Preparing Catalytic Surfaces for Sensing Applications by Immobilizing

Yohann Corvis,† Alain Walcarius,‡ Rick Rink,§ Nadir T. Mrabet,| and Ewa Rogalska*,†. Groupe d'Etude des Vecteurs Supramoléculaires du Médicam...
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Anal. Chem. 2005, 77, 1622-1630

Preparing Catalytic Surfaces for Sensing Applications by Immobilizing Enzymes via Hydrophobin Layers Yohann Corvis,† Alain Walcarius,‡ Rick Rink,§ Nadir T. Mrabet,| and Ewa Rogalska*,†

Groupe d’Etude des Vecteurs Supramole´ culaires du Me´ dicament UMR 7565 CNRS/Universite´ Henri Poincare´ Nancy 1, Faculte´ des Sciences, BP 239, 54506 Vandoeuvre-le` s-Nancy Cedex, France, Laboratoire de Chimie Physique et Microbiologie pour l’Environnement UMR 7564 CNRS/Universite´ Henri Poincare´ Nancy 1, 54600 Villers-le` s-Nancy Cedex, France, Biomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands, and Laboratoire de Pathologie Cellulaire et Mole´ culaire en Nutrition, INSERM EMI 0014/Universite´ Henri Poincare´ Nancy 1, Faculte´ de Me´ decine, BP 184, 54505 Vandoeuvre-le` s-Nancy Cedex, France

Simple and reliable immobilization techniques that preserve the activity of enzymes are of interest in many technologies based on catalysis. Here, two redox enzymes, glucose oxidase from Aspergillus niger and horseradish peroxidase, were immobilized by physisorption on glassy carbon electrodes coated with Schizophyllum commune hydrophobin. Hydrophobins are small, interfacially active proteins that have the remarkable property of adhering to almost any surface. We showed recently that these proteins can be used to immobilize small, electroactive molecules. The results obtained in this work show a way to easily manufacture stable, enzyme-based catalytic surfaces for applications in biosensing. There is an increasing demand for stable catalytic surfaces in different technologies. Selective catalysts are needed in sensors and reactors, and high-throughput catalysts are needed in bioremediation. Enzymes are frequently the catalysts of choice in such applications. The engineering of enzyme-based advanced materials depends, however, to a great extent on adequate methods of enzyme immobilization.1 The existing methods2,3 are delicate, not yet general, and often denaturing for the enzymes. Recently, we have shown that small electroactive molecules could be successfully immobilized on electrodes via hydrophobin HydPt-1 produced in Escherichia coli as a recombinant polypeptide of 13.7 kDa.4 In the present work, SC3 hydrophobin purified from * Corresponding author: (e-mail) [email protected]; (phone) +33 (0)3 83 68 43 45; (fax) +33 (0)3 83 68 43 22. † Groupe d’Etude des Vecteurs Supramole´culaires du Me´dicament, Universite´ Henri Poincare´ Nancy 1. ‡ Laboratoire de Chimie Physique et Microbiologie pour l’Environnement, Universite´ Henri Poincare´ Nancy 1. § Biomade Technology Foundation. | Faculte´ de Me´decine, Universite´ Henri Poincare´ Nancy 1. (1) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat. Biotechnol. 2004, 22, 211-213. (2) Bickerstaff, G. F. Immobilization of Enzymes and Cells; Humana Press: Totowa, NJ, 1997. (3) O Ä ’Fa´ga´in, C. Enzyme Microb. Technol. 2003, 33, 137-149. (4) Bilewicz, R.; Witomski, J.; Van der Heyden, A.; Tagu, D.; Palin, B.; Rogalska, E. J. Phys. Chem. B 2001, 105, 9772-9777.

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Schizophyllum commune5-7 (SC3) was used to bind two redox enzymes, glucose oxidase from Aspergillus niger (GOX; β-Dglucose:oxygen 1-oxidoreductase, EC 1.1.3.4) and horseradish peroxidase (HRP; donor: hydrogen peroxide oxidoreductase, EC 1.11.1.7), to glassy carbon electrodes. Hydrodynamic amperometry and cyclic voltammetry were used to follow the activity of the immobilized enzymes. Transmission electron microscopy (TEM) and contact angle (CA) measurements were used to elucidate the organization of SC3 and the enzymes adsorbed to solid surfaces. Molecular modeling of protein accessible surface areas (ASA) was carried out to probe the polar/nonpolar properties of the enzyme surface with the aim to better understand the protein-protein interactions. Both GOX and HRP are versatile enzymes of considerable commercial importance. Indeed, GOX has applications in the food and fermentation industry, in the textile industry, and as a molecular diagnostic and analytical tool in medical and environmental monitoring applications8-12 while HRP is used as a reagent for organic synthesis and biotransformation as well as in coupled enzyme assays, chemiluminescent assays, immunoassays, and the treatment of wastewaters.13-15 The use of GOX16,17 and HRP18,19 is widespread in biosensors. (5) Wessels, J. G. H. Trends Plant Sci. 1996, 1, 9-15. (6) van Wetter, M.-A.; Schuren, F. H. J.; Schuurs, T. A.; Wessels, J. G. H. FEMS Microbiol. Lett. 1996, 140, 265-269. (7) Wo ¨sten, H. A. B.; Van Wetter, M.-A.; Lugones, L. G.; Van der Mei, H. C.; Busscher, H. J.; Wessels, J. G. H. Curr. Biol. 1999, 9, 85-88. (8) Ro ¨hr, M.; Kubicek, C. P.; Kominek, J. In Biotechnology; Rehm, H. J., Reed, G., Eds.; Verlag Chemie Weiheim: Munich, Germany, 1983. (9) Frew, J. E.; Hill, H. A. O. Philos. Trans., R. Soc. London, Ser. B 1987, 316, 95-106. (10) Turner, A. P. F.; Karube, L.; Wilson, G. S. Biosensors: Fundamentals and Applications; Oxford University Press: Oxford, 1987. (11) Tzanov, T.; Costa, S. A.; Gubitz, G. M.; Cavaco-Paulo, A. J. Biotechnology 2002, 93, 87-94. (12) Wang, S.; Yoshimoto, M.; Fukunaga, K.; Nakao, K. Biotechnol. Bioeng. 2003, 83, 444-453. (13) Ryan, O.; Smyth, M. R.; O Ä ’Fa´ga´in, C. In Horseradish Peroxidase: The Analyst’s Friend; Ballou, D. K., Ed.; Portland Press: London, 1994. (14) Veitch, N. C.; Smith, A. T. Adv. Inorg. Chem. 2001, 51, 107-162. (15) Krieg, R.; Halbhuber, K. J. Cell. Mol. Biol. 2003, 49, 547-563. (16) Raba, J.; Mottola, H. A. Crit. Rev. Anal. Chem. 1995, 25, 1-42. 10.1021/ac048897w CCC: $30.25

