- Email: [email protected]
Mapping of enzyme activity by detection of enzymatic products during AFM imaging with integrated SECM–AFM probes
ARTICLE IN PRESS
Ultramicroscopy 100 (2004) 127–134
Mapping of enzyme activity by detection of enzymatic products during AFM imaging with integrated SECM–AFM probes C. Kranza,*, A Kuenga, A. Lugsteinb, E. Bertagnollib, B. Mizaikoffa a b
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA Solid State Electronics Institute, Vienna University of Technology, Floragasse 7, Vienna A-1040, Austria Received 1 July 2003; received in revised form 15 October 2003; accepted 31 October 2003
Abstract With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy (SECM) with AFM has recently been introduced. Simultaneous contact mode imaging of a micropatterned sample with immobilized enzyme spots and imaging of enzyme activity is shown. In contrast to force spectroscopy the conversion of an enzymatic byproduct is directly detected during AFM imaging and correlated to the activity of the enzyme. r 2004 Elsevier B.V. All rights reserved. Keywords: Atomic force microscopy; Scanning electrochemical microscopy; Combined SECM–AFM; Integrated SECM–AFM probes; Enzyme activity; Self-assembled monolayers; Thin films
1. Introduction Immobilization of active proteins at solid substrate materials is of great interest in fabrication of miniaturized biomedical and environmental sensors and devices. Deposition conditions and procedures [1], have to be carefully monitored, since the immobilization process should not induce a substantial decrease of the protein functionality. AFM is a versatile tool for imaging the structure of proteins however, only few contributions report *Corresponding author. Tel.: +1-404-385-1794; fax: +1404-894-7452. E-mail address: [email protected] (C. Kranz).
on imaging protein activity [2,3]. Several approaches have been described for immobilization of proteins: (i) self-assembly of protein–lipid films [4], (ii) Langmuir–Blodgett techniques [5], (iii) entrapment in polymer films [6–9], and (iv) using o-functionalized thiols, disulfides or silanes [10–14]. The latter approach enables covalent attachment of the protein to the terminal functional group. Special care has to be taken immobilizing enzymes at a substrate surface. Due to the immobilization process a loss of catalytic enzymatic function may occur, which is induced by conformational distortion during the attachment process. Hence, it is crucial to correlate deposition protocols to changes in morphology and biological activity.
0304-3991/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2003.10.004
ARTICLE IN PRESS 128
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
Scanning probe microscopies (SPM) are a versatile tool for imaging biological species. AFM is particularly suitable, since it can be operated at ambient physiological conditions. Proteins have widely been studied by AFM [15]. Besides the investigation of structural [16] and mechanical properties [17,18] of individual proteins, real-time probing of protein–protein interaction [19] has been demonstrated. Only few papers describe direct observation of enzyme activity. Following adsorption onto mica surfaces Radmacher et al. [20] directly observed enzyme activity by detecting height fluctuations of the cantilever. Real-time monitoring of single-binding events between acetyl cholinesterase and the substrate acetylcholine have been shown by recording force spectra [21]. In situ studies of single enzymes and indirect mapping of enzyme activity was demonstrated by imaging phospholipase and the time course of lipid bilayer degradation [22,23]. An alternative approach for studying enzyme activity and kinetics is provided by the detection of molecular byproducts during enzymatic conversion. Scanning electrochemical microscopy is an insitu SPM technique and has gained increasing importance for studying biological systems during the last decade [24]. Scanning a biased microelectrode across the sample surface enables the detection of (electro)active species generated at the sample surface or probing the (electro)active properties of the sample surface. In principle, a Faraday current measured at a biased microelectrode due to hemispherical diffusion of a redox species in solution towards this electrode is disturbed by the sample surface chemistry in the proximity of (several electrode radii above) the sample surface. SECM has extensively been applied to investigate enzyme activity in generator/collector mode [25–27] and feedback mode [28–30] operation. Enzyme immunoassays can be investigated and optimized using SECM [31,32]. Recently, comprehensive reviews on biological applications of SECM have been published [24,33]. A major drawback of SECM compared to AFM and other SPM techniques is the lack of sufficient lateral resolution in conventional SECM imaging due to signal-dependent positioning of the
microelectrode [34]. Additionally, scanning in constant height across the sample surface results in a convolution of the electrochemical response and the topographical information. In general, imaging of enzyme activity in generator/collector mode does not provide topographical information when the substrate of the enzymatic reaction is absent. Consequently, any progress towards improved lateral resolution in mapping of enzyme activity and imaging of biological systems in SECM has to address current independent positioning ideally of nanoelectrodes. Thus, precise control on the distance between the scanned electrode tip and the sample surface has to be ensured. An important aspect enhancing the versatility of scanning probe techniques is the approach to integrate various sensors in a single probe. With the integration of submicro- and nanoelectrodes into AFM probes an elegant way of combining scanning electrochemical microscopy with atomic force microscopy has recently been pioneered [35–38]. Complementary electrochemical and topographical information with high-lateral resolution can be obtained in a single time- and spacecorrelated measurement. Imaging with the integrated SECM-AFM probes in AFM tapping mode was successfully applied to soft samples [39,40]. In this contribution simultaneous imaging of immobilized peroxidase activity in generator/collector mode of SECM and contact mode AFM is demonstrated using bifunctional SECM–AFM probes. The electroactive byproduct of the enzymatic reaction diffuses to the integrated submicroelectrode and is converted leading to a Faraday current, which corresponds to the enzyme activity. The current signal can be simultaneously correlated to the obtained topography.
2. Experimental 2.1. Materials Horseradish peroxidase (EC. 1.11.1.7) was purchased from Sigma (Louis, MO), glutaraldehyde, cystaminium dichloride from Fluka (Buchs, Switzerland), and ferroceniummethyl hydroxide
ARTICLE IN PRESS C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
(FMA) from Aldrich (Milwaukee, WI). All other chemicals were of analytical grade and were obtained from Fisher (Pittsburgh, PA) and Mallinckrodt (Phillipsburg, NY), respectively. Buffers were prepared with deionized water (Millipore). The micro-structured gold samples were obtained from Quantifoil Micro Tools (Jena, Germany).
