Immobilization of Biomolecules on Polyurethane Membrane Surfaces

Dec 19, 2001 - Institute of Microtechnology (IMT), University of Neuchâtel, Rue Jaquet-Droz 1,. CH-2007 Neuchâtel, Switzerland, Centre Suisse d'Elec...
0 downloads 0 Views 148KB Size
Download PDF
90

Bioconjugate Chem. 2002, 13, 90−96

Immobilization of Biomolecules on Polyurethane Membrane Surfaces Patrick Reichmuth,† Hans Sigrist,‡ Martin Badertscher,§ Werner E. Morf,† Nicolaas F. de Rooij,† and Erno¨ Pretsch*,§ Institute of Microtechnology (IMT), University of Neuchaˆtel, Rue Jaquet-Droz 1, CH-2007 Neuchaˆtel, Switzerland, Centre Suisse d’Electronique et de Microtechnique SA (CSEM), Rue Jaquet-Droz 1, CH-2007 Neuchaˆtel, Switzerland, and Swiss Federal Institute of Technology (ETH), Laboratorium fu¨r Organische Chemie, Universita¨tstrasse 16, CH-8092 Zu¨rich, Switzerland. Received May 21, 2001

Lipophilic polymer membranes incorporating binding sites are widely used in various potentiometric, amperometric, and optical sensors. Here, we report on the biofunctional modification of the surface of a Ca2+-selective membrane. A photoactivatable biotin derivative was synthesized and covalently immobilized on a soft polyurethane membrane. The modified polymer was characterized by X-ray photoelectron spectroscopy (XPS) as well as by potentiometric measurements. The selective binding of streptavidin by the photo-cross-linked biotin derivative was demonstrated. The surface coverage obtained with different experimental protocols was analyzed by autoradiography using [35S]streptavidin. The new approach may significantly extend the scope of applicability of potentiometric sensors.

INTRODUCTION

Lipophilic polymeric membranes incorporating ionselective ligands (ionophores) and ion exchangers are widely applied in potentiometric sensors (1, 2). The emf response is in most cases governed by the phase boundary potential that is defined by the bulk properties of the membrane and the sample (1, 3). Plasticized poly(vinyl chloride) is the most widely used membrane matrix, but polymers with low glass transition temperatures such as polyurethanes (4), silicon rubber (5-8), and polyacrylates (9, 10) have also been used without plasticizer. Owing to their relatively good biocompatibility, polyurethanes, especially the commercially available Tecoflex, are the best investigated alternatives to PVC (4, 11-13). Usually, the ion exchanger and the ionophore are simply dissolved in the polymer matrix, but in other cases they have also been covalently bound to the polymer backbone (6, 9, 10, 14-16). To improve the biocompatibility, the surface of potentiometric sensor membranes has been modified by covalent attachment of poly(ethylene oxide) (17), heparin (18), or theophylline (19). So far, however, a surface modification of ion-selective electrode membranes with specific, biofunctional binding sites has not yet been reported. On the other hand, a number of techniques have been used to create selective reaction layers by covalently binding organic compounds to various substrates. One of the most versatile methods is photo-cross-linking using agents that form nitrenes, carbenes, or ketyl radicals upon irradiation (20). For example, Yan et al. used functionalized perfluorophenyl azides to pattern the surface of polystyrene (21, 22). Similarly, Hengsakul et al. used an azidobenzene derivative of biotin for protein * To whom correspondence should be addressed. Phone: +41 16322926.Fax: +4116321164.E-mail: [email protected]. † IMT, University of Neucha ˆ tel. ‡ CSEM, University of Neucha ˆ tel. § ETH Zu ¨ rich.

patterning of polystyrene or nitrocellulose surfaces (23). Several biotin derivatives with photosensitive groups have been described in the literature (24-30). More recently, carbene-generating photobiotins were used for the segregation of micrometer-sized domains of glassy-carbon, quartz, and polymer surfaces (31), for the preparation of bioprobes such as a photoaffinity taxoid probe (32), or for a photoaffinity labeling system for a glycosyltransferase (33). Here we report on the synthesis of a novel photoactive biotin derivative 5 (Figure 1), its immobilization on the surface of an ion-selective polyurethane membrane (Tecoflex, 6 in Figure 1), and its interaction with streptavidin. The long-term goal of these studies is the development of new sensors to monitor guest molecules that are selectively complexed by host molecules immobilized on the sensor surface. The basic idea is to realize systems that yield a response signal due to the alteration of ion fluxes, similar to the principle used in channel-mimetic sensors (34). Instead of using an external current, the response would depend on zero-current transmembrane ion fluxes. The relevance of such fluxes has been demonstrated recently (35-37). At submicromolar sample activities, flux modulation may have a dramatic effect on the emf response of polymer membrane electrodes (38-41). EXPERIMENTAL PROCEDURES

General Methods. Reagents and solvents were reagent-grade and used without further purification, except for THF, which was distilled. Tecoflex SG-80A was purchased from Thermedics Inc. (Woburn, MA). N,NDicyclohexyl-N,N-dioctadecyl-3-oxapentanediamide (ETH 5234), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), and streptavidin from Streptomyces avidinii were obtained from Fluka AG (CH-9471 Buchs, Switzerland). N-[3-[3-(trifluoromethyl)diazirine-3-yl]phenyl]-4-maleimidobutyramide (1, MAD) was synthesized by

10.1021/bc015515+ CCC: $22.00 © 2002 American Chemical Society Published on Web 12/19/2001

Immobilization of Biomolecules on Polyurethane Membrane Surfaces

Figure 1. Synthetic pathway to biotin derivative 5.

