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ANALYST, AUGUST 1992, VOL. 117

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Scanning Probe Microscopies for High-resolution Characterization of Electrochemical Sensors* Plenary Lecture

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Joseph Wang Department of Chemistry, New Mexico State University, Las Cruces, N M 88003, USA

A better understanding of tailored electrodes and electrochemical sensors requires a more detailed picture of their surfaces. N e w scanning probe techniques, such as scanning tunnelling or scanning bioelectrochemical microscopies, offer unique opportunities for high-resolution in situ characterization of tailored electrodebased sensors. Scanning tunnelling microscopy provides valuable information on the topography of pre-treated surfaces, the heterogeneity of composite electrodes, the morphology of electropolymerized films, the packing arrangement of adsorbed monolayers and the microdistribution of immobilized biological components. Scanning bioelectrochemical microscopy is shown to be extremely useful for the mapping of localized biological activity and the monitoring of dynamic biological events. Valuable insights are achieved by correlating the structural features with the preparation/modification conditions and the subsequent sensing performance. Such correlations can facilitate the predictive design of increasingly better sensors. Keywords: Scanning tunnelling microscopy; scanning electrochemical microscopy; modified electrode; biosensor

The ability to control and manipulate the surface properties deliberately can greatly facilitate the development of electrochemical sensors. 1 Tailored electrodes are very attractive for chemical sensing, as they couple the high sensitivity of amperometry with new dimensions of selectivity and stability provided by the surface modifier. Before many of these sensing applications are realized, numerous unanswered questions concerning the microstructures and function of modified electrodes must be addressed. Surface charcterization can play a very important role in understanding the fundamentals and performance of tailored sensor surfaces. Recently developed high-resolution scanning probe microscopies2 offer unique opportunities and challenges for the characterization and optimization of electrochemical sensors based on chemically modified electrodes. The term ‘scanning probe microscopies’ is a generic one for a family of techniques based on different types of interactions between the tip and the surface of interest. The opportunities accruing from use of these techniques for characterizing modified electrodes are described and discussed in the following sections.

Scanning Tunnelling Microscopic Characterization of Tailored Sensor Surfaces In scanning tunnelling miFroscopy (STM), a sharp tip is brought t o within several Angstroms of a sample surface so that a tunnelling current flows when a small bias (2 mV-2 V) is applied between them. The tip is scanned over the surface, while the current is being monitored. The tunnelling current is exponentially related to the tip-to-sample distance, and can be used as a sensitive probe for the structural and electronic properties of interfacial systems. The theoretical and practical aspects of STM are discussed in several reviews.Z4 Scanning tunnelling microscopy has rapidly become a powerful tool in electrochemistry.4 The ability to obtain high-resolution images in real time and space offers unique opportunities for the study of electrode/solution interfaces. Since the first application of STM for in situ observation of electrode surfaces,S several studies have appeared dealing

* Presented at the Meeting on Analytical Applications of Chemically Modified Electrodes, Bristol, UK, January 7-8, 1992.

primarily with the characterization of electrodeposition or dissolution (corrosion) processes. Our activity has focused on the high-resolution probing of amperometric sensors. Such activity has been facilitated by the incorporation of computercontrolled potentiostats and electrochemical cells in commercial STM instruments6 (Fig. 1). In situ electrochemical STM can, therefore, provide valuable insights into the nature of tailored electrodes (unattainable by other techniques). Particularly attractive, for optimum sensor design and performance, is the ability to relate the new surface microstructures with the preparation (modification) conditions and the subsequent amperometric response. Structure-preparation-performance relationships obtained in this laboratory for model tailored surfaces are reported below. Activation Processes Procedures for activating and cleaning of solid electrodes have been developed for obtaining reproducible electroanalytical results.’ It has been demonstrated that STM can be a very powerful tool for studying changes in the surface topography associated with electrochemical pre-treatment of glassy carbon electrodes, and hence for obtaining a better understanding of such activation processes.8 Such ability relies on the exponential tunnelling current-gap relationship characteristics of STM systems. (The tunnelling current can change by a factor of 2 vr more with a change in the tip-surface separation of only 1 A.) Clear changes in the surface roughness have, therefore, been observed under different treatment parameters (potential, frequency, duration, media, etc.). The surface images corresponded well with the cyclic voltammetric data for background and analyte solutions. Additional information Tunnelling current amplifier

