Dense Monolayers of Metal-Chelating Ligands Covalently Attached to

3362

Langmuir 2005, 21, 3362-3375

Dense Monolayers of Metal-Chelating Ligands Covalently Attached to Carbon Electrodes Electrochemically and Their Useful Application in Affinity Binding of Histidine-Tagged Proteins Ronald Blankespoor,† Benoıˆt Limoges,*,§ Bernd Scho¨llhorn,‡ Jean-Laurent Syssa-Magale´,‡,¥ and Dounia Yazidi§ Department of Chemistry, Calvin College, 3201 Burton SE, Grand Rapids, Michigan 49546, Laboratoire d’Electrochimie Mole´ culaire de l’Universite´ Denis Diderot (Paris 7), UMR CNRS 7591, 2 place Jussieu, 75251 Paris Cedex 05, France, and De´ partement de Chimie, Ecole Normale Supe´ rieure, UMR CNRS 8640 - PASTEUR, 24 rue Lhomond, 75231 Paris Cedex 05, France Received November 22, 2004. In Final Form: January 27, 2005 In this work, monolayers of metal complexes were covalently attached to the surface of carbon electrodes with the goal of binding monolayers of histidine-tagged proteins with a controlled molecular orientation and a maintained biological activity. In this novel method, which is simple, versatile, and efficient, the covalent attachment was accomplished in a single step by the electrochemical reduction of aryl diazonium ions that were substituted with a nitrilotriacetic (NTA) or an imminodiacetic (IDA) ligand at the para position. The transient aryl radicals that were generated in the reduction were grafted to the surfaces of glassy carbon, highly oriented pyrolitic graphite, and graphite-based screen-printed electrodes, producing dense monolayers of the ligands. The NTA- and IDA-modified electrodes were shown to efficiently chelate Cu(II) and Ni(II) ions. The presence of the metal was established using X-ray photoelectron spectroscopy and electrochemistry. Surface coverages of the ligands were indirectly determined from the electroactivity of the copper(II) complex formed on the electrode surface. Studies on the effect of electrodeposition time and potential showed that, at sufficiently negative potentials, the surface coverage reached a saturating value in less than 2 min of electrodeposition time, which corresponds to the formation of a close-packed monolayer of ligand on the electrode surface. Once loaded with a metal ion, the modified electrode was able to bind specifically to histidine-tagged proteins such as the horseradish peroxidase (His-HRP) or to an enhanced, recombinant green-fluorescent protein via its N-terminal hexahistidine tail. In the case of His-HRP, the amount of active enzyme specifically immobilized by metal-chelating binding was determined from the analysis of electrocatalytic currents using cyclic voltammetry. The electrochemical grafting makes it possible to accurately controlled and electronically address the amount of deposited ligand on the conductive surfaces of carbon electrodes with any size and shape.

Introduction The immobilization of biological macromolecules (e.g., proteins, enzymes, antibodies, receptor, peptides, DNA, etc.) on a transducer surface in a controlled manner that fully preserves their biological activity is one of the major challenges in making a functioning biosensor.1 In addition, the continuous development of electronic biosensing applications and the growing interest in the construction of electrochemical biosensors arrays2 call for simple, versatile, and efficient methods of immobilizing active biomolecules on electrode surfaces reversibly. Among the large variety of immobilization strategies (physical ad* Author to whom correspondence should be addressed. E-mail: [email protected]. † Calvin College. § Laboratoire d’Electrochimie Mole ´ culaire de l’Universite´ Denis Diderot. ‡ Ecole Normale Supe ´ rieure. ¥ Permanent adress: Faculte ´ des Sciences, Universite´ de Bangui, B. P. 908, Bangui, Central African Republic. (1) (a) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180. (b) Starodub, V. M.; Nabok, A. V.; Starodub, N. F.; Torbicz, W. In Nanostructured Materials and Coatings in Biomedical and Sensor Applications, NATO Science Series, II: Mathematics, Physics and Chemistry; Gogotsi, Y. G., Uvarova, I. V., Eds.; Kluwer Academic: Dordrecht, London, 2003; Vol. 102, pp 311-325. (c) Ferretti, S.; Paynter, S.; Russell, D. A.; Sapsford, K. E.; Richardson, D. J. TrAC, Trends Anal. Chem. 2000, 19, 530.

sorption, covalent attachment, polymer encapsulation, electrostatic or hydrophobic interactions, etc.), methods based on affinity ligands or biospecific recognitions are the most attractive because (i) the activity of the immobilized biomolecule is generally preserved, (ii) the spatial distribution can be well-controlled, (iii) the efficiency of the specific immobilization can be high, making it possible to work with low biomolecule concentrations, (iv) the nonspecific adsorptions are limited, and (v) the stability of the biomolecule is usually improved. The linkage of biomolecules via the biotin/avidin coupling is probably the most popular example of a versatile immobilization strategy,3 and it has been widely used for the preparation of enzyme electrodes which, under certain circumstances, exhibit a fully preserved catalytic activity and an enhanced stability.4 However, this system shares the disadvantages of most immobilization schemes (2) (a) Matsunaga, T.; Lim, T.-K. New Trends Electrochem. Technol. 2003, 2, 485. (b) Ertl, P.; Mikkelsen, S. R. Anal. Chem. 2001, 73, 4241. (c) Moser I.; Jobst G.; Urban G. A Biosens. Bioelectron. 2002, 17, 297. (d) Lenigk, R.; Liu, R. H.; Athavale, M.; Chen, Z.; Ganser, D.; Yang, J.; Rauch, C.; Liu, Y.; Chan, B.; Yu, H.; Ray, M.; Marrero, R.; Grodzinski, P. Anal. Biochem. 2002, 311, 40. (e) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikebukuro, K.; Karube, I. Anal. Chem. 2003, 75, 1116. (3) (a) Wilcheck, M.; Bayer, E. A. In Immobilized Biomolecules in Analysis; Cass, T.; Ligler, F. S., Eds.; Oxford University Press: Oxford, 1998; pp 15-34. (b) Wilcheck, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1.

10.1021/la047139y CCC: $30.25 © 2005 American Chemical Society Published on Web 03/12/2005

Dense Monolayers of Metal-Chelating Ligands

that require chemical modifications of the protein, i.e., the chemical coupling may lead to a significant decrease of the protein activity and the presence of multiple sites on protein for modification results in loss of control over the orientation of the protein during immobilization. The latter point is of significant importance in analytical approaches involving heterogeneous biomolecular recognitions because the random attachment of a protein can alter its binding capacity resulting from steric hindrance of adjacent immobilized proteins or with the surface itself.5 This is also particularly critical for a redox protein in direct electrical contact with a solid electrode, where the efficiency of the direct electron transfer may be strongly influenced by the geometric orientation of the protein on the electrode surface.6 Consequently, the development of methods to control and assess molecular orientation of immobilized proteins onto surfaces has become a focus of considerable research activity in recent years.7-11 One of (4) (a) Anicet, N.; Bourdillon, C.; Moiroux, J.; Saveánt, J.-M. J. Phys. Chem. B 1998, 102, 9844. (b) Anicet, N.; Anne, A.; Moiroux, J.; Saveánt, J.-M. J. Am. Chem. Soc. 1998, 120, 7115. (5) (a) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Mikrochim. Acta 1998, 128, 127. (b) Huang, W.; Wang, J.; Bhattacharyya, D.; Bachas, L. G. Anal. Chem. 1997, 69, 4601. (c) Turkova, J. J. Chromatogr. 1999, 722, 11. (d) Peluso, P.; Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Anal. Biochem. 2003, 312, 113. (6) (a) Hess, C. R.; Juda, G. A.; Dooley, D. M.; Amii, R. N.; Hill, M. G.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 2003, 125, 7156. (b) Le´ger, C.; Jones, A. K.; Albracht, S. P. J.; Arsmtrong, F. A. J. Phys. Chem. B 2002, 106, 13058. (c) Haas, A. S.; Pilloud, D. L.; Reddy, K. S.; Babcock, G. T.; Moser, C. C.; Blasie, J. K.; Dutton, P. L. J. Phys. Chem. B 2001, 105, 11351. (d) Wood, L. L.; Cheng, S.-S.; Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 571. (e) Burgess, J. D.; Hawkridge, F. M. In Electroanalytical Methods for Biological Materials; Brajter-Toth, A., Chambers, J. Q., Eds; Marcel Dekker: New York, 2002; pp 109-142. (f) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877. (g) Song, S.; Clark, R. A.; Bowden, E. F. J. Phys. Chem. 1993, 97, 6564. (7) (a) Lee, J. K.; Kim, Y.-G.; Chi, Y. S.; Yun, W. S.; Choi, I. S. J. Phys. Chem. B 2004, 108, 7665. (b) Luk, Y.-Y.; Tingey, M. L.; Hall, D. J.; Israel, B. A.; Murphy, C. J.; Bertics, P. J.; Abbott, N. L. Langmuir 2003, 19, 1671. (c) Benson, D. E.; Conrad, D. W.; Lorimier R. M.; Trammell S. A.; Hellinga H. W. Science 2001, 1641. (d) Madoz-Gurpide, J.; Abad, J. M.; Fernandez-Recio, J.; Velez, M.; Vazquez, L.; Gomez-Moreno, C.; Fernandez, V. M. J. Am. Chem. Soc. 2000, 122, 9808. (e) Kro¨ger, D.; Liley, M.; Schiweck, W.; Skerra, A.; Vogel, H. Biosens. Bioelectron. 1999, 14, 155. (f) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490. (g) Keller, T. A.; Duschl, C.; Kro¨ger, D.; Se´vin-Landais, A.-F.; Vogel, H.; Cervigni, S. E.; Dumy, P. Supramol. Sci. 1995, 2, 155. (8) (a) Johson, C. P.; Jensen, I. E.; Prakasam, A.; Vijayendran, R.; Leckband, D. Bioconjugate Chem. 2003, 14, 974. (b) Nakamura, C.; Hasegawa, M.; Yasuda, Y.; Miyake J. Appl. Biochem. Biotech. 2000, 84-86, 401. (c) Nieba, L.; Nieba-Axmann, S. E.; Persson, A.; Hamalainen, M.; Edebratt, F.; Hansson, A.; Lidholm, J.; Magnusson, K.; Karlsson, A. F.; Pluckthun, A. Anal. Biochem. 1997, 252, 217. (9) (a) Maly, J.; Illiano, E.; Sabato, M.; De Francesco, M.; Pinto, V.; Masci, A.; Masci, D.; Masojidek, J.; Sugiura, M.; Franconi, R.; Pilloton, R. Mater. Sci. Eng., C 2002, 22, 257. (b) Andreescu, S.; Magearu, V.; Lougarre, A.; Fournier, D.; Marty, J.-L. Anal. Lett. 2001, 34, 529. (10) (a) Gizeli, E.; Glad, J. Anal. Chem. 2004, 76, 3995. (b) Thess, A.; Hutschenreiter, S.; Hofmann, M.; Tampe´, R.; Baumeister, W.; Guckenberger R. J. Biol. Chem. 2002, 227, 36321. (c) Courty, S.; Lebeau, L.; Martel, L.; Lenne, P.-F.; Balavoine, F.; Dischert, W.; Konovalov, O.; Mioskowski, C.; Legrand, J.-F.; Venien-Bryan, C. Langmuir 2002, 18, 9502. (d) Dorn, Y.; Neumaier, K. R.; Tampe´, R. J. Am. Chem. Soc. 1998, 120, 2753. (e) Frey, W.; Schief, W. R.; Pack, D. W.; Chen, C. T.; Chilkoti, A.; Stayton, P.; Vogel, V.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4937. (f) Shnek, D. R.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir 1994, 10, 2382. (g) Kubalek, E. W.; Le Grice, F. J.; Brown, P. O. J. Struct. Biol. 1994, 113, 117. (11) (a) Hodneland, C. D.; Lee, Y.-S.; Min, D.-H.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5048. (b) Wang, J.; Bhattacharyya, D.; Bachas, L. G. In Biomedical Diagnostic Science and Technology; Law, W. T., Akmal, N., Usmani, A. M., Eds; Marcel Dekker: New York, 2002; pp 381-392. (c) Butterfield, D. A.; Bhattacharyya; D.; Daunert, S.; Bachas, L. J. Mem. Sci. 2001, 181, 29. (d) Wang, J.; Bhattacharyya, D.; Bachas, L. G. Biomacromolecules 2001, 2, 700. (e) Madoz, J.; Kuznetzov, B. A.; Medrano, F. J.; Garcia, J. L.; Fernandez, V. M. J. Am. Chem. Soc. 1997, 119, 1043.