129
glutaraldehyde) was immobilized onto the gold structure for 2 h, forming a surface-grafted crosslinked protein gel on the aminated gold spots. After thoroughly rinsing the sample with buffer solution several times the sample was stored at 4 C until analysis. 2.3. Fabrication of integrated SECM–AFM probes
2.2. Sample preparation The microstructured gold/siliconnitride sample was cleaned in H2SO4:H2O2 (70:30 v/v) for a period of 30 s, rinsed three times with water and then immediately immersed into 0.1 M cystaminium dichloride (in acetate buffer; pH 5.5) for 30 min. Cystaminium dichloride forms a aminofunctionalized self-assembled thiol monolayer on the gold spots. After rinsing the sample 5 times with deionized water, the terminated amino function of the self-assembled monolayer was used to covalently attach the enzyme from suspension. The peroxidase (1 mg peroxidase in 100 ml phosphate buffer (pH 7.0) containing 2.5% (v/v)
A detailed description of integrated SECMAFM probe fabrication is given elsewhere [35–41]. Silicon nitride cantilevers without the gold coating at the backside are partially coated with a 100 nm gold layer on the tip side (see Fig. 1a) and subsequently insulated with a 700 nm layer of xylelene polymer (Parylene C) via vapor deposition polymerization. Three-dimensional focused ion beam (FIB) milling enables reproducible exposure of an electroactive area with defined geometry and dimensions integrated above the apex of the original AFM tip. Finally, the original AFM tip is re-shaped correlating the tip height to the dimensions of the integrated electrode.
Fig. 1. (a) Optical micrograph of a modified silicon nitride cantilever: magnification 10 ; (b) scheme of an integrated SECM–AFM probe; (c) SEM–FIB image of an integrated SECM–AFM probe (electrode edge length: 1 mm, re-shaped AFM tip length: 490 nm); (d) cyclic voltammogram recorded at the integrated SECM/AFM probe in 20 mmol l1 ferrocyanide/0.5 mol l1 KCl electrolyte vs. Ag/AgCl, scan rate 50 mV/s.
ARTICLE IN PRESS 130
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
A schematic representation of an integrated SECM-AFM tip is given in Fig. 1b. Hence, the optimum working distance between electrode and sample surface and a high tip aspect ratio for AFM imaging is ensured (Fig. 1c). The integrated electrode shown in Fig. 1c (electrode edge length: 1 mm; thickness of the gold layer: 100 nm; reshaped AFM tip length: 490 nm) was characterized by recording cyclic voltammograms with 20 mmol l1 ferrocyanide in 0.5 mol l1 KCl supporting electrolyte (Fig. 1d). A typical sigmoidal shape of the voltammogram with a limiting current of 1.1 nA was obtained corresponding well with the theory of ring microelectrodes [42]. 2.4. Combined SECM–AFM measurements The SECM–AFM probes were mounted in a standard atomic force microscope (Nanoscope III, Digital Instruments, Santa Barbara, CA) equipped with a liquid cell and a three-electrode set-up with the integrated probe as working electrode. The fluid inlet and outlet of the liquid cell are used to position a Pt-wire as counter electrode and a quasi-Ag/AgCl reference electrode. The AFM head including the electrochemical cell was shielded with a copper housing to reduce noise in the current signal. The electrochemical experiment was controlled by a bi-potentiostat (CH Instruments 832A CH Instruments, Austin, TX). Images were obtained in contact mode of the AFM and generator/collector mode of the SECM for imaging enzyme activity and in the feedback mode of the SECM for the GaAs/gold sample [43].
3. Results and discussion FIB technology allows fabricating high aspect ratio ultra-sharp AFM tips. Hence, the micromachining steps for fabrication of the integrated SECM-probes can be performed with high reproducibility. Furthermore, re-shaped tips with a similar curvature as the initial tips required for high-quality topographic images are provided. The lateral resolution of the electrochemical response is determined by the size of the integrated electroactive area and the distance to the sample surface
[44]. To demonstrate the capability and resolution of microfabricated combined SECM–AFM probes images of periodic Au/GaAs patterns were recorded in AFM contact mode and SECM feedback mode. The pattern consists of 600 nm GaAs grooves with a depth of 100 nm spaced by 400 nm of gold prepared by FIB milling of a gold-coated GaAs substrate. A quasi-reversible redox couple, e.g. ferrocyanide, is added at one oxidation state (reduced) to the solution. The integrated electrode is biased at 600 mV vs. Ag/AgCl, a potential where ferrocyanide is oxidized to ferricyanide. In large distance to the sample surface a steady-state current occurs due to the electrochemical conversion of the mediator. When the SECM–AFM probe is engaged the distance between the electrode and the sample is exclusively determined by the re-shaped AFM tip, which is located in the center of the frame electrode (see Fig. 1c). The distance is adjusted during FIB milling and amounts in general half of the edge length of an integrated frame electrode. In such close proximity of the electrode to the sample surface, the current recorded at the electrode is influenced by the morphology and nature of the sample surface. The insulating GaAs grooves are blocking the diffusion towards the integrated microelectrode and the current decreases (negative feedback). In contrast, the conductive gold surface enables recycling of the redox mediator (reduction of ferricyanide to ferrocyanide) leading to an increased current (positive feedback) compared to the steady state Faraday current in bulk solution. Fig. 2 shows the decreased current above the GaAs regions and the increased current above the gold surface. The obtained resolution is in good agreement with the theory of the feedback mode and demonstrates that features smaller than the integrated electrode dimensions can clearly be resolved. Likewise, the comparison of the topographical and the electrochemical images shows excellent agreement (Fig. 2). The topographic image quality of the remodeled tip is comparable to non-modified AFM Si3N4 cantilevers as already shown in previous studies of our research group [36]. Imaging enzyme activity in contact mode AFM was performed at a micropattern of isolated enzyme spots (Fig. 3). Enzyme spots were created
ARTICLE IN PRESS C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
131
Fig. 3. Scheme of simultaneous SECM–AFM imaging and the involved reactions at the surface of the spot-immobilized peroxidase recorded with an integrated electrode operating in generation/collection mode. The electron donor FMA is oxidized during the enzymatic conversion of H2O2 and diffuses to the integrated electrode where it is amperometrically detected (dashed arrows).
