Photochemical Immobilization of Proteins and Peptides on

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Photochemical Immobilization of Proteins and Peptides on BenzophenoneTerminated Boron-Doped Diamond Surfaces Lionel Marcon,†,‡ Mei Wang,†,‡ Yannick Coffinier,†,‡ Francois Le Normand,§ Oleg Melnyk, Rabah Boukherroub,†,‡ and Sabine Szunerits*,†,‡

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† Institut de Recherche Interdisciplinaire (IRI, USR 3078), Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France, ‡Institut d’Electronique, de Micro electronique et de Nanotechnologie (IEMN, UMR 8520), Cit e Scientifique, Avenue Poincar e - BP. 60069, 59652 Villeneuve d’Ascq, France, §Institut de Physique et Chimie des Mat eriaux/Groupe Surfaces et Interfaces (IPCMS/GSI) UMR 7504 CNRS, Bat 69, 23 rue du Loess, BP 43, 67034 Strasbourg, France, and Institut de Biologie de Lille, UMR CNRS-8525, 1 rue du Pr. Calmette, 59021 Lille, France

Received June 30, 2009 The successful covalent linking of green fluorescence protein and streptavidin to patterned benzophenone-modified boron-doped diamond (BDD) electrodes is demonstrated. Photoreactive benzophenone moieties were covalently grafted to oxidized diamond surfaces via an esterification reaction. Patterned BDD surfaces were obtained using a UV/ ozone lithographic approach either on hydrogen-terminated BDD or on poly(ethylene)-glycol-modified BDD surfaces. UV light (λ=365 nm) irradiation of the patterned BDD surfaces in the presence of green fluorescence protein (GFP) or streptavidin resulted in the covalent immobilization of the proteins. The presence of poly(ethylene) glycol chains reduces significantly the nonspecific adsorption of the proteins. The success of the photoimmobilization of streptavidin was evidenced through biomolecular interaction with avidin. The preservation of the biological activity was furthermore underlined by photoimmobilization of peptides directly onto benzophenone modified BDD using a photomask.

1. Introduction Light-induced reactions between a photoreactive probe on a surface and C-H bonds in the backbone or in side groups of polymeric overcoats and biological molecules such as proteins, oligonucleotides, and enzymes have been intensively investigated in the past decade.1-10 Undeniably, the immobilization of biological molecules on surfaces is a critical step in many bioassays including diagnostic analysis and bioelectronic sensing. One of the main advantages of using photoimmobilization rather than chemical or mechanical immobilization through spotting is almost certainly the possibility of micropatterning the surface with biomolecules using photolithographic approaches. The heterobifunctional reagents employed in photopatterning permit site directed coupling of biomolecules to the surfaces to provide optimal orientation for maximal bioactivity. This has important implications for medical diagnostic assays and high-throughput screens for drug discovery, and genetic screening. Most of these photoreactive probes contain aryl azides, aryl diazarines, or benzophenone. Benzophenone has been reported to be one of *To whom correspondence should be addressed. E-mail: sabine.szunerits@ iri.univ-lille1.fr. Telephone: þ33 3 62 53 17 25. Fax: þ33 3 62 53 17 01. (1) Bartlett, M. A.; Yan, M. Adv. Mater. 2001, 13, 1449–1451. (2) Cosnier, S.; Senillou, A. Chem. Commun. 2003, 3, 414. (3) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Rigsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1997, 12, 1997–2006. (4) Konry, T.; Novoa, A.; Shemer-Avni; Hanuka, N.; Cosnier, S.; Lepellec, A.; Marks, R. S. Anal. Chem. 2005, 77, 1771. (5) Prucker, O.; Naumann, C. A.; Ruhe, C. A.; Knoll, W.; Frank, C. W. J. Am. Chem. Soc. 1999, 121, 8766–8770. (6) Griep-Raming, N.; Krager, M.; Menzel, H. Langmuir 2004, 20, 11811–11814. (7) Elender, G.; Kuhner, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11, 565. (8) Dorman, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64. (9) Ligler, F. S.; Breimer, M.; Golden, J. P.; Niva, D. A.; Dodson, J. P.; Green, T. M.; Haders, D. P.; Sadik, O. A. Anal. Chem. 2002, 74, 713. (10) Balakirev, M. Y.; Portos, S.; Vernaz-Gris, M.; Berger, M.; Arie, J.-P.; Fouque, B.; Chatelain, F. Anal. Chem. 2005, 77, 5474–5479.