Collioud et al. (42). [35S]-Streptavidin from Amersham (CH-8600 Du¨bendorf, Switzerland) had a specific activity of 7.4-74 TBq mmol-1. Gold (99.99%) was obtained from Balzers AG (FL-9496 Balzers, Liechtenstein). Aqueous solutions were prepared with freshly deionized water (18.0 MΩ cm specific resistance) obtained with a NANOpure reagent-grade water system (Barnstead, CH-4009 Basel, Switzerland). All reactions were performed in standard glassware. Evaporation and concentration in vacuo were done at water aspirator pressure, and compounds were dried at 10-2 mbar. Flash column chromatography was made with SiO2 60 (220-440 mesh, 0.035-0.070 mm) from Fluka, TLC aluminum plates coated with SiO2 60 F254 were from E. Merck, and the substances were visualized by UV light at 254 or 366 nm. TLC glass plates coated with SiO2 (Silgur-25 UV254) were from Macherey-Nagel (CH-4702 Oensingen, Switzerland). The following instruments were used: melting points, Bu¨chi melting point apparatus (CH-9239 Flawil, Switzerland); IR spectra, 883 infrared spectrophotometer from Perkin-Elmer; UV/vis spectra, Uvikon 940 from Kontron Instruments; EI-MS, VG Tribid instrument, 70 eV; ESI-MS, Finnigan TSQ 7000 instrument; NMR spectra, Varian Gemini 200 (200 MHz) or Bruker AMX-2-500 (500 MHz) spectrometer at 300 K, with solvent peaks as reference.

Bioconjugate Chem., Vol. 13, No. 1, 2002 91

Syntheses. Synthesis of (3aS,4S,6aR)-11-Bromoundecyl 5-(Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4t-yl)pentanoate (3 in Figure 1). A mixture of biotin (2, 1.350 g, 5.5 mmol), 11-bromo-1-undecanol (6.940 g, 27.6 mmol), p-toluenesulfonic acid (0.105 g, 0.55 mmol), and toluene (34 mL) was stirred and refluxed at 120 °C for 48 h under argon (43). The reaction mixture was cooled to room temperature. Precipitated unreacted biotin was separated by filtration, the solvent was removed in vacuo, and the residue was purified by flash chromatography (MeOH/CH2Cl2 5:95) to give 3 as a white solid (1.970 g, 70%). Anal. (C21H37N2O3SBr) C, H, N, O, S, Br. Mp: 103.5-104.0 °C. IR νmax (cm-1): 3462, 2929, 2853, 1705, 1597. 1H NMR (500 MHz, CD2Cl2, δ): 1.28-1.47 (m, 16H), 1.57-1.74 (m, 6H), 1.85 (m, -CH2CH2Br, 2H), 2.31 (t, J ) 7.4 Hz, -CH2C(O)OCH2-, 2H), 2.71 (d, J ) 12.6 Hz, -CHCH2S-, 1H), 2.90 (dd, J ) 12.6 Hz, J ) 5.0 Hz, -CHCH2S-, 1H), 3.17 (m, -SCH-, 1H), 3.42 (t, J ) 6.9 Hz, -CH2Br, 2H), 4.03 (t, J ) 6.9 Hz, -OCH2-, 2H), 4.30 (m, -NHCHCH2-, 1H), 4.48 (m, -NHCH(CH)2, 1H), 5.43 (s, -NHCH(CH-)2, 1H), 5.73 (s, -NHCHCH2-, 1H). 13C NMR (125 MHz, CD2Cl2, δ): 25.28, 26.33, 28.57, 28.70, 28.78, 29.07, 29.15, 29.65, 29.81, 29.86, 29.88, 33.31 (-CH2CH2Br), 34.31, 34.68, 41.04 (-CH2S-), 55.90 (-SCH-), 60.54 (-NCHCH2), 62.28 (-NCH(CH-)2), 64.82 (-C(O)OCH2-), 163.94 (-NC(O)N), 173.95 (-C(O)O-). EI-MS m/z (relative intensity): M+ 476.1 (1), 418.1 (3), 227.1 (10), 166.1 (19). Synthesis of (3aS,4S,6aR)-11-Sulfanylundecyl 5-(Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4t-yl)pentanoate (4 in Figure 1). A stirred solution of 3 (1.000 g, 2.09 mmol) in THF was cooled to -10 °C, and hexamethyldisilathiane (2.20 mL, 10.45 mmol) and TBAF (10.5 mL, 1.0 M solution in THF with 5% water, 10.45 mmol) were added (44). The resulting reaction mixture was allowed to warm to room temperature while it was stirred. The reaction was carried out under argon and under shielding from ambient light in order to avoid photoinduced side reactions. After 20 min, the reaction mixture was washed with 20 mL of aqueous ammonium chloride (saturated) and 10 mL of CH2Cl2. Recrystallization from CH2Cl2 and hexane gave 4 as white solid (0.673 g, 75% yield). Anal. (C21H38N2O3S2) H, N; C: calcd, 58.57; found, 57.94; S: calcd 14.89; found, 14.31 (the 1H NMR spectrum indicated that some disulfides occurred as impurities in the product). Mp: 99.5-101.0 °C. IR νmax (cm-1): 3462, 2929, 2853, 1705, 1597. UV (CH2Cl2) λmax (log ): 219 (0.48). 1 H NMR (500 MHz, CD2Cl2, δ): 1.28-1.47 (m, 17H), 1.57-1.74 (m, 6H), 1.87 (m, -CH2CH2SH, 2H), 2.31 (t, J ) 7.4 Hz, -CH2C(O)OCH2-, 2H), 2.50 (q, J ) 7.4, -CH2SH, 2H), 2.71 (d, J ) 12.7 Hz, -CHCH2S-, 1H), 2.91 (dd, J ) 12.7, 5.0 Hz, -CHCH2S-, 1H), 3.16 (m, -SCH-, 1H), 4.03 (t, J ) 6.8 Hz, -OCH2-, 2H), 4.30 (m, -NHCHCH2-, 1H), 4.48 (m, -NHCH(CH-)2, 1H), 5.44 (s, -NHCH(CH-)2, 1H), 5.71 (s, -NHCHCH2-, 1H). 13C NMR (125 MHz, CD2Cl2, δ): 24.96 (-CH2SH), 25.28, 26.33 28.71, 28.78 (2C), 29.08, 29.47, 29.66, 29.90 (3C), 34.32, 34.53, 41.04 (-CH2S-), 55.90 (-SCH-), 60.54 (-NCCH2), 62.27 (-NC(CH-)2), 64.82 (-C(O)OCH2-), 163.91 (-NC(O)N-), 173.95 (-C(O)O-). EI-MS m/z (relative intensity): (M + 1)+ 431.2 (49), M+ 430.2 (13), 397.3 (26), 227.1 (76), 166.0 (83). Synthesis of (3aS,4S,6aR)-11-[[3-(3-Trifluoromethyl3H-diaziren-3-yl)phenylamino][1-(4-oxobutyl)pyrrolidine2,5-dioxo-3-yl]thio]undecyl 5-(Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4t-yl)pentanoate (5 in Figure 1). To a solution of 4 (7.32 mg, 17 µmol) in CH2Cl2 (3 mL) at 0 °C was added a solution of 1 (6.23 mg, 17 µmol) in 0.2 mL CH2Cl2, followed by 5 drops of triethylamine. The mixture

