Facile Derivatization of Glassy Carbon Surfaces by N

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Langmuir 1998, 14, 2368-2371

Facile Derivatization of Glassy Carbon Surfaces by N-Hydroxysuccinimide Esters in View of Attaching Biomolecules Agne`s Anne, Bernard Blanc, Jacques Moiroux,* and Jean Michel Save´ant* Laboratoire d’Electrochimie Mole´ culaire, Unite´ Mixte de Recherche Universite´ , CNRS No. 7591, Universite´ Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 05, France Received October 28, 1997. In Final Form: February 9, 1998 The reaction of N-hydroxysuccinimide (NHS) esters with freshly polished glassy carbon surfaces offers a facile and versatile method of derivatization. Surface concentrations larger than 10-10 mol/cm2 can thus be easily achieved. They can be further increased when polishing is carried out in the presence of ammonia, which also improves their reproducibility. It is shown that the derivatization results from the formation of a covalent peptide linkage by reaction of the NHS ester with superficial amino groups on the glassy carbon surface. The peptide linkage is remarkably stable in time and can only be hydrolyzed in very strong basic media. 9-fluorenylmethoxycarbonyl chloride protection, followed by preparation of the NHS ester, by surface derivatization and by mild deprotection allows the grafting of a molecule that contains an amino group located remotely from the electrode surface, thus opening a route to the attachment of a large variety of biomolecules, for which NHS esters are available, in a position where their degradation should be avoided or minimized.

Derivatization of carbon surfaces, with particular attention to the attachment of biomolecules, is an important goal insofar as the carbon substrate is a rather inert material. For example, if the surface is to be used as an electrode, quite positive potentials may be reached on carbon. Several techniques have been described for chemically modifying carbon surfaces.1 One of these consists of the generation of superficial carboxylic groups through corrosive oxidation of the glassy carbon surface.1-5 Biomolecules may then be coupled by reaction of their amino groups with the carboxylic groups using an activating reagent, such as a carbodiimide. Conversely, the surface may be derivatized with amino groups which may then be coupled with carboxylic acid functions.3,6,7 In both cases, chemical and/or electrochemical or radio-frequency oxygen plasma oxidative pretreatments of the electrodes are required and result in a dramatic roughening of the surface.8 Electrochemical derivatization does not entail such a roughening of the electrode surface. Cathodic derivatization of carbon surfaces by phenyl radicals resulting from the reduction of diazonium salts has been described.9 On the anodic side the oxidation of amines10 or of carboxylates11 (Kolbe reaction) also leads to covalent attachment. The presence of appropriate substituents

on the protein to be attached, particularly amine substituents, allows the successive binding of biomolecules. This procedure has been used to immobilize glucose oxidase on carbon electrodes even if the proximity of the enzyme to the electrode surface results in a significant degradation of its activity.9c Cases are rare in which mechanical abrasion of the carbon surface suffices to activate the surface toward direct binding of molecules. This method has been described for the binding of vinylic derivatives.12 We have now found that N-hydroxysuccinimide (NHS) esters bind spontaneously on polished glassy carbon surfaces, giving rise to a strong covalent attachment. Since many NHS esters of biomolecules are currently available or can be easily prepared, this strategy could be used to bind directly these biomolecules to the carbon surface. However, it would entail a risk of degradation as already experienced in the case of glucose oxidase.9c We therefore put forward a two-step strategy aiming at anchoring the molecule of interest at a larger distance from the surface. In a first step, an NHS ester of a molecule containing a protected amine group in the ω-position is reacted with the electrode. The amine group is then deprotected and may then be used to bind the molecule of interest.