activity was obtained in generation/collection mode [26]. The measurement solution contained 2.0 mmol l1 FMA and 0.5 mmol l1 H2O2 and 0.l mol l1 KCI in 0.1 M phosphate buffer solution. The potential of the tip electrode was held at 0.05 V vs. Ag/AgCl. Peroxidase catalyzes the following reaction: 2FMA þ H2 O2 þ 2Hþ -2FMAþ þ 2H2 O: Fig. 2. Simultaneously recorded height and current images in AFM contact mode of a periodic Au/GaAs pattern (400 nm gold stripes spaced at 600 nm) showing (a) topography and (b) simultaneously recorded SECM image with the tip-integrated electrode. Redox mediator: 0.03 mol l1 [Fe(CN)6]4 in 0.5 mol l1 KCl electrolyte solution. The tip was held at a potential of 0.6 V vs. quasi-Ag/AgCl reference electrode (AgQRE). Electrode edge length: 800 nm; tip height: 300 nm; scan rate: 3 Hz.
by chemisorption of a functionalized thiol monolayer (cystaminium chloride) onto gold structures produced by a periodically micropatterned silicon nitride layer deposited onto a gold-coated silicone wafer. The terminal group of the chemisorbed monolayer can be used as anchor group for the covalent attachment of biomolecules [27]. Peroxidase was covalently attached to the gold spots by adding a mixture of glutaraldehyde and peroxidase forming a surface grafted crosslinked protein gel at the aminated surface of the self-assembled monolayer. Electrochemical imaging of peroxidase
H2O2 is the substrate of the enzymatic conversion and is reduced to water with hydroxyl methyl ferrocene (FMA), a metal organic electron donor containing Fe2+ added to the solution. Hence, the oxidized species ferrocinium methylhydroxide (FMA+, containing Fe3+) is continuously generated during the enzymatic conversion (Fig. 3, dashed arrow). FMA+ is diffusing to the integrated electrode and can be amperometrically detected at E=0.05 V vs. Ag/AgCl, a potential where no other electroactive species present in solution can be converted. In general, images obtained in generation/collection mode provide higher sensitivity compared to the feedback mode and are particularly suitable for detection of low enzyme activity [26]. Furthermore, in generation/ collection mode any contribution from the underlying gold substrate is eliminated as Wittstock and Schuhmann suggested [27]. The current response increases with reduced electrode-to-sample distance. Hence, with the described integrated SECM–AFM probe the electrode follows the
ARTICLE IN PRESS 132
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
topography of the sample while the distance between the electroactive area and the sample surface remains constant. If sub-micrometer distances are selected for constant height imaging in conventional SECM, tip crashes may occur due to, e.g. a tilted sample or surface features with dimensions corresponding to the electrode–sample distance. However, in order to improve the lateral resolution in generator/collector mode it is preferable to decrease the distance between the microelectrode and sample surface, which is achieved with the presented concept. Moreover, if the electron donor is not present in solution the topography can not be imaged in conventional SECM experiments due to the omnipresent convolution of topography and electrochemical response. With the presented combination the distance between the integrated electrode and the sample surface can be individually designed and fabricated with respect to the application. To date, micropatterns of covalently attached peroxidase with dimensions of approx. 1–2 mm in diameter
could not be successfully imaged in generator/ collector mode of SECM due to the low concentration of immobilized peroxidase and the comparatively larger working distance during conventional SECM experiments [24]. In order to ensure that the electrochemical image is resulting from enzymatic activity the sample was first imaged in a solution containing 2.0 mmol l1 FMA, 0.l mol l1 KCI in 0.1 mol l1 phosphate buffer solution in absence of H2O2. Contact mode topography and the simultaneously obtained electrochemical image were recorded with the integrated SECM–AFM probe (electrode length: 860 nm; re-shaped tip length: 410 nm) poised at a potential of 0.05 V vs. Ag/AgCl. The topography corresponds with the expected surface properties of the sample (Fig. 4a). As clearly shown in Fig. 4b, in absence of the substrate H2O2 in solution no enzymatic reaction occurs and the current recorded at the electrode during AFM contact mode imaging of the immobilized enzyme spots is negligible. Fig. 4a shows the topography of
Fig. 4. Simultaneously recorded height and current images of peroxidase activity in AFM contact mode. Top view of the height (a) and (c), and simultaneously recorded current (b) in absence of the enzymatic substrate H2O2. (d) The electrochemical response with 0.5 mmol l1 H2O2 in solution. Images are recorded in 2 mmol l1 FMA, 0.1 mol l1 KCl in phosphate buffer (0.1 mol l1, pH 7.0) The tip was held at a potential of 0.05 V vs. Ag/AgCl. Electrode edge length: 860 nm; tip height: 410 nm; scan rate: 1 Hz.
ARTICLE IN PRESS C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
the micro-structured gold sample containing immobilized enzyme spots. Fig. 4c and 4d show the topography and the electrochemical response, respectively, corresponding to the enzymatic activity of the peroxidase in the presence of H2O2 imaged with the same integrated SECM–AFM probe. The periodicity of the enzyme-immobilized patterns in the current image corresponds well with the topography provided by the integrated AFM tip. The dark features around the immobilized enzyme spots result from the reshaped AFM tip height of 410 nm, which compares to the topographical features of the sample surface (height of the silicon nitride layer: 450 nm). Hence, if the reshaped AFM tip is above the center of the immobilized enzyme spot the diffusion of FMA+ to the integrated electrode is intermittently blocked. In order to avoid this effect, integrated SECM–AFM probes with a longer reshaped AFM tip can be selected.
4. Conclusion Simultaneous imaging of enzyme activity during contact mode AFM measurements is demonstrated by integrating an electroactive area into an AFM tip (integrated SECM–AFM probe). Different electrode geometries, such as frame-, ring- and disc-electrodes can be integrated into the AFM-tip at an exactly defined distance above the apex using FIB technology as recently demonstrated by our research group. Ongoing work is focused on chemical modification of the tipintegrated electroactive area, e.g. with immobilized enzymes aiming at the development of scanning probe tip-integrated electrochemical nanosensors for characterization of electrochemical surface processes at soft biological samples with highlateral resolution.
Acknowledgements The National Science Foundation (Grant 0216368 within the program ‘Biocomplexity in . the Environment’) and the ‘Fonds zur Forderung der wissenschaftlichen Forschung’ Austria (Grants
133
P14122-CHE and J2230) are greatly acknowledged. PCT patents on this technology are issued (WO01/94877 and WO01/94926).
