On applicability of laccase as label in the mediated and mediatorless electroimmunoassay: effect of distance on the direct electron transfer between laccase and electrode.

Applicability of laccase as enzyme-label has been investigated. It was shown that the property of laccase to catalyze the oxygen electroreduction at an electrode allows to develop a mediatorless and pseudoreagentless electro-enzyme-immunoassay (EEIA). In this case the electrode acts as an electron-donor substrate. When the bioelectrocatalytic reaction takes place, some electric charge is collected on the electrode. A method of determination of the electrode charge as well as the concentration of oxidized form of the mediator at the electrode surface has been elaborated. For this aim a technique of the measurement of current-surge was employed. Human immunoglobulin G and insulin were taken as model in this investigation. A back titration schemes without any mediator and in the presence of o-carboxybenzoylferrocene as a mediator was applied. The antibody carbon-black and the antigen glassy-carbon electrodes were used. The limits of detection were found to be 0.3 and 1.6 nM, respectively. The advantage of the mediatorless assay is that the charge leakage is imperceptible by open circuit for a long time and the accumulation of the charge occurs linearly with time. The charge accumulation for a long time allows to diminish the limit of detection. However, there is a limitation of the method. The direct electron transfer slows down with increasing the distance between the enzyme molecule and the electrode surface. This effect reduces the sensitivity of the method. The decrease of the electron transfer rate with distance has been estimated. Monolayer of hemoglobin dividing the laccase molecule from the electrode surface decreases the rate by four times. The electron transfer rate for the antibody electrode with associated antigen-laccase conjugate is less than that for the analogous electrode, covered with monolayer of covalently attached laccase, by 210 times. The current-surge peak was expected to decrease with distance by an equation of the form I = I0 exp[-r/r0]. The parameter r0 is equal to 2.2 +/- 0.8 nm. The possibility of the sensitivity increase in the mediatorless mode by 'wiring' through the multilayer film of immunoproteins immobilized on the electrode is discussed.