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Touching Surface-Attached Molecules with a Microelectrode: Mapping the Distribution of Redox-Labeled Macromolecules by Electrochemical-Atomic Force Microscopy Agne`s Anne,† Edmond Cambril,‡ Arnaud Chovin,† and Christophe Demaille*,† Laboratoire d’Electrochimie Mole´culaire, Unite´ Mixte de Recherche Universite´ CNRS No. 7591, Universite´ Paris Diderot-Paris 7, 15 Rue Jean-Antoine de Baı¨f, 75205 Paris Cedex 13, France, and Laboratoire de Photonique et de Nanostructures (LPN/CNRS), Route de Nozay, 91460 Marcoussis, France We report on the development of a mediator-free electrochemical-atomic force microscopy (AFM-SECM) technique designed for high-resolution imaging of molecular layers of nanometer-sized redox-labeled (macro)molecules immobilized onto electrode surfaces. This new AFMSECM imaging technique, we call molecule touching atomic force electrochemical microscopy (Mt/AFM-SECM), is based on the direct contact between surface-anchored molecules and an incoming microelectrode (tip). To validate the working-principle of this microscopy, we consider a model system consisting of a monolayer of nanometer long, flexible, polyethylene glycol (PEG) chains covalently attached by one extremity to a gold surface and bearing at their free end a ferrocene (Fc) redox tag. Using Mt/AFM-SECM in tapping mode, i.e., by oscillating the tip so that it comes in intermittent contact with the grafted chains, we show that the substrate topography and the distribution of the redox-tagged PEG chains immobilized on the gold surface can be simultaneously and independently imaged at the sub-100 nm scale. This novel type of SECM imaging may be found useful for characterizing the surface of advanced biosensors which use electrodegrafted, redox-tagged, linear biochains, such as peptides or DNA chains, as sensing elements. In principle, Mt/ AFM-SECM should also permit in situ imaging of the distribution of any kind of macromolecules immobilized on electrode surfaces or simply conducting surfaces, provided they are labeled by a suitable redox tag. Scanning electrochemical microscopy (SECM) is a near-field technique initially developed to map the chemical reactivity of surfaces.1 In the conventional SECM feedback mode,2 an ultramicroelectrode, used as a local probe and called a “tip”, approaches within a few tip radii distance from an electrochemically active sample surface (substrate) immersed in an electrolyte containing * To whom correspondence should be addressed. E-mail: demaille@ univ-paris-diderot.fr. † Universite´ Paris Diderot-Paris 7. ‡ Laboratoire de Photonique et de Nanostructures (LPN/CNRS). (1) Bard, A. J. In Scanning Electochemical Microscopy; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker: New York, 2001; pp 1-15. (2) Bard, A. J.; Fan, F.-R.; Kwak, J.; Lev, O. Anal. Chem. 1989, 61, 132–138. 10.1021/ac1012464 2010 American Chemical Society Published on Web 07/06/2010
a soluble redox mediator, for example, in its reduced form. The tip is biased at an appropriate potential so that the mediator is oxidized at a diffusion-controlled rate. The tip-generated oxidized form of the mediator reaches the substrate by diffusion where it is reduced and fed back to the tip. This so-called positive feedback redox cycling results in an increase of the tip current that depends jointly on the intrinsic reactivity of the substrate and on the tip-substrate distance. Providing this distance is kept constant, the tip can also be scanned over the substrate surface and the current at the tip is measured as a function of the tip lateral position for obtaining images of the electrochemical reactivity of the sample surface.3,4 Thanks to its spatial resolution capability, this technique is involved in a large variety of applications recently reviewed.5-7 SECM can also be used to characterize surfaces bearing redox species, which can be surface-immobilized either as molecular monolayers or embedded within thin polymeric films. For this application, the tip-generated form of the mediator exchanges electrons with the surface-confined redox species rather than with the underlying substrate surface itself. SECM imaging then allows one to map the 2D distribution of the surfacebound redox species covering the substrate surface. This specific use of SECM has initially been demonstrated using composite substrates bearing thin films of redox polymers8 and also used to image the surface of conducting polymers.9-11 More recently the same technique was used to locate minute amounts of redox tagged proteins films on surfaces.12 Much thinner molecular layers (3) Fan, F.-R. F. In Scanning Electrochemical Microscopy; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker: New York, 2001; pp 111-143. (4) Laforge, F. O.; Velmurugan, J.; Wang, Y.; Mirkin, M. V. Anal. Chem. 2009, 81, 3143–3150. (5) Wittstock, G.; Burchardt, M.; Pust, E. S.; Shen, Y.; Zhao, C. Angew. Chem., Int. Ed. 2007, 46, 1584–1617. (6) Sun, P.; Laforge, F. O.; Mirkin, M. V. Phys. Chem. Chem. Phys. 2007, 9, 802–823. (7) Roberts, W. S.; Lonsdale, D. J.; Griffiths, J.; Higson, S. P. J. Biosens. Bioelectron. 2007, 23, 301–318. (8) Lee, C.; Bard, A. J. Anal. Chem. 1990, 62, 1906–1913. (9) Borgwarth, K.; Heinze, J. In Scanning Electrochemical Microscopy; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker: New York, 2001; pp 201-238. (10) Cheol Jeon, I.; Anson, F. C. Anal. Chem. 1992, 64, 2021–2025. (11) Borgwarth, K.; Ebling, D.; Heinze, J. Electrochim. Acta 1995, 40, 1455– 1460. (12) Cortes-Salazar, F.; Busnel, J.-M.; Li, F.; Girault, H. H. J. Electroanal. Chem. 2009, 635, 69–74.