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the most efficient photophores.8 Benzophenone is stable under ambient light and protic solvents. It can be activated at wavelengths that cause little damage to biomolecules (λ > 340 nm) and can cross-link with higher efficiency than other photoreactive functionalities.11,12 The photoprocess of benzophenone includes a triplet-state excitation forming a diradicaloid. The electron-deficient oxygen interacts with weak C-H bonds, resulting in H-abstraction and radical recombination creating a covalent binding with oligonucleotides and proteins having sterically accessible C-H bonds.13,14 Recently photochemical grafting methods were developed for surfaces such as glass, silicon, gold, platinum, indium tin oxide (ITO) films, and titanium.1-6,15,16 In the case of glass or silica, photoreactive silane anchors are immobilized on the surface.1,5,9,10,17 This allowed attachment of photochemically thin polymer films to the solid interface. Benzophenone-functionalized phosphonic acid was used by Menzel et al. for the modification of titanium surfaces allowing the photochemical linking of poly(vinyl-Nmethylacetamide).6 Photoactive gold surfaces were obtained through derivatization of gold with densely packed disulfide monolayers bearing amine groups to which benzophenone was chemically linked.3 Immunoglobulins were photochemically attached to the surface and characterized using X-ray photoelectron spectroscopy (XPS) and radiolabeling. Cosnier et al. and Senillou and Marks et al. (11) Prestwich, G. D.; Dorman, G.; Elliott, J. Y.; Marecak, D. N.; Chaudhary, A. Photochem. Photobiol. 1997, 65, 112. (12) Ravand, J. L.; Douki, T.; Cadet, J. J. Photochem. Photobiol., B 2001, 63, 88. (13) Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661. (14) Turro, N. J. Molecular Photochemistry; Univ. Sci. Books: Sausalito, CA, USA, 1991. (15) Nakanishi, J.; Kikuchi, Y.; Takarada, T.; Nakayama, H.; Yamaguchi, K.; Maeda, M. J. Am. Chem. Soc. 2004, 126, 16314. (16) Nakanishi, J.; Kikuchi, Y.; Takarada, T.; Nakayama, H.; Yamaguchi, K.; Maeda, M. Anal. Chim. Acta 2006, 578, 100. (17) Ayadim, M.; Soumillion, J. P. Tetrahedron Lett. 1995, 36, 4615.

