 buffer followed by phosphate buffer. Preparation of PS-Biotin-Streptavidin-BiotinHRP Conjugate. A circular micro cover glass was spincoated as described above with 5 % PS solution in xylenes for 2 min and then with 0.5 wt % 1solution in nitromethane on top of the PS film for 1min. The film was baked and irradiated with 254-nm lamps for 5 min to give film 8a. N-(5-Aminopenty1)biotinamide (2.0 mg) was dissolved in 0.1 mL of DMF, and 0.5 mL of NaHC03 buffer was added to form a colorless solution. Film 8a was immersed under this solution at 25 OC for 5 h and washed thoroughly with NaHC03 buffer followed by phosphate buffer. The resulting film 12 was then incubated at 25 "C for 1 h in a premixed solution containing 2.6 pM streptavidin and 0.52 pM biotinated HRP in pH 7.0 phosphate buffer (21). The film was washed thoroughly with phosphate buffer to give film 13. HRP Assays. All HRP solutions and the dilutions were made in pH 7.0 phosphate buffer containing 10.0 mg/mL of BSA. ABTS solutions were prepared in pH 7.0 phosphate buffer and used at a concentration of 1.0 mg/ mL throughout. Hydrogen peroxide was used at a concentration of 0.28 % throughout. Activity of Native HRP. To a cuvette containing 2.0 mL of ABTS solution and 50.0 pL of a HRP solution of various concentration was added 20.0 pL of H202 solution (final concentrations, 1.8 mM ABTS and 0.8 mM HzOz). The absorbance of the resulting solution at 420 nm after

Bioconjugate Chem., Vol. 5, No. 2, 1994

153

incubation at 25 "C for 10 min was read against pH 7.0 phosphate buffer. Assays for Immobilized HRP. To a dish containing 10.0 mL of ABTS solution and film 10 or 11 or 13 was added 100.0 pL of Hz02 solution at 25 "C under stirring. Aliquots of about 2 mL of the resulting solution were taken at about 1min time intervals, and the absorbances at 420 nm were recorded. Aliquots were returned to the dish after each measurement to keep the total volume constant. RESULTS AND DISCUSSION

Syntheses of NHS PFPA esters 2 and 3 were accomplished as shown in eq 1-3. Reaction of 4-azido-2,3,5,6-

+

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NH,CH,CO,Et

F I

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N3

N3 NaOHIMeOHIH,O

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7

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N3 2 n=1

3 n=4

tetrafluorobenzoic acid (9) and glycine ethyl ester hydrochloride with DCC as the coupling reagent gave N-(4azido-2,3,5,6-tetrafluorobenzoy1)glycineethyl ester (5) in 32 % yield. Hydrolysis of 5 with NaOH in methanol gave 6 as a white powder in 99% yield (eq 1). Treatment of 4-azido-2,3,5,6-tetrafluorobenzoyl chloride (9)with 5-aminopentanoic acid under basic conditions followed by acidification gave 5-(4-azido-2,3,5,6-tetrafluorobenzamido)pentanoic acid (7) as a white powder in 87% yield (eq 2). Reaction of 6 and 7 with NHS in the presence of DCC gave N-(4-azido-2,3,5,6-tetrafluorobenzoyl)glycine N-succinimidyl ester (2) and N-succinimidyl5-(4-azido-2,3,5,6tetrafluorobenzamido)pentanoate(3) as white powders in 80% and 90% yields, respectively (eq 3). Reagents, 2,3, and N-succinimidyl4-azido-2,3,5,6-tetrafluorobenzoate (1) (91,form a series of NHS-functionalized PFPAs differing in the lengths of the linker between the PFPA and the NHS ester group. Surface modification of PS thin films with NHSfunctionalized PFPA 1 and 3 was performed as follows. A circular micro cover glass was spin-coated with 5 % PS solution in xylenes. The thickness of the film was measured as 0.5 pm (22). Then a solution of 0.5 wt % 1 or 3 in nitromethane (PS is not soluble in nitromethane) was spin-coated on top of the PS film. In the control

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Bioconjugate Chem., Vol. 5, No. 2, 1994