(1) (a) For reviews see refs 1b and 1c. (b) Murray, R. W. Acc. Chem. Res. 1980, 13, 151. (c) Murray, R. W. Chemically Modified Electrodes. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 263-288. (2) (a) Ianniello, R. M.; Lindsay, T. J.; Yacynych, A. M. Anal. Chem. 1982, 54, 1980. (b) Ianniello, R. M.; Wieck, H. J.; Yacynych, A. M. Anal. Chem. 1983, 55, 2067. (3) Mazur, S.; Matusinovic, T.; Cammann, C. J. Am. Chem. Soc. 1977, 99, 3888. (4) (a) Hummers, W. S.; Offeman, R. E. J. Am. Chem. Soc. 1958, 80, 1339. (b) Watkins, B. F.; Belhing, J. R.; Kariv, E.; Miller, L. L. J. Am. Chem. Soc. 1975, 97, 3549. (5) (a) Bourdillon, C.; Bourgeois, J. P.; Thomas, D. J. Am. Chem. Soc. 1980, 102, 4231. (b) Laval, J. M.; Bourdillon, C.; Moiroux, J. J. Am. Chem. Soc. 1984, 106, 4701. (6) Yacynych, A. M.; Kuwana, T. Anal. Chem. 1978, 50, 640. (7) Oyama, N.; Brown, A. P.; Anson, F. C. J. Electroanal. Chem. 1978, 87, 435. (8) (a) Wang, J.; Martinez, T.; Yaniv, D. R.; McCormick, L. D. J. Electroanal. Chem. 1990, 278, 379. (b) Hoffman, W. P.; Curley, W. C.; Owens, T. W.; Phan, H. T. J. Mater. Sci. 1991, 26, 4545.

Results and Discussion Covalent Derivatization of the Glassy Carbon Electrode by Reaction with an NHS Ester. The NHS esters 1 and 2 which contain electroactive phthalimide (9) (a) Delamar, M.; Hitmi, R.; Pinson, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1992, 114, 5883. (b) Hitmi, R.; Pinson, J.; Save´ant, J.-M. Fr. Patent 91 011172. (c) Bourdillon, C.; Demaille, C.; Hitmi, R.; Moiroux, J.; Pinson, J. J. Electroanal. Chem. 1992, 336, 113. (d) Liu, Y.-C.; McCreery, R. L. J. Am. Chem. Soc. 1995, 117, 11254. (e) Allongue, P.; Delamar, M.; Desbat, B.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1997, 119, 201. (f) Fr. Patent 97 03769. (g) Chen, P.; McCreery, R. L. Anal. Chem. 1996, 68, 3958. (10) Barbier, B.; Pinson, J.; Desarmot, G. J. Electrochem. Soc. 1990, 137, 1757. (11) (a) Andrieux, C. P.; Gonzales, F.; Save´ant, J.-M. J. Am. Chem. Soc. 1997, 119, 4292. (b) Fr. Patent 97 02738. (12) Nowak, A.; Schultz, F. A.; Uman˜a, M.; Abrun˜a, H.; Murray, R. W. J. Electroanal. Chem. 1978, 94, 219.

S0743-7463(97)01171-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/03/1998

Facile Derivation of Glassy Carbon Surfaces

Figure 1. Derivatization of a GC electrode with 1 after polishing in the presence of NH3. (a) Cyclic voltammogram corresponding to saturation, in DMF + 0.1 M n-Bu4 PF6. Scan rate: 10 V/s. (b) Surface concentration versus time of exposure. Scheme 1

and ferrocene groups, respectively, were allowed to react in chloroform with a GC electrode (Scheme 1) freshly polished in the presence of NH3. The electrode was then removed from the chloroform solution, rinsed and ultrasonicated in N,N-dimethylformamide (DMF), and finally transferred into an electrolytic cell containing a pure solution of n-Bu4NBF4 (0.1 M) in DMF. The reductive cyclic voltammogram thus obtained with the electrode allowed to react with 1 shows a reversible surface wave (Figure 1a). The fact that the cathodic peak is more negative than the anodic peak indicates that chargetransfer kinetics and/or ohmic drop interfere.13 The midpoint between the peaks, -1.40 V vs SCE, is a measure of the standard potential of the attached species. It is the same as the previously reported standard potential of the phthalimide/phthalimide anion radical couple in DMF solutions.14 The surface concentration can be obtained by integration of the cyclic voltammogram. At saturation it reaches 4.7

