Enhancing Reliability of Chromogenic Bioassay with Two-Electron TMB Oxidation Enabled by Self-assembled Surfactants Nanozyme

In a standard colorimetric sensing assay based on HRP-H2O2-TMB (3,3’,5,5’-tetramethylbenzidine), the stability of the signal of the chromogenic probe (TMBox) is essential for the accuracy of measurements. Terminators, typically of strong acids, are often added to maintain signal stability. However, additional manipulations may compromise sensing accuracy and the use of strong acids cause safety hazards. Here, a self-assembled sodium dodecyl sulfate (SDS) micellar nanozymes sensing strategy was proposed, which can catalyze the two-electron oxidization of TMB directly to the final state. Unlike the free and bound oxygen intermediates pathways, the micellar nanozymes enabled a microenvironment conducive to two-electron oxidization of TMB, in which the electrostatic attraction between the micellar nanozymes and TMB played crucial roles in substrate activation and intermediates stabilization. It allows micellar nanozyme to remain active even at very high H2O2 concentrations, thus enabling a broader detection range. GOD and the micellar nanozymes were cascaded for glucose detection as an application. As a result, the disadvantage of signal instability in conventional glucose biosensors from natural enzyme systems (HRP and GOD) was avoided.