References [1] D.J. Mueller, H. Janovjak, T. Lehto, L. Kuerschner, K. Anderson, Progr. Biophys. & Mol. Biol. 79 (2002) 1. [2] N.H. Thomson, B.L. Smith, N. Almqvist, L. Schmitt, M. Kashlev, E.T. Kool, P.K. Hansma, Biophys. J. 76 (1999) 1024. [3] B. Waelivaara, A. Askendal, I. Lundstroem, P. Tengvall, J. Coll. Interface. Sci. 187 (1997) 121. [4] Y.F. Dufrene, W.R. Barger, J.D. Green, G.U. Lee, Langmuir 13 (1997) 2271. [5] V. Erokhin, L.A. Feigin, Prog. Colloid Polym. Sci. 85 (1991) 47. [6] A. Akalah, A. Moet, Functionalized Polymers and their Applications, Chapman &Hall, London, 1981. [7] W. Schumann, Microchim. Acta 121 (1995) 1. [8] P.N. Bartlett, J.M. Cooper, J. Electroanal. Chem. 362 (1993) 1. [9] I.S. Gill, F.J. Plou, A. Ballesteros, Stereoselective Biocatalysis (2000) 741. [10] E. Ostuni, L. Yan, G.M. Whitesides, Colloids Surf. B: Biointerfaces 15 (1999) 3. [11] H.H. Weetall, Adv. Mol. Cell Biol. 15A (1996) 161. [12] K. Wadu-Mesthrige, N.A. Amro, G.-Y. Liu, Scanning 22 (2000) 380. [13] L.S. Shlyakhtenko, A.A. Gall, A. Filonov, Z. Cerovac, A. Lushnikov, Y.L. Lyubchenko, Ultramicroscopy 97 (2003) 279. [14] J.J. Gooding, P. Erokhin, D. Losic, W. Yang, V. Policarpio, Y. Liu, F.M. Ho, M. Situmorang, D.B. Hibbert, J.G. Shapter, Anal. Sci. 17 (2001) 3. [15] M.C. Coen, R. Lehmann, P. Groening, M. Bielmann, C. Galli, L. Schlapbach, J. Colloid Interface. Sci. 233 (2001) 180. [16] D.J. Muller, A. Engel, J. Mol. Biol. 285 (1999) 1347. [17] F. Oesterhelt, D. Oesterhelt, M. Pfeiffer, A. Engel, H.E. . Gaub, D.J. Muller, Science 288 (2000) 143. [18] F.M. Rief, M. Gautel, F. Oesterhelt, J.M. Fernadez, H.E. Gaub, Science 276 (1997) 1109. [19] M.B. Viani, L.I. Pietrasanta, J.B. Thompson, A. Chand, I.C. Gebeshuber, J.H. Kindt, M. Richter, H.G. Hansma, P.K. Hansma, Nat. Struct. Biol. 7 (2000) 644. [20] M. Radmacher, M. Fritz, H.G. Hansma, P.K. Hansma, Science 265 (1994) 1577. [21] Z. Yingge, Z. Delu, B. Chunli, W. Chen, Life Sci. 65 (1999) PL253. [22] L.K. Nielsen, J. Risbo, T.H. Callisen, T. Bjornholm, Biochim. Biophys. Acta 1420 (1999) 266. [23] M. Grandbois, H. Clausen-Schaumann, H. Gaub, Biophys. J. 74 (1998) 2398.
ARTICLE IN PRESS 134
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
[24] B.R. Horrocks, G. Wittstock, in: A.J. Bard, M.V. Mirkin (Eds.), Scanning Electrochemical Microscopy, Marcel Dekker, New York, 2001, p. 445. [25] B.R. Horrocks, D. Schmidtke, A. Heller, A.J. Bard, Anal. Chem. 65 (1993) 3605. [26] H. Shiku, Y. Hara, T. Matsue, I. Uchida, T. Yamauchi, J. Electroanal. Chem. 438 (1997) 187. [27] G. Wittstock, W. Schuhmann, Anal. Chem. 69 (1997) 5059. [28] D.T. Pierce, P.R. Unwin, A.J. Bard, Anal. Chem. 64 (1992) 1795. [29] H. Shiku, T. Takeda, H. Yamada, T. Matsue, I. Uchida, T. Yamauchi, Anal. Chem. 67 (1995) 312. [30] C. Kranz, G. Wittstock, H. Wohlschlaeger, W. Schuhmann, Electrochim. Acta 42 (1997) 3105. [31] G. Wittstock, K.J. Yu, H.B. Halsall, T.H. Ridgway, W.R. Heineman, Anal. Chem. 67 (1995) 3578. [32] H. Shiku, T. Matsue, I. Uchida, Anal. Chem. 68 (1996) 1276. [33] G. Wittstock, Fresenius J. Anal. Chem. 370 (2001) 303. [34] A.J. Bard, F.R.F. Fan, M.V. Mirkin, in: A.J. Bard (Ed.), Electroanalytical Chemistry, Marcel Dekker, New York, 18, 1994, p. 268.
[35] C. Kranz, G. Friedbacher, B. Mizaikoff, A. Lugstein, J. Smoliner, E. Bertagnolli, Anal. Chem. 73 (2001) 2491. [36] C. Kranz, B. Mizaikoff, A. Lugstein, E. Bertagnolli, in: M. Taillefert, T.F. Rozan (Eds.), Electrochemical Methods for the Environmental Analysis at Trace Element Biogeochemistry, American Chemical Society Symposium, 2002, p. 320. [37] A. Lugstein, E. Bertagnolli, C. Kranz, B. Mizaikoff, Surf. Interface Anal. 33 (2002) 146. [38] A. Lugstein, E. Bertagnolli, C. Kranz, A. Kueng, B. Mizaikoff, Appl. Phys. Lett. 81 (2002) 349. [39] A. Kueng, C. Kranz, B. Mizaikoff, A. Lugstein, E. Bertagnolli, Appl. Phys. Lett. 82 (2003) 1592. [40] A. Kueng, C. Kranz, B. Mizaikoff, A. Lugstein, E. Bertagnolli, Ang. Chem. Int. Ed. 42 (2003) 3237. [41] E.L.H. Heintz, C. Kranz, B. Mizaikoff, H.-S. Noh, P. Hesketh, A. Lugstein, E. Bertagnolli, Procedings of the IEEE Nanotechnology Conference, 2001. [42] Y. Lee, S. Amemya, A.J. Bard, Anal. Chem. 73 (2001) 2261. [43] J. Kwak, A.J. Bard, Anal. Chem. 61 (1989) 1221. [44] A.J. Bard, F.R.F. Fan, D.T. Pierce, P.R. Unwin, D.O. Wipf, F. Zhou, Science 254 (1991) 68.