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electrochemically deposited thin films of benzophenone-modified polypyrrole on platinum2 and ITO-coated optical fibers,4 allowing the linking of biological receptors such as glucose oxidase, bovine serum albumin (BSA), and hepatitis C virus. Next to these electrical interfaces, boron-doped diamond (BDD) has gained remarkable interest in the biomedical research field18-26 due to its excellent mechanical properties, extreme chemical stability, good electrical conductivity, low background current densities, large potential window in aqueous electrolytes (about -1.35 to 2.3 V/NHE), as well as its biocompatibility.27-29 It has been persuasively shown30 that the bonding stability of DNA to diamond, compared to gold, Si, and glassy carbon, is significantly better, as no degradation of fluorescence intensity was detected. Diamond has thus been used widely for the development of biological platforms. Different surface immobilization schemes have been proposed. A photochemical process between a terminal vinyl group to H-terminated p-type diamond has been proposed by Hamers and co-workers to link DNA.19,25,31 Garrido et al.24 used the same photochemical approach to covalently attach green fluorescence proteins on n-type hydrogen-terminated nanocrystalline diamond films, previously patterned using photolithography. The direct amination of H-BDD and the subsequent immobilization of peptides through semicarbazide linking was recently shown by us.32 Kawarada et al.20 reported on the covalent linking of DNA on aminated surfaces. We have recently shown the possibility to photolink DNA molecules to patterned benzophenone-modified BDD surfaces. The benzophenone termination was obtained through an esterification reaction of oxidized BDD with 3-benzoylbenzoic acid.33 In a continuation of this work, we show in this article the feasibility of performing light-induced reactions between the patterned photoreactive benzophenone probes and the C-H bonds of green fluorescence protein (GFP) and streptavidin. The influence of the surface termination surrounding the benzophenone reactive patterns on nonspecific adsorption is also investigated. Fluorescence imaging was used to characterize the modified surfaces. (18) Carlisle, J. A. Nat. Mater. 2004, 3, 668–669. (19) Lasseter, T. L.; Clare, B. H.; Abbott, N. L.; Hamers, R. J. J. Am. Chem. Soc. 2004, 126, 10220–10221. (20) Zhang, G.-J.; Song, K.-S.; Nakamura, Y.; Ueno, T.; Funatsu, T.; Ohdomari, I.; Kawarada, H. Langmuir 2006, 22, 3728–3734. (21) Yang, W.; Butler, J. E.; Russell, J. N.; Hamers, R. J. Langmuir 2004, 20, 6778–6787. (22) Delabouglise, D.; Marcus, B.; Mermoux, M.; Bouvier, P.; Chane-Tune, J.; Petit, J.-P.; Mailley, P.; Livache, T. Chem. Commun. 2003, 2698–2699. (23) Fortin, E.; Chane-Tune, J.; Mailley, P.; Szunerits, S.; Marcus, B.; Petit, J.-P.; Mermoux, M.; Vieil, E. Bioelectrochemistry 2004, 63, 303–306. (24) Hartl, A.; Schmich, E.; Garrido, J. A.; Hernanod, J.; Catharino, S. C. R.; Walter, S.; Feulber, P.; Kromka, A.; Steinmuller, D.; Stutzmann, M. Nat. Mater. 2004, 1–7. (25) Hamers, R. J.; Butler, J. E.; Lassetera, T.; Nicholsa, B. M.; Russell, J. N.; Tsea, K.-Y.; Yanga, W. Diamond Rel. Mater. 2005, 14, 661–668. (26) Nebel, C. E.; Shin, D.; Rezek, B.; Tokuda, N.; Uetsuka, H.; Watanabe, H. J. R. Soc. Interface 2007, 1–23. (27) Granger, M. C.; Witek, M.; Xu, J. S.; Wang, J.; Hupert, M.; Hanks, A.; Koppang, M. D.; Butler, J. E.; Lucazeau, G.; Mermoux, M.; Strojek, J. W.; Swain, G. M. Anal. Chem. 2000, 72, 3793. (28) Tryk, D. A.; Tsunozaki, K.; Rao, T. N.; Fujishima, A. Diamond Relat. Mater. 2001, 10, 1804. (29) Tang, L.; Tsai, C.; Gerberich, W. W.; Kruckeberg, L.; Kania, D. R. Biomaterials 1995, 16, 483–488. (30) Yang, W.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T. L.; Lasseter, T. L.; Russell, J. N.; Smith, L. M.; Harmers, R. J. Nat. Mater. 2002, 253–257. (31) Strother, T.; Knickerbocker, T.; Russell, J. N.; Butler, J.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 968–971. (32) Coffinier, Y.; Szunerits, S.; Jama, C.; Desmet, R.; Melnyk, O.; Marcus, B.; Gengembre, L.; Payen, E.; Delabouglise, D.; Boukherroub, R. Langmuir 2007, 23, 4494–4497. (33) Szunerits, S.; Shirahata, N.; Actis, P.; Nakanishir, J.; Boukherroub, R. Chem. Commun. 2007, 2793.