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GOX is a flavoenzyme that catalyzes oxidation of β-D-glucose by molecular oxygen to δ-gluconolactone, which subsequently hydrolyzes spontaneously to gluconic acid and hydrogen peroxide. The three-dimensional structure of GOX has been determined recently.20,21 The enzyme contains one tightly, noncovalently bound flavin adenine dinucleotide (FAD) cofactor per monomer and is a homodimer with a molecular mass of 160 kDa, depending on the extent of glycosylation.20 GOX is glycosylated by neutral sugars (mostly mannose-like sugars) and by amino sugars.22 Several reports find that the sugar content may vary from 11 up to 30%.23-26 HRP is an important heme-containing enzyme that has been studied for more than a century. It utilizes hydrogen peroxide to oxidize a wide variety of organic and inorganic compounds. In recent years, new information has become available on the threedimensional structure,27 catalytic intermediates, mechanisms of catalysis, and function of specific amino acid residues.28 Horseradish peroxidase isoenzyme C comprises a single polypeptide of 308 amino acid residues. Its carbohydrate content is somewhat dependent on the source of the enzyme, and values of between 18 and 22% are typical.29 Hydrophobins,30 which are produced by filamentous fungi belonging to the ascomycetes and the basidiomycetes,31,32 play important roles in fungal growth and development. Indeed, they are involved in formation of hydrophobic aerial structures33,34 and attachment of hyphae to hydrophobic surfaces35,36 by self-assembling at hydrophilic-hydrophobic interfaces.35,37,38 Hydrophobins produced by submerged hyphae diffuse in the aqueous environment and self-assemble at the air/aqueous interface. This self-assembly results in a decrease of the water surface tension, allowing hyphae to breach the interface and to grow into the air.7 Aerial hyphae and spores become hydrophobic because the secreted hydrophobins self-assemble at the surface of the cell (17) Wilson, R.; Turner, A. P. F. Biosens. Bioelectron. 1992, 7, 165-185. (18) Ryan, O.; Smyth, M. R.; O Ä ’Fa´ga´in, C. Essays Biochem. 1994, 28, 129-146. (19) Cass, A. E. G.; Smit, M. H. Proc. Conf. Trends Electrochem. Biosens. 1992, 25-42. (20) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153-172. (21) Gidalevitz, D.; Huang, Z.; Rice, S. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2608-2611. (22) Tsuge, H.; Natsuaki, O.; Ohashi, K. J. Biochem. (Tokyo) 1975, 78, 835843. (23) Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1965, 240, 2209-2215. (24) Nakamura, S.; Hayashi, S. FEBS Lett. 1974, 41, 327-330. (25) Kalisz, H. M.; Hecht, H. J.; Schomburg, D.; Schmid, R. D. Biochim. Biophys. Acta 1991, 1080, 138-142. (26) Zoldak, G.; Zubrik, A.; Musatov, A.; Stupak, M.; Sedlak, E. J. Biol. Chem. 2004, 279, 47601-47609. (27) Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nat. Struct. Biol. 1997, 4, 1032-1038. (28) Veitch, N. C. Phytochemistry 2004, 65, 249-259. (29) Welinder, K. G. FEBS Lett. 1976, 72, 19-23. (30) Talbot, N. J. Nature 1999, 398, 295-296. (31) de Vries, O. M. H.; Fekkes, M. P.; Wo¨sten, H. A. B.; Wessels, J. G. H. Arch. Microbiol. 1993, 159, 330-335. (32) Wessels, J. G. H. Adv. Microb. Physiol. 1997, 38, 1-45. (33) Wo ¨sten, H. A. B.; Wessels, J. G. H. Mycoscience 1997, 38, 363-374. (34) Wo ¨sten, H. A. B. Annu. Rev. Microbiol. 2001, 55, 625-646. (35) Wo ¨sten, H. A. B.; Asgeirsdo´ttir, S. A.; Krook, J. H.; Drenth, J. H. H.; Wessels, J. G. H. Eur. J. Cell Biol. 1994, 63, 122-129. (36) Talbot, N. J.; Kershaw, M. J.; Wakley, G. E.; de Vries, O. M. H.; Wessels, J. G. H.; Hamer, J. E. Plant Cell 1996, 8, 985-999. (37) Wo ¨sten, H. A. B.; De Vries, O. M. H.; Wessels, J. G. H. Plant Cell 1993, 5, 1567-1574. (38) Wo ¨sten, H. A. B.; Ruardy, T. G.; van der Mei, H. C.; Busscher, H. J.; Wessels, J. G. H. Colloids Surf. B 1995, 5, 189-195.

