Peptide Immobilization on Amine-Terminated Boron-Doped Diamond

Nov 27, 2006 - CNRS-INPG-UJF, 1130 rue de la piscine, BP 75, 38402 St. Martin d'He`res Cedex, France, Laboratoire des Proce´de´s d'Elaboration de ...
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Langmuir 2007, 23, 4494-4497

Peptide Immobilization on Amine-Terminated Boron-Doped Diamond Surfaces Yannick Coffinier,† Sabine Szunerits,‡ Charafeddine Jama,§ Re´mi Desmet,| Oleg Melnyk,| Bernadette Marcus,‡ Le´on Gengembre,+ Edmond Payen,+ Didier Delabouglise,‡ and Rabah Boukherroub*,† Institut de Recherche Interdisciplinaire (IRI, FRE2963) and Institut d’Electronique, de Microe´ lectronique et de Nanotechnologie (IEMN), Cite´ Scientifique, AVenue Poincare´ - BP. 60069, 59652 VilleneuVe d’Ascq, France, Laboratoire d’Electrochimie et de Physicochimie des Mate´ riaux et des Interfaces (LEPMI), CNRS-INPG-UJF, 1130 rue de la piscine, BP 75, 38402 St. Martin d’He` res Cedex, France, Laboratoire des Proce´ de´ s d’Elaboration de ReVeˆ tements Fonctionnels (PERF) UMR 8008, Baˆ timent C7, Cite´ Scientifique, AVenue Mendeleı¨eV, BP 108, 59652 VilleneuVe d’Ascq, France, Institut de Biologie de Lille, UMR CNRS-8525, 1 rue du Pr. Calmette, 59021 Lille, France, and Unite´ de Catalyse et de Chimie du Solide, UCCS UMR CNRS-8181, UniVersite´ des Sciences et Technologies de Lille, Baˆ t. C3, 59655 VilleneuVe d’Ascq Cedex, France ReceiVed NoVember 27, 2006. In Final Form: January 29, 2007 This paper reports on the formation and characterization of semicarbazide termination on aminated boron-doped diamond (BDD) surfaces, and further preparation of peptide microarray through site-specific R-oxo semicarbazone ligation. Hydrogen-terminated BDD electrodes were first aminated using NH3 plasma treatment and then reacted with triphosgene and Fmoc-protected hydrazine to yield a protected semicarbazide termination. Subsequent deprotection and chemical reaction with glyoxylyl peptides led to the covalent immobilization of the peptides on the surface through site-specific ligation. The resulting surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and fluorescence measurements.

Introduction There is a continuous need and search to interface biomolecules and solid substrates to realize and develop integrated biosensors for real-time monitoring of biomolecular interactions. The possibility of using well-established microfabrication methods for the integration of diverse chemical and biochemical functionalities into microelectronic platforms for applications in genomics and proteomics1,2 or for the construction of libraries of complex molecules2-4 has naturally led to research efforts aimed at immobilizing biomolecules on semiconductor surfaces. Immobilization of biomolecules on semiconductor surfaces requires important criteria such as high stability of the chemically or biologically modified surfaces in physiological media, high affinity for specific binding of biomolecules of interest and the reproducibility and effectiveness of the method. Because of its technological importance, crystalline silicon has extensively been investigated for potential applications in bioelectronics. There have been significant advances in the chemical functionalization of silicon surfaces, and several strategies have been developed to incorporate biomolecules on * To whom correspondence should be addressed. E-mail: rabah. [email protected]. Tel: +33 3 20 19 79 87. Fax: +33 3 20 19 78 84. † IRI and IEMN. ‡ LEPMI. § PERF. | Institut de Biologie de Lille. + Unite ´ de Catalyse et de Chimie du Solide. (1) Wildt, R. M. T. d.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989. (2) Macbeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (3) Crews, C. M.; Spliftgerber, U. Trends Biochem. Sci. 1999, 24, 317. (4) Bishop, A.; Buzko, O.; Heyeck-Dumas, S.; Jung, I.; Kraybill, B.; Liu, Y.; Shah, K.; Ulrich, S.; Witucki, L.; Yang, F.; Zhang, C.; Shokat, K. M. Annu. ReV. Biophys. Biomol. Struct. 2000, 29, 577.

