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Fig.7. In-situ STM images of copper potentiostatic pulse plating on gold. Electrolyte: 0.001 M CuS04 and 0.05 M H2S04 in Millipore water, (a) Clean surface, d = 9 nm. (b) Ten pulses of 0/-100 mV, each 500 ms duration (stripes), d- 44 nm. (c)Copper crystallites created by the process, E = 0 mV, Et = 42 mV, d = 29 nm, It = 4.2 nA.

In studies of electrochemical processes on gold imaged by in-situ STM the surface mobility introduces complications in theoretical modeling [33], In technical applications of in-situ STM the ability of the tip to create well-defined patterns on the surface might be useful in „nanotechnology" or „nanoelectronics". In-situ STM and AFM (atomic force microscopy) have succesfully been introduced in technologically important contexts such a electronic circuit boards, polymer-metal interfaces [34] and pulse plating [35]. An example is shown in Fig. 7(a)-(c). A clean surface was subjected to 10 cycles of square pulses of E = -100 mV for 0.5 s followed by E = 0 mV for 0.5 s. When potentiostatic cycling was activated, imaging was temporarily disturbed (the middle of Fig. 7(b) where 10 horizontal lines appear). However, as the scanning is from below towards the top, it is possible to image the growth of copper between the lines of Fig. 7(b). The final result of the pulse plating process is copper crystallites (Fig. 7(c)). It is well known that potentiostatic or galvanostatic pulse shapes have a profound influence on the morphology of the deposit and the mechanical properties of the coating [36]. The imaging of the process may prove useful in developing metal surface coatings but the image features have yet to be related to macroscopic properties.

3 In-situ STM of adsorbed metalloproteins

Chemical and biological homogeneous electron tunneling (ET) has been mapped in considerable detail [37-43], based on elaborate synthetic work, characterization of many new redox proteins, and new theoretical achievements. Concepts and physical properties in focus have been: (a) the distance and orientation of the donor and acceptor centers, and the nature of the intermediate molecular groups; (b) supramolecular organization in multi-ET patterns; (c) vibrational coherence and (d) fluctuation-induced tunneling and critical phenomena. Interfacial electrochemical ET has not witnessed a parallel evolution due to less feasible structural and theoretical characterization of the heterogeneous, field-exposed, interfacial region. Perspectives for more precise characterization are opening, however, rooted in the availability of new, stable, self-assembled films, and precise deconvolution of the tunnel and nuclear activation factors over broad potential ranges [44, 45], supported by new construction of dielectrically screened energy functional [46, 47] and interfacial ET rate constants [48]. At the same time in-situ STM has opened exciting perspectives for direct imaging of the two-dimensional adsórbate organization. Our research has recently focused on mapping of biological macromolecules by in-situ STM, with reference to concepts and formalism of long-range ET of solute metalloproteins. The activity is prompted by the inherent perspectives of protein mapping at surfaces and by the fact that metalloproteins in homogeneous solution offer some of the most detailed tunnel features.

3.1 In-situ STM patterns of cytochrome c and other metalloproteins

Structural mapping of adsorbate molecules by STM and AFM has been extended to biological macromolecules [49]. These include DNA [50], proteins, and protein complexes [51, and references therin). The natural medium for biological macromolecules is, however, aqueous solution, and water constitutes an integrated element of three-dimensional structure. High-resolution in-situ STM imaging of DNA [50], DNA bases [52], and metalloporphyrins [53] has been achieved recently, but obstacles arise for proteins:

(a) Solute proteins are conformationally labile.

(b) Proteins are collectively mobile on the substrate surface. The mobility patterns are interesting but immobilization is needed to achieve molecular resolution.

(c) Protein mobility, together with the large tunnel distance, poses a risk of mechanical dislodging by the tip during the scanning process.

(d) Metallic surfaces reconstruct in certain potential ranges, resulting in structural features interfering with adsorbate dynamics.

We have investigated in-situ STM of the small single-metal redox proteins cytochrome c (MW «12 kDa) and azurin («14 kDa), and the larger four copper-enzyme laccase (MW « 64 kDa), all involved in natural ET [51, 54, 55]. The choice rested on the following considerations:

(1) Key structural elements of the proteins are transition-metal atoms which are easily reduced or oxidized. The metal atoms provide favourable tunnel routes either as local tunnel barrier indentations, or by accomodating the electron or hole in inelastic STM modes [56, 57].

(2) The proteins are well characterized electrochemically, indicative of facile electron exchange with metallic surfaces of ET through the protein.

(3) Cyt c and azurin are structurally and in other respects very well characterized, and in-situ STM can be referred to many other structural, spectral, and kinetic data. No three-dimensional structure of laccase is available, but the structure of the closely related enzyme ascorbate oxidase is available with high resolution and supports a view of facile ET through the protein, involving all the copper atoms.

Fig.8. Cyt c on gold adsorbed from 10"5 M cyt c and 20 mM MES buffer (pH = 7). Upper left: Organized arrays of »50 nm lateral extent. Constant current mode, It= 1.8 nA. Structures resembling individual cyt c moleculesadsorbed on gold between larger cyt c aggregates. Bottom: Height profile of the structures at the upper right.