Langmuir, Vol. 21, No. 8, 2005 3363

the most versatile and powerful methods is based on metalion chelating agents and their specific affinity for proteins containing histidine residues. This approach has become a standard tool in affinity chromatography for the purification of proteins genetically tagged with a short sequence of five or six consecutive histidine residues at the N- or C-terminus.5c The imidazole moieties of the tag can chelate to the free coordination sites of divalent transition-metal ions such as Cu2+ or Ni2+, which are themselves immobilized onto solid supports via appropriate chelating agents such as the tridentate iminodiacetic acid (IDA) or the tetradentate nitrilotriacetic acid (NTA). Self-assembled monolayers (SAMs) of metal-chelating lipids10 or thiol-functionalized NTA7 on gold supports have been widely investigated for the specific tethering of the oriented layer of histidine-tagged proteins, and the compatibility of gold supports with surface plasmon resonance has allowed quantitative kinetic and thermodynamic studies of specific binding between the immobilized histidine-tagged protein and a partner in solution.7e-g,8 Another benefit of a gold surface is its use as an electrochemical interface for measuring the biological activity of the immobilized protein from an electrical signal. Two clever approaches based on gold electrode functionalized with thiolated NTA have been recently described in which the mediated electrical communication of an enzyme7d or an allosteric protein7c with the gold electrode can be finely tuned through protein orientations on the SAM. It is important to note that the use of gold electrodes covered with self-assembling thiol compounds has a number of disadvantages such as a narrow potential window (range of potential of about 1.2 V in aqueous media) limited in the anodic direction by the faradic current resulting from gold oxide formation and in the cathodic direction by the electrochemical stripping of thiols from the surface.12 Another drawback is the degradation of the chemisorbed monolayer during storage, owing to the oxygen sensitivity of thiol compounds.13 One significant advantage in using carbon electrodes instead of gold is that the former can be used to make robust modified electrodes with a true covalent linkage of the metalchelating species on the surface and with a wider potential window. Several examples of carbon electrodes modified by NTA have been described,9 but the multiple chemical steps of functionalization were laborious and timeconsuming. Moreover, the harsh chemical or electrochemical oxidation methods used to generate oxygenated anchoring groups on carbon electrode surfaces lead to a considerable increase of the capacitive background current, thereby decreasing the sensitivity of the electrode. Finally, the exact nature and number of oxygenated functional groups are difficult to ascertain and control, which may led to ill-defined layers of the attached molecules.14 In this paper, we describe a novel approach to the functionalization of carbon electrodes in a single-step with dense monolayers of chelating metal compounds for the binding of histidine-tagged proteins. This method, which gives acceptable capacitive background currents, has the (12) Beulen, M. W.; Kastenberg, M. I.; van Veggel, C. J. M.; Reinhoudt, D. N. Langmuir 1998, 14, 7463. (13) (a) Brewer, N. J.; Foster, T. T.; Leggett, G. J.; Alexander, M. R.; McAlpine E. J. Phys. Chem. B 2004, 108, 4723. (b) Lee, T.-C.; Hounihan, D. J.; Colorado, R.; Park, J.-S.; Lee, T. R. J. Phys. Chem. B 2004, 108, 2648. (c) Stapleton, J. J.; Harder, P.; Daniel, T. A.; Reinard, M. D.; Yao, Y.; Price, D. W.; Tour, J. M.; Allara, D. L. Langmuir 2003, 19, 8245. (14) (a) Pantano, P.; Morton, T. H.; Kuhr, W. G. J. Am. Chem. Soc. 1991, 113, 1832. (b) Pantano, P.; Kuhr, W. G. Anal. Chem. 1993, 65, 623.

3364

Langmuir, Vol. 21, No. 8, 2005

Blankespoor et al.

advantage of being simple, versatile, and fast. It takes advantage of the covalent attachment of several chelating species to carbon surfaces via the electrochemical reduction of their aryl diazonium salts,15 which makes it possible to accurately control the amount of deposited ligand exclusively on the conductive surface and thereby modify a carbon electrode of any shape and size. The 4-aminophenyl-NTA and 4-aminophenyl-IDA derivatives shown in Scheme 1 are used as the precursors of the diazonium salts and they were electrochemically grafted on the surface of glassy carbon (GC), highly oriented pyrolitic graphite (HOPG), and graphite-based screen-printed electrodes (SPEs). SPEs were selected because they have low background currents, good reproducibility, and high flexibility in the design of their layout. They have the additional advantage of being particularly well-suited for mass production, which makes them useful as disposable biosensors.16 Scheme 1 gives the reactions that were used to covalently attach a metal-chelating molecule to an electrode surface and its subsequent attachment to a histidinetagged protein via Cu(II). This immobilization strategy was first implemented when horseradish peroxidase (HRP) was chemically functionalized on its glycosylated periphery by histidinyl residues. We have selected this enzyme because its catalytic activity can be easily monitored in the electrochemical reduction of H2O2 using a soluble redox mediator to shuttle electrons from the electrode surface to the enzyme active sites. Moreover, as we have recently shown, the surface coverage (ΓHRP) of the deposited active HRP can be precisely determined from the analysis of the catalytic current responses measured by cyclic voltammetry (CV).17 Finally, the recombinant, enhanced green-fluorescent protein (enhanced His-GFP), anchored to an electrode surface via an N-terminal hexahistidine tail, was tested for its specific immobilization by means of incident-light fluorescence microscopy.

Scheme 1. Outline of the Successive Steps Used for the Assemblage of His-Tagged Proteins on Carbon Electrode, Illustrated here in the Case of the 4-Aminophenyl-NTA Liganda

Results and Discussion

a (i) Diazotization of the aniline group with NaNO2 in icecooled HCl; (ii) cathodic functionalization of the carbon electrode immersed in the freshly prepared diazonium solution; (iii) metal loading; and (iv) specific immobilization of the His-tagged protein.

Electrochemical Functionalization of Carbon Electrodes. The starting metal-chelating compounds used for (15) This route has been devised for the functionalization of glassy carbon,15a-d highly oriented pyrolitic graphite,15c carbon fibers,15e-g felt,15h screen-printed carbon films,15i, j and pyrolyzed photoresist films15l-m in acetonitrile and acidic water. (a) Delamar, M.; Hitmi, R.; Pinson, J.; Saveánt, J.-M. J. Am. Chem. Soc. 1992, 114, 5883. (b) Bourdillon, C.; Demaille, C.; Hitmi, R.; Moiroux, J.; Pinson, J. J. Electroanal. Chem. 1992, 336, 113. (c) Allongue, P.; Delamar, M.; Desbat, B.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Saveánt, J.-M. J. Am. Chem. Soc. 1997, 119, 201. (d) D’Amours, M.; Be´langer, D. J. Phys. Chem. B 2003, 107, 4811. (e) Delamar, M.; De´sarmot, G.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Saveánt, J.-M. Carbon 1997, 35, 801. (f) Bath, B. D.; Martin, H. B.; Wightman, R. M.; Anderson, M. R. Langmuir 2001, 17, 7032. (g) Downard, A. J.; Roddick, A. D.; Bond, A. M. Anal. Chim. Acta 1995, 317, 303. (h) Coulon, E.; Pinson, J.; Bourzat, J. D.; Commercon, A.; Pulicani, J. P. Langmuir 2001, 17, 7102. (i) Dequaire, M.; Degrand, C.; Limoges, B. J. Am. Chem. Soc. 1999, 121, 6946. (j) Ruffien, A.; Dequaire, M.; Brossier, P. Chem. Commun. 2003, 912. (k) Anariba, F.; DuVall, S. H.; McCreery, R. L. Anal. Chem. 2003, 75, 3837. (l) Ranganathan, S.; McCreery, R. L. Anal. Chem. 2001, 73, 893. (m) Brooksby, P. A.; Downard, A. J. Langmuir 2004, 20, 5038. (16) SPEs have found successful applications in bioanalytical chemistry16a-c and are the basis of the majority of commercialized personal blood glucose meters.16d-f (a) Alvarez-Icaza, M.; Bilitewski, U. Anal. Chem. 1993, 65, 525A. (b) Hart, J. P.; Wring, S. A. Trends Anal. Chem. 1997, 16, 89. (c) Hilditch, P. I.; Green, M. J. Analyst 1991, 116, 1217. (d) Matthews, D. R.; Holman, R. R.; Bown, E.; Steemson, J.; Watson, A.; Hughes, S.; Scott, D. Lancet 1987, 778. (e) Harn-Shen, C.; Benjamin, K. I.; Chii-Min, H.; Kuang-Chung, S.; Ching Fai, K.; LowTone, H. Diabetes Res. Clin. Pract. 1998, 42, 9. (f) Lehmann, R.; Kayrooz, S.; Greuter, H.; Spinas, G. A. Diabetes Res. Clin. Pract. 2001, 53, 121. (17) Limoges, B.; Saveánt, J.-M.; Yazidi, D. J. Am. Chem. Soc. 2003, 125, 9192.

the electrochemical functionalization of carbon electrode surfaces, i.e., 4-aminophenyl-IDA (3) and 4-aminophenylNTA (6), were synthesized as outlined in Scheme 2. The synthesis of 3 was based upon a literature method used to prepare 1-(4-aminobenzyl)ethylenediamine-N,N,N′,N′tetraacetic acid.18 Since diazonium salts from aniline derivatives are stable in aqueous media for only limited times,we investigated the possibility of directly performing the electrochemical functionalization of the electrode in the solution in which the diazonium salts were generated. This was accomplished by generating the diazonium ion from the aniline derivative in aqueous acid containing NaNO2 at 0 °C and, after a short incubation time, transferring the resulting mixture to a cooled electrochemical cell where the diazonium ion was reduced at the surface of the carbon electrode. The cyclic voltammetric curves recorded at a graphitebased SPE immersed in solutions of the in-situ-generated diazonium salts of 4-aminophenyl-NTA (Figure 1A) and 4-aminophenyl-IDA (Figure 1B) show broad irreversible cathodic peaks at -0.53 and -0.78 V (vs SCE), respec(18) Chmura, A. J.; Schmidt, B. D.; Corson, D. T.; Traviglia, S. L.; Meares, C. F. J. Controlled Release 2002, 78, 249.


