Laccases imaging

Compared to noble metal ORR catalysts, BOD and laccase show a few special features. They can use a second substrate, that is, the organic compound to be oxidized. The organic compound to be oxidized can be selected as a quasi-reversible redox couple, for example, 2,2 -azino-bis(3-ethyl-benzothiazoline-6-sulfonate) (ABTS ) that acts as a one electron donor for the laccase- or BOD-catalyzed reactions and is regenerated electrochemically [120] and thus can be used as mediator in biosensor applications or for SECM imaging ... 

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]. 

FIGURE 3.5 AFM images in liquid (tapping mode) of laccase on a screen-printed carbon electrode (a) height, (h) amphtude, and (c) expanded section showing enzyme molecules marked hy hlack dots. Scale har=50 nm. (Adapted with permission from Ref. [27]. Copyright 2010, Elsevier.)... 

The actual diameter of an object d) smaller than the tip radius has an imaged diameter (D) when the radius of the tip (r) and the height Qi) are considered. Hence, the apparent enzyme diameter of 26 nm, calculated to be an actual radius of 4.5 nm for the enzyme, is in close agreement with an enzyme monolayer thickness of 8nm calculated from force measurements and the reported diameter of the T. versicolor laccase [67]. From values of the observed electric current and electrode surface area (measured by direct electrochemistry and capacitance analysis, respectively) and the enzyme density calculated from the average area per enzyme molecule derived from AFM height images, the current per enzyme molecule was calculated to be 5xlO pA. 

For electrochemical applications, confocal laser scanning microscopy (CLSM) allows a 3D investigation of diffusion layers at a spatial resolution that is compatible with the lateral resolution of SECM without any modification [82]. In 2008, Nogala et al. combined SECM with CLSM and SPM to image laccase embedded in sol-gel film... 

FIGURE 14.15 Combined SPM-CLSM-SECM imaging of laccase aggregate, (a) SFM image (b) CLSM reflection mode image (50x objective) (c) SC/TC mode SECM image. (Reprinted with permission from Ref. [66]. Copyright 2008, Elsevier.)... 

Imaging/Labeling Applications Fibroblasts " galactosylceramide " glucose oxidase (GOx) horseradish peroxidase (HRP) laccase Upids lysozyme crystal nucleic acids paromomycin phosphatidylcholine " proteins sphingomyelin " sphingosines ... 

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