154

et

al.

d

1

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3200 3018

2836

2654

2473

2291

2109

1964

1873

1782

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1691

1600

Wavenumbers (cm-')

Figure 1. FTIR spectra of (a) a PS film, (b) a PS film spincoated with 1 on top, (c) the resulting film baked at 60 "C for 20 min, and (d) the baked film photolyzed with 254-nm lamps for 5 min to give film 8a. Scheme 1

A

1. spin-coat 1 O r 3

9

3. hu 254 nm

9

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a = PS b = P30T

a

R =

9

R = NH(CH,),CO-N

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1 0 X=NH-HRP 1 1 X = NH(CH,),CONH-HRP

experiment using a NaCl disk as the support, an IR spectrum showed the azide absorption at 2138 cm-I after 1 or 3 was spin-coated on the PS film (Figure l b vs la). The film was then baked at 60 "C for 20 min. The baking process removed residual solvent present in the film and likely facilitated the diffusion of the surface-deposited NHS PFPA ester into the PS film, thus increasing the probability of CH bond insertion into the polymer surface of the nitrene intermediate generated in the next step. However, baking did not induce the thermodecomposition of the azide because the intensity of the absorption at 2138 cm-1 after baking (Figure IC)was essentially the same as that before baking (Figure lb). Irradiation of the azidecontaining film with 254-nm lamps for 5 min resulted in complete decomposition of the azide group in 1 or 3 as indicated by the absence of the 2138 cm-l absorption in the IR spectrum of the corresponding control sample (Figure Id vs IC). Photolysis of 1 and 3 generated the highly active nitrene intermediate (9, 11) which then underwent CH bond insertion into the P S surface, giving films 8a and 9a,respectively (Scheme 1). Since an NHSactive ester group is present in 1 and 3, it also becomes covalently attached to the PS surface. As an example of the application of this surface functionalization technique for binding biomolecules to

2

4

6

8

10

12

Time ( m i d

Figure 2. Time course of immobilized HRP reaction using 1 as the surface modification reagent. 1.8 mM ABTW0.8 mM HzOz in 0.1 M pH 7.0 phosphate buffer. A least-squares fit gives absorbance A = 0.0693 + 0.0746t, where t is the time.

polymer surfaces, horseradish peroxidase (HRP) was immobilized on films 8a and 9a. The immobilization was accomplished viaamide formation between the NHS active ester group and an amino group present in HRP. Since the NHS ester is slowly hydrolyzed by water (231,dilution of the HRP solution will result in competition between coupling and loss of the NHS ester group by hydrolysis. A concentration of 50 pM HRP was used in order to minimize the loss of NHS ester caused by hydrolysis ( 2 4 ) . Since only the unprotonated amino groups in HRP are reactive in the coupling reaction,it is necessary to maintain an alkaline pH at which a significant number of amino groups are unprotonated. However, hydrolysis of the NHS ester becomes competitive above pH 9. A pH 8.2 NaHC03 buffer was used since it was demonstrated to give excellent coupling results ( 2 4 ) . Immobilization of HRP on films 8a and 9a was carried out by separately incubating each film in a solution of 50 pM HRP in NaHC03 buffer at 25 "C for 3 h. The resulting films were washed thoroughly with NaHC03 buffer followed by phosphate buffer to give films 10a and lla. In order to determine the immobilization efficiency, activities of the immobilized HRP and the native HRP were measured using the procedure of Groome ( 2 5 ) . The activity of HRP was determined spectrophotometrically at 420 nm and 25 "C using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and hydrogen peroxide (1.8 mM ABTW0.8 mM H.202). In a typical assay for immobilized HRP, film 10a or lla was immersed in 10.0mL of ABTS solution in phosphate buffer (1.0 mg/mL), and 100.0 yL of H2Oz solution (0.28%)was added with stirring. Aliquots of about 2 mL of the solution were taken at about 1-mintime intervals, and absorbances at 420 nm were recorded. A representative curve for the immobilized HRP is shown in Figure 2. Assays of the native HRP were carried out by incubating various HRP solutions of known concentration with 1.8 mM ABTS/0.8 mM HzO2 in pH 7.0 phosphate buffer. Absorbances at 420 nm after 10 min incubation were read against pH 7.0 phosphate buffer. Figure 3 shows a linear relationship between the absorbance and the concentration of HRP. Making the assumption that the immobilized HRP has the same activity as the native HRP ( 2 5 , 2 6 ) ,the amounts of HRP immobilized can be calculated. For example, using 1 as the surface modification reagent, the absorbance at 10 min was calculated to be 0.816 (Figure 2 ) , corresponding to a concentration of 18.1ng/mL (Figure 3). Since the total volume of the assay solution was 10.0 mL, the amount of HRP immobilized on the circular micro cover glass (radius = 10 mm) was 181ng. This corresponds to 0.58 ng/mm2 of surface area.