walls; hyphae that grow over a hydrophobic substrate firmly attach. Hydrophobins are classified as class I or class II based on different hydropathy patterns. SC3 from S. commune is the best studied class I hydrophobin.39 Mature SC3 hydrophobin consists of 112 amino acids with 16-22 mannose residues linked to 14 O-glycosylation sites contained in the N-terminal part of the protein.40 SC3 is the most surface-active protein known, as it lowers water surface tension from 72 to 24 mN m-1 at concentrations of 20-100 µg mL-1.7 Hydrophobins have a high affinity for hydrophobic surfaces.41-43 Indeed, SC3 self-assembles on hydrophobic solids from aqueous solutions forming amphipathic films.37,38,43 The assembled SC3 is highly insoluble and can only be dissociated by formic acid and trifluoroacetic acid at ambient temperature.31,44 Recent results showed that hydrophobin’s conformation is different in bulk, compared to the conformation acquired at an interface. An increase of β-sheet structure was observed in the protein adsorbed at the air/water interface40,45 while the protein was trapped in an intermediate state with a higher percentage of R-helix on a solid hydrophobic surface. This intermediate state could be converted to a final strongly bound state with a higher percentage of β-sheet structure by heating or low pH in the presence of detergent.46 While these data indicate that the adhesion of hydrophobins to surfaces depends on proteins' structural transformations, they are not sufficient to elucidate the precise nature of the interactions involved. The situation is still more complex when interactions between hydrophobins and other proteins are considered. In the present work, we demonstrate that while the interactions involved in the latter process are strongly binding, they are not denaturing for the enzymes immobilized on the film formed with SC3. Indeed, the immobilized GOX was active for at least three months and HRP for at least one month. This finding, offering the possibility of developing new functional materials, shows also the necessity of further research at a deeper mechanistic level on the observed phenomena. EXPERIMENTAL SECTION Surface Modification Using SC3 and Functionalization with Enzymes. SC3 hydrophobin was purified from the culture medium of S. commune as described previously.47 Glassy carbon electrodes (GCE) were coated with SC3 by self-assembly of the protein from aqueous solution (concentration, 0.3 mg mL-1) during ∼30 min. Subsequently, the electrodes were rinsed with distilled water, immersed in a 100 mM phosphate buffer (pH 7.0) (39) Wessels, J. G. H. Annu. Rev. Phytopathol. 1994, 32, 413-457. (40) de Vocht, M. L.; Scholtmeijer, K.; van der Vegte, E. W.; de Vries, O. M.; Sonveaux, N.; Wo¨sten, H. A. B.; Ruysschaert, J. M.; Hadziloannou, G.; Wessels, J. G.; Robillard, G. T. Biophys. J. 1998, 74, 2059-2068. (41) Scholtmeijer, K.; Janssen, M. I.; Gerssen, B.; De Vocht, M. L.; Van Leeuwen, B. M.; Van Kooten, T. G.; Wo¨sten, H. A. B.; Wessels, J. G. H. Appl. Environ. Microbiol. 2002, 68, 1367-1373. (42) Janssen, M. I.; van Leeuwen, M. B. M.; Scholtmeijer, K.; van Kooten, T. G.; Dijkhuizen, L.; Wo ¨sten, H. A. B. Biomaterials 2002, 23, 4847-4854. (43) Wo ¨sten, H. A. B.; Schuren, F. H.; Wessels, J. G. EMBO J. 1994, 13, 58485854. (44) Wessels, J. G. H.; De Vries, O. M. H.; Asgeirsdo´ttir, S. A.; Schuren, F. H. J. Plant Cell 1991, 3, 793-799. (45) Zangi, R.; de Vocht, M. L.; Robillard, G. T.; Mark, A. E. Biophys. J. 2002, 83, 112-124. (46) Wang, X.; De Vocht, M. L.; De Jonge, J.; Poolman, B.; Robillard, G. T. Protein Sci. 2002, 11, 1172-1181. (47) Lugones, L. G.; Wo ¨sten, H. A. B.; Wessels, J. G. H. Microbiology 1998, 144, 2345-2353.