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of redox active species, either redox dendrimers,13 thiol porphyrin monolayers,14 or even redox-tagged DNA strands,15-17 were also imaged in this way. Microarrays of ∼10-100 µm sized spots bearing redox active dendrimers or DNA molecules have been so visualized by SECM.15,16 However, within the micrometer resolution of the SECM tip, no details regarding the distribution of the redox active molecules within the assembled layers could be obtained, i.e., the layers were found electrochemically relatively smooth.17 Moreover, a major limitation for using feedback SECM to map molecular layers of surface-bound redox species arises from the fact that if the substrate is conducting, then the soluble mediator has to be prevented from reaching the substrate surface. Otherwise the mediator exchanges electrons with the substrate rather than with the surface-bound redox species, and the positive feedback current recorded becomes unspecific of the species to be probed. In that context, we report here on the development of a mediator-free electrochemical-atomic force microscopy (AFM-SECM)18-20 technique designed for submicrometer resolution imaging of molecular layers of nanometer-sized redox-labeled (macro)molecules immobilized onto electrode surfaces. The development of this new SECM technique is based on our recent finding that molecular layers of redox-tagged nanometer-sized chains, either DNA (oligonucleotide) or polyethylene glycol chains, end-grafted onto electrode surfaces can be directly electrochemically addressed by an incoming microelectrode-probe in the absence of any redox mediator in solution. In other words, we showed that a local probe can be brought in molecular contact with the chains and their redox heads oxidized or reduced upon direct Brownian collision with the tip. Establishing such a delicate contact required that the probe/molecular layer interaction was finely controlled. This was achieved by mounting the microelectrode as a combined AFM-SECM probe which could be positioned with subnanometer precision within the end-grafted layers.21 So far this type of electrochemical microscopy, which for convenience we label Mt/AFM-SECM for molecule touching AFM-SECM, was only used to record contact mode approach curves, with the aim of probing the motional dynamics of the end-grafted chains. The purpose of the present work is to explore the imaging capabilities of Mt/AFM-SECM for mapping the 2D-distribution of electrodegrafted redox labeled macromolecules at the submicrometer-scale (Scheme 1). EXPERIMENTAL SECTION Chemicals. The linear ferrocene-poly(ethylene glycol)3400-S2 ([Fc-PEG3400-S]2)-disulfide molecules (Mw ) 7800; ∼79 mono(13) Nijhuis, C. A.; Sinha, J. K.; Wittstock, G.; Huskens, J.; Ravoo, B. J.; Reinhoudt, D. N. Langmuir 2006, 22, 9770–9775. (14) Lu, X.; Zhang, L.; Li, M.; Wang, X.; Zhang, Y.; Liu, X.; Zuo, G. ChemPhysChem 2006, 7, 854–862. (15) Yamashita, K.; Takagi, M.; Uchida, K.; Kondo, H.; Takenak, S. Analyst 2001, 126, 1210–1211. (16) Wain, A. J.; Zhou, F. Langmuir 2008, 24, 5155–5160. (17) Gorodetsky, A. A.; Hammond, W. J.; Hill, M. G.; Slowinski, K.; Barton, J. K. Langmuir 2008, 24, 14282–14288. (18) Macpherson, J. V.; Unwin, P. R.; Hillier, A. C.; Bard, A. J. J. Am. Chem. Soc. 1996, 118, 6445–6452. (19) Macpherson, J. V.; Unwin, P. R. Anal. Chem. 2000, 72, 276–285. (20) Kranz, C.; Friedbacher, G.; Mizaikoff, B.; Lugstein, A.; Smoliner, J.; Bertagnolli, E. Anal. Chem. 2001, 73, 2491–2500. (21) Abbou, J.; Anne, A.; Demaille, C. J. Am. Chem. Soc. 2004, 126, 10095– 10108.