92 Bioconjugate Chem., Vol. 13, No. 1, 2002

was allowed to reach room temperature and then was stirred for 7 h. The reaction was shielded from light in order to avoid photoinduced side reactions and was carried out under argon. The resulting mixture was concentrated under reduced pressure, and the residue was purified by chromatography on a silica glass plate (hexane/1,2-dichloroethane/acetonitrile/ethanol 40:30:24: 6) to give 5 as a diastereoisometric mixture in form of a white solid (11.1 mg, 82% yield). 1H NMR (200 MHz, CDCl3, δ): 1.28-1.49 (m, 16H), 1.57-1.74 (m, 8H), 2.04 (m, -NCH2CH2CH2CO-, 2H), 2.33 (m, -CH2COOCH2-, -NCH2CH2CH2CO-, 4H), 2.50 (dd, J ) 18.7, 3.3 Hz, -CH2CH2S-, 2H), 2.69-3.00 (m, -CHCHS-, -SCHCH2CO-, 4H), 3.07-3.24 (m, -SCHCHNH, 1H), 3.58-3.76 (m, -NCH2-, -SCHCO, 3H), 4.07 (t, J ) 6.6 Hz, -OCH2-, 2H), 4.35 (m, -NHCHCH2-, 1H), 4.53 (m, -NHCH(CH-)2, 1H), 4.88 (s, -NHCH(CH-)2, 1H), 5.06 (s, -NHCHCH2-, 1H), 6.94 (d, J ) 7.1 Hz, Ar H, 1H), 7.34 (t, J ) 7.9 Hz, Ar H, 1H), 7.48 (s, Ar H, 1H), 7.67 (d, J ) 7.5 Hz, Ar H, 1H), 8.55 (d, J ) 5.4 Hz, -NH-Ar, 1H). ESI-MS m/z: (M + 1)+ 797, (M + Na)+ 819, (M + K)+ 835. Atomic Force Microscopy (AFM). Glass microscope slides covered with an Au layer were prepared according to a standard procedure for self-assembled monolayers (45). Solution aliquots of pure Tecoflex were cast onto the gold substrate (1.0 × 1.0 cm2) resulting in a membrane of 200 µm thickness. An Autoprobe CP from Park Scientific Instruments was used to image the pure Tecoflex membrane. The images were collected in intermittentcontact mode, in room atmosphere, and at ambient temperature. Commercially available silicon cantilevers with high aspect ratio tips were used. X-ray Photoelectron Spectroscopy (XPS). The gold substrates covered with pure Tecoflex were washed in ethanol and dried for 1 h at room temperature under high vacuum (10-2 mbar). Then 25 µL of a solution of 1 (2.7 mM in ethanol) was deposited. The samples were dried under a vacuum of 20 mbar for 1 h at room temperature. For XPS-studies after photobinding, the samples were irradiated for 20 min using a Stragene UV Stratalinker 350 nm light source with a radiance of 0.95 mW cm-2 and washed in hexane and in ethanol (42, 46). Photolyses were done at ambient temperature (20-25 °C). The surface of dry membrane films was analyzed via XPS under an ultrahigh vacuum of 10-9 mbar. Spectra were collected with a Perkin-Elmer 5700 spectrometer (PerkinElmer, Norwalk, CT) using an Al KR X-ray source (hv ) 1486.7 eV) with an emission power of 350 W to stimulate photoelectron emission. A spherical analyzer with an investigation area of 0.80 mm2 was used, and the hydrocarbon C1s peak was referenced at 285 eV. Samples were analyzed at 45° takeoff angle (the angle between the surface normal and the analyzer lens axes). An average of three measurements was obtained. Data were collected and stored on a Perkin-Elmer computer, which also controlled the spectrometer. Software provided with the instrument was used to interpret the data. Potentiometry. The ion-selective membranes contained 2.0 wt % (25.0 mmol kg-1), ETH 5234, 1.0 wt % (11.0 mmol kg-1) NaTFPB, and Tecoflex. A membrane of 100 µm thickness was obtained by casting a solution of 316.80 mg of the membrane components, dissolved in 2.5 mL THF, into a glass ring (60 mm i.d.) fixed on a glass plate covered with a Teflon foil. For each ISE, a disk of 7 mm diameter was punched from the membrane and glued to plasticized PVC tubing with THF. The inner filling solution was 0.05 M Na2EDTA, 10-3 M CaCl2, and 0.06 M NaOH (pH 8.9, calculated ion activity aCa2+ )

Reichmuth et al.