the surface.5-7 However, there are still technical limitations to overcome before reaching robust and reliable devices-based on silicon. A major drawback of silicon is the lack of chemical stability of the semiconductor/organic interface, which introduces surface active electronic defects. Next to silicon, silicon nitride displays outstanding properties such as mechanical hardness and chemical inertness.8,9 However, the material is highly resistive and thus not suitable in bioelectronics. Recently, boron-doped diamond (BDD) has gained remarkable interest for various applications due to its high chemical stability, good electrical conductivity, large potential window in aqueous electrolytes (about -1.35 to 2.3 V/ NHE), and biocompatibility.10,11 Interfacing diamond surfaces with biological molecules has already been examined in the literature. For example, DNA molecules have successfully been immobilized to a variety of diamond substrates using different chemical strategies.12-18 Ushizawa et al.12 have (5) Boukherroub, R. Curr. Opin. Solid State Mater. Sci. 2005, 9, 66. (6) Buriak, J. M. Chem. ReV. 2002, 102, 1272. (7) Wayner, D. D. M.; Wolkow, R. A. J. Chem. Soc., Perkin Trans. 2 2002, 23. (8) Arafat, A.; Schroe¨n, K.; Smet, L. C. P. M.; Sudho¨lter, E. J. R.; Zuilhof, H. J. Am. Chem. Soc. 2004, 126, 8600. (9) Cattaruzza, F.; Cricenti, A.; Flamini, A.; Girasole, M.; Longo, G.; Mezzi, A.; Prosperi, T. J. Mater. Chem. 2004, 14, 1461. (10) 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. (11) Tryk, D. A.; Tsunozaki, K.; Rao, T. N.; Fujishima, A. Diamond Relat. Mater. 2001, 10, 1804. (12) Ushizawa, K.; Sato, Y.; Mitsumori, T.; Machinami, T.; Ueda, T.; Ando, T. Chem. Phys. Lett. 2002, 351, 105. (13) Takahashi, K.; Tanga, M.; Takai, O.; Okamura, H. Diamond Relat. Mater. 2003, 12, 572. (14) Yang, W.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N.; Smith, L. M.; Hamers, R. J. Nature Mater. 2002, 1, 253. (15) Strother, T.; Knickerbocker, T.; Russell, J. N.; Butler, J.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 968. (16) Hamers, R. J.; Butler, J. E.; Lassetera, T.; Nicholsa, B. M.; Russell, J. N.; Tsea, K.-Y.; Yanga, W. Diamond Relat. Mater. 2005, 14, 661.

10.1021/la063440y CCC: $37.00 © 2007 American Chemical Society Published on Web 03/17/2007