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enzyme solution (concentration, 1.0 and 0.7 mg mL-1 with GOX and HRP, respectively) during ∼30 min, and rinsed again with distilled water. The functionalized GCEs were stored dry at 4 °C. The enzymes were purchased from Sigma: glucose oxidase from Aspergillus niger type X-S, reference G 7141 and horseradish peroxidase type VI-A, reference P 6782. Electrochemical Detection of GOX and HRP Activity. The hydrodynamic amperometry and cyclic voltammetry were performed with an EG&G potentiostat model 283, Princeton Applied Research. All experiments were carried out in a 35-mL cell comprising a three-electrode system with an Ag|AgCl reference, a platinum auxiliary, and a rotating GCE working electrode (0.07 cm2; 1700 rpm for the hydrodynamic amperometry). The electrolyte used was a 50 mM phosphate buffer, pH 7.0. The applied potential in hydrodynamic amperometry experiments was 1.2 V with GOX and - 0.2 V with HRP. A scan rate of 50 mV s-1 was used in all cyclic voltammetry experiments. Hydroquinone (QH2) used as HRP substrate was from Prolabo. Enzyme Activity Determination. Prior to the electrochemical experiments involving functionalized GCE, the specific activity of GOX and HRP was determined spectrophotometrically at 405 nm using dissolved enzymes. GOX and HRP solutions (concentration, ∼1 mg mL-1) were added to the spectrophotometric cell containing 50 mM phosphate buffer, 2,2′-azino-bis(3-ethylbenzothiazoline6-sulfonic acid) diammonium salt (ABTS; molar extinction coefficient 32.4 mM-1 at 405 nm), and glucose (total volume, 2.13 mL). Final enzyme concentration was 4.3 × 10-2 µg mL-1 GOX/14 µg mL-1 HRP with GOX activity measurements and 1.1 × 10-1 µg mL-1 HRP/20 µg mL-1 GOX with HRP activity measurements. Final glucose and ABTS concentrations were 3.3 mM and 47.9 µM, respectively. The specific activity of the enzymes immobilized on the glassy carbon electrodes was determined using the same method but SC3/GOX- or SC3/HRP-coated electrodes were introduced in the solution, instead of the dissolved enzymes. The UV-visible measurements were performed using UV-2110PC (Shimadzu) and 10-mm optical pathway polystyrene cells (Elvetec). The enzyme substrates used in the spectrophotometric experiments were from Sigma. Transmission Electron Microscopy. TEM of negatively stained proteins was performed using a Zeiss EM 902 microscope operated at 80 keV. SC3 was adsorbed to amorphous carboncoated TEM grids from water solution (concentration, 0.3 mg mL-1) for 15 min, and the grids were rinsed with distilled water. The enzymes were adsorbed on the SC3-coated grids from 100 mM phosphate buffer, pH 7.0 (concentrations, 1.0 and 0.7 mg mL-1 with GOX and HRP, respectively) for 15 min, and the grids were rinsed again with distilled water. Sodium phosphotungstic acid (2%) was used to stain the proteins for 10 s. Excess stain was blotted with filter paper, and the grid was allowed to dry before microscopic examination. Photomicrographs were taken at magnifications of 150×, 3000×, and 30000×. Electron energy loss spectroscopy (EELS) measurements allowed calculating the thickness (t) of the protein films from the obtained t/λ values, where λ is the mean electron free path. For the experiments performed with a Philips CM 20/STEM microscope operated at 160 keV and with carbon-based samples, the calculated λ value was 143 nm.48 1624

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Contact Angle Measurements. Sessile water-drop CA measurements were performed at room temperature on bare and modified GCE, and TEM grids, using a homemade camera-based static contact angle apparatus. Glassy carbon electrodes were thoroughly cleaned before each measurement using a procedure described elsewhere.49 The adsorption of SC3 and enzymes to GCE and TEM grids was carried out from pure water solutions. The reported values are the averages of three measurements of reverse osmosis-purified water. Accessible Surface Area Calculations. Accessible surface areas50 were calculated with the SurVol analytical procedure,51 implemented in the Brugel molecular-modeling package52,53 on a Silicon Graphics O2 R10000 station. For this purpose, the crystallographic X-ray structure of monomeric GOX obtained from the Protein Data Bank (PDB code, 1GAL;25 with FAD and with bound sugars, mannose (Man) and N-acetylglucosamine (NAG), and HRP (PDB code, 1H57;54 with bound calcium and heme, but with no sugars present) were used after elimination of crystallographic water molecules. ASA were computed using a 2.809 ( 0.047 Å (n ) 10) probe sphere radius. This probe size was determined from gyration radius analysis of the terminal NAG in glycosylated chains linked to several GOX proteins, and it was chosen to mimic interactions between hydrophobin SC3 and either GOX or HRP that are also likely to involve sugars. Moreover, this probe choice permits taking into account possible SC3/GOX and SC3/HRP interactions involving large enough, hence stable contact interfaces, while excluding the amino acid residues located in narrow clefts.55,56 Similarly, protein atom sizes play a significant role both quality-wise and quantity-wise in ASA calculations. In this study, we chose the unified atoms as defined in the work of Li and Nussinov.57 SurVol makes use of the radical plane approach to detect individual surfaces, can distinguish cavity versus external and continuous versus discontinuous surfaces, and generate several tables corresponding to each predefined ensemble (e.g., “polar” corresponds here to the ensemble containing the collection of the following amino acid residues: D, E, G, H, K, N, Q, R, S, T). GOX is known as a homodimer, but the contents of the asymmetric unit (ASU) of its X-ray structure (1GAL) define a single copy of a monomeric macromolecule and do not provide any symmetry operation data to generate a dimer;58 the structure is provided with bound FAD and five short N-linked sugar chains per subunit. The ASU of HRP (1H57) contains an unglycosylated monomer with bound calcium and heme. RESULTS AND DISCUSSION Transmission electron microscopy experiments gave us some insight in the organization of SC3 hydrophobin, GOX, and HRP (48) Egerton, R. F. Electron Energy Loss Spectroscopy in the Electron Microscope; Plenum Press: New York, 1986. (49) Kamau, G. N.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1985, 57, 545-551. (50) Lee, B.; Richards, F. M. J. Mol. Biol. 1971, 55, 379-400. (51) Alard, P., Ph.D. Dissertation. Universite´ Libre de Bruxelles, Faculte´ des Sciences, Brussels, Belgium, 1991. (52) Delhaise, P.; Bardiaux, M.; Wodak, S. J. Mol. Graphics 1984, 2, 103-106. (53) www.algonomics.com. (54) Berglund, G. I.; Carlsson, G. H.; Smith, A. T.; Szoeke, H.; Henriksen, A.; Hajdu, J. Nature 2002, 417, 463-468. (55) Mrabet, N. T. Biochemistry 1992, 31, 2690-2702. (56) Mrabet, N. T. Methods 1992, 4, 14-24. (57) Li, A.-J.; Nussinov, R. Proteins: Struct., Funct., Genet. 1998, 32, 111-127. (58) http://pqs.ebi.ac.uk/pqs-bin/macmol.pl?filename)1gal.