2.3 × 10-12 M). A diaphragm separated the internal filling solutions from the reference half cell (Ag/AgCl in 0.1 M KCl. The ISEs were conditioned for 2 d in a solution of 5 × 10-5 M 2-morpholinoethanesulfonic acid (MES) and 5 × 10-5 M NaOH (pH 6.5). Measuring solutions were prepared by successive automatic dilution of stock solutions with a Liquino 711 and two Dosino 700 instruments (Metrohm AG, CH-9010 Herisau). Potentials were measured with a 16-channel electrode monitor EMF16 (Lawson Labs Inc., Malvern, PA 19355) at room temperature (20-21 °C) in solutions stirred with a rod stirrer. An Ag/AgCl reference electrode (Metrohm, type 6.0729.100) with a 1 M KCl bridge electrolyte was used. All EMF values were corrected for the liquid-junction potential at the sample/bridge electrolyte interface using the Henderson equation (3). For the preparation of ISEs with immobilized 5, ISE membranes were washed in deionized water and dried under vacuum (20 mbar) for 1 h at room temperature. Then 40 µL of an acetonitrile (MeCN) solution of 5 (0.28 mM) were deposited on each membrane. The samples were dried for 1 h under a vacuum of 20 mbar at room temperature. For photobinding, the ISEs were irradiated for 20 min using a Stragene UV Stratalinker 350 nm light source with a light intensity of 0.95 mW cm-2 and washed in MeCN. Photolyses were done at ambient temperature (20-25 °C). The conditioned ISEs were set, before the streptavidin incubation, into a solution of 0.02% polyoxyethylenesorbitan monolaurate (Tween 20) for 20 min. A 0.1 M MES buffer of pH 6.1 (with NaOH) was used for the streptavidin binding experiments. The concentration of streptavidin in the MES buffer was 42 µg mL-1 (0.7 µM), and the reaction of this solution with the surface was allowed to proceed for 1 h. The membranes were rinsed with water in order to remove unbound streptavidin and set back into the conditioning solution for 1 h before the potentiometric measurements. Radiochemistry. Glass slides (0.8 × 0.8 cm2) were covered with a membrane which had the same composition as the one used in the potentiometric experiments. All samples were washed in acetonitrile (MeCN) and dried under vacuum (10-2 mbar) for 1 h at room temperature. Samples of series A and B: deposition of 25 µL of a solution of 5 (2.3 mM in MeCN), drying for 10 min under high vacuum of 0.05 mbar at room temperature, UV irradiation, washing in MeCN. Samples of series C: deposition of 25 µL of 5 (2.3 mM in MeCN), drying for 20 min under 0.05 mbar at r.t., UV-irradiation, washing in MeCN. Samples of series D, E, and F: deposition of 25 µL 5 (2.3 mM in MeCN), drying for 80 min under 0.05 mbar at rt, UV irradiation, washing in MeCN. Samples of series G: deposition of a pure MeCN drop, drying for 80 min under 0.05 mbar at rt, UV irradiation, washing in MeCN. Samples of series H and I: only washing in MeCN. All photobinding experiments were carried out employing a high-pressure mercury lamp (Osram HBO 350 W) mounted in a SUSS LH 1000 lamp house equipped with a shutter to control exposure times. The samples were irradiated for 200 s with a light intensity of 1.14 W cm-2 at 365 nm at ambient temperature (20-25 °C). After the photoimmobilization, the glass slides with the membrane were conditioned in water for 2 d. A 0.1 M MES buffer of pH 6.1 (with NaOH) was used for the streptavidin binding. The protein concentrations in the MES buffer were 21 µg mL-1 (0.35 µM) streptavidin (not radiolabeled) and 3.1 ng mL-1 (52 pM) [35S]-streptavidin with an activity of 15.4 × 103 Bq mL-1. After being rinsed with water in order to remove unbound streptavidin,

Immobilization of Biomolecules on Polyurethane Membrane Surfaces

Bioconjugate Chem., Vol. 13, No. 1, 2002 93

Table 1. Elemental Analysis of Tecoflex Membranes with and without MAD analytical method

C (%)

O (%)

N (%)

F (%)

combustion analysisa XPSa XPSb

78.57 78.08 77.97

19.05 20.98 19.52

2.38 0.95 1.80

0.35

a

b

MAD was not photoimmobilized on the membrane surface. MAD was photoimmobilized on the membrane surface.

Table 2. Surface Densities of [35S]-Streptavidin on Tecoflex Membranes

seriesa A B C D E F G H I

vacuum time cross-linking (min) of 5 10 10 20 80 80 80 80 0 0

yes yes yes yes yes yes no no no

treatment with Tween

density of streptavidin (ng cm-2)b

no treatment before streptavidin before streptavidin no treatment after streptavidin before streptavidin no treatment no treatment before streptavidin

311 ( 43 45 ( 9 42 ( 19 134 ( 33 152 ( 81 30 ( 9 17 ( 3 26 ( 17 22 ( 14

a The capital letters correspond to the series described in the Experimental Procedures and Figure 3. b The radiolabel densities were quantified by autoradiography. Error bounds correspond to standard deviations obtained from five measurements (four in series F).

the samples were set back into the conditioning solution for 1 h, and dried at room temperature for 15 h. As shown in Table 2, some sample series were set into a solution of 0.02% Tween 20 for 20 min before or after the streptavidin incubation. Autoradiography of the dried glass slides with the membranes was done with storage screens from Molecular Dynamics (Sunnyvale, CA), which were exposed for 12 h. For quantification, a PhosphorImager from Molecular Dynamics and an external one-point calibration (n ) 5) with [35S]-streptavidin was used. All work with radioactivity was performed in a certified laboratory. RESULTS AND DISCUSSION

In this work, the surface of an ion-selective polyurethane membrane was modified by photocross-linking of biotin. First, the morphology of a pure Tecoflex membrane without 1 was mapped out using atomic force microscopy (AFM). The surface structure of the Tecoflex membrane was featureless with a root-mean-square roughness of about 0.7 nm. The height differences between the lower and higher spots were around 3 nm. Nurdin et al. (47) found for a similar polyurethane surface, after drying it in the vacuum, a peak-to-valley distance of about 15 nm. The plain and homogeneous surface morphology of the investigated polyurethane is well suited for the planned application. In the next step, X-ray photoelectron spectroscopy (XPS) was used to obtain evidence for the cross-linking of 1 to the polyurethane membrane surface. Tecoflex membranes with and without 1 were examined by XPS for providing a qualitative and quantitative analysis of their surface. No fluorine was detected in the case of membranes without 1 (Table 1). There are small differences between the surface concentrations determined from the XPS analysis (Table 1, second row) and the bulk membrane composition found from the elemental analysis (Table 1, first row). This may be the consequence of a phase separation that takes place at the few outermost molecular layers of the polyurethane, where little or no hard segments were found in earlier studies (48, 49). For the

