The reaction of horseradish peroxidase (HRP) and its fluorogenic substrate enables the sensitive analysis of various targets, making it frequently used in high-throughput techniques. Fluorescence-activated droplet sorting (FADS) is an ultrahigh-throughput analytical method that shows great potential in single-cell analysis and enzyme engineering. However, the application of HRP fluorescence assays in FADS has encountered difficulties due to the defects of nonspecific reactivity and relatively poor droplet retention of traditional HRP substrates. In this study, a new HRP fluorescent substrate, AQHR, was constructed via modifying the resorufin scaffold. Compared with the commercial substrate ADHP, AQHR has enhanced hydrophilicity and red-shifted absorption and emission spectra . In addition, AQHR exhibited excellent reactivity to HRP/H2O2 and greatly elevated anti-interference capability in comparison with ADHP. In the present study, the advantages of AQHR were confirmed by multienzyme cascade fluorescence assays and FADS screening of carboxylesterase (CES) mutants. AQHR is expected to become a promising substrate for HRP and be widely used in FADS and other analytical fields.
Li et al. (Mon,) studied this question.