Michaela Nebel

Scanning electrochemical microscopy in neuroscience.

This article reviews recent work involving the application of scanning electrochemical microscopy (SECM) to the study of individual cultured living cells, with an emphasis on topographical and functional imaging of neuronal and secretory cells of the nervous and endocrine system. The basic principles of biological SECM and associated negative amperometric-feedback and generator/collector-mode SECM imaging are discussed, and successful use of the methodology for screening soft and fragile membranous objects is outlined. The drawbacks of the constant-height mode of probe movement and the benefits of the constant-distance mode of SECM operation are described. Finally, representative examples of constant-height and constant-distance mode SECM on a variety of live cells are highlighted to demonstrate the current status of single-cell SECM in general and of SECM in neuroscience in particular.

4D shearforce-based constant-distance mode scanning electrochemical microscopy.

4D shearforce-based constant-distance mode scanning electrochemical microscopy (4D SF/CD-SECM) is designed to assess SECM tip currents at several but constant distances to the sample topography at each point of the x,y-scanning grid. The distance dependent signal is achieved by a shearforce interaction between the in-resonance vibrating SECM tip and the sample surface. A 4D SF/CD-SECM measuring cycle at each grid point involves a shearforce controlled SECM tip z-approach to a point of closest distance and subsequent stepwise tip retractions. At the point of closest approach and during the retraction steps, pairs of tip current (I) and position are acquired for various distances above the sample surface. Such a sequence provides x,y,I maps, that can be compiled and displayed for each selected data acquisition distance. Thus, multiple SECM images are obtained at known and constant distances above the sample topography. 4D SF/CD-SECM supports distance-controlled tip operation while continuous scanning of the SECM tip in the shear-force distance is avoided. In this way, constant-distance mode SECM imaging can be performed at user-defined, large tip-to-sample distances. The feasibility and the potential of the proposed 4D SF/CD-SECM imaging is demonstrated using on the one hand amperometric feedback mode imaging of a Pt band electrode array and on the other hand the visualization of the diffusion zone of a redox active species above a microelectrode in a generator/collector arrangement.

Visualization of Oxygen Consumption of Single Living Cells by Scanning Electrochemical Microscopy: The Influence of the Faradaic Tip Reaction†

The respiration activity of an individual living cell is an indicator of its metabolic vitality. Closely positioned microelectrodes have been suggested for determination of the respiration activity by monitoring the local oxygen concentration. Although first attempts for visualization of the oxygen consumption rate of single living cells by means of scanning electrochemical microscopy (SECM) were already described in 1998, evaluation of the respiratory activity of individual cells remains challenging and the complexity is often underestimated. In particular, the dimensions of the cell itself lead to limitations of conventionally used constant-height mode SECM investigations. Apart from convolution of the oxygen reduction current at the SECM tip with topographic effects, constant-height mode experiments require working distances comparable or below the height of the cell body, thus increasing the risk of tip crash. Attempts to overcome these restrictions include among others positioning of the tip to distances outside the feedback range, embedding of the cells into cavities, or efforts to subtract topographic contributions after cell death. Moreover, as living cells are irregular in dimension, the tip-to-cell distance varies with the tip position. Therefore, constant-distance mode (cd-mode) SECM techniques are inherently advantageous for this purpose. In particular, coupling SECM with scanning probe techniques, such as atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) as well as shearforce and impedance-based techniques, led to efficient strategies to control the tip-to-sample separation. Recently, we described a shearforce-based cd method (4D SF/ CD-SECM) that is able to work at various tip-to-sample separations. It can hence detect complete diffusion profiles in the surroundings of sources or sinks of redox-active species. Although SECM distance control systems are available, the detection of the respiration activity of single living cells remains challenging. Owing to the small rate of oxygen consumption by a single cell, only small current variations ontop of a high background current are measured. Even more importantly, a biological cell acts as an immiscible liquid– liquid interface in a SECM experiment. Lipophilic redox mediators are known to undergo transmembrane diffusion processes and can be utilized to investigate intracellular redox activity. However, concentration changes in the vicinity of the cellular membrane, for example by the tip reaction, may induce local concentration gradients and cause a diffusional exchange of redox species over the lipid bilayer in a socalled SECM-induced transfer (SECM-IT) mode. The high solubility of oxygen in lipids promotes this transmembrane diffusion and oxygen can easily cross the cell membrane. This diffusion process superimposes the detection of cell respiration. As a result, in most reports addressing detection of cell metabolism based on the detection of variations in the local oxygen concentration, the positioned microelectrode does not act as a passive observer but actively influences the oxygen concentration inside the gap between tip and cell, resulting in imaging artifacts that have not previously been addressed. Even though mentioned occasionally, this effect was neglected in SECM investigations of respiration activity at living cells. Herein, we address the influence of the oxygen reduction rate at the SECM tip on imaging the respiration activity at living cells. We provide strategies to avoid limitations resulting from a strong tip reaction using a potential pulse profile at the tip with a time dependent data acquisition in the shearforce-based cd-mode of SECM. Commonly, the detection of the local oxygen concentration in close proximity to the cell body is performed by means of a variation of the generator-collector mode of SECM with the tip being continuously polarized at oxygen reduction potential. The tip competes with the respiring living cell for the available oxygen inside the gap between SECM tip and cell surface. Crossing the cell body during a SECM line scan should therefore lead to a decrease of the tip current owing to a locally lowered oxygen concentration caused by cell [*] Dr. M. Nebel, S. Gr tzke, Prof. Dr. W. Schuhmann Lehrstuhl f r Analytische Chemie, Elektroanalytik & Sensorik and Center for Electrochemical Sciences, CES Ruhr-Universit t Bochum Universit tsstrasse 150, 44780 Bochum (Germany) E-mail: wolfgang.schuhmann@rub.de

Microelectrochemical visualization of oxygen consumption of single living cells.