Figure 2. Response of ISEs based on the calcium-selective ionophore ETH 5234, NaTFPB and Tecoflex: A, without B and C with surface modification with 5; C, after incubation with a streptavidin solution. Inner filling solution: 0.05 M Na2EDTA, 10-3 M CaCl2, and 0.06 M NaOH (pH 8.9). Sample solution: CaCl2 in a buffer of 5 × 10-5 M 2-morpholinoethanesulfonic acid (MES) and 5 × 10-5 M NaOH (pH 6.5). Electrode slopes (mV dec-1 in the range 10-5 M bis 10-7 M): A, 25.8; B, 24.3; C, 25.0.

membrane with 1, the detection of fluorine is a clear evidence for the successful photoimmobilization on the membrane surface (46). On the basis of the positive results of the abovedescribed preliminary experiments, a biotin derivative with a covalently attached photo-cross-linking unit was designed and synthesized. The synthetic pathway to the novel photobiotin 5 with the carbene-generating trifluoromethylaryldiazirine group is shown in Figure 1. Brooks et al. demonstrated recently that photo-crosslinking of a related photobiotin (31) was more efficient than for the nitrene derivative used in their earlier studies (31). The photo-cross-linking unit is a derivative of N-[3-[3-(trifluoromethyl)diazirine-3-yl]phenyl]-4maleimidobutyramide (1, MAD), which has been shown to have excellent properties in several earlier studies (20). To obtain optimal binding characteristics of streptavidin, a spacer with a carbon chain of 11 CH2 groups is interposed between the biotin moiety and the photocrosslinker (24, 25). The intermediate product 4, a thiolbearing biotin derivative was prepared by a simpler procedure and in a higher yield than for similar compounds described previously (26-30). The influence of the surface modification on the potentiometric response behavior of membrane-based sensors was investigated for Ca2+-selective electrodes. A complete coverage of the membrane with a dense layer of proteins may be expected to result in a different response signal of the respective ISE. The experiments were performed using calcium-selective electrodes based on Tecoflex membranes. The biotin derivative 5 was photoimmobilized onto the surface of the membranes containing the Ca2+-selective ionophore ETH 5243 and the ion exchanger NaTFPB (see the Experimental Procedures). To achieve detection limits in the nanomolar range (39, 50), the inner filling solution of these ISEs contained a high sodium activity and a low calcium activity established with EDTA as ion-buffering system. The calibration curves of an untreated membrane (A) and one with immobilized biotin (B) were found to be virtually identical (Figure 2). This is even true for membrane B after incubation in a streptavidin solution (curve C). In all cases, it is possible to measure calcium activities down to the nanomolar range. It appears that the surfaceimmobilized biotin-streptavidin conjugate does not influence the potentiometric response of these sensors.

94 Bioconjugate Chem., Vol. 13, No. 1, 2002

Figure 3. Autoradiographic measurements on Tecoflex filmscoated (8 × 8 mm) glass slides, after incubation into a solution of [35S]-streptavidin. The darkness is proportional to the density of bound or adsorbed [35S]-streptavidin. Contact times before photo-cross-linking (in min): A, B, 10; C, 20; D-F, 80; blank samples: GI. Tween 20 was applied before streptavidin for samples B, C, and F and after streptavidin for membranes E. The density values shown in the five columns were determined from repeated experiments under identical conditions.

Evidently, the exchange of calcium ions at the interface between membrane and sample solution is not blocked by the binding of streptavidin. This implies that the streptavidin layer on the membrane surface cannot be very dense. To confirm this hypothesis, the surface coverage is analyzed experimentally in the following section. The distribution of covalently bound biotin accessible for conjugation with streptavidin was monitored by autoradiography using the soft β-emitter [35S]-streptavidin as radiolabel. The experiments were performed on Tecoflex membranes that had the same composition as the ones used for the ISE measurements. From the data shown in Figure 3 and Table 2, it is evident that the membranes with photoimmobilized biotin (A - F) bind a significantly larger amount of [35S]-streptavidin than the ones without biotin (G and H). Consequently, the cross-linked biotin derivative is able to bind [35S]-streptavidin on the polyurethane surface, and its biological activity is apparently not affected by the photochemical immobilization process. Although some adsorption of streptavidin occurs even for membranes without biotin, the effect is in all cases

Reichmuth et al.