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2. Experimental Section 2.1. Materials. 4-Benzoylbenzoic acid, dimethyl sulfoxide (DMSO), dimethylaminopyridine, N,N-dicyclohexylcarbodiimide (DCC), toluene, sodium acetate, and 1,3-dimethylimidazolium-bis(trifluoromethylsulfonyl)imide were obtained from Aldrich and used without further purification. Phosphate buffer saline (PBS, 0.01 M, pH 7.2, including 0.138 M NaCl, 0.0027 M KCl) tablets and Tween 20 were obtained from Sigma. mPEGsilane-5000 (O-Methyl-O0 -[2-(trimethoxysilylethyl)]polyethyleneglycol) was purchased from Nektar Transforming Therapeutics. Green fluorescence protein (GFP, protein G conjugate with Alexa Fluor 488, λabs = 495 nm, λem = 519 nm, molecular weight = 20 000 Da, 2-6 dye molecules per protein) and biotin-conjugated quantum dots (Qdots) 605 were obtained from Invitrogen. Stock solutions of GFP of 1 mg mL-1 in PBS were made and kept at 4 °C. Streptavidin from Streptomyces avidinii was purchased from Sigma. The influenza hemagglutinine peptide (HA-peptide, HA1 fragment 98-106, Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) was obtained from Aldrich. Stock solutions of HA-peptide of 1 mg mL-1 in PBS were made and kept at 4 °C. Rhodamine anti-HA was purchased from Invitrogen, and stock solutions of 10 μg mL-1 were used. 2.2. Boron-Doped Diamond Films. Two different types of diamond samples were used. For atomic force microscopy (AFM) and microdroplet formation, polished freestanding polycrystalline diamond films were purchased from Windsor Scientific (Slough, England). These boron-doped polycrystalline chemical vapor deposited diamond layers were grown to a thickness greater than 500 μm by adding diborane to the methane and hydrogen source gases supplied to a microwave chemical vapor deposition (CVD) reactor. The samples were polished both on the nucleation and the growth side to a mirror finish. The electrodes were supplied as 55 mm2 plates. The resulting samples have a bulk electrical resistance of about 0.75 mΩ. The average boron doping level of the material was reported to be about 51020 B cm-3 as determined by secondary ion mass spectroscopy (SIMS). Prior to use, samples were cleaned in 3:1 (v/v) concentrated H2SO4/H2O2 (30%) for 15 min followed by copious rinsing with Milli-Q water and then slightly polished using diamond past (0.3 μm) to obtain a smooth particle-free surface. (Caution! Piranha solution reacts violently with organic materials; it must be handled with extreme care, followed by copious rinsing with deionized water.) Polycrystalline boron-doped diamond films (1.5-2 μm thick) deposited on silicon substrates, in a hot filament-assisted chemical vapor deposition reactor supplied with diborane and methane in hydrogen, were provided by CSEM (Neuchatel, Switzerland) and used for photochemical grafting experiments. The doping level of boron was determined to be NA ∼3  1019 B cm-3 by SIMS measurements. Hydrogenation of the surface of the BDD samples was performed in an ultrahigh vacuum (UHV) CVD chamber using the hot-filament chemical vapor deposition (HF CVD) mode described elsewhere.34 The conditions were the following: 100 sccm H2 for 10 min and P=15 mbar with tungsten filaments (two pairs of tungsten filaments placed 5 and 10 mm above the substrate, respectively) at 180 W (around 2450 K). The surface of the substrate was heated on the back side by using an infrared heater in order to keep a constant temperature of 973 K. Following this treatment, the sample was cooled to room temperature under gaseous hydrogen. 2.3. Functionalization of H-Terminated BDD. Oxidation of Diamond. A low pressure mercury arc lamp (UVO cleaner,

no. 42-220, Jelight, P = 1.6 mW cm2, distance from sample = 3 mm, t = 60 min) was used to photochemically oxidize asreceived BDD samples as reported previously.35 (34) Arnault, J. C.; Demuynck, L.; Speisser, C.; Le Normand, F. Eur. Phys. J. B 1999, 11, 327–343. (35) Boukherroub, R.; Wallart, X.; Szunerits, S.; Marcus, B.; Bouvier, P.; Mermoux, M. Electrochem. Commun. 2005, 7, 937–940.

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Figure 1. Chemical grafting involved for the fabrication of benzophenone-modified boron-doped diamond arrays: (a) photolithographic oxidation of as-grown BDD, (b) photolitographic decomposition of mPEG-silane chains and formation of OH-BDD patterns, (c) esterification of OH-BDD patterns with benzophenone, and (d) photoimmobilization of biomolecules on benzophenone-terminated regions.

