Discovering a Dihydrofluorescein Analogue as a Promising Fluorescence Substrate to HRP

Horseradish peroxidase (HRP) combined with its fluorescence substrates is attracting increasing attention for biochemical analysis. Amplex red is the most widely used fluorescence substrate to HRP; however, it suffers from some drawbacks, such as nonspecific responsiveness toward carboxylesterases. Discovering a new small molecular fluorescence substrate with improved sensitivity and selectivity for HRP is thus desired. Herein, three dihydrofluorescein derivatives (DCFHs) are presented to serve as HRP substrates through fluorescence turn-on methods. The most promising one, 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthene-3,6-diol (DCFH-1), exhibited excellent sensitivity in the detection of HRP. Moreover, DCFH-1 does not respond to carboxylesterase, thus holding advantages over Amplex red. In the further study, the detection reagent in the commercial ELISA kits was replaced with DCFH-1 to establish a new fluorescence ELISA, which works very well in the quantification of inflammatory cytokine biomarkers from in vitro models.

Automated summary

Horseradish peroxidase (HRP) combined with its fluorescence substrates, such as Amplex red, is widely used in biochemical analysis. However, Amplex red has limitations due to nonspecific responsiveness. In this study, three new dihydrofluorescein derivatives were identified as improved HRP substrates. The most promising derivative, DCFH-1, exhibited excellent sensitivity and selectivity for HRP detection, outperforming Amplex red. DCFH-1 was further used in a fluorescence ELISA for the quantification of inflammatory cytokine biomarkers, yielding successful results.