Langmuir, Vol. 21, No. 8, 2005 3365

Scheme 2. Synthesis of IDA- and NTA-Terminated Aniline Derivatives

Figure 1. Three successive cyclic voltammograms recorded at a SPE immersed in a 1.3 mM (A) 4-aminophenyl-NTA (6) or (B) 4-aminophenyl-IDA (3) in 0.1 N HCl containing 1.1 equiv of NaNO2 (3-fold dilution of the solution of diazotization, see Experimental Section). Scan rate, 50 mV/s; temperature, 4 °C.

tively. In each case, the irreversible cathodic peak tends to disappear upon continuous cycling of the potential, with a progressive shift of the peak potential toward more negatives potentials. This behavior is characteristic of the reduction of an aryl diazonium ion to a highly reactive aryl radical which rapidly forms a covalent bond to the surface,15a,c,19 thereby rendering the reduction of diazonium ion more and more difficult with the progressive growth of the grafted layer. As expected, the cathodic peak potential of the aryl diazonium ion from 6, which has an electron-withdrawing amide group, is more positive than the one from 3, making the reduction of aryl diazonium ion from 6 easier. It is worth noting that the decrease of the peak current upon continuous cycling is faster with the NTA diazonium ion than with the one containing IDA, suggesting a greater propensity of self-inhibition with NTA at the surface. We will come back to this point later. Similar results were obtained at GC and HOPG electrodes. Characterization of the Functionalized Electrodes. With the aim of probing the presence of ionizable groups on the electrode surface derivatized by NTA or IDA, we recorded the voltammetric curves of aqueous solutions of ferricyanide (FeCN63-) under different conditions (Figure 2). At pH 7.4, the reversible voltammetric response of FeCN63- observed at an unmodified electrode (CV A, E° ) 0.18 V, ∆Ep ) 130 mV) was strongly attenuated at the NTA-grafted electrode (CV B). Moreover, the large increase of the peak potential difference (∆Ep ≈ 900 mV) (19) Generation of an aryl radical through a concerted electron transfer with the cleavage of dinitrogen followed by formation of a covalent bond with the surface species is a proposed mechanism for the electrochemical grafting of aryl diazonium at carbon surfaces. Andrieux, C. P.; Pinson, J. J. Am. Chem. Soc. 2003, 125, 14801.

Figure 2. CV curves recorded at (A) unmodified SPE and (B, C) NTA-modified SPE immersed (A, B) in a phosphate buffer saline of pH 7.4 containing 0.1 M KCl and 1 mM FeCN63- and (C) in HCl of pH 1.0 containing 1 mM FeCN63-. Scan rate, 0.05 V/s. The NTA-modified electrodes were prepared using the following electrochemical grafting parameters: tElectrodep. ) 90 s and EElectrodep. ) -1.0 V.

shows a large decrease in the apparent rate of electron transfer at the electrode surface. Soaking the electrode in HCl 0.1 N (CV C)20 or in a buffer solution (pH 7.4) containing CuCl2 (1 mM, not shown) restored the voltammetric response of FeCN63-. These results imply an electrostatic repulsion between FeCN63- and the negatively charged electrode surface at neutral pH, an effect which disappears as the carboxylates groups in the ligand are protonated or chelated to a metal ion. The presence of the ligand and Cu2+ on the electrode surface was confirmed by X-ray photoelectron spectroscopy (XPS). Figure 3 shows the XPS spectrum of a HOPG electrode derivatized by IDA and pretreated with Cu(II). Peaks are observed for C, N, O, and Cu. The high resolution of the C1s peak at high binding energy exhibits the characteristic components (shoulders) of the IDA group. As expected, these shoulders are absent in the C1s peak at an unmodified HOPG surface pretreated in the same way with Cu(II), and no peak for copper is present. Although the Cu/C ratio has a contribution from the carbon surface and from the carbon contained in the grafted ligand, the Cu2p/C1s atomic ratio is on average 0.02. If one assumes a carbon atom surface density equal to that of a basal plane graphite coverage of 7.3 × 10-9 mol/cm2, the XPS results yield a copper coverage of 1.43 × 10-10 mol/ cm2. It will be shown later that this is close to the surface coverage found electrochemically. The binding energy of the N1s peak is the same at modified and unmodified HOPG. The shoulder of this peak, located at a higher (20) The positive shift of the standard potential of FeCN63- is related to its protonation in acidic media.

3366


Figure 3. XPS analysis of a IDA-grafted HOPG electrode first loaded with Cu2+ by incubation for 30 min in a phosphate buffer (pH 7.4) containing 1 mM CuCl2 and next rinsed with water and dried before analysis.

Figure 4. Cyclic voltammetric curves recorded in phosphate buffer (pH 7.4) at a (A) SPE or (B) GC electrode derivatized by (blue solid line) IDA or (red solid line) NTA and preconditioned in a solution of 1 mM CuCl2 in phosphate buffer (pH 7.4) for 30 min. The dashed CV curves were obtained at electrodes functionalized with NTA before loading with Cu2+. Scan rate: 50 mV/s. All of the electrodes were derivatized by conducting three consecutive cyclic scans (50 mV/s) from 0.0 to -1.0 V in the diazonium solution.

binding energy, is characteristic of a tetrahedral (sp3) nitrogen that we attribute to the protonated tertiary amino group of the ligand. The atomic ratio of the Cu2p/N1sshoulder is 0.87, indicating that nearly all of the grafted NTA groups have been complexed to Cu2+. The absence of a cyclic voltammetric oxidation or reduction wave from the functionalized electrodes immersed in deaerated aqueous buffer (pH 7.4 or 5) indicates that the surface-attached NTA or IDA groups are not electroactive in the potential window of the aqueous buffer. However, after the functionalized electrodes were incubated in a solution of copper ions (Cu2+) and re-immersed in a deoxygenated buffer, the CVs show a broad reduction wave and a relatively well-defined oxidation peak in the reverse scan (Figure 4). Reduction and oxidation waves were observed from both of the grafted ligands, and the peak currents decreased with repetitive cyclic scans, suggesting that Cu is lost. At an unmodified electrode pretreated with copper or at an electrode immersed under open circuit for several minutes in the diazonium solution and loaded in the same way with Cu2+, no peak in the CVs were observed. All of these results suggest that the reduction and oxidation waves come from the complexes of Cu-NTA or Cu-IDA attached to the electrode surface. We attribute the reduction wave to the irreversible reduction of the chelated Cu(II) into Cu(0) and the oxidation wave to the redissolution of the electrodeposited

Blankespoor et al.

Figure 5. Surface coverages of chelated copper (9, 0) at NTAmodified SPEs and (b) IDA-modified SPEs as a function of the conditions of (A) electrodeposition time and (B) potential applied during the electrochemical modification of the electrode surface. In A, the influence of the electrodeposition time was investigated by applying a constant electrodeposition potential of (9, b) -1.0 and (0) -0.65 V. In B, the electrodeposition time at a each controlled potential was fixed at 90 s. Error bars: standard deviation from the average measurements at two or three SPEs.

metallic copper to free Cu2+.21 Some of these assumptions are supported by voltammetric experiments in homogeneous solution containing a stoichiometric mixture of 4-aminophenyl-IDA and CuCl2 (see Figure S1 and comments in Supporting Information). The electroactivity of the copper complexes gives us the opportunity to indirectly determine the surface coverage of the grafted ligand and to study the influence of the electrodeposition time and electrolysis potential on the formation of the layer. Surface coverages (ΓCu2+) were estimated from charge integration of the re-oxidation wave (ΓCu2+ ) Q/nFS where Q is the integrated charge, F is the Faraday constant, S the geometric area of the electrode, and n ) 2 for the two-electron reduction) at a low scan rate in such a way that all of the electroactive copper ions chelated on the electrode surface are oxidized.22 Although a kinetic study of the copper binding revealed that a 10 min incubation period in 1 mM CuCl2 was long enough to complex all of the available grafted ligands on the electrode surface, an incubation time of 30 min was used for all of the studies. The influence of the grafting electrodeposition time (tElectrodep.) and potential (EElectrodep.) on ΓCu2+ are shown in Figure 5 for both ligands. Comparison of the curves (Figure 5A) reveals that ΓCu2+ rapidly approaches a limiting value independent of the polarization time without a significant kinetic difference between the grafted ligands. More interestingly, the limiting ΓCu2+ is potential dependent and increases as the grafting potential is shifted in the cathodic direction (compare open-square with filled-square data in Figure 5A). Finally, when similar grafting conditions are selected for both ligands (i.e., by applying a deposition potential which is 150-200 mV more negative than the reduction peak potential of the diazonium ion), the limiting ΓCu2+ value obtained for IDA-modified electrodes is significantly higher than for NTA-modified electrodes. Variations of the limiting ΓCu2+ values with the electrodeposition potential are plotted in Figure 5B for the two ligands. The data verify the increase of the limiting plateau value of ΓCu2+ with the application of more and more negative potentials and point toward the attainment of a saturation value at sufficiently negative potentials for both ligands. The plots also confirm that, when the electrodeposition potential is positive to the foot of the voltammetric reduction wave of the NTA or IDA diazonium ions, there is no binding of copper ions on the electrode surface. More


intriguing was the dependence of the slope of the plots in Figure 5B with the nature of the grafted species. The results described here are consistent with a self-inhibition mechanism in which the progressive loading of the electrode has the effect, for steric and/or repulsive reasons, of blocking the incoming diazonium cations and thereby slowing down the charge-transfer kinetics as the surface coverage increases.15c As a result, the peak potentials become more negative during the reduction of the diazonium ion in continuous cyclic voltammetry (Figure 1). Another consequence of this self-inhibition is that the coverage of the electrode surface is stopped at a value significantly lower than that of a saturated monolayer when the controlled potential is not sufficiently negative (the rate of reduction drop to a value close to zero). This also explains why increasingly negative electrodeposition potentials are required to compensate for the apparent decrease in rate constant from the self-inhibition. A similar dependence of the surface coverage on the deposition potential and self-limiting film formation was previously observed in the case of the electrochemical grafting of the 4-nitrophenyldiazonium ion in acetonitrile.23 Finally, it should be noted that there is a marked difference in the self-inhibition rates of the two grafted diazonium ions. It is clear from the CVs in Figure 1 that self-inhibition is more pronounced with the NTA diazonium ion than with the IDA diazonium ion. This is corroborated by the results in Figure 5B, where the increase of the copper surface coverage with the electrodeposition potential is found to be slower for NTA-functionalized electrodes than for IDA electrodes. The difference in self-inhibition rates for the two ligands could arise from an inherently higher reactivity of the NTA aryl radical with the carbon surface. A more likely reason for the difference is that the attached NTA ligand has a more pronounced steric and/or repulsive effect on the incoming diazonium ions to be grafted than IDA. The ΓCu2+ saturation values observed at very negative applied potentials (Figure 5B) could be attributed to maximal surface coverages. If one assumes that a surface is flat and homogeneous and its coverage by a grafted ligand approximates a close-packed layer, it is possible to determine if a film is close to a monolayer or not. This is an important point that we have addressed given the propensity of diazonium reagents to form multilayers.24 However, the assumption of a flat and homogeneous surface is not valid for SPEs because their surfaces are composed of small, heterogeneous, conductive graphite particles embedded in an insulating binder of polystyrene. Consequently, even if some aryl radicals are grafted to the polystyrene binder surrounding the graphite particles, only the copper complexes directly attached to the conductive zones has a chance to be electrochemically detected. Copper surface coverage measurements were then made by conducting experiments with HOPG electrodes. The HOPG has the advantage of having a very flat surface with a geometric area approximately the same as (21) It is worth noting that the cathodic peak potential for the electrode functionalized by NTA (-0.60 V) was slightly more negative than the cathodic peak potential at the IDA-modified electrode (-0.73 V). This more negative reduction potential of the tetradentate NTA-copper complex is in agreement with the increase of the coordination number, which should stabilize the complex and make its reduction more difficult compared with the tridentate IDA-copper complex. (22) The peak area was independent of the scan rate for scan rates ranging from 0.01 to 0.1 V/s. (23) Downard, A. J. Langmuir 2000, 16, 9680. (24) Multilayer films have been proposed to form via attack of electrogenerated radicals on groups already attached to the electrode surface.15c,25a,b (a) Kariuki, J. K.; McDermott, M. T. Langmuir 1999, 15, 6534. (b) Kariuki, J. K.; McDermott, M. T. Langmuir 2001, 17, 5581.