PEG-ylation of Oxidized BDD (PEG-BDD). Poly(ethylene) glycol (PEG) linking to oxidized diamond samples was performed in a solution of 100 mg mPEG-silane in 10 mL of dry toluene. The reaction was carried out at room temperature for 6 days under nitrogen atmosphere. The resulting surface was rinsed with toluene, dichloromethane, water, and ethanol and then dried in a stream of argon. Preparation of Benzophenone-Terminated BDD (BP-BDD) Surfaces. 3-Benzoylbenzoic acid (2 mmol) and dicyclohexylcarbodiimide (2 mmol) were dissolved in CH2Cl2 (10 mL). The resulting solution was reacted with oxidized BDD and dimethylaminopyridine (0.66 mmol) for 48 h at room temperature under argon. The benzophenone-modified BDD was sonicated for 5 min in Milli-Q water and methanol and then dried under an argon stream. 2.4. Patterning of BDD Interfaces. Oxidized patterns were formed on hydrogenated and PEG-modified BDD surfaces by UV irradiation through an optical mask (50  50 μm2 or 400  400 μm2 openings) using a low pressure mercury arc lamp (UVO cleaner, no. 42-220, Jelight, P = 1.6 mW cm2, distance from sample=3 mm, t=60 min). Benzophenone termination in the oxide patterns was obtained by exposing the patterned BDD interface to 3-benzoylbenzoic acid as described above. 2.5. Photoimmobilization. Green fluorescence protein (GFP) and streptavidin from Streptomyces avidinii were photoimmobilized on BP-BDD by depositing 10 mL of a diluted protein solution (1 mg mL-1 in PBS) manually on the patterned BDD surfaces and illuminating the sample in air through a filter (λ=365 ( 5 nm) for Langmuir 2010, 26(2), 1075–1080

30 min using a xenon lamp (Hamamatsu Japan). The intensity of the light was measured using a Nova II (PD300-UV, Japan) photodiode and was determined as being 5 mW cm-2. Afterward, the interfaces were sonicated for 30 min in Tween 20 (0.05 v/v %) in PBS (0.01 M, pH 7.2) three times to remove the excess of protein. The fluorescence of GFP was used to detect the presence of the protein. In the case of streptavidin, the surface was further incubated with biotin-conjugated Qdots 605 for 1 h at room temperature and then washed three times with Milli-Q water and methanol. The Qdots-conjugate stock solution was diluted 1:5 in incubation buffer supplied by the manufacturer prior to incubation. Red fluorescent Qdots were selected because their specific excitation/emission spectra make them distinguishable from any kind of background fluorescence. Photoimmobilization of HA-peptide was performed on benzophenone-terminated BDD interfaces by illuminating the sample in air through a photomask at λ =365 ( 5 nm for 30 min and 5 mW cm-2 using a xenon lamp (Hamamatsu Japan). Afterward, the interfaces were sonicated for 30 min in Tween 20 (0.05 v/v %) in PBS (0.01 M, pH 7.2) three times to remove the excess of protein.

2.6. Instrumentation. Fluorescence Imaging of Photoimmobilized GFP. Fluorescence images were captured with a Coolsnap ES2 camera (Photometrics, Tucson, AZ) using Nikon Elements software under an Eclipse 80i (Nikon Instruments, Tempe, AZ) optical microscope equipped with a DAPI excitation (365/50 nm) and a Cherry emission (630/75 nm or 475/30 nm) filter set. DOI: 10.1021/la903012v

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Figure 2. Topographic and surface potential images of photolithographically patterned H-BDD using UV/ozone through a photomask: (A) topographic image and (B) surface potential image (the dark squares correspond to oxidized regions, and the bright areas to hydrogenated regions).

Figure 3. Formation of water (A) and hydrophilic ionic liquid (B) droplet arrays on the hydrophilic areas of a patterned BDD interface.

Kelvin Probe Force Microsopy (KFM). Kelvin Probe images were recorded under a stream of argon using a multimode model atomic force microscope (Veeco, Santa Barbara, CA) equipped with a Nanoscope III controller (Digital Instruments). Goldcoated silicon cantilevers (force constant=2.1 N m-1; resonance frequency = 27.75 kHz; Q-factor = 189.1) were used. An AC bias voltage of 2 V was applied through the tip at a frequency of 25.5 kHz between the probe and sample. Images were acquired at a probe scan rate of 0.15 Hz.

3. Results and Discussion 3.1. Patterning of Hydrogen-Terminated BDD Surfaces. The strategy for the fabrication of micropatterns on polycrystalline BDD is based on a photolithographic approach. It consists of tightly sealing an optical mask with micrometric openings against the as-grown BDD interface and exposing the sample to UV/ ozone for 60 min. After UV/ozone treatment, the H-BDD surface is locally oxidized, producing a mix of different surface terminations including carbonyl, ether, and surface hydroxyl groups. In the following, only the surface hydroxyl groups will be used, thus presented exclusively in Figure 1a. The patterned BDD surface was analyzed using KFM. KFM not only allows determination the surface topography but in addition delivers images of the surface work function on a nanometer scale depending on the 1078 DOI: 10.1021/la903012v