Amine-Terminated Boron-Doped Diamond Surfaces

studied DNA covalent attachment to oxidized diamond powder through ester linkage using diffuse reflectance infrared spectroscopy. The excellent properties of CVD diamond chips for the preservation of valuable gene samples were demonstrated through covalent binding of DNA molecules to CVD diamond chip in a multistep process.13 Hamers et al.14-16 have used a two-step chemical approach to incorporate amine groups on hydrogen-terminated diamond surfaces and the subsequent linkage of DNA molecules to the surface. The authors showed that DNA-modified diamond surfaces are chemically more stable than DNA-modified silicon surfaces under the conditions of hybridization and denaturation. More recently, Zhang et al.17 reported a one-step method for direct amination of polycrystalline diamond and subsequent DNA micropatterning on the aminated surfaces. Besides DNA linking to diamond surfaces, there are only few reports in the literature dealing with protein-diamond interfaces. Hamers et al.19 used a photochemical approach to covalently attach ethylene glycol oligomers to hydrogenterminated p-type diamond films and investigated the ability of the oligo(ethylene glycol)-modified surfaces to resist biofouling. Garrido et al.20 used the same photochemical method to covalently attach proteins on n-type hydrogen-terminated nanocrystalline diamond films and showed that the immobilized proteins retain their functionality. Recently, we have described a simple method for direct amination of hydrogen-terminated BDD electrodes using NH3 plasma and showed that the aminated surface exhibits interesting electrochemical characteristics compared to oxidized BDD surface.21 Here, we explore the chemical reactivity of the aminated BDD surfaces for the preparation of peptide microarray through site-specific R-oxo semicarbazone ligation. The coupling chemistry combines the high reactivity of the glyoxylyl peptides toward semicarbazide groups with mild experimental conditions and chemoselectivity. Experimental Section Peptide Synthesis. The synthesis of glyoxylyl peptides 1 and 2 was performed on a 0.2 mmol scale using the Fmoc/tert-butyl strategy on a Novasyn TG resin (Novabiochem) modified by an isopropylidene tartrate-based linker. The preparation of peptide amide 3 was performed on a 0.2 mmol scale using the 9-fluoromethoxy-carbonyl (Fmoc)/ tert-butyl strategy on a Rink amide resin ([5-(4-Fmocaminomethyl-3,5-dimethoxyphenoxy)valeric acid]-polyethyleneglycol-polystyrene) (Fmoc-PAL-PEG-PS) resin).22 Preparation of H-Terminated and Aminated BDD Surfaces. Polycrystalline diamond layers were synthesized on a silicon high purity p-type wafer by microwave plasma-enhanced chemical vapor deposition (PECVD) in a conventional reactor.23 The dopant source was boron oxide set in a Pt crucible placed on the substrate holder near the silicon substrate. The dopant concentration in the diamond layers, as estimated from Raman spectroscopy measurements is in the range 1019 to 1020 B cm-3. The film resistivity was e0.1 Ω. cm as measured with a four-point probe. Amine-terminated surfaces were obtained by NH3 plasma treatment of hydrogen-terminated BDD surfaces.21 (17) Zhang, G.-J.; Song, K.-S.; Nakamura, Y.; Ueno, T.; Funatsu, T.; Ohdomari, I.; Kawarada, H. Langmuir 2006, 22, 3728. (18) Kru¨ger, A. Angew. Chem., Int. Ed. 2006, 45, 6426. (19) Lasseter, T. L.; Clare, B. H.; Abbott, N. L.; Hamers, R. J. J. Am. Chem. Soc. 2004, 126, 10220. (20) Hartl, A.; Schmich, E.; Garrido, J. A.; Hernanod, J.; Catharino, S. C. R.; Walter, S.; Feulber, P.; Kromka, A.; Steinmuller, D.; Stutzmann, M. Nature Mat. 2004, 1-7. (21) Szunerits, S.; Jama, C.; Coffinier, Y.; Marcus, B.; Delabouglise, D.; Boukherroub, R. Electrochem. Commun. 2006, 8, 1185. (22) Coffinier, Y.; Olivier, C.; Perzyna, A.; Grandidier, B.; Wallart, X.; Durand, J.-O.; Melnyk, O.; Stie´venard, D. Langmuir 2005, 21, 1489. (23) Mermoux, M.; Fayette, L.; Marcus, B.; Rosman, N.; Abello, L.; Lucazeau, G. Diamond Relat. Mater. 1995, 4, 745.

Langmuir, Vol. 23, No. 8, 2007 4495 Preparation of Semicarbazide-Terminated Diamond Surfaces. The NH2-terminated surfaces were treated with 0.1 M triphosgene/ 0.8 M diisopropylethylamine solution in 1,2-dichloroethane for 2 h under sonication followed with a 22 mM Fmoc-NH2NH2 solution in DMF containing 1% ethanol for 2 h under sonication. Finally, removal of the Fmoc groups was performed with 0.2% piperidine and 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF (30 min). The substrates were washed with DMF, water (two times), and methanol and dried under reduced pressure. The semicarbazide functional group corresponds to -NH-C(dO) -NH-NH2. Peptide Microarray. Peptides 3×-FLAG-NH2 (3xDYKDDDDK-NH2: 3×Asp-Tyr-Lys-Asp-Asp-Asp-Asp-LysNH2), 3×-FLAG-COCHO (3xDYKDDDDK-COCHO: 3xAspTyr-Lys-Asp-Asp-Asp-Asp-Lys-COCHO) and HA-COCHO (3×YPYDVPDYA-COCHO: H-Tyr-Pro-Tyr-Asp-ValPro-Asp-Tyr-Ala-COCHO) (10-4 M), dissolved in 0.1 M, pH 5.5, sodium acetate buffer were printed five times each as nanoliter drops on the diamond substrate using a 4 piezo tips Perkin-Elmer BioChip Arrayer I. The printed wafers were incubated at 37 °C in a humid chamber (60% relative humidity) overnight then soaked in a saturated solution (0.1% Tween 20 (by volume), 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS, 0.01 M, pH 7.2)) for 60 min under sonication. A washing solution containing 0.05% Tween 20 (by volume) in PBS (0.01 M, pH 7.2) was used three times to remove excesses of peptides and BSA. Incubation was performed using 100 µL of 10-2 mg/mL labeled (tetramethylrhodamine) antibody anti-peptide FLAG diluted in the saturated solution. The slides were incubated 100 min at 37 °C in a humid chamber, washed four times with the washing solution, deionized water, and ethanol, and then dried in air on the bench. Surface Characterization. X-ray photoelectron spectroscopy (XPS) measurements were performed with an ESCALAB 220 XL spectrometer from vacuum Generators. A monochromatic Al KR X-ray source (1486.6 eV) was operated in the CAE (constant analyzer energy) mode (CAE ) 100 eV for survey spectra and CAE ) 40 eV for high-resolution spectra), using the electromagnetic lens mode. No flood gun source was needed due to conducting character of the substrates. The angle between the incident X-rays and the analyzer is 58°. The detection angle of the photoelectrons is 90°, as referenced to the sample surface. Array imaging was performed using the Cy3 channel of an Affymetrix 418 array scanner at a resolution of 10 µm, and the fluorescence was quantified using “ScanArray Express” software (Perkin-Elmer).