Bioconjugate Chem., Vol. 5, No. 2, 1994 155

Immobilization of Biomolecules to Polymer Surfaces

-

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E

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-

0.8

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0.6

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0.4

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20

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Figure 3. Relationship between absorbance and concentration of native HRP. 1.8 mM ABTS/0.8 mM HzOz in 0.1 M pH 7.0 phosphate buffer. Cuvettes were incubated at 25 "C for 10 min before reading the absorbances at 420 nm against pH 7.0 phosphate buffer blank. The least-squares fit gives absorbance A = O.O746C, where C is the concentration.

From the four samples each of films 10a or l l a , the amount of HRP immobilized was calculated to be 0.5 f 0.1 ng/mm2for film 10a and 1.0 f 0.2 ng/mm2for film lla. The amount of HRP immobilized using 3 was twice as much as that using 1 as the surface modification reagent. In the case of 3-functionalized PS film 9a, the NHS ester group is separated from the polymer surface by four methylene units as compared to l-functionalized PS film 8a. The NHS ester separated from the PS surface by an extended spacer would be expected to experienceless steric interference by the PS matrix, and consequently the reaction with the large HRP molecules should be more efficient. A HRP molecule has been described as having an average molecular weight of 40 000 and a radius of 2.67 nm in the hydrated state (27). Considering a monolayer of densely packed HRP spheres on a flat surface, the maximum amount of HRP molecules laid on the surface is calculated to be 2.7 ng/mm2 (28). Therefore, the surface coverage of immobilized HRP is about 19 % of the maximum coverage calculated using 1 and about 37% using 3 as the surface modification reagent, indicating a reasonable immobilization efficiency. Two control experiments were carried out. In the first experiment, a PS film prepared by spin-coating 5% PS xylenes solution on a piece of micro cover glass was baked and photolyzed. After incubating in 50 pM HRP solution at 25 OC for 3 h and washing thoroughly with NaHC03 buffer followed by phosphate buffer, the film was subjected to the enzyme assay as described above. The result showed no enzyme activity,indicating that there was no detectable nonspecific adsorption of HRP molecules on the PS surface. In the second control experiment, the methyl PFPA ester 4 instead of the NHS PFPA ester 1 or 3 was used as the surface modification reagent. A piece of micro cover glass was spin-coated with 5% PS xylenes solution and then witha solution of0.5 w t % methyl4-azido-2,3,5,6tetrafluorobenzoate (4) (9) in nitromethane on top of the PS film. The resulting film was baked, photolyzed, incubated in 50 pM HRP solution at 25 "C for 3 h, and rinsed thoroughly with NaHC03 buffer followed by phosphate buffer. No enzyme activity was detected, indicating that there was no reaction between the HRP molecule and the "unactivated" methyl PFPA estermodified PS surface. Thus, HRP was likely immobilized on films 8a and 9a through covalent amide formation between the NHS active ester group and the amino group present in HRP.

0

I

I

10

20

I 30

Time (day)

Figure 4. Storage stability of immobilized HRP at 4 "C in pH 7.0 phosphate buffer of film (a) loa, (b) lla, and (c) 13.

