Laccase was detected histochemically in Pycnoporus cinnabarinus fungal hyphae by growing the organism on cellophane or microporous polycarbonate membranes overlaid on malt extract agar. Membrane sections were stained with a reagent of 2,6-dimethoxyphenol and colloidal gold for 30 min and irradiated for 24 s at maximum output in a microwave oven. Laccase was detected as purple-blue deposits at the apical tip region in high concentrations, and at lower levels on the exterior, sub-apical hyphal sheath.

Histochemical detection of laccase in Pycnoporus cinnabarinus using microwave-enhanced colloidal gold microcrystallization

The problems related to the mechanisms of electron transfer between electron-conducting structures (metals, semiconductors or organic conductors) and biopolymer molecules are central and most important in biolelectronics. Our interest in this area was to study the enzyme behavior at the interface “ionic conductor (electrolyte solution or solid electrolyte) - electron conductor (metal, carbon or semiconductor)”. In the experiments with various enzymes, we have shown that the direct electron transport between a conductor and the active site of redox enzyme is feasible. The rates of electron transfer, which may well proceed by the tunneling mechanism, can exceed the rates of subsequent enzymatic steps. In this case, we can think of an electric “contact” between semiconductor and enzyme active site. Experimentally, the processes of electron transfer between enzyme active site and conductor are most convenient to observe in electrochemical systems upon enzyme immobilization on electron conductor surface. In this case, the enzyme can act as electrocatalyst “pumping out” the electrons from conductor or donoring them into the conductor. This phenomenon was first revealed for a blue copper-containing oxidase (lacease) reducing molecular oxygen to water; the source of electrons was the conductor matrix [Berezin I.V et al. 1978; Berezin I.V et al., 1980]. The phenomenon of acceleration of electrode processes in direct “contact” of the enzyme and conductor was termed bioelectrocatalysis. Later, this phenomenon was observed for a number of other enzymes and for the enzymes entrapped in the matrices of organic semiconductors and organic metals [Varfolomeev S.D. et al, 1978; Hill H.A.O. et al, 1981]. This review is an endeavour to sum up these studies and to demonstrate the applicability of direct electron transport for creation of reagentless biosensors.

S. D. Varfolomeyev

Papers

Electric “Contacts” between Conductors and Protein Active Sites