Results and Discussion The site-specific immobilization of peptides on semicarbazideterminated BDD surfaces proceeds in a multistep process according to Scheme 1.24-27 First, amine termination was obtained by NH3 plasma treatment of the hydrogen-terminated BDD surface at room temperature.21 The NH2-terminated surface was next treated with a triphosgene/diisopropylethylamine solution followed with a Fmoc-NH-NH2 solution in DMF to yield a protected semicarbazide termination on the BDD surface. Finally, removal of the Fmoc groups was performed using a solution of piperidine and DBU in DMF to yield semicarbazide terminated BDD surface. The resulting surfaces are stable and can be stored for several weeks at room temperature in a dust-proof container without any apparent degradation. (24) Melnyk, O.; Duburcq, X.; Olivier, C.; Urbe`s, F.; Auriault, C.; GrasMasse, H. Bioconjugate Chem. 2002, 13, 713. (25) Olivier, C.; Hot, D.; Huot, L.; Olivier, N.; El-Mahdi, O.; Gouyette, C.; Huynh-Dinh, T.; Gras-Masse, H.; Lemoine, Y.; Melnyk, O. Bioconjugate Chem. 2003, 14, 430. (26) Duburcq, X.; Olivier, C.; Desmet, R.; Halasa, M.; Olivier, C.; Grandidier, B.; Heim, T.; Stie´venard, D.; Auriault, C.; Melnyk, O. Bioconjugate Chem. 2004, 15, 317. (27) Duburcq, X.; Olivier, C.; Malingue, F.; Desmet, R.; Bouzidi, A.; Zhou, F.; Auriault, C.; Gras-Masse, H.; Melnyk, O. Bioconjugate Chem. 2004, 15, 307.

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Coffinier et al.

Scheme 1. Schematic Illustration of the Formation of Semicarbazide Termination on Hydrogen-terminated BDD Surfaces and Subsequent Immobilization of Glyoxylyl Peptides through Semicarbazone Ligation

X-ray photoelectron spectroscopy (XPS) was used to analyze the surface composition of the semicarbazide-terminated BDD surface. XPS survey (not shown) exhibited peaks at 285, 399, and 532.6 eV due to C1s, N1s and O1s, respectively. The highresolution XPS spectrum of C 1s peak of the semicarbazideterminated BDD surface displays signals due to C1s from the bulk and from the surface C-N and C-O features at 285, 286.5, and 287.5 eV, respectively (Figure 1). However at this stage, a contribution from C-O features resulting from partial surface oxidation of the hydrogenated diamond during NH3 plasma treatment is not excluded.21 The N1s XPS spectrum shows a significant and unsymmetrical peak centered at 399 eV, which is in agreement with the semicarbazide chemical composition (Figure 2). The broadening of the N 1s peak may also arise from the presence of CdN and C-N-C formed during surface amination.21 Furthermore, the XPS spectroscopy was used to evaluate the conversion yield of the amine to semicarbazide

Figure 1. High-resolution XPS spectrum of C1s of semicarbazideterminated BDD surface.

Figure 2. High-resolution XPS spectrum of N1s of semicarbazideterminated BDD surface.