Langmuir, Vol. 14, No. 9, 1998 2369

× 10-10 mol/cm2. The variation of the coverage with the time of exposure is represented in Figure 1b. As expected, the rate of fixation of the phthalimide derivative increases upon raising the concentration of 1 in the reaction mixture. The surface coverage is very stable. It is affected neither by ultrasonication in DMF nor by storage in the same solvent for 8 days. The only way to clean a derivatized electrode is to polish it. These observations strongly suggest that the binding of the phthalimide group to the surface is covalent. The surface derivatization may also be carried out without the presence of NH3 in the polishing mixture. The surface concentrations are then significantly smaller and less reproducible, ranging from (0.4 to 2) × 10-10 mol/ cm2 at saturation. Since NHS esters are potential carbocations, it may be envisaged that the reaction involves the attack of the phenyl rings of graphite in a Friedel-Crafts manner. This possibility seems, however, unlikely since all our attempts to derivatize highly oriented pyrolytic graphite surfaces proved unsuccessful. The fact that the presence of NH3 in the polishing mixture increases the surface concentration suggests that the derivatization involves the formation of a superficial peptide link resulting from the coupling of the NHS ester with superficial amine groups created by the reaction with NH3 of dangling bonds formed by abrasion. This was confirmed by experiments where the NHS ester 2, instead of 1, was attached to the surface according to the same procedure. The cyclic voltammogram of a GC electrode derivatized with 2 exhibits a standard potential of 0.69 V vs SCE less positive than the solution standard potential, 0.82 V vs SCE of 2, which contains an ester functionality, as expected from the lesser electron-withdrawing character of the amide substituent as compared to the ester substituent. The same standard potential is obtained when the grafting of 2 is carried out in the absence of NH3, indicating that attachment involves superficial amino groups, in this case too rather than hydroxyl groups that would lead to an ester linkage. This conclusion is confirmed by examining how the bond between the surface and the attached group is hydrolyzed. Both the amide and ester groups may be hydrolyzed in basic media although not under the same conditions. As will be shown later on, the deprotection of an attached amine can be achieved by dipping the derivatized electrode in a piperidine/DMF (20/80) mixture during 72 h, without alteration of the surface concentration of electroactive species. In such a basic medium and after such a duration it is likely that the hydrolysis or aminolysis of an ester should proceed appreciably. Efficient hydrolysis of the Fc-CO-X-derivatized electrode in the case of 2 (Scheme 1) was effective only after reaction with 0.1 M N(n-Bu)4OH in methanol/ DMF (10/90), a medium in which DMF itself gets slowly hydrolyzed, i.e., a medium well suited for the hydrolysis of amides. The electrode was assayed in cyclic voltammetry to monitor the reaction of hydrolysis. The halfreaction time was 18 h. Since the glassy carbon used for preparing the electrode does not contain nitrogen, the formation of amine groups (13) Laviron, E. Voltammetric Methods for the Study of Adsorbed Species. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1982; Vol. 12, pp 53-157. (14) (a) Evans, J. F.; Blount, H. N. J. Am. Chem. Soc. 1978, 100, 4191. (b) Evans, J. F.; Blount, H. N. J. Electroanal. Chem. 1979, 102, 289. (c) Evans, J. F.; Blount, H. N. J. Phys. Chem. 1979, 83, 1970. (d) Svanholm, U.; Parker, V. D. Acta Chem. Scand. B 1973, 27, 1454. (e) Evans, J. F.; Blount, H. N. J. Org. Chem. 1976, 41, 516.

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Anne et al.

