Inner Filter Effect-Based Sensor for Horseradish Peroxidase and Its Application to Fluorescence Immunoassay

Being an important model peroxidase, horseradish peroxidase (HRP) has been thoroughly understood, and the detection of HRP is not only directly related to peroxidase-triggered catalytic process, but also linked to the development of HRP-based enzyme-linked immunosorbent assay (ELISA). Herein, we have reported an unconventional fluorescent sensor for convenient assay of HRP activity based on the HRP-catalyzed specific conversion of p-phenylenediamine (PPD) into chromogenic PPDox with H₂O₂ as the oxidizing agent, accompanied by the fluorescence quenching effect on fluorescein. By combining UV-vis absorption spectrum, isothermal titration calorimetry, and fluorescence lifetime analysis, we have confirmed the inner filter effect as a main quenching mechanism in our proposed fluorescent assay. According to the intrinsic sensitivity of fluorescent sensor and high selectivity, our PPD/fluorescein-based sensing system can be utilized for real-time monitoring of the HRP activity in real biological samples. Furthermore, the unambiguous response mechanism and excellent sensing performance encourage us to extend such HRP assay into the HRP-based fluorescent ELISA, which has a broad prospect of application in fluorescent diagnosis of hepatocellular carcinoma (HCC) by sensing alpha-fetoprotein, the well-known serologic HCC marker.