Fig.8. Cyt c on gold adsorbed from 10"5 M cyt c and 20 mM MES buffer (pH = 7). Upper left: Organized arrays of »50 nm lateral extent. Constant current mode, It= 1.8 nA. Structures resembling individual cyt c moleculesadsorbed on gold between larger cyt c aggregates. Bottom: Height profile of the structures at the upper right.

Figure 8 shows representative images of polycrystalline or flame-annealed gold surfaces in contact with 10"3 M cyt c in 20 mM MES buffer (pH = 7.0) [51]. The surface structure is quite different from the structure in the absence of cyt c. The view shows arrays of individual structural elements about 50 nm in lateral extent, corresponding to »100 molecules. Similar patterns are observed for azurin. The structures are fluctuationally mobile. This mobility impedes molecular resolution but, subject to adequate persistence, much smaller structures of the size of individual molecules are clearly visible between the larger structures (Fig. 8). The smaller structures have not so far been found for azurin for which, on the other hand, ex-situ AFM on flame-annealed gold has shown structures of similar size [55]. The height (0.5 + 0.1 nm) and lateral extent (3.3 ± 0.5 nm) for the molecular-size cyt c structures are close enough to the crystallographic dimensions to indicate that Fig. 9 could well show molecular resolution.

In-situ STM of laccase has so far been attempted using only basal-plane pyrolytic graphite surfaces [54]. No adsorption could be detected. This is perhaps not surprising as laccase voltammetry requires edge-plane graphite and/or pre-adsorption of promoters. Low-resolution ex-situ micron-scale laccase structures could be recorded during evaporation of laccase solution where individual molecular-size structures, possibly dislodged by mechanical tip contact, could also be observed.

3.2 In-situ tunneling through metalloproteins as a three-center multiphonon electron transfer process

In-situ STM has been approached theoretically. Focus has been on (a) solvent polarization effects on the metallic electronic structure [58, 59]; (b) local pseudopotentials [59]; (c) electron tunnel routes through networks of „quantum dots" [60]; (d) time correlation effects and noise [60]; (e) resonance tunneling [62, 63], and (f) tunneling through intermediate local molecular levels as in other three-level coherent thermal and optical processes [56, 57, 64],

We discuss briefly the latter view. The intermediate state is representative of the metal centers in metalloproteins or large transition-metal complexes, spatially separated from the electrodes by protein or intramolecular ligand frames. The electron-or hole-transferring redox level has also much lower energy than surrounding protein or solvent. Figure 9 shows the level dynamics. The local level, at the equilibrium nuclear configuration q = 0, is initially well above the Fermi level of both tip and substrate. Nuclear fluctuations take the level close to the Fermi level of the negatively biased electrode. This lowers the tunnel barrier, even to the extent of temporary population but at the expense of nuclear activation. A variety of three-level STM patterns then arise of which the following are particularly important:

(1) The populated level begins to relax vibrationally after the first ET step, but the second step, i.e., electron transfer from the populated intermediate level to the positively biased electrode, occurs before full relaxation. This pattern corresponds to coherent two-step ET, of which strictly resonating three-level transfer would be a limiting case.

(2) The tunnel interactions may be weak so that full vibrational relaxation occurs after the first step. Renewed fluctuations induce the second step so that the overall process turns into two sequential independent single-ET processes.

The view in Fig. 9 can be given a quantitative frame which incorporates distance and voltage relations [56, 57, 64], In-situ STM here offers an additional dimension

Fig. 9. Molecular adsórbate level between two continuous metallic-level distributions. Tunneling is from the negatively (right) to the positive (left) biased electrode and feasible when nuclear fluctuations take the adsórbate level from the initial location above both Fermi levels, sRF and elf, respectvely, to a location between the Fermi levels, q with different subscripts indicates nuclear configurations at which tunneling in the coherent two-step ET mode can occur [53, 55].

compared with electrochemical ET. STM can thus be monitored at variable bias voltage, but fixed overpotential. The latter is the substrate-molecule potential difference relative to a reference potential. The tunnel current can also be recorded at variable overpotential and fixed bias potential. In the latter case the electrode potentials vary in parallel. It has been noted [64] that the current-overpotential relation exhibits close formal relations to the excitation profile of resonance Raman spectroscopy while the current-bias voltage relation correspondingly resembles the resonance Raman spectrum [64].

The view discussed above offers frames for in-situ STM phenomena once uniform adsórbate organization can be achieved. This is not at present the case for metalloproteins but a recent investigation of in-situ current-voltage relations of protoporphyrin IX and Fe-protoporphyrin IX at highly oriented pyrolytic graphite is extremely illuminating [53]. In particular, the current-overpotential relation has anmaximum when the local molecular level passes the Fermi levels at small bias voltage. This is in line with vibrationally coherent two-step ET and different from both electrochemical single-step ET and sequential two-step ET as a current plateau would be expected at high overvoltage in either of these limits.

Acknowledgments. The financial support from the National Danish Research Foundation is gratefully acknowledged.

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Electrochemical Nanotechnology

In-situ Local Probe Techniques at Electrochemical Interfaces Edited by W. J. Lorenz and W. Plieth © WILEY-VCH Verlag GmbH, 1998

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