Enzyme-mediated feedback imaging

In FB mode, the UME tip is held at a potential where electrolysis of a soluble redox mediator proceeds under diffusion-controlled conditions and the surface does not communicate with the tip. FB mode requires that the tip be positioned at a distance d as close as possible to the surface. This requires that the probe approach cmrents be adjusted accordingly during the initial semp of the system. When the UME tip is positioned at a quasi-infinite distance (oo ) from the surface (where d 0 times the tip radii), the FB current is defined by Equation 14.2 [9]  

Because of its low sensitivity compared with the GC mode, FB mode requires a highly active enzyme. This method of imaging involves the replacement of the enzyme cofactor or electron acceptor by a mediator. To be able to determine the enzymatic activity using the enzyme-mediated positive FB mode in SECM, the system must satisfy the following condition (Equation 14.3) [28]  

TABLE 14.1 Common EEC Anode Enzymes Studied Using Enzyme-Mediated FB Imaging Mode in SECM 

Enzyme Substrate/Type of UME Reaction Mediator References 

FIGURE 14.6 GOx-modified beads arranged and images using FB mode. (Reprinted with permission from Ref. [33]. Copyright 2(X)1, Wiley-VCH Verlag GmbH.) 

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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). 

Even feedback imaging of BOD and laccase is possible with [Fe(CN)eP as mediator and Au tips (Pt tips are not useful because the ORR would occur at the tip) [24], The sample should preferably be an insulator to exclude interfering electron transfer reactions between the mediator and the substrate that do not involve the enzymes. This working mode has advantages with respect to lateral resolution and possibilities for quantitation. It has been used for the analysis of spot homogeneity and a comparison of the pH dependence of laccase and BOD (Figure 11.22) [24]. 

Immobilized enzymes can be investigated in the feedback and in the GC mode. In the feedback mode oxidoreductases can be imaged. They use the SECM mediator as electron donor (or acceptor). Table 37.1 gives an overview about the enzymes investigated. 

A related technique was demonstrated by Shiku et al. (91), who formed alkylsilane monolayers at glass substrates and used electrochemical generation of OH radicals at the tip via the Fenton reaction to locally destroy the SAM. Diaphorase could then be patterned on the surface by physical adsorption to the undamaged hydrophobic areas or via covalent linkages to the radical-attacked areas. The former process was imaged in feedback mode with a ferrocenyl mediator and showed a decreased current over the disk of destroyed SAM and a constant background of enzyme activity over the rest... 

Diaphorase enzyme and diaphorase/albumine mixtures immobilized on the surface of a glass plate were imaged by Yamada [123] and coworkers using platinum disc tips. FcMeOH redox mediator and NADH reagent were used in these studies. The FcMeOH was oxidized at the platinum tip. The immobilized diaphorase catalyzed the oxidation of NADH by FcMeOH" the oxidized form of FcMeOH. So, if the tip was near to the catalytic surface, then the enzyme catalyzed reaction increased the local concentration of FcMeOH. That positive feedback generated increase of the current showed the active enzyme spots on the sample surface. 

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