Scheme 2

Figure 2. (a) Cyclic voltammogram of a GC electrode derivatized with 2 after 40 h of exposure, in DMF + 0.1 M n-Bu4PF6 + 0.01 M HBF4. Scan rate: 10 V/s. Full line: before deprotection. Dashed line: during deprotection. Dotted line: after deprotection. (b) Surface coverage versus time of exposure.

on the surface, in the absence of NH3, must occur during polishing most probably by means of species contained in the polishing mixture. Protection, Attachment, and Deprotection of a Molecule Containing an Amine Functionality Located far from the Anchoring Point. The preceding experiments have shown the effectiveness of NHS esters as binding agents for derivatizing GC surfaces. We now wish to attach, by means of an NHS ester, a molecule containing an amine functionality at a remote position from the surface which could then serve to anchor a biomolecule. It is necessary that the amine be protected before the NHS ester is prepared. It is also useful to introduce a redox label in the system in order to easily characterize the reality and the extent of the derivatization. We selected the ferrocene group for this purpose. As a protecting agent, we chose 9-fluorenylmethoxycarbonyl chloride (FMOC) for it is known that deprotection of FMOC amine derivatives proceeds smoothly in moderately basic media15 compatible with the stability of the ferrocene moiety. The whole system is represented in Scheme 2. Exposure of a GC electrode, freshly polished in the presence of NH3, to a chloroform solution of 3 (Scheme 2), followed by rinsing and ultrasonication in DMF, and finally transfer into an electrolytic cell con(15) (a) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404. (b) Carpino, L. A. Acc. Chem. Res. 1987, 20, 401. (c) Myers, A. G.; Gleason, J. L.; Yoon, T.; Kung, D. W. J. Am. Chem. Soc. 1997, 119, 656.

taining a pure electrolyte DMF solution resulted in the voltammogram shown in Figure 2a (full line) where the reversible wave of the ferrocene/ferrocenium couple is clearly visible. Integration of the wave again allows the estimation of the surface coverage. The variation of the coverage with the time of exposure is shown in Figure 2b. Saturation is reached more slowly than with 1 and corresponds to a lesser number of molecules on the surface ((0.5 to 1.3) × 10-10 mol/cm2), presumably because the grafted group is larger than that with 1. The FMOC group was removed by immersion of the derivatized electrode in a 20:80 piperidine-DMF mixture. To follow the deprotection of the amino group, an acid, 0.01 M HBF4, was added to the solution where the cyclic voltammetric characterization was carried out for distinguishing the protected from the unprotected amine. In solution, in the presence of the acid, [N-(ferrocenylmethyl)6-amino]hexanoic acid and its FMOC-protected derivative, [N-(ferrocenylmethyl)-N-FMOC-6-amino]hexanoic acid, both exhibit a reversible cyclic voltammetric wave with standard potentials of 0.62 and 0.51 V vs SCE, respectively. This difference results from the fact that the unprotected (aminomethyl)ferrocene is protonated, whereas the FMOCprotected molecule is not. As is already observed with another (aminomethyl)ferrocene molecule, protonation renders the standard potential more positive.16 We thus expect deprotection to cause a positive shift of the surface wave of the grafted ferrocene group. This is indeed what is observed, as can be seen in Figure 2a where the voltammogram passes from the full to the dotted curve as (16) Bourdillon, C.; Demaille, C.; Moiroux, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1993, 115, 2.