smaller than for membranes with biotin. While the surface distribution of the labeled streptavidin strongly varies from experiment to experiment (rows in Figure 3), the variation of the total amount within a given series of experiments is much smaller (Table 2, last column). The nonuniform distribution of the labeled streptavidin on membranes A-F must be caused by irregular photoimmobilization of 5, since the [35S]-streptavidin coverage on membranes G-I without immobilized biotin is found to be much more homogeneous, as shown in Figure 3. The heterogeneity of the coverage by 5 after deposition and vacuum treatment can even be observed with a magnifying glass. Previously, nonuniform immobilization of photobiotin was also reported for the case of solid substrates (51). AFM images also indicated that the distribution of streptavidin on biotinylated surfaces is nonuniform, and that the proteins tend to cluster (52). More than 80% of the adsorbed proteins were found in clusters of three or more molecules (28). Similarly, it was found that only a part of biotin is able to bind avidin if it is immobilized on a surface (51). One of the reasons for the heterogeneous photoimmobilization may be the low photocoupling efficiency. For example, it was found in previous studies that the photocoupling efficiency of MAD-[35S]Cys to polystyrene is only 7% (42). In the present system, the diffusion of the biotin derivative 5 into the polyurethane membrane before cross-linking may also have an effect. The diffusion coefficient of organic compounds in such membranes is on the order of 10-8 cm2 s-1 (1). Assuming that compound 5 is sufficiently soluble in Tecoflex, one would expect a mean diffusion distance of 35 and 49 µm after 10 and 20 min, respectively. The amount of biotin available for the surface reaction would decrease accordingly. The measured dependence of the streptavidin density on the contact time before photo-cross-linking suggests such an influence. Figures 3 A vs D, as well as Figure 3 B and C vs F, indicate that an effect is well observable after contact times of 80 min, but the differences are small between 10 and 20 min. To reduce nonspecific adsorption of proteins, the corresponding substrates are occasionally treated with a surfactant (53). Experiments were done in these lines with the nonionic surfactant Tween 20. If the surfactant is applied before the contact of the biotinylated membrane with [35S]-streptavidin, the surface concentration of bound proteins is heavily reduced (see A vs B and D vs F in Figure 3 and Table 2). However, as demonstrated by the experiments H and I, this decrease is not due to a reduction of the nonspecific adsorption. It might rather be a consequence of an accumulation of Tween 20 at the membrane surface (54, 55) and its competitive binding to the protein (56). In striking contrast, a treatment of the membranes after conjugation with [35S]-streptavidin does not affect the surface concentration at all (see D and E in Figure 3 and Table 2). Before performing the radiochemistry experiments, the membranes were kept 2 d in distilled water. As indicated by control experiments without conditioning (not shown) this did not significantly influence the measured [35S] activity. Thus, once the biotin derivative is bound to the surface, it forms a rather stable layer despite the presence of a soft polymer substrate. Using the density of a two-dimensional crystalline streptavidin (C2,2,2) monolayer (262 ng cm-2) (30, 57) the percentage of the protein bound relative to the amount required for a compact monolayer can be calculated. It is 120% for membrane A and 50-60% for membranes D and E with long contact times before photoimmobilization.

Immobilization of Biomolecules on Polyurethane Membrane Surfaces CONCLUSIONS

We demonstrated that biotin can be covalently immobilized on the surface of a soft polyurethane and that the immobilized biotin is capable of binding streptavidin. Films made from Tecoflex, the applied polyurethane, seem to be ideally suited as substrate because of their very plain surface. While the total amount of streptavidin bound to the biotinylated polyurethane is rather close to that required for a compact monolayer, the uniformity varied significantly. Such a variation was also observed earlier with hard substrates (51). ACKNOWLEDGMENT

This work was financially supported by the Swiss National Science Foundation, the National Institutes of Health (Grant No. R01-GM59716), and Orion Research, Inc. (Beverly, MA). We gratefully acknowledge the help of Martin Pu¨ntener and Andre´ Mu¨ller concerning the syntheses, Andrea Rainelli for some preliminary measurements, Prof. Roel Prins for permission to use his AFM instrument, and Prof. Nicholas Spencer for performing the XPS measurements. LITERATURE CITED (1) Bakker, E., Bu¨hlmann, P., and Pretsch, E. (1997) Carrierbased ion-selective electrodes and bulk optodes. 1. General characteristics. Chem. Rev. 97, 3083-3132. (2) Bu¨hlmann, P., Pretsch, E., and Bakker, E. (1998) Carrierbased ion-selective electrodes and bulk optodes. 2. Ionophores for potentiometric and optical sensors. Chem. Rev. 98, 15931687. (3) Morf, W. E. The Principles of Ion-Selective Electrodes and of Membrane Transport; Elsevier: New York, 1981. (4) Cha, G. S., Liu, D., Meyerhoff, M. E., Cantor, H. C., Midgley, A. R., Goldberg, H. D., and Brown, R. B. (1991) Electrochemical performance, biocompatibility, and adhesion properties of new polymer matrices for solid-state ion sensors. Anal. Chem. 63, 1666-1672. (5) Pick, J., Toth, K., Pungor, E., Vasak, M., and Simon, W. (1973) A potassium-selective silicone-rubber membrane electrode based on a neutral carrier. Anal. Chim. Acta 64, 477-480. (6) Reinhoudt, D. N., Engbersen, J. F. J., Brzo´zka, Z., van den Vlekkert, H. H., Honig, G. W. N., Holterman, H. A. J., and Verkerk, U. H. (1994) Development of K+-selective chemically modified field effect transistors with functionalized polysiloxane membranes. Anal. Chem. 66, 3618-3623. (7) Oh, B. K., Kim, C. Y., Lee, H. J., Rho, K. L., Cha, G. S., and Nam, H. (1996) One-component room-temperature vulcanizing-type silicone rubber-based calcium-selective electrodes. Anal. Chem. 68, 503-508. (8) Malinowska, E., Oklejas, V., Hower, R. W., Brown, R. B., and Meyerhoff, M. E. (1996) Enhanced electrochemical performance of solid-state ion sensors based on silicon rubber membranes. Sens. Actuators B33, 161-167. (9) Heng, L. Y., and Hall, E. A. H. (2000) Producing “selfplasticizing” ion-selective membranes. Anal. Chem. 72, 42-51. (10) Malinowska, E., Gawart, L., Parzuchowski, P., Rokicki, G., and Brzo´zka, Z. (2000) Novel approach of immobilization of calix[4]arene type ionophore in “self-plasticized” polymeric membrane. Anal. Chim. Acta 421, 93-101. (11) Wang E., and Meyerhoff, M. E. (1993) Evaluation of polyurethane-based membrane matrixes for optical ionselecitve sensors. Anal. Lett. 26, 1519-1533. (12) Lindner, E., Cosofret, V. V., Ufer, S., Buck, R. P., Kao, W. J., Neuman, M. R., and Anderson, J. M. (1994) Ion-selective membranes with low plasticizer content: Electroanalytical characterization and biocompatibility studies. J. Biomed. Mater. Res. 28, 591-601. (13) Yun, S. Y., Hong, Y. K., Oh, B. K., Cha, G. S., Nam, H., Lee, S. B., and Jin, J.-I. (1997) Potentiometric properties of ion-selective electrode membranes based on segmented polyether urethane matrices. Anal. Chem. 69, 868-873.

