Electron transfer (ET) kinetics through n-dodecanethiol (C12SH) self-assembled monolayer on gold electrode was studied using cyclic voltammetry (CV), scanning electrochemical microscopy (SECM) and electrochemical impedance spectroscopy (EIS). An SECM model for compensating pinhole contribution, was used to measure the ET kinetics of solution-phase probes of ferrocyanide/ferricyanide (Fe(CN)(6)(4-/3-)) and ferrocenemethanol/ferrociniummethanol (FMC0/+) through the C12SH monolayer yielding standard tunneling rate constant (k(ET)(0)) of (4 +/- 1) x 10(-11) and (3 +/- 1) x 10(-10) cm s(-1) for Fe(CN)(6)(4-/3-) and FMC0/+ respectively. Decay tunneling constants (beta) of 0.97 and 0.96 angstrom(-1) for saturated alkane thiol chains were obtained using Fe(CN)(6)(4-) and FMC respectively. Also, it was found that methylene blue (MB) molecules are effectively immobilized on the C12SH monolayer and can mediate the ET between the solution-phase probes and underlying gold substrate. SECM-mediated model was used to simultaneously measure the bimolecular ET between the solution-phase probes and the monolayer-immobilized MB molecules, as well as tunneling ET between the monolayer-immobilized MB molecules and the underlying gold electrode, allowing the measurement of k(B1) = (5 +/-1) x 10(6) and (4 +/- 2) x 10(7) cm(3) mol(-1) s(-1) for the bimolecular ET and k(ET)(0)/MB = (1 +/- 0.3) x 10(-3) and (7 +/- 3) x 10(-2) s(-1) for the standard tunneling rate constant of ET using Fe(CN)(6)(4-)/(3-) and FMC0/+ probes respectively. (C) 2010 Elsevier Ltd. All rights reserved.