Ultramicroscopy 100 (2004) 127–134
Mapping of enzyme activity by detection of enzymatic products during AFM imaging with integrated SECM–AFM probes C. Kranza,*, A Kuenga, A. Lugsteinb, E. Bertagnollib, B. Mizaikoffa a b
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA Solid State Electronics Institute, Vienna University of Technology, Floragasse 7, Vienna A-1040, Austria Received 1 July 2003; received in revised form 15 October 2003; accepted 31 October 2003
Abstract With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy (SECM) with AFM has recently been introduced. Simultaneous contact mode imaging of a micropatterned sample with immobilized enzyme spots and imaging of enzyme activity is shown. In contrast to force spectroscopy the conversion of an enzymatic byproduct is directly detected during AFM imaging and correlated to the activity of the enzyme. r 2004 Elsevier B.V. All rights reserved. Keywords: Atomic force microscopy; Scanning electrochemical microscopy; Combined SECM–AFM; Integrated SECM–AFM probes; Enzyme activity; Self-assembled monolayers; Thin films
1. Introduction Immobilization of active proteins at solid substrate materials is of great interest in fabrication of miniaturized biomedical and environmental sensors and devices. Deposition conditions and procedures [1], have to be carefully monitored, since the immobilization process should not induce a substantial decrease of the protein functionality. AFM is a versatile tool for imaging the structure of proteins however, only few contributions report *Corresponding author. Tel.: +1-404-385-1794; fax: +1404-894-7452. E-mail address: [email protected] (C. Kranz).
on imaging protein activity [2,3]. Several approaches have been described for immobilization of proteins: (i) self-assembly of protein–lipid films [4], (ii) Langmuir–Blodgett techniques [5], (iii) entrapment in polymer films [6–9], and (iv) using o-functionalized thiols, disulfides or silanes [10–14]. The latter approach enables covalent attachment of the protein to the terminal functional group. Special care has to be taken immobilizing enzymes at a substrate surface. Due to the immobilization process a loss of catalytic enzymatic function may occur, which is induced by conformational distortion during the attachment process. Hence, it is crucial to correlate deposition protocols to changes in morphology and biological activity.
0304-3991/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2003.10.004
ARTICLE IN PRESS 128
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
Scanning probe microscopies (SPM) are a versatile tool for imaging biological species. AFM is particularly suitable, since it can be operated at ambient physiological conditions. Proteins have widely been studied by AFM [15]. Besides the investigation of structural [16] and mechanical properties [17,18] of individual proteins, real-time probing of protein–protein interaction [19] has been demonstrated. Only few papers describe direct observation of enzyme activity. Following adsorption onto mica surfaces Radmacher et al. [20] directly observed enzyme activity by detecting height fluctuations of the cantilever. Real-time monitoring of single-binding events between acetyl cholinesterase and the substrate acetylcholine have been shown by recording force spectra [21]. In situ studies of single enzymes and indirect mapping of enzyme activity was demonstrated by imaging phospholipase and the time course of lipid bilayer degradation [22,23]. An alternative approach for studying enzyme activity and kinetics is provided by the detection of molecular byproducts during enzymatic conversion. Scanning electrochemical microscopy is an insitu SPM technique and has gained increasing importance for studying biological systems during the last decade [24]. Scanning a biased microelectrode across the sample surface enables the detection of (electro)active species generated at the sample surface or probing the (electro)active properties of the sample surface. In principle, a Faraday current measured at a biased microelectrode due to hemispherical diffusion of a redox species in solution towards this electrode is disturbed by the sample surface chemistry in the proximity of (several electrode radii above) the sample surface. SECM has extensively been applied to investigate enzyme activity in generator/collector mode [25–27] and feedback mode [28–30] operation. Enzyme immunoassays can be investigated and optimized using SECM [31,32]. Recently, comprehensive reviews on biological applications of SECM have been published [24,33]. A major drawback of SECM compared to AFM and other SPM techniques is the lack of sufficient lateral resolution in conventional SECM imaging due to signal-dependent positioning of the
microelectrode [34]. Additionally, scanning in constant height across the sample surface results in a convolution of the electrochemical response and the topographical information. In general, imaging of enzyme activity in generator/collector mode does not provide topographical information when the substrate of the enzymatic reaction is absent. Consequently, any progress towards improved lateral resolution in mapping of enzyme activity and imaging of biological systems in SECM has to address current independent positioning ideally of nanoelectrodes. Thus, precise control on the distance between the scanned electrode tip and the sample surface has to be ensured. An important aspect enhancing the versatility of scanning probe techniques is the approach to integrate various sensors in a single probe. With the integration of submicro- and nanoelectrodes into AFM probes an elegant way of combining scanning electrochemical microscopy with atomic force microscopy has recently been pioneered [35–38]. Complementary electrochemical and topographical information with high-lateral resolution can be obtained in a single time- and spacecorrelated measurement. Imaging with the integrated SECM-AFM probes in AFM tapping mode was successfully applied to soft samples [39,40]. In this contribution simultaneous imaging of immobilized peroxidase activity in generator/collector mode of SECM and contact mode AFM is demonstrated using bifunctional SECM–AFM probes. The electroactive byproduct of the enzymatic reaction diffuses to the integrated submicroelectrode and is converted leading to a Faraday current, which corresponds to the enzyme activity. The current signal can be simultaneously correlated to the obtained topography.
2. Experimental 2.1. Materials Horseradish peroxidase (EC. 1.11.1.7) was purchased from Sigma (Louis, MO), glutaraldehyde, cystaminium dichloride from Fluka (Buchs, Switzerland), and ferroceniummethyl hydroxide
ARTICLE IN PRESS C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
(FMA) from Aldrich (Milwaukee, WI). All other chemicals were of analytical grade and were obtained from Fisher (Pittsburgh, PA) and Mallinckrodt (Phillipsburg, NY), respectively. Buffers were prepared with deionized water (Millipore). The micro-structured gold samples were obtained from Quantifoil Micro Tools (Jena, Germany).