Bioconjugate Chem., Vol. 13, No. 1, 2002 95 (14) Daunert, S., and Bachas, L. G. (1990) Ion-selective electrodes using an ionophore covalently attached to carboxylated poly(vinyl chloride). Anal. Chem. 62, 1428-1431. (15) Kusy, R. P., Whitley, J. Q., Buck, R. P., Cosofret, V. V., and Lindner, E. (1994) Syntheses and kinetics of piperazinemodified poly(vinyl chloride)s for use as fixed-site proton carrier membranes. Polymer 35, 2141-2147. (16) Kimura, K., Matsuba, T., Tsujimura, Y., and Yokoyama, M. (1992) Unsymmetrical Calix[4]arene Ionophore/Silicone Rubber Composite Membranes for High-Performance Sodium IonSensitive Field-Effect Transistors. Anal. Chem. 64, 2508-2511. (17) Espadas-Torre, C., and Meyerhoff, M. E. (1995) Thrombogenic properties of untreated and poly(ethylene oxide) modified polymeric matrixes useful for preparing intraarterial ion-selective electrodes. Anal. Chem. 67, 3108-3114. (18) Brooks, K. A., Allen, J. R., Feldhoff, P. W., and Bachas, L. G. (1996) Effect of surface-attached heparin on the response of potassium-selective electrodes. Anal. Chem. 68, 1439-1443. (19) Kuijpens, J. M. H., Kardaun, G. A., Blezer, R., Pijpers, A. P., and Koole, L. H. (1995) Immobilization of theophilline on medical-grade polyurethane inhibits surface-induced activation of blood platelets. J. Am. Chem. Soc. 117, 8691-8697. (20) Sigrist, H., Collioud, A., Clemence, J., Gao, H., Sanger, M., and Sundarababu, G. (1995) Surface immobilization of biomolecules by light. Opt. Eng. 35, 2339-2348. (21) Yan, M., Cai, S. X., Wybourne, M. N., and Keana, J. F. W. (1993) Photochemical functionalization of polymer surfaces and the production of biomolecule-carrying micrometer-scale structures by deep-UV litography using 4-substituted perfluoropheny azides. J. Am. Chem. Soc. 115, 814-816. (22) Yan, M., Cai, S. X., Wybourne, M. N., and Keana, J. F. W. (1994) N-hydroxysuccinimide ester functionalized perfluoropheny azides as novel photoactive heterobifunctional crosslinking agents. The covalent immobilization of biomolecules to polymer surfaces. Bioconjugate Chem. 5, 151-157. (23) Hengsakul, M., and Cass, A. E. G. (1996) Protein patterning with a photoactivable derivative of biotin. Bioconjugate Chem. 7, 249-254. (24) Torres-Rodriguez, L. M., Roget, A., Billon, M., and Bidan, G. (1998) Synthesis of a Biotin Functionalized Pyrrole and Its Electropolymerization: Toward a Versatile Avidin Biosensor. Chem. Commun. 21, 1993-1994. (25) Wang, Y. L., and Bobbitt, D. R. (1994) Binding Characteristics of Avidin and Surface Immobilized Octylbiotin Implications for the Development of Dynamically Modified Optical-Fiber Sensors. Anal. Chim. Acta 298, 105-112. (26) Dubrovsky, T. B. (2000) Protein Architecture, pp 25-54, Marcel Dekker: New York. (27) Busse, S., Kashammer, J., Kramer, S., and Mittler, S. (1999) Gold and Thiol Surface Functionalized Integrated Optical Mach-Zender Interferometer for Sensing Purposes. Sens. Actuators B 60, 148-154. (28) Perez Luna, V. H., O Brien, M. J., Opperman, K. A., Hampton, P. D., Lopez, G. P., Klumb, L. A., and Stayton, P. S. (1999) Molecular Recognition Between Genetically Engineered Streptavidin and Surface-Bound Biotin. J. Am. Chem. Soc. 121, 6469-6478. (29) Knoll, W., Zizlsperger, M., Liebermann, T., Arnold, S., Badia, A., Liley, M., Piscevic, D., Schmitt, F. J., and Spinke, J. (2000) Streptavidin Arrays as Supramolecular Architectures in Surface-Plasmon Optical Sensor Formats. Colloids Surf. A 161, 115-137. (30) Jung, L. S., Nelson, K. E., Campbell, C. T., Stayton, P. S., Yee, S. S., Perez-Luna, V., and Lopez, G. P. (1999) Surface Plasmon Resonance Measurement of Binding and Dissociation of Wild-Type and Mutant Streptavidin on Mixed BiotinContaining Alkylthiolate Monolayers. Sens. Actuators B 54, 137-144. (31) Brooks, S. A., Dontha, N., Davis, C. B., Stuart, J. K., O’Neill, G., and Kuhr, W. G. (2000) Segregation of micrometerdimension biosensor elements on a variety of substrate surfaces. Anal. Chem. 72, 3253-3259. (32) Lin, S., Fang, K., Hashimoto, M., Nakanishi, K., and Ojima, I. (2000) Design and synthesis of a novel photoaffinity taxoid as a potential probe for the study of paclitaxel-microtubules interactions. Tetrahedron Lett. 41, 4287-4290.