Figure 2. Contact angle measurements of pure water on (A) bare GCE surface and (B) bare amorphous carbon TEM grids. Contact angles are 88.7° and 87.8°, respectively.

Figure 1. TEM micrographs of the films formed with SC3, GOX, and HRP adsorbed on a carbon microscopy grid. (A-C) SC3. (D-F) GOX. (G-I) HRP. (J-L) GOX adsorbed on the SC3 film. (M-O) HRP adsorbed on the SC3 film. All samples were contrasted using 2% phosphotungstic acid (w/v). Scale: the width of the snapshots in the left, middle, and right column is 570, 28.6, and 2.9 µm, respectively.

adsorbed on amorphous carbon. The TEM micrograph obtained at 150× magnification shows that the adsorbed SC3 formed a film covering the whole surface of the carbon grid (Figure 1A). The morphology of the SC3 film was visualized at 3000× and 30000× magnification (Figure 1B,C). Formation of films on the microscopy grid was also observed with GOX (Figure 1D-F) and HRP (Figure 1G-I). However, these films do not cover uniformly the surface of the grid, as shown on the micrographs taken at low magnification (Figure 1D,G). Indeed, these films break, furl, and peel off from the surface (Figure 1E,H). At higher magnifications, a rather homogeneous local morphology of the enzyme films was observed (Figure 1F,I). In the case of the enzymes adsorbed to the SC3coated grids, the stability of the films is clearly increased compared to the enzymes adsorbed directly on the grid, while the film morphology does not differ significantly (Figure 1J-L,M-O). The results obtained suggest that SC3 adheres more strongly to the carbon grid compared to GOX and HRP. On the other hand, the enzymes form more stable structures when adsorbed to the SC3

films compared to the bare carbon grid surface. The EELS measurements of the thickness of the films formed with SC3, GOX, and HRP gave values of 202, 208, and 323 Å, respectively; the thickness of the SC3/GOX film was 281 Å and that of SC3/ HRP was 376 Å. The thickness of the enzyme layers adsorbed to SC3 estimated by comparing the latter two values with the thickness of the SC3 film was 79 Å with GOX and 173 Å with HRP. The TEM observations showing that the GOX and HRP films detach easily from the carbon grid but not from the SC3-modfied grid indicate that these enzymes have a higher propensity for protein-protein interactions compared to protein-surface interactions; this let us think that SC3, which adheres strongly to solid surfaces, could be used as an enzyme-immobilizing agent with the aim of preparing functional materials. Glassy carbon electrodes were chosen for enzyme immobilization because their surface hydrophobic/hydrophilic properties are comparable to these of the amorphous carbon microscopy grids used in TEM studies. Indeed, the contact angle measurements indicate that both surfaces are relatively hydrophobic (Figure 2), with CA value of ∼88°. The permeability of the SC3 film was checked using cyclic voltammetry. To this end, glassy carbon electrodes were modified with SC3 by adsorption of the protein from an aqueous solution. Cyclic voltammetry results showed that the SC3 layer is permeable to H2O2 (Figure 3). Surprisingly, the electrode covered with SC3 shows a higher sensibility toward H2O2 oxidation compared to the bare one; while 0.2 mM H2O2 could be detected with the modified GCE (Figure 3B), the bare GCE only allowed the detection of H2O2 above 0.9 mM. The higher sensitivity of the modified GCE may be due to the diminished capacitive current in the presence of the SC3 layer or to a local accumulation of H2O2 at the electrode surface. In the next step, GOX was adsorbed from a 1.0 mg mL-1 aqueous solution to the electrode coated with the SC3 film, and its catalytic activity was followed with hydrodynamic amperometry (Figure 4). The current response of the system, corresponding Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Figure 4. Electrochemical test of the immobilized GOX activity. Increasing concentrations of glucose (the injections of 150 µL of 1 M glucose solution are indicated with arrows) were detected with hydrodynamic amperometry using a rotating glassy carbon modified with SC3 and functionalized with GOX as a working electrode. The immediate current response of the system indicates a good transport of H2O2 through the SC3 layer. The reference curve 1 was obtained with an electrode on which GOX was adsorbed in the absence of SC3; curves 2-6 were obtained with the SC3/GOX electrode used on the 1st, 27th, 36th, 68th, and 99th day after the functionalization, respectively.