shape and the diameter of the probing tip. Because of the different electron affinities of oxidized and hydrogenated diamond surfaces, a difference in surface potential can be observed. Figure 2 shows the KFM image of the H-BDD patterned surface obtained by applying an AC voltage of þ2 V at a frequency of 25.5 kHz between the gold-coated silicon cantilever and the BDD sample. The oxide patterns (dark areas in Figure 2) are clearly resolved and reveal a potential difference between the oxidized and the hydrogenated regions (bright areas in Figure 2) of about 150 mV on average. The micropatterns could be furthermore visualized by using the difference in wetting properties of the hydrogenated and oxidized areas. H-BDD is hydrophobic (contact angle of 92°), while oxidized BDD is hydrophilic.35 Water and hydrophilic liquids are preferentially condensed on the oxidized regions of the patterned diamond. Figure 3 shows optical images of water and ionic liquid (1,3-dimethylimidazolium-bis(trifluoromethylsulfonyl)imide) droplet arrays formed on the patterned BDD surface. The low vapor pressure of the ionic liquid prevents evaporation of the liquid, and the ionic liquid array remains stable for several days.36 (36) Ranke, J.; Stolte, S.; Stormann, R.; Arning, J.; Jastorff, B. Chem. Rev. 2007, 107, 2183–2206.

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Figure 4. Fluorescence images after photoimmobilization of proteins on the patterned benzophenone-BDD surface: (A) GFP (the surrounding area is H-BDD), (B) GFP (the surrounding area is PEG-BDD), and (C) specific binding of biotin-conjugated Qdots 605 to streptavidin immobilized on PEG-BDD patterned surface. Parameters for photochemical linking: t=30 min, λ=365 nm, and P=5 mW cm-2.

3.2. Chemical Functionalization of OH-BDD Patterns with Benzoylbenzoic Acid. The oxidized regions of the patterned BDD have a surface composition consisting of ether (C-O-C), carbonyl (CdO), and hydroxyl (OH) groups. The presence of OH groups after UV/ozone treatment of H-BDD has been evidenced by the successful coupling of the oxidized BDD surface with an organosilane35 or molecules bearing an acid group through an esterification reaction.33,37 The reactivity of the hydroxyl groups toward benzoylbenzoic acid, activated using N,N-dicyclohexylcarbodiimide (DCC), is used here to modify OH-BDD with benzophenone units (Figure 1c).33 As reported previously, the benzophenone-terminated BDD surface shows a water contact angle of 56° with a surface coverage of Γ=(1.5 þ 0.3)1014 molecules cm-2.33 The ester-type linkage was stable in the pH range 3-10, while outside the linkage was it labile due to protonation of the ester linkage. As biomoleular interactions are mainly performed at pH 6-9, this is not a further limitation. In our recent communication, we discussed the successful photochemical linking of oligonucleotide strands to the benzophenonemodified BDD. The underlying mechanism is outlined in Figure 1d. Benzophenone and its derivatives absorb a photon at around 350 nm, resulting in the promotion of one electron from a nonbonding sp2-like n-orbital on oxygen to an antibonding π*orbital of the carbonyl group. The actual electron-deficient oxygen n-orbital becomes electrophilic and therefore interacts with weak C-H σ-bonds, resulting in a hydrogen abstraction to complete the half-filled n-orbital. When amines or similar heteroatoms are in the vicinity of the excited carbonyl, an

electron-transfer step may occur, followed by proton abstraction from an adjacent group. While perhaps the most effective H-donors include backbone C-H bonds in amino acids, methylene groups of peptides and proteins are also good candidates for providing abstractable hydrogens. 3.3. Photochemical Grafting of Green Fluorescence Protein (GFP) on the Patterned BDD Surface. The protein selected for photoimmobilizaton was GFP, as it offers the possibility of an easy and direct visualization of the protein attachment. GFP contains a chromophore that absorbs blue light (λmax between 395 and 470 nm) and re-emits it as green fluorescence between 509 and 540 nm.38 Figure 4A shows the fluorescence image of a photolithographically patterned hydrogenated BDD surface (Figure 1a) consisting of benzophenone-terminated and hydrogenated regions (5050 μm2) after photoimmobilization of GFP molecules. While the photochemical linking of GFP is evidenced by the larger fluorescence intensity in the areas where benzophenone was present, significant nonspecific interaction between GFP and the surrounding H-BDD was observed. Consequently, the patterned regions are poorly defined. Nonspecific binding could be successfully diminished by appropriate surface functionalization. Protein repelling PEG films were used to minimize nonspecific interactions following the reaction scheme outlined in Figure 1b. After oxidation of H-BDD, the hydroxyl groups on BDD were used for the covalent grafting of a trimethoxysilane based PEG. UV/ozone treatment of the PEGterminated BDD interface through an optical mask allows a local decomposition of the PEG chains and recovery of the initial

