Oxygen-free in situ scanning tunnelling microscopy

doi:10.1016/j.jelechem.2006.02.011

Journal of Electroanalytical Chemistry

Volume 599, Issue 2, 15 January 2007, Pages 213-220

https://doi.org/10.1016/j.jelechem.2006.02.011 Get rights and content

Abstract

Scanning tunneling microscopy under full electrochemical potential control (in situ STM) has been used extensively as an efficient method to characterize microstructures at solid/liquid interfaces at the atomic and molecular levels. However, under ambient conditions oxygen may interfere in open in situ STM systems by diffusion through the solutions. Such interference can be serious for oxygen sensitive systems both for the target molecules themselves and for chemical linker molecules bound to the electrode surface for example, via Au–S bonding. This is strikingly illustrated in the present report. We present here oxygen-free in situ STM, in which the samples are contained in an argon atmosphere which removes oxygen in the system. Interference from oxygen is profoundly reduced in this setup and demonstrated by three examples. Two of these are in situ STM of the intermediate-size sulfur containing organic molecule cysteamine and the biomolecule homocysteine. The third example is the iron–sulfur protein ferredoxin (Pyrococcus furiosus). Monolayers of these molecules on Au(1 1 1) are imaged to molecular resolution and the images compared in the presence and absence of oxygen. In all the cases, the STM image quality of the monolayer structures has improved significantly in the absence of oxygen. Reasons for the drastic effects of dioxygen based on both chemical surface reactivity and electron scattering effects are discussed. These observations are important in general for STM of oxygen sensitive chemical and biological samples.

Introduction

Since its invention in the early 1980s, scanning tunneling microscopy (STM) has been demonstrated to be an efficient surface sensitive technique for real space imaging of surface structures at the atomic scale. Moreover, STM has been brought to operate not only in ultra-high vacuum (UHV), but also in air and liquids [1]. The merit of operation in liquid attracts increasing attention because this has opened the option of investigating solid/liquid interfaces as well as biological molecules in situ. This is highly important as electrochemical phenomena occur at electrode/electrolyte interfaces and aqueous buffer solutions are essential to keep biological molecules active. In situ STM was developed by combining STM with electrochemistry in the late 1980s [2], [3]. Reference and counter electrode are contained in the STM cell together with electrolyte solution, while the STM tip is insulated except at the outermost end [2], [3]. In this configuration, both the substrate and STM tip are fully controlled by the electrochemical potential. This provides not only the scene for studying electrochemically related reactions but also a means to study tunneling mechanisms. In situ STM is also denoted as a four-electrode system [2] since the tip can be regarded as an additional electrode apart from the electrochemical (working, reference and counter) three-electrode system. This in situ probe technique offers potential applications in fields of chemistry, physics, and biology. In situ STM has been successfully employed to explore many surface-related aspects with atomic or molecular resolution, such as underpotential metal deposition (UPD), anion adsorption, corrosion, metal dissolution and nano-pattern deposition, and self-assembled monolayers [4], [5], [6].

Application of in situ STM to biological molecules has come to offer biotechnological perspectives in contexts of bioelectrochemistry, biosensors, biochips and bionanotechnology. This is based on the achievement of well-controlled orientation of metalloproteins and enzymes immobilized on solid surfaces [7], [8]. Both outstanding concepts and technical challenges, however, remain. Immobilization of biological molecules on solid surfaces with biological function retained is a general problem which has, however, been improved profoundly by employing molecular surface architectures, such as self-assembled monolayers (SAM) of functionalized thiol-containing molecules on metal surfaces [9], [10], [11], [12], [13], [14]. On the conceptual side mechanisms of the tunneling current through biological macromolecules is not understood to the same extent as for small molecules. Tunnelling is, however, demonstrated possible and resonance or coherent electron transfer mechanisms are suggested by theoretical studies [15], [16].

A technical obstacle is that oxygen can interfere with biological systems. Oxygen interference has shown to be an unexpected limitation in the application of in situ STM in biological contexts. This has prompted us to develop oxygen-free in situ STM. In the present report, Ar is used in the in situ STM system to remove and minimize the influence of oxygen. As examples, three important bio-related systems, cysteamine, homocysteine and the iron–sulfur protein, Pyrococcus furiosus ferredoxin [Fe₃S₄] on Au(1 1 1)-surfaces have been successfully investigated by both in situ STM and oxygen-free in situ STM. Image resolution and quality are significantly improved by using oxygen-free in situ STM in these three cases. Reasons for the improvement based on oxygen interference with the Au–S surface chemistry and on a role of oxygen in the STM tunneling mechanism are discussed.

Section snippets

Chemicals

3-Mercaptopropionic acid (MPA, >98%, Merck), cysteamine (>98%, Fluka) and homocysteine (Fluka, ⩾95%) were used without further purification. Phosphate buffer (PB) was prepared by mixing KH₂PO₄ (suprapur, 99.995%, Merck) and K₂HPO₄ (suprapur, 99.99%, Merck). NaAc (pH = 6.0) was prepared by mixing NaAc (>99%, Suprapur, Merk) with HAc (>99.7%, Aldrich). P. furiosus ferredoxin (Pf Fd) [Fe₃S₄] was expressed by Hans E.M. Christensen at the Department of Chemistry using gene technology in Escherichia

Results and discussion

Fig. 2 shows representative in situ STM images of the Au(1 1 1)-electrode surface in the presence (A) and absence of oxygen (B), respectively. Herringbone lines with periodic distance 6.4 nm and angle 60° are observed clearly. This is a typical pattern for clean and reconstructed Au(1 1 1)-surfaces as reported in the literature [22]. This means that neither Ar nor oxygen affects the tunneling current from STM tip to bare Au(1 1 1), and is further an indication of insignificant adsorption of oxygen or

Conclusions

Similarity between gas/solid and liquid/solid interfaces for many small and intermediate size molecules is observed by UHV STM and in situ STM with sharp contrast and high resolution, even though the environments are drastically different for the two cases. Water, dioxygen molecules and one atmosphere pressure appearing in the liquid/solid interface are close to natural environment. For this reason, in situ STM offers wide perspectives for applications. The aqueous medium and possible counter

Acknowledgements

Assistance from the workshop at the Department of Chemistry, Technical University of Denmark, and financial support from The Danish Science Research Council for Technology and Production Sciences is acknowledged. We thank Dr. Hans E.M. Christensen, Department of Chemistry for providing the Pyrococcus furiosus ferredoxin samples.

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Oxygen-free in situ scanning tunnelling microscopy

Abstract

Introduction

Section snippets

Chemicals

Results and discussion

Conclusions

Acknowledgements

Surf. Sci.

J. Electroanal. Chem.

Curr. Opin. Chem. Biol.

Surf. Sci.

Trends Biotechnol.

Electrochem. Commun.

Progr. Surf. Sci.

Electrochim. Acta

Adv. Inorganic Chem.

Chem. Rev.

J. Phys. Chem. A

Errata

Science

Angew. Chem. Int. Ed.

Chem. Rev.

Progr. Surf. Sci.

J. Phys. Chem. B

J. Phys. Chem.

Langmuir

J. Phys. Chem. B

J. Phys. Chem. B