/-

Solution

Tip

Reference electrode c

1

4 v, = T -L

Fig. 1 In situ electrochemical scanning tunnelling microscopy

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on activation processes can be obtained by atomic force microscopy (AFM), which affords knowledge of the formation of insulating layers (e.g., oxides) during anodic pretreatments.9 Analogous STM and AFM studies should be useful for probing sensor passivation processes, involving the formation of inhibitory layers. 1 0

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ElectropolymerizationProcesses The fabrication of many electrochemical sensors relies on electropolymerization processes.11.12 The electrochemical preparation approach permits precise control of the surface microstructures and hence of the sensor performance. Scanning tunnelling microscopy can be used to investigate the morphology of electropolymerized films (on the nanometre scale), and it provides valuable insights into the correlation between the anodization conditions and the growth patterns. For example, it has been shown that the morphology of phenolic films is strongly dependent on the anodization conditions. 10 While a nucleation-and-growth process (leading to discrete, disordered aggregates) appears for potentialscanning experiments, a layer-by-layer growth process characterizes the fixed-potential polymerization (e.g., Fig. 2). Additional structural differences were observed via the use of different anodization potentials, scan rates or phenolic monomers. Scanning tunnelling microscopy has also been used to obtain structural-preparation correlations for poly(aniline) coatings, where the high-resolution images indicate differences in the electropolymerization kinetics. 13

Composite Sensor Surfaces Studies on the heterogeneity of solid electrode surfaces represent another example of utilizing STM for characterizing amperometric sensors. In particular, composite (array-like) electrodes possess signal-to-noise advantages when compared with traditional electrodes consisting of a single conducting phase.13 Such improved detectability depends strongly on the distribution of the conductor within the material. Because STM can be applied only to electrical conductors, scans taken over ‘large’ (micrometre) surfaces can provide useful information on the spatial variation of the conducting and insulating regions. Such tunnelling-on/tunnelling-off effects have been used in this laboratory to characterize the microdistribution of the conducting region in complex composite materials, such as modified carbon paste,” carbon foam composite16 and graphite-epoxyl7 electrodes.

Fig. 2 Three-dimensional STM view of a poly(pheno1) film on a glassy carbon surface. following electropolymerization at 0.9 V for 1 min; reproduced, with permission, from ref. 10

Visualization of Biosensors Scanning tunnelling microscopy images of biocomponents on electrochemical transducers can offer valuable insights into the operation of amperometric biosensors. High-resolution STM visualization of biosensors can provide useful insights into the immobilization of biocomponents on electrode surfaces. Careful correlation of the resulting surface microstructures with the amperometric response can facilitate the fabrication of biosensors under optimum conditions. One promising immobilization avenue is the entrapment of enzymes in electropolymerized films. Scanning tunnelling microscopy (under potentiostatic control) has been shown in this laboratory to offer distinct views of glucose oxidase within a poly(pyrro1e) coating.18 Such imaging capability is attributed to changes in the local work function associated with the presence of the insulating enzyme clusters within the conducting polymeric matrix. The resulting ‘black hole’ (e.g., Fig. 3) thus reflects the microdistribution of the enzyme on the surface. Such distribution, and the subsequent amperometric response, are strongly dependent on parameters of the immobilization/electropolymerizativn procedure. Similar structural/response correlations should facilitate the predictive design of biosensors based on other bicomponen t/immobilization/transducer systems. Adsorbed Monolayers Self-organized monolayers represent a versatile and powerful approach to tailored interfaces, as they provide new levels of selectivity and can serve as model systems for biomembranes. Scanning tunnelling microscopy can provide valuable insights into the behaviour of monolayers on surfaces. Specifically, Widrig et aL.19 have used STM to reveal the packing arrangements of n-alkanethiolate monolayers spontaneously adsorbed on gold surfaces. In addition, nano-scale defects within organic monolayers can be imaged by a combination of underpotential deposition and STM.20 Similar activity is currently being devoted to STM investigations of LangmuirBlodgett monolayers and bilayers of phospholipids and fatty acids.