Langmuir, Vol. 21, No. 8, 2005 3367 Table 1. Surface Coverage (pmol/cm2) of NTA or IDA Copper Complex at Different Functionalized Carbon Electrodes functionalized electrodea

ΓCu2+,directb

ΓCu2+,indirectc

NTA-modified SPE IDA-modified SPE NTA-modified GC IDA-modified GC NTA-modified HOPG IDA-modified HOPG

87 ( 4 120 ( 25 218 ( 60 225 ( 60 368 ( 95 364 ( 20

150 ( 20 230 ( 50 290 ( 115 360 ( 100

a All of the electrodes were modified by applying a constant potential of -1.4 V for 90 s in the diazonium solution followed by immersion into 1 mM Cu2+ in phosphate buffer. b Surface coverages estimated from integration of the voltammetric anodic peak of copper recorded directly at the functionalized electrode. c Surface coverages obtained from the indirect ASV determination of copper ions released in acidic solution.

the actual surface area. The experiments were also done at GC electrodes, although their surfaces are rough and pitted. Table 1 compares the maximal surface coverages of copper complexes obtained at the different carbon electrodes. For simplicity and because the actual surface areas of SPE and GC are not known, the data in Table 1 are all based on the geometric areas of the electrodes. In Table 1 are also reported the maximal surface coverages of Cu2+ determined indirectly by anodic stripping voltammetry (ASV) at a thin mercury-film-coated microelectrode, after acidic release of the metal ions within a small drop of 1 N aqueous HCl deposited over the modified electrode surface (see Experimental Section for details). The ΓCu2+ values measured indirectly by this method at IDA- and NTA-modified HOPG electrodes are in good agreement with those from direct electrochemical detection. It shows that most of the copper ions bonded to the modified HOPG electrode surface are electrochemically accessible in cyclic voltammetry. This result is consistent with the formation of a monolayer of ligand instead of multilayered film. This conclusion is based on the fact that the reduction of the surface copper ions is not a simple reversible electron exchange process and hence site-to-site electron hopping cannot be a mechanism for charge transfer from the electrode surface to the outer parts of a multilayered film. This means that copper ions which are located too far from the electrode surface for efficient electron tunneling are electroinactive and are therefore not detected voltammetrically. Consequently, in the case of a multilayered film the amount of copper ions determined by voltammetry should be less than the amount of copper indirectly determined by ASV after acidic dissolution. But this is not the case here. The results in Table 1 also show that the maximal surface coverages of Cu-NTA and Cu-IDA at the GC and HOPG electrodes are not very different, indicating that the maximal coverage is not strongly influenced by the nature and roughness of the carbon surface. Assuming a homogeneous distribution of the grafted molecules with the ligand and tether perpendicular to the surface in an ideal close-packed monolayer, calculated theoretical values for ΓCu2+ of 4.4 × 10-10 and 3.0 × 10-10 mol/cm2 are obtained for IDA and NTA, respectively.25 Given the maximal experimental surface coverage values for IDA- or NTA-modified HOPG electrode (ca. 3.6 × 10-10 mol/cm2), it is reasonable to conclude that the covalently attached IDA and NTA ligands on the GC and HOPG surfaces are dense monolayers. A somewhat (25) The theoretical surface coverages of the grafted IDA and NTA molecules were calculated with the Alchemy 2000 software package (Tripos, Inc.).

3368


Blankespoor et al.

ΓCu2+,eq )

Figure 6. Binding isotherms of Cu2+ at SPE derivatized with (9, b) 4-aminophenyl-NTA and (0, O) 4-aminophenyl-IDA at two different pHs: (9, 0) phosphate buffer at pH 7.5 and (b, O) citrate buffer at pH 5.5. The surface concentration of copper(II) was determined at equilibrium from integration of the oxidation wave of copper in CV experiments. The experimental plots were fitted to Langmuir isotherm (solid lines). All of the electrodes were modified by conducting three consecutive cyclic scans (50 mV/s) of the electrode from 0.0 to -1.0 V in the diazonium solution followed by immersion in aqueous copper(II) solution for 2 h.

lower surface coverage value per monolayer (2.5 × 10-10 mol/cm2) has been recently determined for films consisting of 2-5 layers of nitrophenyl groups electrochemically grafted on flat surfaces of pyrolized photoresist plates.15m The same workers have also found that films prepared in aqueous acidic solutions have lower limiting thicknesses than those prepared in acetonitrile, approaching in some cases the single layer.15m The results obtained here are consistent with this earlier finding, and given that attachment can only occur when phenyl radicals diffuse to the surface, it seems unlikely that the film can grow in multilayers.24 The maximal surface coverage found here is also close to the surface coverage of Cu2+ determined at the gold electrode covered with a self-assembled monolayer of dithioctic acid terminated with an NTA group.7d In contrast to the results found with HOPG electrodes, the ΓCu2+ values for NTA- and IDA-modified SPEs from the indirect ASV method are almost double those from the direct voltammetric determination (Table 1). This is what would be expected from a SPE surface that is not totally electrochemically active (i.e., the surface is composed of a random distribution of closely spaced insulating and conductive microscopic zones), and as a result, only the copper complexes directly attached to the conductive sites can be detected using cyclic voltammetry (thereby giving the actual area of the delimited printed disk electrode that is electroactive). This also signifies that the excess of copper determined by ASV comes from ligands grafted on the insulating polystyrene binder (reflecting thus the actual area of the printed electrode) and that some of the aryl radicals generated at graphite sites are sufficiently long-lived to diffuse to the surrounding polystyrene binder for covalent coupling. Affinity Binding of Copper for the Ligand-Functionalized Electrode. Owing to the ease of monitoring the binding of Cu2+ to the NTA- or IDA-functionalized electrode, the apparent association constants (Kapp) of copper complexes attached to the electrode were determined at equilibrium from Langmuir isotherm plots measured at two different pHs (Figure 6). The plots were fitted with eq 1.

ΓSKappCCu2+ 1 + KappCCu2+

(1)

where ΓCu2+,eq and ΓS are, respectively, the equilibrium and saturated surface concentrations of copper(II), and CCu2+ is the concentration of Cu2+ in the bulk solution. The apparent association constants are given in Table 2. Not surprisingly, on the basis of data in the literature for the homogeneous binding of copper(II) to IDA or NTA ligands,26 copper(II) binds more strongly to IDA- than NTA-derivatized electrode by about 1 order of magnitude. Moreover, the affinity binding increases with increasing pH for both ligands. This pH dependency presumably comes from the protonation of the tertiary amine of the ligand which competes with complex formation. The absolute association constants (Kabs) of the attached copper(II) complexes can be calculated using the pKa values of the protonated amines27 (eq 2).

pKabs ) pKapp - pH + pKa

(2)

Surprisingly, the calculated pKabs values from the NTAor IDA-modified electrodes are 2-3 orders of magnitude lower than the binding values obtained from homogeneous solutions of these ligands with copper(II).26,28 The pKapp obtained with NTA was also abnormally low compared with that measured by impedance spectroscopy at a selfassembled monolayer of thiol-terminated NTA (pKapp ) 8.3 at pH 7.4) for which no significant difference between the surface and homogeneous binding of copper(II) was observed.28a Although there is no obvious explanation for the much lower pKapp of copper(II) than at a gold electrode coated by thiol-terminated NTA, it is worth noting that a long spacer between the NTA group and the anchoring thiol was used for the preparation of the SAM on a gold surface, placing the NTA groups far from the gold surface. In the present case, the grafted ligands are much closer to the hydrophobic carbon surface and they probably have less mobility. The large differences in surface and bulk pKapp values are not unexpected given the large shifts in apparent dissociation constants that have been observed for mixed acid monolayers,29 an effect that was attributed to unfavorable solvation of the ionizable group at the monolayer interface.29c Large pKa shifts relative to solution were also observed in studies of amino groups attached to hydrophobic surfaces.30 For example, it has been shown that the pKa of an amine group linked to a thiolalkane self-assembled on a gold surface is 6-7 pK units lower than in homogeneous solution.30a This was attributed to the relatively hydrophobic environment of the amine group in the self-assembled layer. Such an effect could also be present here, and a decrease of the pKa of the tertiary amine would explain the decrease of the pKabs. Other (26) Martell, A. E.; Smith, P. M. Critical Stability Constants; Plenum Press: New York, 1974; Vol. 6. (27) The pKa of the tertiary amines contained in the 4-aminophenylNTA and 4-aminophenyl-IDA were determined by acid-base titration in homogeneous solution. (28) (a) Stora, T.; Hovius, R.; Dienes, Z.; Pachoud, M.; Vogel, H. Langmuir 1997, 13, 5211. (b) Ando, T. Bull. Chem. Soc. Jpn. 1962, 35, 1395. (29) (a) Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006. (b) Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114. (c) Creager, S. E.; Clarke, J. Langmuir 1994, 10, 3675. (d) Lee, T. R.; Carey, R. I.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 741. (e) Bryant, M. A.; Crooks, R. M. Langmuir 1993, 9, 385. (30) (a) Smalley, J. F.; Chalfant, K.; Feldberg, S. W.; Nahir, T. M.; Bowden, E. F. J. Phys. Chem. B 1999, 103, 1676. (b) Chatelier, R.; Drummond, C.; Chan, D.; Vasic, Z.; Gengenbach, T.; Griesser, H. Langmuir 1995, 11, 4122.


