Decoupling of the Confused Complex in Oxidation of 3,3′,5,5′-Tetramethylbenzidine for the Reliable Chromogenic Bioassay

Regulation of the reaction pathways is a perennial theme in the field of chemistry. As a typical chromogenic substrate, 3,3′,5,5′-tetramethylbenzidine (TMB) generally undertakes one-electron oxidation, but the product (TMB_ox1) is essentially a confused complex and is unstable, which significantly hampers the clinic chromogenic bioassays for more than 50 years. Herein, we report that sodium dodecyl sulfate (SDS)-based micelles could drive the direct two-electron oxidation of TMB to the final stable TMB_ox2. Rather than activation of H₂O₂ oxidant in the one-electron TMB oxidation by common natural peroxidase, activation of the TMB substrate by SDS micelles decoupled the thermodynamically favorable complex between TMB_ox2 with unreacted TMB, leading to an unusual direct two-electron oxidation pathway. Mechanism studies demonstrated that the complementary spatial and electrostatic isolation effects, caused by the confined hydrophobic cavities and negatively charged outer surfaces of SDS micelles, were crucial. Further cascading with glucose oxidase, as a proof-of-concept application, allowed glucose to be more reliably measured, even in a broader range of concentrations without any conventional strong acid termination.