Figure 3. Voltammograms showing H2O2 redox process. Dotted lines, bare GCE; solid line, SC3-coated GCE. Bulk concentrations of H2O2 were (A) 0.0, (B) 0.2, (C) 0.6, and (D) 4.3 mM.

to the oxidation of the H2O2 produced by the enzymatic glucose oxidation, showed that GOX was immobilized on the electrode and that it stayed active. The apparent Michaelis-Menten constant (KMapp) determined for the immobilized GOX using a modified Lineweaver-Burk equation59,60 was 33.6 mM. This value is close 1626 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

to the data reported in the literature (37 mM) for the dissolved enzyme,61 indicating that there is no loss of enzyme affinity for the substrate upon immobilization. On the other hand, the amperometry experiments performed repeatedly over 99 days using the same electrode proved that no loss of enzyme activity occurred during this period. The stability, the instantaneous response, the detection limit of ∼1 mM, and the sensitivity of ∼0.9 µA mM-1 of the functionalized system suggest that it may be a good candidate for developing an electrochemical glucose sensor. It should be noted that no GOX activity was observed when the enzyme was adsorbed to the bare electrode (Figure 4, curve 1). This result may be due to the enzyme desorption from the bare electrode. Indeed, the TEM observations showing GOX films peeling off the microscopy grid (Figure 1D) support this proposal. However, denaturation of GOX adsorbed to the bare GCE cannot be excluded. A second redox enzyme, the horseradish peroxidase, was used to catalyze the reaction of QH2 oxidation. The results obtained with voltammetry and hydrodynamic amperometry (Figure 5) show that HRP can be successfully immobilized on GCE via SC3. The voltammograms presented in Figure 5A show that in the presence of H2O2 the QH2 oxidation peak decreases, while the reduction peak of its oxidized form (quinone) increases at the HRP-modified GCE electrode surface. This indicates that there is consumption of QH2 and formation of quinone. These results are in accord with the HRP catalytic mechanism described in the literature.62-64 Indeed, H2O2 is reduced by the native form of the (59) Shu, F. R.; Wilson, G. S. Anal. Chem. 1976, 48, 1679-1686. (60) Kamin, R. A.; Wilson, G. S. Anal. Chem. 1980, 52, 1198-1205. (61) Tiller, J. C.; Rieseler, R.; Berlin, P.; Klemm, D. Biomacromolecules 2002, 3, 1021-1029. (62) Chance, B. Science 1949, 109, 204-208. (63) Lobel, K. D.; Hench, L. L. J. Biomed. Mater. Res. 1998, 39, 575-579.

Figure 5. Electrochemical test of the immobilized HRP activity. (A) Voltammograms showing the QH2 redox process using an extemporaneously prepared electrode. Measurements were recorded in 50 mM phosphate buffer (curve 1) or in a buffer solution of 0.5 mM QH2 (curve 2), 0.5 mM QH2 and 2 mM H2O2 (curve 3), or 0.5 mM QH2 and 2 mM H2O2 after 10 min of immersion (curve 4). (B) Hydrodynamic amperometry responses recorded at increasing concentrations of hydrogen peroxide (the injections of H2O2 are indicated with arrows). The curves 1-3 were obtained with a SC3/HRP electrode used on the 1st, 8th, and 19th day after the functionalization, respectively.

peroxidase, HRP{Fe(III)}, which is oxidized to HRP{Fe(V)}. Then, QH2 reduces HRP{Fe(V)} to is native state, forming quinone. The current-H2O2 concentration dependency obtained from hydrodynamic amperometry experiments performed at an applied potential of -0.2 V indicates that the sensitivity of the system toward H2O2 was 1.3, 0.3, and 0.1 µA mM-1 for the 1st, 8th, and 19th day of experiments, respectively. The decrease of HRP activity with time may be due to its intrinsic instability in the presence of a reactive oxygen species, hydrogen peroxide.65,66 Indeed, on the 36th day after immobilization on a glassy carbon electrode HRP was active when used for the first time (results not shown). The latter observation indicates that this electrode has excellent storage properties and could be possibly used as a (64) Ferapontova, E. E.; Grigorenko, V. G.; Egorov, A. M. Biochemistry (Moscow) 2001, 66, 832-839. (65) Bockle, B.; Martinez, M. J.; Guillen, F.; Martinez, A. T. Appl. Environ. Microbiol. 1999, 65, 923-928. (66) Olsen, L. F.; Hauser, M. J. B.; Kummer, U. Eur. J. Biochem. 2003, 270, 2796-2804.

single-use sensor. KMapp of 0.9 mM determined for the immobilized HRP indicates that there is no loss of enzyme affinity for the substrate upon immobilization (KMapp ) 0.7 mM for the dissolved enzyme61). An important parameter to be taken into account in elaborating modified electrodes is the quantity of the immobilized catalyst. However, determination of nanogram amounts of adsorbed proteins is a major challenge from the experimental point of view; predicting the adsorption of proteins on surfaces using analytical models is presently under development.67 Here, the quantity of the enzymes adsorbed on the electrode was estimated using EELS results and approximating the proteins by rigid spheres with a partial specific volume of 0.73 mL g-1.68 The calculated diameters of SC3, GOX (dimer), and HRP were 32, 72, and 45 Å, respectively (cross sections 806, 4051, and 1607 Å2, respectively). Comparison of the thickness of the SC3/GOX film obtained from the TEM experiments and of the GOX diameter suggests that the enzyme forms a monolayer when adsorbed to the SC3 film. Consequently, the number of GOX molecules adsorbed on the electrode (surface 0.07 cm2) via SC3 is ∼1.7 × 1011, corresponding to 46 ng of the protein. In the case of HRP, comparison of the film thickness with the enzyme diameter suggests that it forms four layers. Consequently, 1.7 × 1012 HRP molecules (116 ng) would be adsorbed. One more intriguing question concerning immobilized enzymes is how the immobilization influences the enzyme kinetics. However, while enzyme kinetics can be easily determined using, for example, spectrophotometry, electrochemistry is not the method of choice in such measurements. Consequently, we compared the efficiency of the dissolved- and immobilized-enzyme electrochemical systems. This was done by comparing the quantity of the enzyme necessary to trigger an instantaneous current response at a given substrate concentration. We considered the response as instantaneous for the values of ∆i/∆t > 1.4 µA s-1 for GOX and ∆i/∆t < -0.08 µA s-1 for HRP. The results obtained with dissolved GOX and HRP using hydrodynamic amperometry are presented in Figures 6 and 7. In the case of the dissolved GOX, the instantaneous response of the system was obtained for the final concentration 1.6 × 102 µg mL-1 enzyme, that is 5.5 × 106 ng total quantity of the enzyme present in the 35-mL electrochemical cell (Figure 6). Comparison of this value with the calculated quantity of GOX adsorbed on the electrode via SC3 (46 ng) shows that 1.2 × 105 times more GOX molecules was needed to obtain an instantaneous response of the system using the dissolved enzyme. In the case of HRP, an instantaneous response of the dissolved enzyme system was obtained for the HRP concentration of 5.7 × 10-1 µg mL-1, that is 2.0 × 104 ng total enzyme quantity present in the cell (Figure 7). Compared to 116 ng of HRP adsorbed on the SC3-modified electrode, 173 times more of the dissolved enzyme was used. It is worth noting that the current versus time dependency evolves linearly with the enzyme quantity both with GOX and HRP (insets of Figures 6 and 7). The latter result indicates that the initial slope of the amperometric curves reflects the reaction rate and that the process is not diffusion-limited. To compare the intrinsic properties of the immobilized and dissolved enzymes, the enzyme kinetics were measured spectro(67) Nicolau, D. V. J.; Nicolau, D. V., 2nd Asia-Pacific Bioinformatics Conference, Chen, Y,-P, P,, Ed., Dunedin, New Zealand, 2004. (68) Beck, K. FEBS Lett. 1989, 249, 1-4.