Langmuir, Vol. 21, No. 8, 2005 3369

Table 2. pKapp and pKabs of the Binding of Cu2+ to Electrochemically Grafted NTA and IDA pKapp chelator

pH 7.4

pH 5.5

pKa

pKabs

IDA NTA

6.3 5.7

5.0 4.3

9.4 9.8

8.6 ( 0.4 8.4 ( 0.4

Scheme 3. Coordination of the 1,1′-Bis(methylimidazole)ferrocene (Shown at Left) to an NTA-Modified Electrode Loaded with Ni2+

effects such as the high local concentration of IDA or NTA and the presence of a charge double layer can cause the formation constants for metal complexation at the interface to differ from those in bulk solution. Formation of Copper and Nickel Complexes with an Electroactive Imidazole Probe. Among the diverse divalent transition-metal ions typically employed in metalaffinity chromatography, nickel(II) and copper(II) are the most widely used. We decided, therefore, to also investigate the binding affinity of Ni2+ to NTA- and IDA-modified electrodes. In contrast to Cu(II), we were unable to detect the Ni(II) complexes of NTA or IDA using cyclic voltammetry in aqueous buffer. An alternative approach was then taken to indirectly establish the presence of chelated nickel(II) on the electrode surface. This was accomplished using the electroactive probe 1,1′-bis(methylimidazole)ferrocene (7) which is expected to bind to Ni2+ via its imidazole ligands, as depicted in Scheme 3. To validate this approach, experiments were first carried out with copper ions at the NTA-modified electrode. The cyclic voltammograms in Figure 7 shows that after the addition of the 1,1′-bis(methylimidazole)-ferrocene to the electrochemical cell, the reversible current response of the ferrocene group (E° ) 0.38 V, Figure 7A) was significantly enhanced at an NTA-functionalized SPE pretreated with Cu2+ (red trace) compared with the response at an NTA electrode not loaded with copper (green trace). Concomitantly, the cathodic and anodic waves associated with the reduction and oxidation of the copper complex anchored on the electrode surface (Figure 7B) were totally suppressed in the presence of the ferrocene probe (red trace). Peak currents of the oxidation or reduction waves of the ferrocene vary linearly with the scan rate (0.02-2.0 V), as expected for a species that is attached to the electrode surface. In the absence of pretreatment by Cu2+, the intensities of the reversible waves of the ferrocene derivative at NTA-grafted electrode were indistinguishable from those observed at an unmodified electrode. Similar results were obtained with the IDA electrode. All of these observations point toward the formation of a ternary complex on the electrode surface with the binding of the 1,1′-bis(methylimidazole)-ferrocene to the available coordination sites of the Cu(II)-NTA complex. However, the 1,1′-bis(methylimidazole)-ferrocene-Cu(II)-NTA complex appears to be quite labile. After the modified electrode was rinsed and scanned in a pure buffer, no peaks were observed for the ferrocene derivative. It is not very surprising that this complex is quite labile because a low affinity of the ligand imidazole for the Cu(II)-IDA complex was previously reported (pKapp ≈ 3.5).31 Although replacement of Cu(II) by Ni(II) in the layer was also observed to give a clear enhancement of the current response of

Figure 7. CV curves recorded at NTA-functionalized SPEs pretreated with 1 mM (red and black traces) Cu2+ or (blue trace) Ni2+, and immersed (black traces) in a pure phosphate buffer (pH 7.4) or (red and blue traces) in a phosphate buffer containing 1 µM 1,1′-bis(methylimidazole)ferrocene (7). The green trace was obtained at NTA-functionalized SPE without metal ion, in a 1 µM of 7. Scan rate, 50 mV/s.

ferrocene 7 (blue trace), it was approximately one-half the current obtained from an equivalent amount of copper(II). After the CVs recorded in the presence of Ni(II) and Cu(II) (blue and red traces in Figure 7) were corrected for the small diffusion contribution of the bulk ferrocene measured at the NTA electrode free of metal ion (green trace in Figure 7), the resulting anodic waves were integrated and the surface concentrations of 1,1′-bis(methylimidazole)-ferrocene anchored on the electrode were determined. Values of 16.6 × 10-11 and 8.2 × 10-11 mol/cm2 were obtained at Cu-NTA- and Ni-NTA-coated electrodes, respectively, which is close to the value (8.7 × 10-11 mol/cm2) found directly from the integration of the anodic peak current in the case of the copper complex (black trace in Figure 7). The NTA- and IDA-modified SPEs, once loaded with copper and dried, were tested for their storage stability upon prolonged exposure to air at room temperature, and whatever the nature of the grafted complex, the voltammetric responses of copper drop to half their initial values after ca. 40 days. This is in contrast to the very good stability of carbon electrodes derivatized with covalently bound 4-nitrophenyl groups, which gave no significant decrease in current of the voltammetric reduction wave of the nitro group after several months of storage under similar conditions. These results suggests a slow degradation of the Cu-NTA and Cu-IDA layers upon longterm storage. Since the metal ions can be released from the electrode by immersion in acidic solution, we have also examined the feasibility of repetitive loading of the modified electrodes with Cu2+. From the constancy of the current response of copper recorded after each cycle of metal-ion release/loading, it was concluded that the modified electrodes could be reused almost 4-5 times before significant deterioration of the signal occurred. Enzyme-Modified Electrode and Binding Specificity. HRP, chemically functionalized by histidine residues, was used as an analytical tool to probe the potential of our modified electrode for the binding of His-tagged proteins. The HRP enzyme has the advantage of being relatively robust and stable, and once immobilized on an electrode surface, its catalytic activity can be easily monitored by cyclic voltammetry in the presence of its natural H2O2 substrate and an artificial redox mediator that shuttles electrons from the electrode surface to the enzyme active sites. Moreover, despite a complex mech(31) (a) Sinha, P. C.; Saxena, V. K.; Nigam, N. B.; Sriastava, M. N. Indian J. Chem. 1989, 28A, 335. (b) Todd, R. J.; Johson, R. D.; Arnold, F. H. J. Chromatogr. A 1996, 725, 225.

3370


Figure 8. Electrocatalytic responses obtained at SPEs derivatized by the diazonium salt (blue and red curves) of 4-aminophenyl-NTA or (green curve) 4-aminohyppuric acid, and preloaded (blue and green curves) or not (red curve) with Cu2+ (electrode immersed for 30 min in 0.5 mM Cu2+) and HisHRP (immersion for 1 h in 20 nM His-HRP containing 2.5 µM DETAPAC). The CV curves were recorded in phosphate buffer (pH 7.4) containing 20 µM [Os(bpy)2pyCl]2+ and 2 mM H2O2. Scan rate, 10 mV/s; temperature, 20 °C.

anism of substrate inhibition/cosubstrate reactivation,32 we have shown that the enzyme surface coverage (ΓHRP) of the deposited active HRP can be precisely determined from the analysis of the catalytic current responses in cyclic voltammetry using the reversible single electron donor [OsII(bpy)2pyCl]+.17 The reactions involved in the electrocatalytic reduction of H2O2 by HRP in the presence of [OsIII(bpy)2pyCl]2+ (OsIII/OsII couple) are given in Scheme S1 of the Supporting Information. Since the native HRP is naturally glycosylated and does not possess surface-accessible histidines, we chemically modified HRP by covalently attaching histidines to the carbohydrates surrounding the protein. This was accomplished by the periodate oxidation of the carbohydrate shell into aldehyde groups followed by Schiff base condensation with the amino group of histidine. An average number of 5-6 histidine residues were attached to each HRP molecule in this manner, as was determined from amino acid analysis. The activity of the resulting Histagged HRP (His-HRP) was measured, as previously,32 using both steady-state spectrophotometry and cyclic voltammetry in homogeneous solution (see the Experimental Section), the two measurable rate constants of the rate-determining step of the main catalytic cycle (reaction 3 in Scheme S1) were found to be k3 ) k3,1k3,2/(k3,-1 + k3,2) ) k3,2/K3,M) (6.6 ( 1.3) × 106 M-1 s-1 and k3,2 ) 235 ( 75 s-1, giving a Michaelis constant K3,M ) 35 ( 5 µM. The K3,M value is in good agreement with the one previously obtained for the native HRP (25 ( 10 µM), whereas the rate constant k3 is ∼15% lower, indicating that the activity of the His-tagged HRP is ∼85% of that of the native enzyme. It is reasonable to conclude, therefore, that the introduction of the histidinyl residues on the periphery of the enzyme does not significantly change the catalytic properties of the enzyme. Evidence for the specific binding of the His-HRP at an electrode functionalized by a monolayer of Cu(II)-NTA is shown in Figure 8, where the catalytic current responses are recorded in the presence of the H2O2 substrate, the [OsIII(bpy)2pyCl]2+ redox mediator, and a small amount of diethylenetriaminepentaacetic acid (DETAPAC) to scavenge metal ion impurities that could complex with the NTA group. When the NTA-functionalized SPE was successively pretreated with a solution of Cu(II) and a diluted solution of His-HRP followed by immersion in a (32) Dequaire, M.; Limoges, B.; Moiroux, J.; Saveánt, J.-M. J. Am. Chem. Soc. 2002, 124, 240.

Blankespoor et al.

electrochemical cell containing a slight excess of H2O2 (2 mM) and a relatively low concentration of [OsIII(bpy)2pyCl]2+ (20 µM), a characteristic and well-defined catalytic wave of H2O2 reduction, showing a typical hysteresis effect,17 was recorded. Modified electrodes that were not pretreated with Cu(II) did not bind His-HRP since only the reversible waves of the mediator with no discernible catalytic current were recorded. Conversely, NTA-modified electrodes loaded with copper were unable to bind native HRP. To further confirm that the binding was due to the formation of a complex between the chelate and the histidinyl residues in the tagged protein, the NTA ligand was replaced by 4-aminohippuric acid (Scheme 1), a mimicking molecule that possesses a terminal carboxylate group and that can be electrochemically grafted from its parent diazonium salt. Figure 8 shows that even after the hippuric acid-modified electrode was treated with copper(II) followed by His-HRP, no significant catalytic current was obtained. Furthermore, the same procedure, carried out using an unmodified electrode, shows the absence of nonspecific binding of His-HRP on the hydrophobic surface of the carbon electrode. Additional evidence for the specificity of the binding comes from experiments in which 100 mM imidazole, DETAPAC, or ethylenediaminetetracetic acid (EDTA) was added to solutions where HRPloaded electrodes were immersed. In each instance, desorption of the protein occurred, as indicated by the drop of the catalytic current in CV experiments (not shown). All of the above experiments were also conducted using glassy carbon and HOPG electrodes, and all of the results were consistent with those using SPEs. Using the framework of the HRP mechanism (Scheme S1) and assuming that the metal-binding of the His-HRP on the electrode surface results in an enzyme layer that exhibits a nearly fully preserved activity, as was shown previously for HRP assembled through an avidin-biotin linkage, one can estimate the amount of deposited active enzyme, ΓHRP, from the maximum plateau current, ip,cat, of the catalytic S-shaped wave observed in Figure 8, according to eq 317

ip,cat ) ip - id )

2FSk3CP0ΓHRP CP0 CS0 1+ + K3,M k6 k5 0 + C k4 k4 P

(3)

where F is the faraday constant, S the electrode surface area, C0P the bulk mediator concentration, C0S the bulk H2O2 substrate concentration, and id the diffusion current of the mediator in the absence of H2O2. For the calculation, the homogeneous constants k3 and K3,M determined above in homogeneous solution for the His-HRP were used in eq 1, as well as the rate constant ratios k6/k4 ) 3.3 × 10-4 M-1 and k5/k4 ) 69.3 previously determined for HRP.17 Using these data and the experimental concentrations of C0P ) 20 µM and C0S ) 2 mM, a proportionality coefficient of 1.3 × 106 mol/(cm2‚µA) between ip,cat and ΓHRP was calculated. The maximum plateau current (5.9 µA) in Figure 8 allowed us to calculate a HRP surface concentration of 4.5 pmol/cm2, which is of comparable magnitude to the surface coverage (3.8 pmol/cm2) found previously for a HRP monolayer immobilized through an avidinbiotin linkage onto an electrode.17 Using the ΓCu2+ value of 150 pmol/cm2 found for the NTA-grafted SPE (Table 1), there were approximately 30 Cu-NTA complexes per molecule of His-HRP. However, even though there are multiple points of attachment to the His-HRP, which


