Abstract
H2O2 and horseradish peroxidase (HRP) are commonly used together in bioassays. HRP is required to accelerate the reaction between a chromogenic substrate (e.g., 3,3′,5,5′‐tetramethylbenzidine, TMB) and H2O2, and thus amplifies the signal. Herein, molecular oxygen for enzyme‐free and H2O2‐free oxidation is explored, still using the same colorimetric reaction. Restricted by spin selection rules, the ground‐state triplet oxygen needs to be converted to the singlet state to oxidize TMB. Phloxine B (PB) is used as the photosensitizer because of its excellent performance and safety. Under green light irradiation, each PB has a turnover of approximately 51 TMB molecules in 20 min, making PB a “molecular enzyme mimic” for signal amplification. With its small size, multiple PB molecules are loaded on a graphene oxide nanosheet to design a modified enzyme‐linked immunosorbance (ELISA) assay (termed photosensitization immunosorbent assay, PISA), improving the 1:1 enzyme/target ratio to n:1. PISA is more sensitive for carcinoembryonic antigen than a commercial ELISA kit, and successfully measures the antigen in the serum of multiple cancer patients. This simple and green method of oxidation coupled with the small size of the photosensitizer and graphene oxide may enable many other applications in biosensor development, smart materials, and energy harvesting.