(37) Das, M. R.; Wang, M.; Szunerits, S.; Gengembre, L.; Boukherroub, R. Chem. Commun. 2009, 2753.

(38) Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C. Science 1994, 263, 802.

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Figure 5. Fluorescence images after photoimmobilization of HApeptide through a photomask on benzophenone-terminated BDD surface. Parameters for photochemical linking: t=30 min, λ=365 nm, and P=5 mW cm-2.

oxidized surface (patterned BDD surface consisting of PEG/ oxide regions). The newly generated oxide patterns were transformed into a benzophenone termination through an esterification reaction between the surface hydroxyl groups and benzoylbenzoic acid (PEG/BP patterns). Photoimmobilization of GFP at λ=365 nm for 30 min with a light intensity of 5 mW cm-2 resulted in largely improved fluorescence images (Figure 4b). While the role of the surrounding PEG chains on the success of specific photolinking is clear, the influence of the photochemical irradiation on the biological activity of GFP is not evident from this experiment. 3.4. Photochemical Grafting of Streptavidin and Detection of Streptavidin/Biotin Interaction. To illustrate the preservation of the protein biological activity, streptavidin was photoimmobilized on the patterned benzophenone-terminated BDD modified with PEG chains (PEG/BP). The biological activity can be assessed by incubating the streptavidin-modified BDD interfaces with biotin-conjugated quantum dots (Qdots 605). Figure 4C shows the fluorescence image of the incubated interface. The strong fluorescence signal indicates that biotin is linked to the photochemically immobilized streptavidin and the biological function of streptavidin was preserved during the photochemical activation

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process. In a control experiment, oxidized BDD interfaces were photochemically irradiated in the presence of streptavidin in a similar way as on the benzophenone-terminated BDD surface. No fluorescence signal could be detected in this case, underlying the role of the benzophenone photolinker. 3.5. Photochemical Grafting of Influenza Hemagglutinine Peptides (HA-Peptides). A final test for the compatibility and generality of the photoimmobilization strategy on benzophenone-terminated BDD interfaces was performed using influenza hemagglutinine peptides (HA-peptides).32 The peptides were locally photografted on benzophenone-modified BDD interfaces using a lithographic photomask. The biological activity was confirmed by incubating the HA-peptide-modified interfaces with fluorescence labeled antibody anti-HA peptides. Figure 5 shows the fluorescence image of the incubated interface. The fluorescence signal coming from the areas where HA-peptides were photochemically linked is stronger than that from the unreacted benzophenone-terminated areas. The determined fluorescence intensity compares well to fluorescence intensities observed for the same reaction on aminated diamond surfaces.32 This indicates that the grafting density of the biomolecules using the photochemical approach is similar.

4. Conclusion The photoimmobilization of two proteins, green fluorescence protein and streptavidin, on patterned benzophenone-terminated BDD was demonstrated. Nonspecific adsorption could be diminished by modifying oxidized BDD interfaces with protein repelling poly(ethylene glycol) units. The preservation of the protein biological activity upon its photoimmobilization was demonstrated by further interaction of streptavidin with biotin-conjugated quantum dots. Furthermore, influenza hemagglutinine peptides were photochemically immobilized directly onto benzophenon-modified BDD through a photomask. The fluorescence intensity measured was comparable to fluorescence intensities observed for the same reaction on amine-terminated diamond. This underlines one of the advantages of benzophene-modified interfaces being the possibility of acting on microscopic selected areas via a litographic approach. Acknowledgment. The Agence Nationale de la Recherche (ANR), the Centre National de la Recherche Scientifique (CNRS), and the Nord-Pas-de Calais region are gratefully acknowledged for financial support. Naoto Shirahata is acknowledged for help in KFM measurements.

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