Langmuir, Vol. 21, No. 8, 2005 3371

where A is the immobilized metal complex, B the enzyme target, AB the enzyme-metal complex, and AI symbolizing a deactivation pathway of the enzyme complex. Considering the simplest case of a Langmuirian kinetic law and taking into account the mass transport, the following differential equations can be written

dΓAB ) dt

Figure 9. Kinetic binding of His-HRP (normalized surface coverage, θHis-HRP, as a function of incubation time, t) at CuNTA-coated SPEs. (A) Experimental data at several bulk concentrations of His-HRP: (2)2, (1) 20, (b) 40, and (9) 200 nM. (B) Simulated kinetic binding curves.

should result in a more stable layer than with the 1,1′bis(methylimidazole)-ferrocene, the catalytic current response of the enzyme electrode dropped to half its initial value after 2 days of storage in a phosphate buffer. This lack of stability can be attributed to a relatively weak affinity binding of the histidinyl residues of HRP for the metal complex. With the aim of estimating the affinity binding of HisHRP to the metal complex attached to the electrode, the amount of anchored His-HRP was monitored as a function of the incubation time and His-HRP concentration. For this purpose, a series of SPEs coated with a monolayer of Cu-NTA were immersed in different concentrations of diluted His-HRP solutions without agitation, and at specified incubation times, the electrodes were quickly removed and transferred to an electrochemical cell containing 20 µM [OsIII(bpy)2pyCl]2+ and 2 mM H2O2. The catalytic activity of the immobilized enzyme was then measured to monitor indirectly the progress of the specific binding. The resulting kinetic binding curves are displayed in Figure 9. As expected, the higher the His-HRP concentrations, the faster were the kinetic bindings and greater the amount of immobilized enzyme. However, after prolonged incubation times, the surface coverage of HisHRP does not reach a limiting value which would indicate a saturated monolayer, but instead it goes through a maximum after which it asymptotically decayed. This result suggests a competitive displacement of the copper ions from the electrode surface by bulk His-HRP and DETAPAC. This assertion is supported by the fact that a much slower decrease of the enzyme activity was systematically observed for HRP electrodes removed from the enzyme solution and stored in a pure phosphate buffer solution. The presence of the DETAPAC chelator in the enzyme solution was found to contribute to the decay since its replacement by a weaker chelator such as EDTA leads to a slower decrease, but with the undesired effect of slightly increasing the nonspecific bindings. Nevertheless, even in the absence of an added chelator that can compete with the His-HRP, the decay of the enzyme coverage was persistent. A reaction scheme that can account phenomenologically for the initial increase in kinetic binding followed by its decay (Figure 9) is as follows

kfC0ΓA - kbΓAB + k-iΓAI - kiΓAB )

D (C - C0) (4) δ b

dΓAI ) kiΓAB - k-iΓAI dt

(5)

where Γi are the surface concentrations of the i subscript species; Cb and C0 are the concentrations of B in the bulk solution and at the electrode surface, respectively, D is the diffusion coefficient of B, and δ is the thickness of the steady-state diffusion-convection layer which results from the natural Brownian thermal agitation in the absence of hydrodynamic convection. A rough estimate of the latter parameter is 100 µm at room temperature in a phosphate buffer. Numerical resolution of the two differential eqs 4 and 5 allows one to predict the fractional coverage-time curve (see Supporting Information). Using the δ value of 100 µm, a diffusion coefficient of 2 × 10-7 cm2/s1, which is reasonable for the HRP protein having a molecular mass of 45 000, and adjusting iteratively the unknown parameters ΓS, kf, kb, ki, and k-i to fit the simulated coveragetime curves to the experimental data (Figure 9), we were able to get a rough estimate of ΓS) 8 pmol/cm2 and an affinity binding constant K ) kf/kb) 5 × 107 M-1. The estimated value of saturation surface coverage closely corresponds to the theoretical value that can be calculated for a compact monolayer (8.5 pmol/cm2) of HRP assuming a projected area of ∼20 nm2 per molecule. The affinity binding of the His-HRP is on the same order of magnitude as the binding constant recently found for a His-tagged antibody toward a Ni-NTA lipid bilayer on an acoustic waveguide device (K ) 2 × 107 M-1).10a This level of binding, however, is not as high as that found in the affinity of avidin for biotin, but it is higher than the specific interaction of the sugar residues of HRP with a selfassembled monolayer of boronic acid (K ) 4.9 × 106 M-1).33 Immobilization of the Recombinant Enhanced His-Tagged Green-Fluorescent Protein. To demonstrate the potential use of our modified electrodes for the specific immobilization of a genetically His-tagged protein, we investigated the specific immobilization of an enhanced green-fluorescent protein (GFP) tagged by an N-terminal hexahistidine tail. The enhanced His-GFP is a mutant form of the wild-type GFP, which was shown to fluoresce (at 510 nm) 35 times more brightly than native GFP.34 We took advantage of this strong fluorescence by imaging His-tagged GFP on the electrodes functionalized by CuNTA with an incident-light fluorescence microscope, as shown in Figure 10. The fluorescence image of an NTA electrode treated with Cu2+ and enhanced His-GFP show an obvious green fluorescence, indicating the presence of the protein on the electrode surface, whereas no fluores(33) Abad, J. M.; Velez, M.; Santamaria, C.; Guisan, J. M.; Matheus, P. R.; Vazquez, L.; Gazaryan, I.; Gorton, L.; Gibson, T.; Fernandez, V. M. J. Am. Chem. Soc. 2002, 124, 12845. (34) Cormack, B.; Valdivia, R.; Falkow, S. Gene 1996, 173, 33.

3372


Blankespoor et al.

carbon felt that, owing to their high specific area, might be used for heavy metal decontamination. Experimental Section

Figure 10. (B) Fluorescence images (10× magnification) of the surface of NTA-functionalized SPEs pretreated successively for 30 min with 1 mM Cu2+ and then for 5 min with 500 µg/mL of His-tagged enhanced GFP in PB containing 50 µM EDTA. (A) As in B, but without Cu2+ pretreatment.

cence could be detected in the absence of copper pretreatment. The fluorescence was also observed to be relatively homogeneously distributed on the surface of the SPE, which shows that the ligands are not uniquely grafted on the graphite particles but are also found on the surrounding polystyrene binder of the conductive ink. Conclusions We have shown for the first time that it is possible to covalently attach in a single-step dense monolayers of NTA and IDA ligands to the surface of carbon electrodes via the electrochemical reduction of their aryl diazonium salts in aqueous acid. When these monolayers are loaded with metal ions such as Cu(II) or Ni(II), the modified electrodes are able to specifically bind histidine-tagged proteins derived from horseradish peroxidase and a recombinant, enhanced green-fluorescent protein. We have shown that there are a number of advantages in using this method to concentrate active biomolecules at a surface. First, the ligands needed to accomplish the electrochemical attachment are easy to synthesize and the procedure anchoring them to the surface is simple, efficient, and fast. This is a significant advantage over the preparation of gold electrodes with self-assembled monolayers of thiols, which generally requires hours of incubation in the thiol solutions.7a,b,f A second advantage of our method is the covalent linkage of the metal-chelating species that provides a stable monolayer and a tether with a large potential window. This makes it possible to accurately determine the amount of deposited ligand exclusively on the conductive surface regardless of the size and shape of the electrode. A final advantage of this method of surface modification is that it should be useful in reversibly immobilizing with a controlled molecular orientation a wide variety of histidine-tagged proteins at a surface without significant loss of biological activity. It should find application, therefore, in the assemblage of three-dimensional structures of redox multi-enzyme complexes, and also in areas such as proteomics, drug screening, and electrochemical biosensors. We hope that this work will stimulate studies aimed at a better understanding of the direct electrical communication between the electrode surface and redox proteins. Toward that goal, we are now using NTA- and IDA-grafted electrodes to favor the direct electron transfer between histidine-tagged redox proteins and carbon surfaces when the former adopt a controlled molecular orientation on the carbon surface. Finally, the NTA- and IDA-grafted electrodes might find applications in analytical chemistry for the selective detection of copper(II) traces and also in the environmental field, e.g., by the preparation of NTA- or IDA-grafted

Reagents. Lyophilized HRP (mainly composed of isoenzyme C) was purchased from Sigma (type VI; RZ ) 3.1). CuCl2‚2 H2O and NiCl2‚6 H2O were obtained from Aldrich. The chromogenic cosubstrate 3,5,3′,5′-tetramethylbenzidine, DETAPAC, and EDTA were supplied by Aldrich. The [OsII(bpy)2pyCl]PF6 was synthesized as previously described.35 Its oxidized form, ([OsIII(bpy)2pyCl](PF6)2), was obtained by chemical oxidation with AgPF6 (Aldrich). Hydrogen peroxide (50%) was supplied by Prolabo (reagent-grade product). Its concentration was determined by permanganate titration. Sodium periodate, L-histidine, sodium cyanoborohydride, and NaNO2 were reagent p.a. grade and were purchased from Aldrich. The enhanced His-tag GFP was prepared and purified by the group of Pr. Xavier Santarelli according to their published procedure.36 The phosphate buffer (PB, 4.3 mM NaH2PO4, 15.1 mM Na2HPO4, and 50 mM NaCl, pH 7.4, leading to an ionic strength of 0.1 M) and all of the other aqueous solutions were prepared using water purified by a Milli-Q water purification system from Millipore. Chemical Derivatization of Horseradish Peroxidase by Histidinyl Residues (His-HRP). Histidine was covalently coupled to the shell of carbohydrates surrounding horseradish peroxidase according to a previously described procedure.37 Briefly, the carbohydrates of the HRP (1.25 mL of a 2 mg/mL solution in pH 5.5 acetate buffer) were oxidized with sodium periodate (1.25 mL of a 27 mM aqueous solution freshly prepared) for 90 min in the dark at room temperature. The reaction was stopped by removing excess periodate by gel filtration on a PD10 column. The oxidized HRP contained in the first 3.5 mL eluted from the column with PB (pH 7.4) was collected, and L-histidine was added (20.9 mg). The reaction mixture was left in the dark for 2 h at room temperature. The Schiff base formed was reduced by sodium cyanoborohydride (10 µL of a 5 M solution in PB) in the dark for 60 min at 4 °C. The excess cyanoborohydride and histidine were remove by centrifugation (14 000g for 10 min) of the mixture through the membrane of a microconcentrator (15 000 MW cutoff, Nanosep, Pall). The centrifugation was repeated six times using PB for washing. The concentration of modified horseradish peroxidase was determined spectrophotometrically using the Soret extinction coefficient of 102 mM-1 cm-1 at 403 nm and its activity assayed as previously reported using cyclic voltammetry32 or by monitoring the absorbance change at 450 nm resulting from the HRP conversion of the 3,5,3′,5′-tetramethylbenzidine (1 mM) to a yellow diphenoquinoimine in the presence of H2O2 (1 mM).17,38 The average number of histidine residues attached per HRP molecule was determined from an amino acid analysis after hydrolysis in 6 M HCl for 72 h at 110 °C. Electrochemical Equipment. Cyclic voltammetry and chronoamperometry were carried out with a PST 20 Autolab potentiostat (Eco-Chemie) interfaced with a PC computer. Graphite-based screen-printed electrodes with a 0.096 cm2 sensing disk surface area were prepared from a homemade graphite-based ink composed of graphite particles (Ultra Carbon, UCP 1M, Johnson Matthey) and polystyrene (3:2 weight ratio).39 A fresh surface of highly oriented pyrolytic graphite (HOPG) plate (Carbon Lorraine) was prepared by removing several graphite layers with an adhesive tape. This surface was covered with an electrical-insulating, self-adhesive tape with an eyelet (5 mm diameter, i.e., 0.2 cm2) that formed a low-volume electrochemical cell to which droplets of 30-50 µL could be (35) Kober, E. M.; Caspar, J. V.; Sullivan, B. P.; Meyer, T. J. Inorg. Chem. 1988, 27, 4587. (36) Dieryck, W.; Noubhani, A. M.; Coulon, D.; Santarelli X. J. Chromatogr. B 2003, 786, 153. (37) Chaga, G. Biotechnol. Appl. Biochem. 1994, 20, 43. (38) Porstmann, B.; Porstmann, T. In Non Isotopic Immunoassay; Ngo, T. T., Ed.; Plenum Press: New York, 1988; pp 57-84. (39) Bagel, O.; Limoges, B.; Scho¨llhorn, B.; Degrand, C. Anal. Chem. 1997, 69, 4688.