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Scheme 1. Working Principle of Molecule Touching (Mt) AFM-SECM for Imaging Molecular Layers of Nanometer-Sized Redox Tagged Macromolecules, Such As Linear PEG or DNA Chains
mer units per chain) were custom-synthesized as described elsewhere.22 All chemical and solvents were analytical grade and used without further purification. All aqueous solutions were made with Milli-Q purified water (Millipore). The electrolyte solution was filtered immediately before use on a 0.22 µm nylon Cameo filter. Preparation of the Flat Gold Template-Stripped (TS) Surfaces. Flat gold surfaces were produced by template-stripping of a 200 nm thick gold layer deposited on mica, as previously described.21,23 These template stripped (TS) gold surfaces present large flat areas characterized by a peak to peak roughness in the order of ∼1-2 nm (see Figure S1 in the Supporting Information). The starting gold on the mica surface was purchased from Ssens. Fabrication of the Patterned Gold Surfaces. The patterned gold surfaces, consisting of an array of interdigitized gold bands on Si/SiO2, were fabricated using an electron-beam lithography technique and lift-off resist process, as previously described.24 Briefly, a 1 µm thick insulating layer of silicon oxide (SiO2) was formed on a silicon wafer using the plasma-enhanced chemical vapor deposition (PECVD) process. The metallic gratings are defined by EBL (VISTEC EBPG5000plus Gaussian electron beam lithography system operating at 100 kV) on a layer of a negativetone resist (ma-N 2403, Micro-Resist), spin-coated on the SiO2 layer with a 250 nm thickness. Resist development is performed in a MIF 726 developer, follow-up by the evaporation of a titanium/ gold layer (5 nm/20 nm), which is finally lifted-off in acetone. Titanium was used to improve the gold adhesion onto the nonconductive SiO2 layer. The patterned gold surfaces were characterized by scanning electron microscopy and tapping mode AFM imaging (Figure S2 in the Supporting Information). The width of the gold bands was found to vary slightly from surface to surface from 500 up to 700 nm. The patterned surfaces were systematically cleaned using an UV-ozone cleaner (exposition time 2 min) before grafting of the Fc-PEG chains. Estimating the Effective Surface Area of the Template Stripped and Patterned Gold Substrates. As previously detailed,25 the effective surface area of the template stripped (TS) (22) Anne, A.; Demaille, C.; Ce´dric, G. ACS Nano 2009, 3, 819–827. (23) Hegner, M.; Wagner, P.; Semenza, G. Surf. Sci. 1993, 291, 39–46. (24) Anne, A.; Cambril, E.; Chovin, A.; Demaille, C.; Goyer, C. ACS Nano 2009, 3, 2927–2940. (25) Anne, A.; Demaille, C.; Moiroux, J. Macromolecules 2002, 35, 5578–5586.
substrate was measured after the Fc-PEG chains had been cathodically stripped, by integration of the reductive stripping peak of the oxide film grown by oxidation of gold in 0.5 M H2SO4. We did not use this method for the patterned substrate for fear of damaging the gold band electrodes. We preferred to estimate the effective surface area of the gold band electrodes of the patterned substrates from the double layer capacitance of the substrate, as measured by cyclic voltammetry (CV) (at +0.02 V/SCE, 1 M NaClO4, 2 V/s), and using a value of ∼7 µF/cm2 for the specific capacity of gold. This later value was measured from the CV recorded at a template stripped surface. Preparation of the Fc-PEG Monolayers on Gold Substrate Surfaces. The substrates were covered with a fresh aqueous solution containing ∼0.5 mM of custom-synthesized Fc-PEG3400disulfide molecules, for ∼2 h, at ambient temperature, under argon, and protected from light. This resulted in the covalent end-grafting of Fc-PEG chains on the gold surface via the formation of a stable gold-“sulfur” bond. The modified gold surfaces were carefully washed with Milli-Q water, then 1 M aqueous NaClO4. Fabrication of Combined AFM-SECM Tips. The tips were fabricated according to a procedure already largely detailed elsewhere.26 Briefly, a 60 µm-diameter gold wire is flattened, and its extremity is successively bent and etched, so as to obtain a flexible cantilever bearing a conical tip with a spherical apex ∼100-300 nm in diameter. The probe is fully insulated by deposition of an electrophoretic paint and glued onto an AFM chip. The apex is selectively exposed in order to act as a current-sensing nanoelectrode. During the fabrication, only the probes presenting a very small leakage current