The detection of cellular respiration activity is important for the assessment of the status of a biological cell. Due to its non-invasive character and high spatial resolution scanning electrochemical microscopy (SECM) is a powerful tool for single cell measurements. Common limitations of respiration studies performed by SECM are discussed and strategies provided to further adapt SECM detection schemes to the specific requirements for the investigation of single cell respiration. In particular the combination of a potential pulse technique in the redox competition mode of SECM with a shearforce-based constant-distance positioning of the SECM tip is proposed for characterising the impact of the tip reaction during SECM imaging. The adjustment of the driving force of the tip reaction and the selection of the time for data acquisition after applying the potential pulse allowed a successful visualization of cell respiration activity.

Constant-distance mode SECM as a tool to visualize local electrocatalytic activity of oxygen reduction catalysts.

Summary Multidimensional shearforce-based constant-distance mode scanning electrochemical microscopy (4D SF/CD-SECM) was utilized for the investigation of the activity distribution of oxygen reduction catalysts. Carbon-supported Pt model catalyst powders have been immobilized in recessed microelectrodes and compared to a spot preparation technique. Microcavities serve as platform for the binder-free catalyst sample preparation exhibiting beneficial properties for constant-distance mode SECM imaging concerning modified surface area and catalyst loading. The integration of the redox competition mode of SECM into the detection scheme of the 4D SF/CD mode is demonstrated for specifically adapting high-resolution SECM experiments to powder-based catalyst preparations.

High-Resolution Analysis of Photoanodes for Water Splitting by Means of Scanning Photoelectrochemical Microscopy

In pursuance of efficient tools for the local analysis and characterization of novel photoelectrocatalytic materials, several SECM-based techniques have been developed, aiming on the combined benefit of a local irradiation of the analyzed sample and a microelectrode probe for the localized electrochemical analysis of the surface. We present the development and application of scanning photoelectrochemical microscopy (SPECM) for the laterally resolved characterization of photoelectrocatalytic materials. Particularly, the system was developed for the photoelectrochemical characterization of n-type semiconductor-based photoanodes for water splitting. By using the tip microelectrode simultaneously for local irradiation and as an electrochemical probe, SPECM was capable to simultaneously provide information about the local photocurrent generated at the sample under irradiation and to detect the photoelectrocatalytically evolved oxygen at the microelectrode. In combination with a novel means of irradiation of the interrogated sample, local analysis of semiconductor materials for light-induced water splitting with improved lateral resolution is achieved.

Using Cavity Microelectrodes for Electrochemical Noise Studies of Oxygen‐Evolving Catalysts

Cavity microelectrodes were used as a binder-free platform to evaluate oxygen evolution reaction (OER) electrocatalysts with respect to gas bubble formation and departure. Electrochemical noise measurements were performed by using RuO2 as a benchmark catalyst and the perovskite La0.58 Sr0.4 Fe0.8 Co0.2 O3 as a non-noble metal OER catalyst with lower intrinsic conductivity. Changes in the current during the OER originate from variations in electrolyte resistance during the formation of the gas phase and partial coverage of the active area. Fluctuations observed in current and conductance transients were used to establish the contribution from the ohmic overpotential and to determine the characteristic frequency of oxygen evolution. The proposed quantitative determination of gas bubble growth and departure opens up the route for a rational interface design by considering gas bubble growth and departure as a main contributing factor to the overall electrocatalytic activity at high current densities.

Thermoresponsive amperometric glucose biosensor.

The authors report on the fabrication of a thermoresponsive biosensor for the amperometric detection of glucose. Screen printed electrodes with heatable gold working electrodes were modified by a thermoresponsive statistical copolymer [polymer I: poly(ω-ethoxytriethylenglycol methacrylate-co-3-(N,N-dimethyl-N-2-methacryloyloxyethyl ammonio) propanesulfonate-co-ω-butoxydiethylenglycol methacrylate-co-2-(4-benzoyl-phenoxy)ethyl methacrylate)] with a lower critical solution temperature of around 28 °C in aqueous solution via electrochemically induced codeposition with a pH-responsive redox-polymer [polymer II: poly(glycidyl methacrylate-co-allyl methacrylate-co-poly(ethylene glycol)methacrylate-co-butyl acrylate-co-2-(dimethylamino)ethyl methacrylate)-[Os(bpy)2(4-(((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)methyl)-N,N-dimethylpicolinamide)](2+)] and pyrroloquinoline quinone-soluble glucose dehydrogenase acting as biological recognition element. Polymer II bears covalently bound Os-complexes that act as redox mediators for shuttling electrons between the enzyme and the electrode surface. Polymer I acts as a temperature triggered immobilization matrix. Probing the catalytic current as a function of the working electrode temperature shows that the activity of the biosensor is dramatically reduced above the phase transition temperature of polymer I. Thus, the local modulation of the temperature at the interphase between the electrode and the bioactive layer allows switching the biosensor from an on- to an off-state without heating of the surrounding analyte solution.