The generality of this methodology for polymer surface modification was extended to an organic conducting polymer, poly(3-octylthiophene) (P30T). P30T was prepared chemically from 3-octylthiophene using Fee13 according to a known procedure (19). The functionalization of P30T surface was performed by spin-coating a circular micro cover glass with 2 % P30T solution in chloroform followed by a solution of 0.5 wt % 1 or 3 in methanol on top of the P30T film. Methanol was used as the solvent because it spread on P30T film better than nitromethane and it does not dissolve the polymer film. In a control experiment using a NaCl disk as the support, the IR spectrum showed a strong azide absorption at 2138 cm-l after spin-coating of 1or 3 onto P30T. The film was then irradiated with 254-nm lamps for 5 min to give film 8b or 9b. The IR spectra of the corresponding control samples showed the complete disappearance of the azide absorption in 8b and 9b. Immobilization of HRP on films 8b and 9b and the enzyme activity assays of the immobilized HRP on films 10b and l l b were carried out in the same manner as described above for films 8a, 9a and loa, 1la. Similarly, by making the assumption that immobilized HRP has the same activity as the native HRP (25,26),the amount of HRP immobilized was calculated as 0.2 f 0.1 ng/mm2for film 10b and 0.3 f 0.1 ng/mm2 for film l l b . Again, the latter was larger than the former, the same result as observed in the case of using PS as the polymer substrate. A control experiment was carried out to determine whether there was nonspecific adsorption of HRP molecules on the P30T surface. AP30T film prepared by spin-coating 2 % P30T solution in chloroform was baked, photolyzed, and incubated in 50 pM HRP solution at 25 OC for 3 h. After being rinsed thoroughly with NaHC03 buffer followed by phosphate buffer, the film was subjected to the enzyme activity assay. No detectable enzyme activity was observed,indicating that there were no HRP molecules nonspecifically adsorbed on the P 3 0 T film. The stability of HRP immobilized on the PS film upon storage was also studied. Films 10a and 1la were stored at 4 "C in pH 7.0 phosphate buffer. HRP on film 10a lost essentially all of its initial activity after 1.8 days. HRP on film lla retained 100% of its initial activity after 1.8 days but lost 31% of its initial activity after 4.8 days (Figure 4). It has been reported that enzymes tend to lose their biological activities after immobilization on solid matrices due to the masking of their active sites by the solid matrix or multipoint attachments which restrict the local movements necessary for the activities (29). In the case of film loa, there was only a short spacer between HRP and PS. The polymer matrix may mask the active sites and/or alter the three-dimensional structure of HRP at its active sites

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Bioconjugate Chem., Vol. 5, No. 2, 1994

Scheme 2

8a

12

0

Streptavidin-biotin-HRP

ACKNOWLEDGMENT

streptavidin I

biotin - H R P

biotinated HRP. Some free streptavidin molecules are present in the solution of streptavidin-biotin-HRP and will not contribute to the activity of film 13 after conjugation. An enzyme activity assay of film 13 stored at 4 OC showed that HRP immobilized in this manner retained 100% of its initial activity after 6 days and 38% of its initial activity after 28 days (Figure 4). In conclusion, we present the synthesis of NHS functionalized PFPAs 2 and 3 and a general method using bifunctional cross-linking reagents such as 1 and 3 for the covalent modification of polymer surfaces. Application of this methodology for bioconjugation was demonstrated through covalently immobilization of HRP to the NHS PFPA ester-functionalized PS and P30T surfaces. Utilization of the multilayer system, biotin-streptavidinbiotin-HRP, greatly enhanced the storage stability of the immobilized HRP. This new bioconjugation strategy may find application for the construction of novel micrometerscale biosensors based on field-effecttransistors and optical waveguides.