Facile Derivation of Glassy Carbon Surfaces

deprotection proceeds. Full deprotection typically takes 280 min. Conclusions The reaction of NHS esters with glassy carbon surfaces is a facile and versatile method of derivatization. It simply requires the exposure of a surface freshly polished in the presence of NH3 to a solution of NHS ester in chloroform. Surface concentrations larger than 10-10 mol/cm2 can thus be easily achieved. They can be further increased when polishing is carried out in the presence of ammonia. The derivatization results probably from the formation of a covalent peptide linkage by reaction of the NHS ester with superficial amino groups on the glassy carbon surface. The peptide linkage is remarkably stable in time and can only be hydrolyzed in very strong basic media. FMOC protection, followed by preparation of the NHS ester, by surface derivatization, and by mild deprotection, allows the grafting of a molecule that contains an amino group located remotely from the electrode surface. Since the NHS esters of many biomolecules are available,17 a route is thus opened to the attachment of a large variety of these molecules in the position where their degradation is expected to be avoided or minimized. Experimental Section Chemicals. Besides, 1-3, all chemicals were from Aldrich and Fluka. They were of the highest purity available and used as received. The chromatographic stationary phases were from Merck. N-Succinimidyl 2-phthalimidoacetate (1) was prepared as described in the literature18 except that the carboxylic acid, N-hydroxysuccinimide, and carbodiimide concentrations were equal and were 10 times smaller than those in ref 18. N-Succinimidyl ferrocenecarboxylate (2) was prepared as described in the literature.19 [N-Succinimidyl [N-(Ferrocenylmethyl)-N-(9-fluorenylmethoxycarbonyl)-6-amino]hexanoate (3). [N-(Ferrocenylmethyl)-6amino]hexanoic acid was prepared in a first step. A procedure was sketched earlier for this preparation; however, the authors did not report precisely on the excess of NaBH4 which must be used for the reduction of the intermediary imine and on how they isolated and characterized the product.20 Ferrocenecarboxaldehyde (2.31 g, 10.8 mmol) in DMF (32 mL) was condensed with 6-aminohexanoic acid (12.8 mmol) in the presence of water (4 mL) and NaOH (1.6 mL of a 30% aqueous solution). The reaction mixture was heated at 80 °C for 3 h. Successive aliquots of NaBH4 (total amount ) 1.63 g, 43.2 mmol) were added carefully at room temperature, and the mixture was left overnight under stirring. The residue after vacuum evaporation was dissolved in water (40 mL) and 2 M H3PO4 was added slowly until pH 3. During the acidification a brown viscous liquid separated and was carefully eliminated. Ferrocenemethanol was also eliminated by two successive extractions with toluene (100 mL, each time). The remaining aqueous solution was stirred vigorously while its pH was increased up to 6 or 7 by the addition of solid Na2CO3. [N-(Ferrocenylmethyl)-6-amino]hexanoic acid precipitated then and was filtered. The yellow solid (53% yield) was characterized by 1H NMR; it decomposed above 210 °C. 1H NMR (17) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, CA, 1996. (18) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J. Am. Chem. Soc. 1964, 86, 1839. (19) Takenaka, S.; Uto, Y.; Kondo, H.; Ihara, T.; Takagi, M. Anal. Biochem. 1994, 218, 436. (20) Shoham, B.; Migron, Y.; Riklin, A.; Willner, I.; Tartakovsky, B. Biosens. Bioelectron. 1995, 10, 341.