129
glutaraldehyde) was immobilized onto the gold structure for 2 h, forming a surface-grafted crosslinked protein gel on the aminated gold spots. After thoroughly rinsing the sample with buffer solution several times the sample was stored at 4 C until analysis. 2.3. Fabrication of integrated SECM–AFM probes
2.2. Sample preparation The microstructured gold/siliconnitride sample was cleaned in H2SO4:H2O2 (70:30 v/v) for a period of 30 s, rinsed three times with water and then immediately immersed into 0.1 M cystaminium dichloride (in acetate buffer; pH 5.5) for 30 min. Cystaminium dichloride forms a aminofunctionalized self-assembled thiol monolayer on the gold spots. After rinsing the sample 5 times with deionized water, the terminated amino function of the self-assembled monolayer was used to covalently attach the enzyme from suspension. The peroxidase (1 mg peroxidase in 100 ml phosphate buffer (pH 7.0) containing 2.5% (v/v)
A detailed description of integrated SECMAFM probe fabrication is given elsewhere [35–41]. Silicon nitride cantilevers without the gold coating at the backside are partially coated with a 100 nm gold layer on the tip side (see Fig. 1a) and subsequently insulated with a 700 nm layer of xylelene polymer (Parylene C) via vapor deposition polymerization. Three-dimensional focused ion beam (FIB) milling enables reproducible exposure of an electroactive area with defined geometry and dimensions integrated above the apex of the original AFM tip. Finally, the original AFM tip is re-shaped correlating the tip height to the dimensions of the integrated electrode.
Fig. 1. (a) Optical micrograph of a modified silicon nitride cantilever: magnification 10 ; (b) scheme of an integrated SECM–AFM probe; (c) SEM–FIB image of an integrated SECM–AFM probe (electrode edge length: 1 mm, re-shaped AFM tip length: 490 nm); (d) cyclic voltammogram recorded at the integrated SECM/AFM probe in 20 mmol l1 ferrocyanide/0.5 mol l1 KCl electrolyte vs. Ag/AgCl, scan rate 50 mV/s.
ARTICLE IN PRESS 130
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
A schematic representation of an integrated SECM-AFM tip is given in Fig. 1b. Hence, the optimum working distance between electrode and sample surface and a high tip aspect ratio for AFM imaging is ensured (Fig. 1c). The integrated electrode shown in Fig. 1c (electrode edge length: 1 mm; thickness of the gold layer: 100 nm; reshaped AFM tip length: 490 nm) was characterized by recording cyclic voltammograms with 20 mmol l1 ferrocyanide in 0.5 mol l1 KCl supporting electrolyte (Fig. 1d). A typical sigmoidal shape of the voltammogram with a limiting current of 1.1 nA was obtained corresponding well with the theory of ring microelectrodes [42]. 2.4. Combined SECM–AFM measurements The SECM–AFM probes were mounted in a standard atomic force microscope (Nanoscope III, Digital Instruments, Santa Barbara, CA) equipped with a liquid cell and a three-electrode set-up with the integrated probe as working electrode. The fluid inlet and outlet of the liquid cell are used to position a Pt-wire as counter electrode and a quasi-Ag/AgCl reference electrode. The AFM head including the electrochemical cell was shielded with a copper housing to reduce noise in the current signal. The electrochemical experiment was controlled by a bi-potentiostat (CH Instruments 832A CH Instruments, Austin, TX). Images were obtained in contact mode of the AFM and generator/collector mode of the SECM for imaging enzyme activity and in the feedback mode of the SECM for the GaAs/gold sample [43].
3. Results and discussion FIB technology allows fabricating high aspect ratio ultra-sharp AFM tips. Hence, the micromachining steps for fabrication of the integrated SECM-probes can be performed with high reproducibility. Furthermore, re-shaped tips with a similar curvature as the initial tips required for high-quality topographic images are provided. The lateral resolution of the electrochemical response is determined by the size of the integrated electroactive area and the distance to the sample surface
[44]. To demonstrate the capability and resolution of microfabricated combined SECM–AFM probes images of periodic Au/GaAs patterns were recorded in AFM contact mode and SECM feedback mode. The pattern consists of 600 nm GaAs grooves with a depth of 100 nm spaced by 400 nm of gold prepared by FIB milling of a gold-coated GaAs substrate. A quasi-reversible redox couple, e.g. ferrocyanide, is added at one oxidation state (reduced) to the solution. The integrated electrode is biased at 600 mV vs. Ag/AgCl, a potential where ferrocyanide is oxidized to ferricyanide. In large distance to the sample surface a steady-state current occurs due to the electrochemical conversion of the mediator. When the SECM–AFM probe is engaged the distance between the electrode and the sample is exclusively determined by the re-shaped AFM tip, which is located in the center of the frame electrode (see Fig. 1c). The distance is adjusted during FIB milling and amounts in general half of the edge length of an integrated frame electrode. In such close proximity of the electrode to the sample surface, the current recorded at the electrode is influenced by the morphology and nature of the sample surface. The insulating GaAs grooves are blocking the diffusion towards the integrated microelectrode and the current decreases (negative feedback). In contrast, the conductive gold surface enables recycling of the redox mediator (reduction of ferricyanide to ferrocyanide) leading to an increased current (positive feedback) compared to the steady state Faraday current in bulk solution. Fig. 2 shows the decreased current above the GaAs regions and the increased current above the gold surface. The obtained resolution is in good agreement with the theory of the feedback mode and demonstrates that features smaller than the integrated electrode dimensions can clearly be resolved. Likewise, the comparison of the topographical and the electrochemical images shows excellent agreement (Fig. 2). The topographic image quality of the remodeled tip is comparable to non-modified AFM Si3N4 cantilevers as already shown in previous studies of our research group [36]. Imaging enzyme activity in contact mode AFM was performed at a micropattern of isolated enzyme spots (Fig. 3). Enzyme spots were created
ARTICLE IN PRESS C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
131
Fig. 3. Scheme of simultaneous SECM–AFM imaging and the involved reactions at the surface of the spot-immobilized peroxidase recorded with an integrated electrode operating in generation/collection mode. The electron donor FMA is oxidized during the enzymatic conversion of H2O2 and diffuses to the integrated electrode where it is amperometrically detected (dashed arrows).
activity was obtained in generation/collection mode [26]. The measurement solution contained 2.0 mmol l1 FMA and 0.5 mmol l1 H2O2 and 0.l mol l1 KCI in 0.1 M phosphate buffer solution. The potential of the tip electrode was held at 0.05 V vs. Ag/AgCl. Peroxidase catalyzes the following reaction: 2FMA þ H2 O2 þ 2Hþ -2FMAþ þ 2H2 O: Fig. 2. Simultaneously recorded height and current images in AFM contact mode of a periodic Au/GaAs pattern (400 nm gold stripes spaced at 600 nm) showing (a) topography and (b) simultaneously recorded SECM image with the tip-integrated electrode. Redox mediator: 0.03 mol l1 [Fe(CN)6]4 in 0.5 mol l1 KCl electrolyte solution. The tip was held at a potential of 0.6 V vs. quasi-Ag/AgCl reference electrode (AgQRE). Electrode edge length: 800 nm; tip height: 300 nm; scan rate: 3 Hz.
