Scanning electrochemical microscopy of light-switchable redox enzymes cascades.

Bioelectronics is an active and progressive area of research associated with biochemistry, electrochemistry, and biotechnology. The combinations of these aspects led to a wide range of diverse applications, ranging from medical diagnostics and therapeutics, to alternative energy sources. Redox proteins and enzymes emerged as substances of interest in bioelectronics as it offers prominent and selective catalytic properties which are achieved through allosteric regulation. However, one fundamental challenge needs to be addressed to fully exploit the potential of enzymes in bioelectronics. That is, the development of a means to obtain spatial and temporal control over enzyme’s catalytic activity. Inspired by Nature’s photosynthesis, this thesis focuses on the development of light-addressable redox enzyme system. This is achieved through the use of artificial photosensitive chromophores which act as a switch to trigger the electron transfer within the enzyme complexes to activate or deactivate the enzyme catalytic ability.The photo/electrochemical properties of the light-addressable construct components include, two different artificial photosensitisers, two redox enzymes and their corresponding co-factors, are systematically studied by using electrochemical, microscopic, spectroscopic, and X-ray techniques. Several surface immobilisation techniques are evaluated by using CV, X-ray photoelectron spectroscopy and scanning electrochemical microscopy (SECM). Spatial control over the enzyme immobilisations were achieved by using microcontact printing of alkanethiol on gold surfaces. It was found that immobilisation strategy plays an important role in enzyme surface orientation and catalytic activity. The immobilisation of cyt c via maleimide-cysteine residueviiilinkage suggests enhanced catalytic activity with the ability to dock their native electron-transfer partner CcP.Two different light-addressable systems were developed and constructed on ITO surfaces, Ru-Symmetry-cyt c-CcP and Ir-Symmetry-GOx. The activity of the two enzyme systems are investigated by colourimetric assay, cyclic voltammetry, SECM and nuclear magnetic resonance spectroscopy. The results show that in the presence of photochemical stimuli, both systems exhibit the capacity to activate the enzyme catalytic activity and to react with their respective substrates. On the other hand, in the absence of the photochemical stimuli, the electron transfer by the photosensitive chromophores is suppressed, turning off the enzyme catalytic activity. Finally, Ir-Symmetry-GOx with CcP enzymatic cascade system was established and the activity was investigated. These results demonstrate temporal control over the light-addressable enzyme system, and thus achieving ‘on-demand’ enzyme activation