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 show n that the property of laccase to catalyze the oxygen electroreduction at a n electrode allows to develop a mediatorless and pseudoreagentless electro- enzyme-immunoassay (EEIA). In this case the electrode acts as an electron-d onor substrate. When the bioelectrocatalytic reaction takes place, some ele ctric charge is collected on the electrode. A method of determination of th e electrode charge as well as the concentration of oxidized form of the med iator at the electrode surface has been elaborated. For this aim a techniqu e of the measurement of current-surge was employed. Human immunoglobulin G and insulin were taken as model in this investigation. A back titration sch emes without any mediator and in the presence of omicron -carboxybenzoylfer rocene 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 a nd the accumulation of the charge occurs linearly with time. The charge acc umulation for a long time allows to diminish the limit of detection. Howeve r, there is a limitation of the method. The direct electron transfer slows down with increasing the distance between the enzyme molecule and the elect rode surface. This effect reduces the sensitivity of the: method. The decre ase of the electron transfer rate with distance has been estimated. Monolay er of hemoglobin dividing the laccase molecule from the electrode surface d ecreases the rate by four times. The electron transfer rate for the antibod y electrode with associated antigen-laccase conjugate is less than that for the analogous electrode, covered with monolayer of covalently attached lac case. by 210 times. The current-surge peak was expected to decrease with di stance by an equation of the form I = I-0 exp[- r/r(0)]. The parameter r(0) 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 immunoprot eins immobilized on the electrode is discussed. (C) 2001 Elsevier Science B .V. All rights reserved.