Developing a fluorescence substrate for HRP-based diagnostic assays with superiorities over the commercial ADHP

The combination of horseradish peroxidase (HRP) and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays. 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP or Amplex red) is the most sensitive fluorogenic substrate known for HRP in current market, however, it suffers from some drawbacks, such as non-specific reactivity to carboxylesterase and limited fluorescence stability. In the present study, a novel HRP substrate 10-cyclopropylcarbonyl-dichloro-dihydroxyphenoxazine (AR-2), has been prepared, which exhibited improved sensitivity than ADHP in sensing HRP. Moreover, the fluorescence of AR-2/HRP demonstrated improved tolerance to physiological relevant pH fluctuation as compared to ADHP/HRP. Successful detection of uric acid/ urate oxidase reaction indicated excellent application prospect of AR-2/HRP for monitoring H2O2-generating biochemical reactions. More interestingly, an enzyme-linked immunosorbent assay (ELISA) using AR-2 as the fluorescence reporter has been successfully used in detecting IgG against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from human serum samples. Overall, AR-2 exhibits improved performances over the commercial ADHP, which will be an ideal alternative to ADHP in HRP-based fluorescence biochemical analysis and immunoassays.