Channel-Splitting Photoassisted Enzyme Biofuel Cells: A High-Confluent and Self-Powered Platform for Electrochemistry–Photoelectrochemistry-Coupled Ratiometric Bioassays

The integration of photoelectrochemical (PEC) and electrochemical (EC) signals is attractive for ratiometric EC bioassays. However, their different signal acquisition patterns will bring additional deviations and different magnitude values for weakening the self-calibration reliability. Herein, we present a photoassisted enzyme biofuel cell (PA-EBFC) as the self-powered and high-confluent system for PEC-EC-coupled ratiometric bioassays. The target can trigger the introduction of Au nanoparticle-decorated TiO₂ nanospheres (TiO₂@Au NSs) onto an anode, which can provide steric hindrance for suppressing the glucose oxidation over Au nanobowls while endowing photoresponsive ability. Moreover, the contributions of photoresponsive ability and steric hindrance to the open-circuit voltage (<i>E</i>^OCV) of PA-EBFC have been counteracted by optimizing the Au content of TiO₂@Au NSs, generating a constant <i>E</i>^OCV under illumination (<i>E</i>_light^OCV) and a target-related decrease in <i>E</i>^OCV without illumination (<i>E</i>_dark^OCV). Using microRNA-486-5p (miR-486-5p) as a model analyte, the entropy-driven DNA circuit has also been integrated for improving the sensitivity. The developed platform has displayed a good negative correlation between the <i>E</i>_dark^OCV/<i>E</i>_light^OCV and the logarithm of miR-486-5p concentration, along with a limited detection of 0.48 fM. This study demonstrates that splitting the nonillumination to illumination channels of PA-EBFC could be a promising strategy to upgrade EC-PEC-coupled ratiometric assays.