Design of a Fluorescent Probe for Hydrogen Peroxide Detection in Escherichia coli Fermentation Broth Based on ESIPT Mechanism

In this paper, two novel fluorescent probes, HBTMI‐B and HBTOI‐B, were designed and synthesized to detect endogenous hydrogen peroxide (H 2 O 2 ) in the fermentation broth of the bioengineered bacterium Escherichia coli ( E. coli DH5α ). By fluorescence spectroscopy experiments, the probes showed good sensitivity and selectivity in the reaction, with the fluorescence color shifting from violet to blue‐green and an obvious red‐shift at 300‐μM H 2 O 2 concentration. For the theoretical calculations, the reaction mechanisms of the probe molecules during the reaction with H 2 O 2 were investigated by using density‐functional theory (DFT) and time‐dependent density‐functional theory (TD‐DFT). In the S 1 state, the reaction barriers of the proton transfer were found to be 1.83 and 3.21 kcal/mol, respectively. Titration experiments further confirmed the high sensitivity of HBTMI‐B and HBTOI‐B at trace amounts of H 2 O 2 , with detection limits of 3.27 × 10 −7  M and 3.65 × 10 −7  M, respectively. In addition, biocompatibility tests showed that the two probes were non‐toxic to E. coli DH5α in a certain concentration range, proving their potential in biological experiments. Ultimately, the experiments validated the effectiveness of these two probes for the detection of endogenous H 2 O 2 in bioengineered fermentation broths, which provides a new tool for the detection of hydrogen peroxide in the field of bioengineering.