Dense Monolayers of Metal-Chelating Ligands deposited without spreading out. A glassy carbon electrode (disk area of 0.071 cm2) sealed in epoxy resin was polished to a mirror finish with suspensions of alumina (0.05 µm) and sonicated for a few minutes in ethanol before use. A saturated calomel electrode (SCE) was employed as a reference electrode. The counter electrode was a platinum wire. With the exception of voltammetric measurements at HOPG electrodes, which were carried out at room temperature, voltammetric experiments were performed in a water-jacketed electrochemical cell maintained at 20 ( 0.5 °C with a circulating water bath. When necessary, solutions in the electrochemical cell were deaerated with argon gas. Electrochemical Functionalization of the Electrodes. The general procedure used for electrode modification is as follows. The diazonium reagent was generated by combining 44 µL of an ice-cold solution of NaNO2 in water (7.6 mg/mL) with an ice-cold 1 N HCl solution containing 4 µmol of the aniline derivative. The reaction was allowed to proceed for 5 min before 1 mL of the resulting mixture was transferred to an electrochemical cell containing 2 mL of 0.1 N HCl at 4 °C.40 An SPE or GC electrode was then lowered into the diazonium solution at 4 °C, and a constant negative potential was applied for a specified time to covalently attach NTA or IDA to the electrode surface. After modification, the electrode was carefully rinsed with water. Since the cathodic peak current of the diazonium ion did not decrease appreciably over several hours at 4 °C, a series of electrodes could be modified using the same solution. Protein Immobilization Procedures. All experiments were performed at room temperature. The IDA- or NTA-modified electrodes were saturated with metal ions by dipping the electrodes in PB containing 1 mM CuCl2 or NiCl2 for 30 min, followed by thorough washing with Milli-Q water. The loaded electrodes could be left on the benchtop for days before use. The specific immobilization of His-HRP on the surface of metalchelating electrodes was carried out by dipping the modified electrodes in 2 mL of PB (pH 7.4) containing His-HRP (concentration ranging from 2 to 200 nM) and 2.5 µM DETAPAC for different incubation times (10 min to 25 h). Indirect Determination of Copper(II) Chelated on the Surface of NTA- or IDA-Electrodes by Anodic Stripping Voltammetry. After the NTA- or IDA-modified carbon electrode (SPE, GC, or HOPG electrodes) was loaded with copper(II) and rinsed, a small drop of 1 M HCl (50 µL) was placed on its surface to detach copper (II) from the surface and dissolve it in the acidic drop. The amount of copper(II) contained in the drop was then determined at a microband electrode coated with a thin mercury film using the technique of square wave anodic stripping voltammetry. The carbon microband electrode was prepared from a screen-printed electrode as previously described,41 and the mercury film was deposited on the carbon microband by applying a potential of -1.0 V for 15 min in a 0.1 M HCl solution containing 100 mg/L mercury(II). Next, the potential was set at -0.1 V for 1 min and the freshly prepared thin mercury film-coated microband electrode was then ready for trace measurement of copper(II). After the immersion of a small SCE reference electrode, a platinum wire counter electrode and the thin mercury filmcoated microband electrode into the drop of 50 µL, the stripping signals was recorded in square-wave voltammetry using the following parameters: deposition potential of -1.3 V, deposition time of 2 min, square-wave amplitude of 50 mV, frequency of 100 Hz, and step potential of 4 mV. The well-defined copper peak located at -0.25 V was used as the analytical response, and the concentrations of copper(II) contained in the 50 µL drop were determined from a standard calibration curve obtained under the same experimental conditions. X-Ray Photoelectron Spectroscopy (XPS). XPS signals were recorded using a VG Scientific ESCALAB 250 system equipped with a micro-focused, monochromatic Al KR X-ray source (1486.6 eV) and a magnetic lens that increases the electron acceptance angle and, hence, the sensitivity. An X-ray beam with a size of 650 µm was used at a power of 20 mA × 15 kV. The (40) The diazotization of aniline is a fast and quantitative reaction. Considering that the aniline derivatives were totally converted into diazoniums, the final concentration of the diazoniums in the electrochemical cell should be 1.3 mM. (41) Authier, L.; Grossiord, C.; Brossier, P.; Limoges, B. Anal. Chem. 2001, 73, 4450.

Langmuir, Vol. 21, No. 8, 2005 3373 spectra were acquired in the constant analyzer energy mode, with a pass energy of 150 and 40 eV for the survey and the narrow regions, respectively. The carbon plates were mounted on conducting, double-sided adhesive tapes as a precaution for any static charge that might build up without the charge compensation system turned on. The pressure in the analysis chamber was typically 2 × 10-9 mbar. The Avantage software, version 1.85, was used for digital acquisition and data processing (quantification of peak fitting). The surface compositions (in atomic %) were determined from the integrated peak areas of C1s, N1s, and Cu2p and their respective sensitivity factors. The fractional concentration of a particular element A was computed using the following equation:

%A )

IA/sA

∑(I /s ) n

× 100%

n

where In and sn are the integrated peak areas and the manufacturer’s sensitivity factors, respectively. Fluorescence Measurements. The electrode surfaces were imaged with an inverted epifluorescence microscope (Olympus IX51) equipped with a 100 W Hg arc lamp for epi-illumination. A fluorescence filter set with an excitation filter at 480 nm (slit width of 40 nm) and an emission barrier filter at 510 nm were selected. Images were collected with a digital camera. Synthesis of 4-Aminophenyl-IDA (3). N,N-Di-(carbo-tbutoxymethyl)-2-(4-nitrophenyl)ethanamine (1). Into a roundbottom flask equipped with magnetic stirring and continuous bubbling with N2 gas, dry DMF (40 mL) was added to 3.04 g (15 mmol) of 2-(4-nitrophenyl)ethylamine hydrochloride (Aldrich). After the solid dissolved, 31.4 mL (150 mmol) of diisopropylethylamine (DIPEA, Aldrich) was introduced with stirring followed by the dropwise addition over 30 min of a solution prepared by combining 10 mL of tert-butyl bromoacetate (135 mmol, Aldrich) with 17 mL of dry DMF. To this yellow-brown mixture was added 2.5 g of potassium iodide (15 mmol), and the solution color immediately faded. The solution was allowed to stand in the dark at room temperature under N2 for 17 h. The progress of the reaction was followed by thin-layer chromatography (TLC) on silica gel using a 2:1 mixture of cyclohexaneethyl acetate, and visualization was accomplished using ninhydrin. Once the reaction was complete, the solution was filtered and the solvent removed under reduced pressure (rotary evaporator). The brown oil residue was next dissolved in 150 mL diethyl ether and washed successively with 3 × 75 mL of water, 40 mL of a saturated solution of NaCl, 40 mL of a solution of sodium thiosulfate, and finally 50 mL of water. The organic phase was dried over MgSO4 and filtered, and the solvent was removed under reduced pressure. The resulting viscous brown oil was chromatographed on silica gel and eluted with cylohexane-ethyl acetate (1:1). A single yellow band was collected and evaporated to dryness, giving 5.18 g (13.1 mmol, 87%) of 1 as an amber oil. 1H NMR (200 MHz, CDCl ), δ: 1.47 (s, 18H), 2.97 (m, 4H), 3.46 3 (s, 4H), 7.40 (d, 2H, J ) 4.4 Hz), 8.14 (d, 2H, J ) 4.4 Hz). MS (EI), m/z (relative intensity): 395 (5, [M + H]), 339 (5), 283 (18), 258 (15), 237 (100), 202 (16), 191 (20), 150 (26), 146 (86). Anal. Calcd for C20H30O6N2: C, 60.89; H, 7.67; N, 7.10. Found: C, 59.08; H, 7.64; N, 6.71. N,N-Di-(carbo-t-butoxymethyl)-2-(4-aminophenyl)ethanamine (2). To a solution of 5.06 g (12.8 mmol) of 1 in 200 mL of methanol was added 0.5 g of 10% Pd-C. The suspension was stirred for 22 h at room temperature under a slight positive pressure of hydrogen gas. The reaction was followed by TLC on silica gel using a 1:1 mixture of cyclohexane-ethyl acetate containing a few drops of aqueous ammonia (28%). The starting nitro compound has an Rf value of 0.96, whereas the aniline product has an Rf value of 0.6 (visualized using ninhydrin). Once the reaction was completed, the solution was filtered and the solvent was removed under reduced pressure. The resulting viscous liquid was chromatographed on silica gel and eluted with cylohexane-ethyl acetate (1:1). The first fraction was collected, and removal of the solvent gave 3.65 g (10 mmol, 78%) of the desired compound 2 as a pale yellow solid: mp 75 ( 1 °C.1H NMR (200 MHz, CDCl3), δ: 1.47 (s, 18H), 2.81 (m, 4H), 3.49 (s, 4H), 6.61 (d, 2H, J ) 4.4 Hz), 7.00 (d, 2H, J ) 4.4 Hz). 13C NMR