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Figure 6. Hydrodynamic amperometry detection of the dissolved GOX activity. The experiments were performed as a function of GOX quantity at a constant glucose concentration of 9.4 mM; other experimental conditions were as described in the legend of Figure 4. GOX concentration (in µg mL-1): (black) 2.5 × 10-2 (cyan) 5.7, (blue) 3.4 × 101, (green) 7.4 × 101, (magenta) 1.1 × 102, and (red) 1.6 × 102. Inset: current versus time dependency calculated for enzyme quantities with which ∆i/∆t < 1.4 µA s-1.

Figure 8. Immobilized enzyme kinetics followed spectrophotometrically. (A) GOX. (B) HRP. After the removal of the enzyme-functionalized GCE from the spectrophotometric cell, no increase of the optical density was observed.

Figure 7. Hydrodynamic amperometry detection of the dissolved HRP activity. The experiments were performed in function of HRP quantity at constant QH2 and H2O2 concentrations of 0.5 mM and 92 µM, respectively; other experimental conditions were as described in the legend of Figure 5. HRP concentrations (in µg mL-1): (black) 1.4 × 10-3; (cyan) 1.1 × 10-2; (blue) 2.2 × 10-2; (green) 3.1 × 10-2; (magenta) 4.5 × 10-2; (orange) 2.7 × 10-1; (red) 5.7 × 10-1. Inset: current versus time dependency calculated for enzyme quantities with which ∆i/∆t > - 0.08 µA s-1.

photometrically. The specific activities obtained with the dissolved enzymes were 17.8 µmol min-1 mg-1 with GOX and 6.0 µmol min-1 mg-1 with HRP. The immobilized enzyme specific activities, calculated using kinetics shown in Figure 8, and the immobilized enzyme quantities, estimated as explained before, were 3.8 and 0.5 µmol min-1 mg-1 with GOX and HRP, respectively. The specific activity of the immobilized enzyme was 4.7 times lower in the case of GOX and 12 times lower in the case of HRP compared to the dissolved enzymes. It should be noted, however, that approximating the protein shapes by spheres and assuming that the entire GCE surface is occupied by the enzyme molecules has probably led to overestimating the quantity of the adsorbed 1628 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

enzymes. Consequently, the calculated specific activities of the immobilized enzymes may be underestimated. These results, together with the affinity constant (KMapp) values obtained from the electrochemical experiments, show that the described immobilization method preserves the enzymes in their active form. To get some insight into the interactions involved in enzyme immobilization, contact angle measurements were performed using bare and protein-coated GC (Figure 9). As expected, the hydrophobicity of the glassy carbon (Figure 9A) decreases after coating with GOX (Figure 9B) or HRP (Figure 9C). There is a clear difference between GOX and HRP, as in the latter case, the GCE surface becomes more hydrophilic (60.9° with HRP compared to 70.5° with GOX). However, change of the hydrophobic into a hydrophilic nature of GCE is most flagrant when it is coated with SC3 (Figure 9D), as the CA decreases by more than half. When GOX is immobilized on the latter surface (Figure 9E), its hydrophobicity increases to a value comparable with this of the GC/GOX surface. The same tendency is observed with HRP (Figure 9F), but in this case, the hydrophobicity of the surface is lower compared to the GC/HRP surface. In the absence of a 3-D structure of SC3, the interpretation of the CA results can be only tentative. According to the literature, the affinity of SC3 for different surfaces depends on its capacity to rearrange its conformation and orientation in such a way as to

Figure 9. Contact angle measurements. (A) Bare GC, 88.7°; (B) GC/GOX, 70.5°; (C) GC/HRP, 60.9°; (D) GC/SC3, 41.0°; (E) GC/SC3/GOX, 71.6°; (F) GC/SC3/HRP, 55.9°