Scanning Bioelectrochemical Microscopy Scanning bioelectrochemical microscopy (SBECM) was developed in this laboratory21 to allow in situ mapping of biologically active surfaces. This scanning probe microscopic technique is a variant of scanning electrochemical microscopy,2’which involves use of a similar experimental setting as STM, but relies on a different sensing mechanism (the

Fig. 3 Three-dimensional STM view of glucose oxidase entrapped within a poly(pyrro1e) coating on a glassy carbon surface; reproduced. with permission, from ref. 18

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ANALYST, AUGUST 1992, VQL. 117

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tion-conditions/structuraVperformance correlations for providing the foundation for nanometre-scale surface modification. Chemical modification of this sort would play an increasing role in the future fabrication of microsensors.

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Outlook

Fig. 4 Scanning bioelectrochemical microscopy of biological surfaces. Reactant (R) diffuses into the biological surface where the reaction occurs, producing a product (P) which is measured at the microelectrode (after reaching it by diffusion), resulting in another product (P’)

monitoring of Faradaic, rather than tunnelling, currents). It is based on placing a microelectrode tip in close proximity to the biological specimen (in contact with a markedreactant solution) in order to probe amperometrically/voltammetricallythe biological consumption of the electroactive species or biogeneration of a detectable product (Fig. 4). For example, mapping of the localized glucose oxidase activity in a carbon paste - biosensor has been accomplished by incrementally moving the carbon fibre tip over the surface and monitoring the oxidation of the enzymically produced hydrogen peroxide. In addition to two-dimensional X-Y images of the surface biocatalytic activity, SBECM can be used for probing the kinetics of biological surface reactions. This is accomplished by repetitive and rapid measurements of biologically consumed (or generated) marker, performed at a fixed location above the biosurface, but at short time intervals (10 s ) . Square-wave voltammetry or chronoamperometry are particularly suitable for this task. The dynamics of hydrophobic partitioning of drugs into lipid layers, of metal uptake by an alga-containing surface or the enzymic activity of tissue (mushroom) surfaces has thus been explored.23 Such ability to reveal the dynamics of biological processes is, perhaps, the greatest power of SBECM. The non-destructivehon-damaging SBECM operation is very attractive for studying delicate biological structures (in their active state). Future SBECM work will be aimed at improving its resolution (through the use of nanometre tips to yield molecule-resolved images), and expanding its utility for investigating the structure/dynamics of biomembranes, interactions with deoxyribonucleic acid (DNA) and immunological reactions of affinity-based biosensing surfaces. There is no doubt that, on improving its resolution, SBECM will play an increasing role in studies of biological processes, in general, and for understanding the performance of biosensors, in particular.

Microlithography In addition to i n situ characterization, it is possibIe to use scanning probe techniques for the modification of surfaces with high resolution. We are currently exploring the utility of STM and SBECM for the microfabrication of chemical sensors. In particular, we are interested in coupling the controlled (programmed) movement of the tip with modulation of the bias voltage for electrodepositing reactive structures of nanometre dimensions. These could include extremely narrow and pre-defined enzyme ‘lines’, pre-defined membrane barriers (molecular sieves) or microelectrode arrays. Alternatively, it could be possible to use the tip to remove, locally, material from the surface. We hope to couple the lithographic and imaging capabilities of these techniques with amperometric testing, and to use the resulting prepara-

The techniques of STM and SBECM are excellent tools for probing the structure of chemically modified electrodes. However, extreme caution is often required for the proper interpretation of complex surface objects and for obtaining reproducible images. Many important opportunities remain for further advances in the use of STM, SBECM, AFM and related scanning probe microscopic techniques for the characterization of electrochemical sensors and tailored electrodes. The author believes that the most valuable contributions of these imaging techniques will be the in situ investigation of molecular interactions and recognition. These and other studies will undoubtedly result in a rational design of new interfaces with predictable properties. Such developments are both stimulating and encouraging for those working in the field of modified electrodes. The author thanks D. Yaniv, L. McCormick, T. Martinez, N. Naser, R . Li and L. H . Wu for their valuable contributions, and the National Institutes of Health and the American Chemical Society for financial support.

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