by chemisorption of a functionalized thiol monolayer (cystaminium chloride) onto gold structures produced by a periodically micropatterned silicon nitride layer deposited onto a gold-coated silicone wafer. The terminal group of the chemisorbed monolayer can be used as anchor group for the covalent attachment of biomolecules [27]. Peroxidase was covalently attached to the gold spots by adding a mixture of glutaraldehyde and peroxidase forming a surface grafted crosslinked protein gel at the aminated surface of the self-assembled monolayer. Electrochemical imaging of peroxidase
H2O2 is the substrate of the enzymatic conversion and is reduced to water with hydroxyl methyl ferrocene (FMA), a metal organic electron donor containing Fe2+ added to the solution. Hence, the oxidized species ferrocinium methylhydroxide (FMA+, containing Fe3+) is continuously generated during the enzymatic conversion (Fig. 3, dashed arrow). FMA+ is diffusing to the integrated electrode and can be amperometrically detected at E=0.05 V vs. Ag/AgCl, a potential where no other electroactive species present in solution can be converted. In general, images obtained in generation/collection mode provide higher sensitivity compared to the feedback mode and are particularly suitable for detection of low enzyme activity [26]. Furthermore, in generation/ collection mode any contribution from the underlying gold substrate is eliminated as Wittstock and Schuhmann suggested [27]. The current response increases with reduced electrode-to-sample distance. Hence, with the described integrated SECM–AFM probe the electrode follows the
ARTICLE IN PRESS 132
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
topography of the sample while the distance between the electroactive area and the sample surface remains constant. If sub-micrometer distances are selected for constant height imaging in conventional SECM, tip crashes may occur due to, e.g. a tilted sample or surface features with dimensions corresponding to the electrode–sample distance. However, in order to improve the lateral resolution in generator/collector mode it is preferable to decrease the distance between the microelectrode and sample surface, which is achieved with the presented concept. Moreover, if the electron donor is not present in solution the topography can not be imaged in conventional SECM experiments due to the omnipresent convolution of topography and electrochemical response. With the presented combination the distance between the integrated electrode and the sample surface can be individually designed and fabricated with respect to the application. To date, micropatterns of covalently attached peroxidase with dimensions of approx. 1–2 mm in diameter
could not be successfully imaged in generator/ collector mode of SECM due to the low concentration of immobilized peroxidase and the comparatively larger working distance during conventional SECM experiments [24]. In order to ensure that the electrochemical image is resulting from enzymatic activity the sample was first imaged in a solution containing 2.0 mmol l1 FMA, 0.l mol l1 KCI in 0.1 mol l1 phosphate buffer solution in absence of H2O2. Contact mode topography and the simultaneously obtained electrochemical image were recorded with the integrated SECM–AFM probe (electrode length: 860 nm; re-shaped tip length: 410 nm) poised at a potential of 0.05 V vs. Ag/AgCl. The topography corresponds with the expected surface properties of the sample (Fig. 4a). As clearly shown in Fig. 4b, in absence of the substrate H2O2 in solution no enzymatic reaction occurs and the current recorded at the electrode during AFM contact mode imaging of the immobilized enzyme spots is negligible. Fig. 4a shows the topography of
Fig. 4. Simultaneously recorded height and current images of peroxidase activity in AFM contact mode. Top view of the height (a) and (c), and simultaneously recorded current (b) in absence of the enzymatic substrate H2O2. (d) The electrochemical response with 0.5 mmol l1 H2O2 in solution. Images are recorded in 2 mmol l1 FMA, 0.1 mol l1 KCl in phosphate buffer (0.1 mol l1, pH 7.0) The tip was held at a potential of 0.05 V vs. Ag/AgCl. Electrode edge length: 860 nm; tip height: 410 nm; scan rate: 1 Hz.
ARTICLE IN PRESS C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
the micro-structured gold sample containing immobilized enzyme spots. Fig. 4c and 4d show the topography and the electrochemical response, respectively, corresponding to the enzymatic activity of the peroxidase in the presence of H2O2 imaged with the same integrated SECM–AFM probe. The periodicity of the enzyme-immobilized patterns in the current image corresponds well with the topography provided by the integrated AFM tip. The dark features around the immobilized enzyme spots result from the reshaped AFM tip height of 410 nm, which compares to the topographical features of the sample surface (height of the silicon nitride layer: 450 nm). Hence, if the reshaped AFM tip is above the center of the immobilized enzyme spot the diffusion of FMA+ to the integrated electrode is intermittently blocked. In order to avoid this effect, integrated SECM–AFM probes with a longer reshaped AFM tip can be selected.
4. Conclusion Simultaneous imaging of enzyme activity during contact mode AFM measurements is demonstrated by integrating an electroactive area into an AFM tip (integrated SECM–AFM probe). Different electrode geometries, such as frame-, ring- and disc-electrodes can be integrated into the AFM-tip at an exactly defined distance above the apex using FIB technology as recently demonstrated by our research group. Ongoing work is focused on chemical modification of the tipintegrated electroactive area, e.g. with immobilized enzymes aiming at the development of scanning probe tip-integrated electrochemical nanosensors for characterization of electrochemical surface processes at soft biological samples with highlateral resolution.
Acknowledgements The National Science Foundation (Grant 0216368 within the program ‘Biocomplexity in . the Environment’) and the ‘Fonds zur Forderung der wissenschaftlichen Forschung’ Austria (Grants
133
P14122-CHE and J2230) are greatly acknowledged. PCT patents on this technology are issued (WO01/94877 and WO01/94926).