3374


(50 MHz, CDCl3), δ: 28.1, 33.9, 56.0, 56.5, 80.9, 115.2, 129.5, 129.9, 129.9, 144.4, 170.6. Anal. Calcd for C20H32O4N2: C, 65.90; H, 8.86; N, 7.69. Found: C, 65.90; H, 9.00; N, 7.71%. N,N-Di-(carboxymethyl)-2-(4-aminophenyl)ethanamine (3). The ester groups contained in 2 were hydrolyzed by slowly adding 80 mL of a solution of HCl (30%) over 2.0 g (5.5 mmol) of compound 2 cooled in an ice bath. Once the solid was completely dissolved, the solution was heated at 40 °C for 48 h. The progress of the reaction was monitored by TLC (silica gel) using methanolethyl acetate (1:1) containing few drops of aqueous ammonia (28%) as the eluant. The monoacid product has an Rf value of 0.63, whereas the desired diacid 3 does not migrate. When the hydrolysis was complete, the acidic solution was freeze-dried to give 1.56 g (4.8 mmol, 87%) of 3 as an off-white hygroscopic powder. 1H NMR (200 MHz, D2O), δ: 3.19 (t, 2H), 3.67 (t, 2H), 4.18 (s, 4H), 7.44 (m, 4H). 13C NMR (50 MHz, D2O + DMSO-d6), δ: 30.5, 56.2, 58.2, 125.0, 130.4, 132.0, 138.3, 169.1. Anal. Calcd for C12H18O4N2Cl2‚1.5 H2O: C, 40.90; H, 6.53; N, 7.95; Cl, 20.16. Found: C, 39.02; H, 6.30; N, 7.68; Cl, 21.65. Synthesis of 4-Aminophenyl-NTA (6). p-Nitrobenzoic Acid, NHS Ester (4). To 80 mL of anhydrous THF with stirring was added 4.00 g (24.0 mmol) of p-nitrobenzoic acid, 5.44 g (26.4 mmol) of 1,3-dicyclohexylcarbodiimide (DCC), and 3.04 g (13.2 mmol) of N-hydroxysuccinimide (NHS). A solid separated out almost immediately. After being stirred overnight, the mixture was cooled in an ice bath and vacuum filtered, giving 10.8 g of an off-white solid. The solid was combined with 100 mL of water and stirred at room temperature for 20 min to remove a small amount of unreacted NHS, which is soluble in water. After the solid was collected by vacuum filtration, it was combined with 200 mL of acetone and stirred at room temperature for 30 min to separate the NHS ester from the urea, which is only slightly soluble in acetone. After the urea was removed by vacuum filtration, the acetone in the filtrate was evaporated in a rotary evaporator leaving 4.20 g of solid. Recrystallization from heptane-toluene gave 3.88 g (61%) of the NHS ester as light yellow plates. An analytically pure sample of the NHS ester 4 was obtained using flash chromatography (silica gel, 9:1 methylene chloride-acetone): mp 213-4 °C. IR (KBr) 3118, 2992, 2946, 1771, 1740, 1531, 1353, 1197, 1066, 1008, 862, 712 cm-1. 1H NMR (400 MHz, d6-acetone) δ: 8.39-8.42 (m, 2H), 8.46-8.50 (m, 2H), 2.99 (s, 4H). 13C NMR (400 MHz, d6-acetone) δ: 169.64, 160.95, 152.04, 131.83, 130.84, 124.51, 25.75. Anal. Calcd for C11H8N2O6: C, 50.01; H, 3.05; N, 10.60. Found: C, 49.66; H, 3.55; N, 10.72. (S)-N-[5-(p-Nitrobenzoylamino)-1-carboxypentyl]iminodiacetic Acid (5). To 25 mL of dry DMF was added 1.050 g (ca. 3.61 mmol) of (S)-N-(5-amino-1-carboxypentyl)iminodiacetic acid (NRNR-bis(carboxymethyl)-L-lysine hydrate, ca. 90%, Fluka). The mixture was heated at 70 °C for 1 h under argon, resulting in the partial dissolution of the lysine derivative. After 1.050 g (3.97 mmol) of the NHS ester of p-nitrobenzoic acid was added to the DMF solution, the mixture was heated at 100 °C for 2 h, during which time the lysine derivative and NHS ester completely dissolved. After cooling, the bulk of the DMF was removed by vacuum distillation (bp 30-40 °C), leaving a viscous, yellow oil. The oil was combined with 25 mL of water and 2.0 mL of ethanol and heated to 80-85 °C. Upon cooling, a gummy solid separated out that was collected by decanting the mother liquor. After being dryed at 70 °C (0.10 Torr), the off-white solid (0.985 g) was dissolved in 50 mL of acetone near its boiling point. A small amount of an insoluble solid was removed by gravity filtration, and the acetone in the filtrate was removed under reduced pressure in a rotary evaporator. The residue was recrystallized from water-ethanol to give 650 mg (40%) of the desired compound 5 as an off-white solid. An analytically pure sample was obtained by recrystallization from toluene-acetonitrile followed by recrystallization from water. The solid softened and slowly began to melt at 155 °C. IR (KBr) 3328, 3028, 2957, 2871, 1735, 1636, 1600, 1524, 1414, 1352, 1347, 1257, 867, 846, 721 cm-1. 1H NMR (400 MHz, d6-acetone) δ: 8.27-8.31 (m, 2H), 8.10-8.14 (m, 2H), 3.70 (d, J ) 18 Hz, 2H), 3.62 (d, J ) 18 Hz, 2H), 3.51 (dd, J ) 6.0 and 8.8 Hz, 1H), 3.44 (q, J ) 6.0 Hz, 2H), 1.83-1.92 (m, 1H), 1.60-1.78 (m, 4H), 1.51-1.60 (m, 1H); 13C NMR (400 MHz, d6acetone) δ: 173.73, 173.61, 165.12, 149.64, 141.07, 128.76, 123.58, 65.56, 54.87, 39.68, 29.66, 29.07, 23.85. MS (CI): 412 (M + H).

Blankespoor et al. Anal. Calcd for C17H21N3O9: C, 49.64; H, 5.15; N, 10.21. Found: C, 48.75; H, 5.22; N, 9.98. (S)-N-[5-(p-Aminobenzoylamino)-1-carboxypentyl]iminodiacetic Acid (6). To a solution of 237 mg (0.660 mmol) of 5 in 25 mL of methanol was added 25 mg of 10% Pd-C, and the resulting mixture was hydrogenated for 1 h at 40-45 psi. After removal of the catalyst by filtration, the methanol was removed under reduced pressure in a rotary evaporator leaving an off-white solid. Further drying was accomplished in a vacuum desiccator giving 207 mg (95%) of compound 6. IR (KBr) 1655, 1642, 1630, 1618, 1611, 1599, 1553, 1509, 1401, 1342, 1336, 1330, 1322, 1316, 1194, 1051, 862, 846 cm-1. 1H NMR (400 MHz, D2O) δ: 7.85 (d, J ) 7.6 Hz, 2H), 7.48 (d, J ) 7.6 Hz, 2H), 3.91-4.00(m, 5H), 3.41 (broad s, 2H), 1.87-2.00 (m, 2H), 1.54-1.70 (m, 4H). 13C NMR (400 MHz, D2O) δ: 172.24, 170.29, 169.66, 134.42, 134.08, 129.08, 123.00, 67.91, 55.22, 39.58, 28.16, 26.78, 23.55. MS (CI): 382 (M + H). Anal. Calcd for C17H23N3O7: C, 53.53; H, 6.08; N, 11.02. Found: C, 52.95; H, 6.26; N, 10.82. Synthesis of 1,1′-Bis(N-imidazolylmethyl)ferrocene (7). 7 was prepared from 1,1′-bis(pyridiniummethyl)ferrocene dibromide using the procedures previously published for the synthesis of the 1-ferrocenylmethylimidazole.42 The starting compound, 1,1′-bis(pyridiniummethyl)ferrocene dibromide, was synthesized according to a published procedure43 and converted to 1,1′-bis(pyridiniummethyl)ferrocene hexafluorophosphate by adding slowly at room temperature a saturated aqueous solution of potassium hexafluorophosphate to a saturated aqueous solution of 1,1′-bis(pyridiniummethyl)ferrocene dibromide until no further precipitation occurred. The mixture was cooled at 0 °C for 2 h and filtered to afford the desired hexafluorophosphate salt of the ferrocene derivative. A mixture of 700 mg (1.3 mmol) of 1,1′-bis(N-methylpyridinium)ferrocene hexafluorophosphate and 900 mg (6.5 mmol) of imidazole in 30 mL of acetonitrile was refluxed for 48 h. The solvent was next evaporated under reduced pressure, and the remaining viscous liquid residue was dissolved in water. An aqueous solution of 1 N HCl was added, adjusting the pH below 3, and the solution was extracted two times with dichloromethane. Then, the pH of the aqueous phase was adjusted to 9-10 with NaOH (3 N) followed by the extraction of the yellow aqueous phase with dichloromethane. The collected organic fractions were dried over anhydrous magnesium sulfate. Evaporation of the solvent and subsequent chromatographic purification on silica gel (20% methanol in dichloromethane) afforded 226 mg (51%) of 7. 1H NMR (400 MHz, CDCl3) δ: 4.16 (s, 8H, Cp), 4.78 (s, 4H, CH2), 6.89 (s, 2H, imidazole), 7.00 (s, 2H, imidazole), 7.46 (s, 2H, imidazole H-2). 13C NMR (100 MHz, CDCl3) δ (ppm): 59.7 (CH2), 69.1 (Cp), 69.4 (Cp), 83.3 (CpC-1), 118.7 (imidazole), 128.9 (imidazole),137 (imidazoleC-2). MS (CI+, NH3) m/z (relative intensity): 347 (43, [M + H]), 199 (44, [M + H - C3H4N2]), 147 (100). 7 was further purified by precipitation of its protonated form from an acidic aqueous solution of hydrochloric acid (1 M) to which a saturated aqueous solution of potassium hexafluorophosphate was added slowly at room temperature. After the mixture was cooled at 0 °C for several hours, the precipitate was filtered to afford the pure hexafluorophosphate salt of 7: mp 173-175 °C (decomposition). 1H NMR (250 MHz, DMSO-d6) δ: 3.4 (s, broad, 2H, N-H), 4.33 (d, 4H, J ) 1.5 Hz, Cp), δ 4.35 (d, 4H, J ) 1.5 Hz, Cp), 5.24 (s, 4H, CH2), 7.68 (s, 2H, imidazole), 7.77 (s, 2H, imidazole), 9.14 (s, 2H, imidazoleH-2). 13C NMR (100 MHz, DMSO-d6) δ: 49.6 (CH2), 70.7 (Cp), 70.8 (Cp), 82.8 (Cp C-1), 121.0 (imidazole), 122.5 (imidazole),135.6 (imidazoleC-2). MS (FAB+), m/z (relative intensity): 347.1 (10, [M - H - PF6]), 279 (14, [M - C3H4N2]), 169.2 (100). Anal. Calcd for C18H20F12FeN4P2: C, 33.88; H, 3.16; N, 8.78. Found: C, 33.46; H, 3.18; N, 8.60.

Acknowledgment. The authors would like to acknowledge Dr. Moahamed M. Chehimi for XPS experi(42) (a) Howard, J.; Thomas, J.-L.; Hanlon K.; McGuirk, D. Synth. Comm. 2000, 30, 1865. (b) Bildstein, B.; Malaun, M.; Kapocka, H.; Ongania, K.-H.; Wurst, K. J. Organomet. Chem. 1998, 552, 45. (43) Georgopoulou, A. S.; Mingos, D. M. P.; White, A. J. P.; Williams, D. J.; Horrocks, B. R.; Houlton, A. J. Chem. Soc., Dalton Trans. 2000, 17, 2969.


Langmuir, Vol. 21, No. 8, 2005 3375

ments, He´le`ne Pelle´ for its technical assistance during synthesis, and Professor Xavier Santarelli for his generous gift of the His-tag enhanced GFP.

IDA and CuCl2, mechanism of HRP, and numerical resolution of eqs 4 and 5. This material is available free of charge via the Internet at http://pubs.acs.org.

Supporting Information Available: Voltammetric experiments in homogeneous solution containing 4-aminophenyl-

LA047139Y

Dense Monolayers of Metal-Chelating Ligands Covalently Attached to

Recommend Documents