Medium Effect of Bicontinuous Microemulsion on Cobaltocene-Mediated Electroreduction of Coenzyme NAD

In this paper, a phosphate buffer (0.10 M, pH 7.5)-n-hexadecane bicontinuous microemulsion (BME) stabilized by the nonionic surfactant C₁₂E₄ was for the first time used as the medium to investigate its effect on the electrochemical behavior of the cobaltocene redox couple (Cc (III)/Cc (II)) as electron mediator and the Cc-mediated electroreduction of coenzyme NAD⁺. The results indicate that Cc (III)/Cc (II) exhibits a better electrochemical reversibility (ΔE_p ≈ 60 mV, i_pa/i_pc ≈ 1.07) and a little more positive shift of the formal potential in the above BME than in the aqueous phosphate buffer medium. With BME as the medium, Cc (II) can effectively mediate the electroreduction of NAD⁺ even without diaphorase (DH), although DH can accelerate the mediated reaction, especially at a high level of NAD⁺. The electrochemical behavior of Cc (III)/Cc (II) and the efficiency of the Cc (II)-mediated NAD⁺ electroreduction are significantly affected by the composition of the BME. With the increase of the oil volume fraction (0.45-0.60), the formal potential of Cc (III)/Cc (II) is positively shifted, the peak current is gradually increased, and the amplification of the reduction current induced by the mediated reaction between Cc (II) and NAD⁺ becomes pronounced. With the increase of the surfactant content, the formal potential of Cc (III)/Cc (II) shifts positively, the peak current varies little, and the electrocatalytic reduction current becomes small. All the medium effects of the BME could be well explained in terms of the effective electrode area occupied by the oil domain, the microstructural size of BME, and the local concentrations of both Cc (II) in the oil domain and NAD⁺ in the water domain. The present work is helpful for the application of a BME in the fields of NADH-based bioelectrosynthesis.