96 Bioconjugate Chem., Vol. 13, No. 1, 2002 (33) Hatanaka, Y., Hashimoto, M., Nishihara, S., Narimatsu, H., and Kanaoka, Y. (1996) Synthesis and characterization of a carbene-generating biotinylated N-acetylglucosamine for photoaffinity labeling of β-(1f4)-galactosyltransferase. Carbohydr. Res. 294, 95-108. (34) Bu¨hlmann, P., Aoki, H., Xiao, K. P., Amemiya, S., Tohda, K., and Umezawa, Y. (1998) Chemical sensing with chemically modified electrodes that mimic gating at biomembranes incorporating ion-channel receptors. Electroanalysis 10, 11491158. (35) Sokalski, T., Ceresa, A., Zwickl, T., and Pretsch, E. (1997) Large improvement of the lower detection limit of ionselective polymer membrane electrodes. J. Am. Chem. Soc. 119, 11347-11348. (36) Morf, W. E., Badertscher, M., Zwickl, T., de Rooij, N. F., and Pretsch, E. (1999) Effects of ion transport on the potential response of ionophore-based membrane electrodes: A theoretical approach. J. Phys. Chem. B 103, 11346-11356. (37) Bakker, E., and Pretsch, E. (2001) Potentiometry at trace levels. Trends Anal. Chem. 20, 11-19. (38) Sokalski, T., Zwickl, T., Bakker, E., and Pretsch, E. (1999) Lowering the detection limit of solvent polymeric ion-selective electrodes. 1. Modeling the influence of steady-state ion fluxes. Anal. Chem. 71, 1204-1209. (39) Sokalski, T., Ceresa, A., Fibbioli, M., Zwickl, T., Bakker, E., and Pretsch, E. (1999) Lowering the detection limit of solvent polymeric ion-selective membrane electrodes. 2. Influence of composition of sample and internal electrolyte solution. Anal. Chem. 71, 1210-1214. (40) Ye, Q., and Meyerhoff, M. E. (2001) Rotating electrode potentiometry: Lowering the detection limits of nonequilibrium polyion-sensitive membrane electrodes. Anal. Chem. 73, 332-336. (41) Vigassy, T., Morf, W. E., Badertscher, M., Ceresa, A., de Rooij, N. F., and Pretsch, E. (2001) Making use of ion fluxes through potentiometric sensor membranes: ISEs with step responses at critical ion activities. Sens. Actuators B 76, 476481. (42) Collioud, A., Cle´mence, J. F., Sa¨nger, M., and Sigrist, H. (1993) Oriented and Covalent Immobilization of Target Molecules to Solid Supports: Synthesis and Application of a Light-Activatable and Thiol-Reactive Cross-Linking Reagent. Bioconjugate Chem. 4, 528-536. (43) Crisp, G. T., and Gore, J. (1997) Biotin Derivates as Gelators of Organic Solvents. Synth. Commun. 27, 2203-2216. (44) Hu, J., and Fox, M. A. (1999) A Convenient Trimethylsilylthioxy-Dehalogenation Reaction for the Preparation of Functionalized Thiols. J. Org. Chem. 64, 4959-4961. (45) Fibbioli, M., Bandyopadhyay, K., Liu, S.-G., Echegoyen, L., Enger, O., Diederich, F., Bu¨hlmann, P., and Pretsch, E. (2000) Redox-active self-assembled monolayers as novel solid contacts for ion-selective electrodes. Chem. Commun. 339-340.

Reichmuth et al. (46) Leonard, D., Chevolot, Y., Bucher, O., Sigrist, H., and Mathieu, H. J. (1998) ToF-SIMS and XPS Study of Photoactivatable Reagents Designed for Surface Glycoengineering - Part I. N-(m-(3-(trifluoromethyl)diazirine-3-yl)phenyl)-4maleimido-butyramide (MAD) on Silicon, Silicon Nitride and Diamond. Surf. Interface Anal. 26, 783-792. (47) Nurdin, N., and Descouts, P. (1995) Effect of Toluene Extraction on Biomer TM Surface: II. An Atomic Force Microscopy Study. J. Biomater. Sci. Polymer. 7, 425-438. (48) Chen, Z., Ward, R., Tian, Y., Eppler, A. S., Shen, Y. R., and Somorjai, G. A. (1999) Surface Composition of Biopolymer Blend Biospan-SP/Phenoxy and Biospan-F/Phenoxy Observed with SFG, XPS, and Contact Angle Goniometry. J. Phys. Chem. B 103, 2935-2942. (49) Hearn, M. J., Briggs, D., Yoon, S. C., and Ratner, B. D. (1987) SIMS and XPS Studies of Polyurethane Surfaces 2. Polyurethanes with Fluorinated Chain Extenders. Surf. Interface Anal. 10, 384-391. (50) Ceresa, A., Sokalski, T., and Pretsch, E. (2001) Influence of Key Parameters on the Lower Detection Limit and Response Function of Solvent Polymeric Membrane Ion-Selective Electrodes. J. Electroanal. Chem. 501, 70-76. (51) Brooks, S. A., Ambrose, W. P., and Kuhr, W. G. (1999) Micrometer Dimension Derivatization of Biosensor Surfaces Using Confocal Dynamic Patterning. Anal. Chem. 71, 25582563. (52) Dontha, N., Nowall, W. B., and Kuhr, W. G. (1997) Generation of Biotin/Avidin/Enzyme Nanostructures with Maskless Photolithography. Anal. Chem. 69, 2619-2625. (53) Wahlgren, M., Welin-Klintstro¨m, S., and Karlsson, C. A.C., Eds. Interactions Between Proteins and Surfactants at Solid Interfaces; Marcel Dekker, 1998. (54) Ye, Q., Vincze, A., Horvai, G., and Leermakers, F. A. M. (1998) Partial blocking of ion transport at the interface of an ion-selective liquid membrane electrode by neutral surfactants. Experiments and computer simulation. Electrochim. Acta 44, 125-132. (55) Malinowska, E., and Meyerhoff, M. E. (1998) Influence of nonionic surfactants on the potentiometric response of ionselective polymeric membrane electrodes designed for blood electrolyte measurements. Anal. Chem. 70, 1477-1488. (56) Duhamel, R. C., and Whitehead, J. S. (1990) In AvidinBiotin Technology (Wilchek, M., Bayer, E. A., Eds.) Vol. 184, pp 210-207, Academic Press, San Diego. (57) Darst, S. A., Ahlers, M., Meller, P. H., Kubalek, E. W., Blankenburg, R., Ribi, H. O., Ringsdorf, H., and Kornberg, R. D. (1991) Two-Dimensional Crystals of Streptavidin on Biotinylated Lipid Layers and Their Interactions with Biotinylated Macromolecules. Biophys. J. 59, 387-396.

BC015515+