Figure 10. Accessible surface analysis. (A) GOX monomer and (B) HRP surface properties. Polar, nonpolar, and aromatic patches represented by bordered pie slices are given in blue, green, and yellow, respectively. (C) GOX monomer and (D) HRP amino acid-type distribution within the total ASA. Blue, green, and yellow bars represent polar, nonpolar, and aromatic residues. The amino acid contributions are presented relative to glycine (G, no side chain; GOX ASA 908.655 Å2, n ) 24; HRP ASA 319.180 Å2, n ) 8) to better visualize the relative side-chain accessibility.

establish either hydrophobic or hydrophilic interactions with the environment; as a consequence of SC3 adsorption, the hydrophobic/ hydrophilic properties of surfaces are inverted.37,43,69 Our experiments show that GOX and HRP have a similar, albeit less pronounced capacity. Indeed, both the hydrophobicity of the bare GC and hydrophilicity of the SC3-coated GC decrease following GOX and HRP adsorption. These results indicate that GOX and HRP establish hydrophobic interactions with the GC surface while the interactions with the SC3 layer are polar. (69) Wo ¨sten, H. A. B.; de Vocht, M. L. Biochim. Biophys. Acta 2000, 1469, 7986.

Accessible surface analysis was performed to get more insight into the molecular surface properties of GOX and HRP and their interactions with SC3. For this purpose, ASA were computed using the traditional rolling-sphere approach.50 Probing the GOX and HRP surface shows that the polarity of the 2.809 Å probeaccessible area is ∼75% of the total protein ASA with both enzymes (Figure 10 A,B). Indeed, GOX presents a large uninterrupted polar surface patch that is accessible to the probe, while there exist much smaller and discontinuous nonpolar or aromatic patches. The total polar GOX area is 12 830.184 Å2 representing 77.0% of the total protein ASA, with four patches, of which the largest one Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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represents 76.4% of the total protein ASA. The total nonpolar GOX area is 3146.824 Å2 (18.9%, 38 patches) and total aromatic GOX area is 689.534 Å2 (4.1%, 25 patches). 1GAL contains five glycosylated chains consisting of NAG, Man, or both with a total ASA of 2401.936 Å2 of which one single sugar chain accounts for 50.6%. The total polar HRP area is 8771.627 Å2 (73.2%, 2 patches), with a major continuous patch representing 72.1% of the total ASA. The total nonpolar HRP area is 2665.703 Å2 (22.3%, 18 patches) and total aromatic area is 537.263 Å2 (4.5%, 11 patches). The relative accessibility of different amino acids is presented in Figure 10C and D. Interestingly, the polar surface of HRP is positively charged, while that of GOX is mostly negatively charged. It should be noted that the glycosylated HRP used in the bench experiments may be still more polar compared to the unglycosylated structural model used in the ASA calculations; a higher surface polarity is also expected in the case of the GOX homodimer used in the experiments, compared to the partly glycosylated monomer used in modeling.70 It is reasonable to suppose that the large polar enzyme surfaces are involved in the interactions with the polar surface formed with the SC3 adsorbed to the electrode (Figure 9D), while the nonpolar surfaces of the SC3-adsorbed enzymes are exposed. Taken together, the modeling results converge with these obtained from the CA measurements and support our proposal that the most significant part of the enzyme-SC3 binding is provided by polar interactions. It can be supposed that the nonpolar interactions involved in the adsorption of GOX and HRP on the bare GCE, while too weak to allow stable retention, are denaturing for the enzymes.71 CONCLUSIONS The capacity of SC3 to act as an agent retaining enzymes on GCE is specific for this protein. Indeed, neither replacing of SC3 with proteins such as bovine serum albumin and lysozyme nor a direct adsorption of the enzymes to the bare GC surface allowed their lasting immobilization in an active form. While the upper (70) Lo Conte, L.; Chothia, C.; Janin, J. J. Mol. Biol. 1999, 285, 2177-2198. (71) Voros, J. Biophys. J. 2004, 87, 553-561.2

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limit of the life span of the immobilized enzymes was not checked systematically, it was shown that the immobilized GOX kept its activity on the 99th day of repeated use while HRP was active on the 36th day after immobilization when used for the first time. The affinity for the substrate (KMapp) is comparable for the immobilized and dissolved GOX and HRP. The kinetic measurements indicate that the enzyme specific activity is lower for the immobilized, compared to the dissolved enzymes; however, the immobilized enzyme activity is probably underestimated. The contact angle measurements and accessible surface analysis suggest that the major contribution to the interactions involved in the immobilization of the enzymes on the SC3 layers comes from polar amino acids. Looking to the future, the approach presented in this paper has a bearing for preparing stable enzyme-based catalytic surfaces in an easy, rapid, and reliable way. Within less than 1 h, without resorting to covalent chemistry, enzymes can be stably immobilized on a solid surface. One more advantage of the proposed method of enzyme immobilization is the SC3 biocompatibility, which offers the possibility of medical and food industry applications. The generality of the method will be checked using other enzymes and solid surfaces. ACKNOWLEDGMENT We thank Prof. G. Robillard and Dr. K. Schottmeijer for helpful discussions and advice on the manuscript. We also thank Dr. J. Ghanbaja and Dr. H. Ayatti for their help with TEM experiments. We are grateful to Dr. M. Petrissans and M. Hakkou for giving us access to the contact angle apparatus. Ph.D. fellowship (Bourse Docteur Inge´nieur) from BiOMaDe Technology Foundation and the Centre National de la Recherche Scientifique (Y. C.) is gratefully acknowledged. N.T.M. acknowledges the financial support from La Ligue contre le Cancer and L'Institut National pour la Sante´ et la Recherche Me´dicale. Received for review July 28, 2004. Accepted December 29, 2004. AC048897W