References [1] D.J. Mueller, H. Janovjak, T. Lehto, L. Kuerschner, K. Anderson, Progr. Biophys. & Mol. Biol. 79 (2002) 1. [2] N.H. Thomson, B.L. Smith, N. Almqvist, L. Schmitt, M. Kashlev, E.T. Kool, P.K. Hansma, Biophys. J. 76 (1999) 1024. [3] B. Waelivaara, A. Askendal, I. Lundstroem, P. Tengvall, J. Coll. Interface. Sci. 187 (1997) 121. [4] Y.F. Dufrene, W.R. Barger, J.D. Green, G.U. Lee, Langmuir 13 (1997) 2271. [5] V. Erokhin, L.A. Feigin, Prog. Colloid Polym. Sci. 85 (1991) 47. [6] A. Akalah, A. Moet, Functionalized Polymers and their Applications, Chapman &Hall, London, 1981. [7] W. Schumann, Microchim. Acta 121 (1995) 1. [8] P.N. Bartlett, J.M. Cooper, J. Electroanal. Chem. 362 (1993) 1. [9] I.S. Gill, F.J. Plou, A. Ballesteros, Stereoselective Biocatalysis (2000) 741. [10] E. Ostuni, L. Yan, G.M. Whitesides, Colloids Surf. B: Biointerfaces 15 (1999) 3. [11] H.H. Weetall, Adv. Mol. Cell Biol. 15A (1996) 161. [12] K. Wadu-Mesthrige, N.A. Amro, G.-Y. Liu, Scanning 22 (2000) 380. [13] L.S. Shlyakhtenko, A.A. Gall, A. Filonov, Z. Cerovac, A. Lushnikov, Y.L. Lyubchenko, Ultramicroscopy 97 (2003) 279. [14] J.J. Gooding, P. Erokhin, D. Losic, W. Yang, V. Policarpio, Y. Liu, F.M. Ho, M. Situmorang, D.B. Hibbert, J.G. Shapter, Anal. Sci. 17 (2001) 3. [15] M.C. Coen, R. Lehmann, P. Groening, M. Bielmann, C. Galli, L. Schlapbach, J. Colloid Interface. Sci. 233 (2001) 180. [16] D.J. Muller, A. Engel, J. Mol. Biol. 285 (1999) 1347. [17] F. Oesterhelt, D. Oesterhelt, M. Pfeiffer, A. Engel, H.E. . Gaub, D.J. Muller, Science 288 (2000) 143. [18] F.M. Rief, M. Gautel, F. Oesterhelt, J.M. Fernadez, H.E. Gaub, Science 276 (1997) 1109. [19] M.B. Viani, L.I. Pietrasanta, J.B. Thompson, A. Chand, I.C. Gebeshuber, J.H. Kindt, M. Richter, H.G. Hansma, P.K. Hansma, Nat. Struct. Biol. 7 (2000) 644. [20] M. Radmacher, M. Fritz, H.G. Hansma, P.K. Hansma, Science 265 (1994) 1577. [21] Z. Yingge, Z. Delu, B. Chunli, W. Chen, Life Sci. 65 (1999) PL253. [22] L.K. Nielsen, J. Risbo, T.H. Callisen, T. Bjornholm, Biochim. Biophys. Acta 1420 (1999) 266. [23] M. Grandbois, H. Clausen-Schaumann, H. Gaub, Biophys. J. 74 (1998) 2398.
ARTICLE IN PRESS 134
C. Kranz et al. / Ultramicroscopy 100 (2004) 127–134
[24] B.R. Horrocks, G. Wittstock, in: A.J. Bard, M.V. Mirkin (Eds.), Scanning Electrochemical Microscopy, Marcel Dekker, New York, 2001, p. 445. [25] B.R. Horrocks, D. Schmidtke, A. Heller, A.J. Bard, Anal. Chem. 65 (1993) 3605. [26] H. Shiku, Y. Hara, T. Matsue, I. Uchida, T. Yamauchi, J. Electroanal. Chem. 438 (1997) 187. [27] G. Wittstock, W. Schuhmann, Anal. Chem. 69 (1997) 5059. [28] D.T. Pierce, P.R. Unwin, A.J. Bard, Anal. Chem. 64 (1992) 1795. [29] H. Shiku, T. Takeda, H. Yamada, T. Matsue, I. Uchida, T. Yamauchi, Anal. Chem. 67 (1995) 312. [30] C. Kranz, G. Wittstock, H. Wohlschlaeger, W. Schuhmann, Electrochim. Acta 42 (1997) 3105. [31] G. Wittstock, K.J. Yu, H.B. Halsall, T.H. Ridgway, W.R. Heineman, Anal. Chem. 67 (1995) 3578. [32] H. Shiku, T. Matsue, I. Uchida, Anal. Chem. 68 (1996) 1276. [33] G. Wittstock, Fresenius J. Anal. Chem. 370 (2001) 303. [34] A.J. Bard, F.R.F. Fan, M.V. Mirkin, in: A.J. Bard (Ed.), Electroanalytical Chemistry, Marcel Dekker, New York, 18, 1994, p. 268.
[35] C. Kranz, G. Friedbacher, B. Mizaikoff, A. Lugstein, J. Smoliner, E. Bertagnolli, Anal. Chem. 73 (2001) 2491. [36] C. Kranz, B. Mizaikoff, A. Lugstein, E. Bertagnolli, in: M. Taillefert, T.F. Rozan (Eds.), Electrochemical Methods for the Environmental Analysis at Trace Element Biogeochemistry, American Chemical Society Symposium, 2002, p. 320. [37] A. Lugstein, E. Bertagnolli, C. Kranz, B. Mizaikoff, Surf. Interface Anal. 33 (2002) 146. [38] A. Lugstein, E. Bertagnolli, C. Kranz, A. Kueng, B. Mizaikoff, Appl. Phys. Lett. 81 (2002) 349. [39] A. Kueng, C. Kranz, B. Mizaikoff, A. Lugstein, E. Bertagnolli, Appl. Phys. Lett. 82 (2003) 1592. [40] A. Kueng, C. Kranz, B. Mizaikoff, A. Lugstein, E. Bertagnolli, Ang. Chem. Int. Ed. 42 (2003) 3237. [41] E.L.H. Heintz, C. Kranz, B. Mizaikoff, H.-S. Noh, P. Hesketh, A. Lugstein, E. Bertagnolli, Procedings of the IEEE Nanotechnology Conference, 2001. [42] Y. Lee, S. Amemya, A.J. Bard, Anal. Chem. 73 (2001) 2261. [43] J. Kwak, A.J. Bard, Anal. Chem. 61 (1989) 1221. [44] A.J. Bard, F.R.F. Fan, D.T. Pierce, P.R. Unwin, D.O. Wipf, F. Zhou, Science 254 (1991) 68.