Redox-mediated dsDNA-dye photooxidase mimic enable catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine by dissolved O2 at neutral pH for improved biosensing

Catalytic oxidation of 3,3′,5,5′-Tetramethylbenzidine (TMB, an excellent chromogenic substrate) at neutral pH is critically important for amplified bioanalysis. Although some nanozymes exhibited the peroxidase activity at neutral pH, it is difficult to modulate their activity for homogeneous detection of biomolecules. In this work, we developed a redox-mediated dsDNA-dye photooxidase mimic that enables catalytic oxidation of TMB by dissolved O2 at neutral pH for improved biosensing. During illumination, the double-stranded DNA-SYBR Green I (dsDNA-SG) photogenerated singlet oxygen (1O2) can oxidized Mn2+ to Mn3+ that can efficiently oxidize TMB to produce a distinct blue within 4 min under neutral conditions. The catalytic oxidation of TMB can be readily modulated by the formation or dissociation of dsDNA during the sensing. After investigating a series of redox mediators, we found that only the Mn3+/Mn2+ redox mediator can lead to the oxidation of TMB at neutral pH. The maximum reaction rate of Mn2+-mediated dsDNA-SG photooxidase mimic under neutral conditions (pH 7.0) was 1.7 × 10−4 mM/s, even higher than that of horseradish peroxidase (HRP, 8.0 × 10−5 mM/s). The redox-mediated dsDNA-SG photooxidase mimic was used for detection of APE1 at pH 7.0 with over 130-fold higher sensitivity than that at 4.0, owing to the high enzymatic activity of APE1 at neutral pH. Meanwhile, we further extended this photooxidase mimic for the sensitive detection of DNA (LOD, 8 pM) and heavy metal ions at neutral pH. The redox-mediated dsDNA-dye photooxidase mimic with the ease of modulating its enzymatic activity and working at neutral pH is quite appealing for biosensing.