Enzymes, activity, imaging electrodes

The concentrations of the enzyme substrate have to be selected with some care. In the GC experiment, it should be well above the Michaelis-Menten constant of the enzyme for this substrate. The presence of the UME with its sheath will also limit the diffusion of the enzyme substrate towards the active regions. This may lead to an underestimation of enzyme activity on the surface or to great distorsion in recorded images. Using electrodes with a small RG is a good idea for GC experiments. This question is explained in Procedure 52 (see in CD accompanying this book). 

Formation and imaging of microscopic enzymically active spots on an alkanethiolate-covered gold electrode by scanning electrochemical microscopy. Analytical Chemistry, 69 (24), 5059-5066. 

In the transient RC mode, both the UME tip and the enzyme-modified surface sample compete for the same analyte present in the microenvironment between the tip and the sample. Here, both tip and sample are held at the same potential, enabling both of them to electrochemically convert the analyte. When tip and sample are not along the same vertical axis, and are sufficiently separated laterally, the currents at the tip and sample are determined by the bulk analyte concentration and kinetics at the respective electrodes. However, as the tip moves closer to the sample and/or when it is directly above the sample, both electrodes (tip and sample) compete for the limited quantity of the analyte present between them. This results in a reduced tip current, as the analyte concentration available at the tip is reduced by its reaction at the sample. The decrease in the tip current is imaged as enzymatic redox activity in RC mode of SECM [63]. RC mode does not impose any limitation on the quality of the SECM activity image. Therefore, there are no restrictions to both the sample size and the minimum size of the UME tip. 

Vertically aligned CNT-modified electrodes are based on a more elaborated technique than other methods, and microscopic images are used to characterize the integrity of this type of electrode. The technique has been applied for the immobilization of enzymes and DNA, and the sensors based on this technique have shown a lower detection limit than those based on other methods. More research activities using this technique, particularly with low density CNT arrays, are expected in the near future because of its sensitivity and versatility. 

In this chapter, we review the recent progress in the development of different metal oxide nanoparticles with various shapes and size for fabrication of biosensors. The development of metal oxide nanomaterials surface film for direct electron exchange between electrodes and redox enzymes and proteins will be summarizing. The electrochemical properties, stability and biocatalytic activity of the proposed biosensors will be discussed. The biocompatibility of the metal oxide nanomaterials for enzymes and biomolecules will be evaluated. We will briefly describe some techniques for the investigation of proteins and enzymes when adsorbed to the electrode surfaces. Cyclic voltammetry, impedance spectroscopy, UV-visible spectroscopy and surface imaging techniques were used for surface characterization and bioactivity measuring. 

Butt and coworkers studied the voltammetry of the (Escherichia coli) decaheme class of nitrite reductases [244] (Figure 2.13). Well-defined although unstable catalytic multi-electron voltammetric reduction of nitrite by the enzyme immobihzed on bare Au(lll) electrode surfaces is notable. The decaheme nitrite reductase is a second case for single-molecule in situ STM of a redox metalloenzyme, but image interpretation is presently not at the level for CuNiR [227]. Molecular-scale structures can be observed on the Au(lll) electrode surface under conditions where the enzyme is electrocatalytically active, with both the natural dimer and surface-dissociated monomer enzyme structures identified. Molecular conductivities (in situ STM contrasts) of the enzyme and the active enzyme-substrate states are, however, not very distinctive. 

Enzyme-mediated feedback can be used to image enzyme patterns. To successfully image enzymatic features, lip fouling from oxide formation or adsorption from solution constituents must be avoided. The enzyme reaction at the substrate must not be inhibited by solution species. It must also be able to sustain a level of regeneration activity of the mediator that can compete with its mass transport from the bulk electrode to the tip. In the case of a glucose oxidase catalyzed reaction, a digital simulation of the positive feedback observed from this enzyme quantitatively expresses this limitation (143). 

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