Langmuir, Vol. 14, No. 9, 1998 2371 (2% ND4OD in D2O, 200 MHz): δ 1.43 (2H, m), 1.65 (4H, m), 2.28 (2H, t), 2.79 (2H, t), 3.76 (2H, s), 4.36 (7H, s), 4.43 (2H, s). The FMOC protection of the amine was carried out as follows. [N-(Ferrocenylmethyl)-6-amino]hexanoic acid (1.05 g, 3 mmol) and Na2CO3 (0.70 g, 6 mmol) were dissolved in water (40 mL) plus dioxane (40 mL). Once the homogeneity was obtained by stirring, the solution was cooled at 0 °C and a dioxane (20 mL) solution of 9-fluorenylmethoxycarbonyl chloride (0.78 g, 3 mmol) was added under stirring.14a,21 The reaction mixture was then held at 0 °C for 1 h and at room temperature for 2 h. After dilution with water (100 mL), the aqueous phase was purified by two successive extractions with pentane (100 mL, each time), acidified to pH 3 with 2 M H3PO4, and extracted again with ethyl acetate (100 mL). The organic phase was washed with NaClsaturated water and dried over MgSO4 before evaporation under vacuum. The weight of the remaining viscous orange liquid corresponded to 50% yield. A solid form could be obtained either after slow evaporation of a diethyl ether solution or after persistent drying over P2O5. The product can be purified by column chromatography over silica gel with CHCl3/CH3OH (95/ 5) eluent and characterized by its 1H NMR (in CDCl3) and mass spectra. The 1H NMR spectrum showed that there exist two rotamers, at similar concentrations in CDCl3. There is no free rotation around the axis of the N-COO bond. 1H NMR (1:1 rotamer ratio, CDCl3, 200 MHz): δ 1.2-1.8 (6H, m), 2.29 (1H, t), 2.32 (1H, t), 2.92 (1H, t), 3.12 (1H, t), 3.84 (1H, s), 3.89 (1H, s), 4.0-4.3 (10H, m), 4.52 (1H, d), 4.63 (1H, d), 7.3-7.5 (4H, m), 7.60 (1H, d), 7.70 (1H, d), 7.79 (1H, d), 7.89 (1H, d). The NHS ester was then produced by reaction of the preceding FMOCprotected amino acid (820 mg, 1.5 mmol) with equimolar amounts of N-hydroxysuccinimide and dicyclohexylcarbodiimide in 1,2dimethoxyethane (20 mL) at 0 °C for 20 h. Dicyclohexylurea precipitated and was eliminated by filtration. The residue obtained after solvent evaporation was recrystallized from 2-propanol and purified by column chromatography over silica gel with CHCl3/CH3OH (98/2) eluent. The yield was 70%. In thin-layer chromatography the ferrocene group could be revealed in the presence of the vapor of a 70% aqueous solution of nitric acid through the appearance of the blue-green color of the ferricenium cation. The NHS ester group was revealed by successive reactions with a methanolic solution of hydroxylamine and an aqueous acidic solution of FeCl3.22 1H NMR (1:1 rotamer ratio, CDCl3, 200 MHz): δ 1.2-1.8 (6H, m), 2.56 (1H, t), 2.60 (1H, t), 2.88 (4H, s), 2.95 (1H, t), 3.17 (1H, t), 3.90 (1H, s), 4.0-4.3 (10H, m), 4.59 (1H, d), 4.67 (1H, d), 7.3-7.5 (4H, m), 7.60 (1H, d), 7.70 (1H, d), 7.79 (1H, d), 7.89 (1H, d). Carbon Electrodes. The glassy carbon (Tokai Corp.) electrodes were prepared from 3-mm-diameter rods embedded in epoxy resin (Torr Seal, Varian). The geometric area of the disk electrode thus obtained was 0.07 cm2. They were polished with diamond powder suspensions, to which NH3 was added so as to reach a 1 M concentration, down to 1 µm, and rinsed ultrasonically for 2 min in dichloromethane. The epoxy resin surrounding did not affect our results; nonembedded rods gave similar covalent attachments. Clean highly oriented pyrolytic graphite (HOPG from Union Carbide, ZYB grade) surfaces were obtained by removing a few graphite layers with adhesive tape and were used as electrodes according to the procedure reported previously.9e Instrumentation. Chromatographic, spectroscopic, and electrochemical instruments were the same as those previously described.23 The cyclic voltammograms were recorded at 20 °C.

LA971171T (21) Myers, A. G.; Gleason, J. L.; Yoon, T.; Kung, D. W. J. Am. Chem. Soc. 1997, 119, 656. (22) Stahl, E. Thin Layer Chromatography, 2nd ed.; SpringerVerlag: Berlin, Germany, 1988. (23) Anne, A.; Moiroux, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1993, 115, 10224.