13 R = biotin-streptavidin-biotin-HRP

by stretching the molecule. Multipoint attachment may also occur during the immobilization process; thus, the initial activity of the immobilized HRP was lower with 10a compared to lla and the activity decreased rapidly over a 1-2-day period. In the case of film 1 la,HRP was separated from the polymer matrix by four additional methylene units. The four methylene units act as a spacer to reduce the chance of masking of the active sites by the polymer matrix, resulting in a higher initial activity and enhanced storage stability of the immobilized HRP. A multilayered system containing biotin-streptavidinbiotin-HRP was constructed on the PS surface in order to test whether further separation of HRP from the surface would lead to greater storage stability. Biotin (vitamin H) is known to bind strongly and specifically to the bacterial protein streptavidin with a binding constant of 1015 M-1. The binding properties are only slightly influenced when biotin is functionalized (30). Since streptavidin has four binding sites for biotin situated on two opposite sides of the tetrameric protein, streptavidin can thus be used to link two different functions in the assembly (31). The PS-biotin-streptavidin-biotin-HRP assembly was constructed as shown in Scheme 2. Functionalization of the PS surface with 1 to give film 8a was performed in the same manner as described earlier. A solution of N45aminopenty1)biotinamide in DMF (2.0 mg/O.l mL) was prepared and was added to 0.5 mL of 0.1 M pH 8.2 NaHC03 buffer. Film 8a was immersed in the resulting solution at 25 "C for 5 h. After thorough rinsing with NaHC03 buffer followed by phosphate buffer, the resulting film 12 was incubated at 25 "C for 1 h in a premixed solution of 2.6 pM streptavidin and 0.52 pM biotinated HRP in pH 7.0 phosphate buffer. The film was then rinsed with the buffer to give film 13. Using the same method as described above to obtain the linear relationship between absorbance and the concentration of biotinated HRP, the amount of biotinated HRP immobilized on film 13 was calculated to be 0.25 ng/mm2.1 The lower initial activity of film 13 as compared to that of film 10 is likely due to the fact that excess streptavidin was used in the conjugation step with 1 A film was prepared in a similar manner as film 13 but using 3 as the surface modification reagent. The amount of immobilized biotinalted H R P was calculated t o be 0.63 ng/mm2.

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(22) Cai, S. X., Kanskar, M, Wybourne, M. N., and Keana, J. F. W. (1992)Introduction of functionalgroups into polymer films via deep-UV photolysis or electron-beam lithography: modification of polystyrene and poly(3-octylthiophene) by a functionalized perfluorophenyl azide. Chem. Mater. 4 , 879. (23) Lomants, A. J., and Fairbanks, G. (1976) Chemical probes of extended biological structures: synthesis and properties of the cleavable protein crosslinking reagent [%SIdithiobis(succinimidy1)propionate.J. Mol. Biol. 104,243. (24) Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3, 2. (25) Groome, N. P. (1980) Superiority of ABTS over Trinder reagent as chromogen in highly sensitive peroxidase assays for enzyme linked immunoadsorbent assay. J.Clin. Chem. Clin. Biochem. 18,345. (26) Nakane, P. K., and Kawaoi, A. (1974) Peroxidase-labeled antibody a new method of conjugation. J. Histochem. Cytochem. 22, 1084. (27) Steiner, H., and Dunford, H. B. (1978) Ionic strength dependence of the oxidation of iodide and ferrocyanide by compound I of horseradish peroxidase. Eur. J.Biochem. 82, 543. (28) Each sphere generates a void area of 1/2 X 5.34 X (0.87 X 5.34) - 3 X 1/6 X ~ ( 2 . 6 7=) ~1.15 nm2.Therefore, the number of HRP molecules densely packed on a flat surface of 1mm2 is (1X 106)2/(~(2.67)2+ 1.15) = 4.25 X 1010,whichcorresponded to 4.25 X 101°/(6.02 X loz3)X 40 000 X los = 2.7 ng of HRP/ mm2. (29) Solomon, B., Hafas E., Koppel, R., Schwartz, F., and Fleminger, G. (1991) Highly active enzyme preparations immobilized via matrix-conjugated anti-Fc antibodies. J. Chromatogr. 539,335. (30) Bayer, E. A., and Wilchek, M. (1980) The use of the avidinbiotin complex as a tool in molecular biology. Methods of Biochemical Analysis (D. Glick, Ed.) Vol. 26, p 1-46, John Wiley &. Sons, New York. (31) Haussling, L., Michel, B., Ringsdorf, H., and Rohrer, H. (1991)Direct observation of streptavidin specificallyadsorbed on biotin-functionalized self-assembled monolayer with the scanning tunneling microscope. Angew. Chem.,Znt. Ed. Engl. 30,569.