Figure 3. Fluorescence images of peptide microarrays made from semicarbazide-modified diamond surfaces after incubation in the presence of tetramethylrhodamine labeled antibody anti-peptide FLAG (10-2 mg/mL). The peptides were spotted five times at a concentration of 10-4 M.

termination. In theory, the transformation of amine to semicarbazide groups adds two nitrogen and one carbon atoms. The N/C theoretical ratio is 1 and 3 for amine and semicarbazide-terminated surfaces, respectively. From the XPS results, the N/C experimental ratio was calculated and found to be 0.0252 for the amineterminated diamond surface. Based on the result and assuming all the amine groups are converted to semicarbazide, the N/C experimental ratio should be 0.0756. However, XPS analysis gave a N/C ratio of 0.0274 for the semicarbazide-terminated BDD surface, corresponding to a conversion yield of 36%. The low conversion may be due to the lack of chemical reactivity of the NdC and C-N-C groups formed during surface amination. The increase of the N/C ratio provides a good indication of the presence of semicarbazide functional groups on the surface. The successful formation of semicarbazide-terminated BDD surfaces was further demonstrated by the chemoselective reaction with glyoxylyl peptides through the formation of an R-oxo semicarbazone linkage. Two different glyoxylyl peptides (peptide 1: 3×FLAG-COCHO and peptide 2: 3×HA-COCHO) and a peptide amide (peptide 3: 3×FLAG-NH2) were printed in a microarray format using a 4 piezo tips Perkin-Elmer BioChip Arrayer I. The fluorescence images obtained using the Cy3 channel of an Affymetrix 418 array scanner, after incubation in the presence of tetramethylrhodamine labeled antibody antipeptide FLAG are shown in Figure 3. The fluorescence intensity obtained from the spots printed with peptide 1 is much higher than that observed from peptide 3, under the same experimental conditions. The result is in agreement with specific covalent ligation of peptide 1 with the semicarbazide surface and physisorption of peptide 3 on the surface. Based on the pKa

Amine-Terminated Boron-Doped Diamond Surfaces

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Figure 4. Fluorescence intensity of peptide microarrays (median value after background correction) prepared from semicarbazide-modified diamond surfaces and incubated in the presence of antibody anti-peptide FLAG labeled with tetramethylrhodamine (10-2 mg/mL). The ratio of fluorescence intensity between peptides 1 and 3 is 4.

values of semicarbazide (3.65 at 25 °C) and alkylamines (10.65 for methylamine at 25 °C),28 the semicarbazide surface is expected to be uncharged at physiological pH whereas the peptide 3 is protonated. This favors electrostatic interactions between the semicarbazide-terminated surface and peptide 3. A low fluorescence signal was observed when the semicarbazide-terminated BDD surface was reacted with the control glyoxylyl peptide 2 (without a FLAG fragment) and incubated in the presence of antibody anti-peptide FLAG labeled with tetramethylrhodamine. This is consistent with a high specific interaction of the FLAG fragment and very low nonspecific adsorption of the antibody on the surface. Water was also spotted on the semicarbazideterminated BDD surface after each peptide deposition. A fluorescence signal close to background intensity was observed in this case, which is in accordance with the absence of peptide carryover during microarray preparation. Figure 4 displays the relative fluorescence intensity for the three different peptides after incubation in the presence of tetramethylrhodamine labeled antibody anti-peptide FLAG and copious rinsing with the Tween solution. The ratio of fluorescence intensity between peptides 1 and 3 is 4. The result is comparable to the data obtained with semicarbazide-functionalized Si(111) surfaces,22 but lower than that obtained for semicarbazide(28) Patai, S., Ed. InThe Chemistry of the Hydrazo, Azo, and Azoxy Groups; JohnWiley & Sons: New York, 1975; p 161.

terminated glass slides.25 This difference may be due to the presence of CdN and C-N-C on the surface that are not converted to semicarbazide groups. However, it is hard to draw any conclusions at this stage without more experimental data.

Conclusions In conclusion, semicarbazide-functionalized layers can be readily prepared on NH2-terminated BDD surfaces. The semicarbazide layer formation was evidenced by site-specific immobilization of glyoxylyl peptides through R-oxo semicarbazone linkage, and fluorescence experiments. The R-oxo semicarbazone native ligation applies also for other biomolecules and ligands, and provides a straightforward method for the preparation of new bioactive diamond surfaces and complex structures. The covalent immobilization of biomolecules on BDD surfaces combined with the semiconducting electrical properties of diamond opens opportunities for potential applications in bioelectronics. 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. LA063440Y