A Nanoenzyme-Sensitized Photoelectrochemical Biosensing Platform Integrated with a Two-Step Radical Polymerization Signal Amplification Strategy for Ultrasensitive Detection of PTP1B Activity

Dephosphorylation is an essential process in cellular signaling with protein phosphatases playing a critical role in cellular functions and disease mechanisms. Herein, a novel and ultrasensitive photoelectrochemical (PEC) biosensing platform for detecting protein tyrosine phosphatase 1B (PTP1B) activity was developed, based on the sensitization effect of a magnetic ZnFe₂O₄@ZrMOF nanoenzyme integrated with a two-step radical polymerization signal amplification strategy. The PTP1B-specific phosphorylated peptide (p-peptide) was immobilized on a 96-well plate and then coupled to the ZnFe₂O₄@ZrMOF nanoenzyme through coordination between its phosphate groups and Zr⁴⁺ ions. When PTP1B was present, the p-peptide was specifically recognized and dephosphorylated, causing the release of ZnFe₂O₄@ZrMOF. After magnetic separation, the detached ZnFe₂O₄@ZrMOF nanoenzyme, with peroxidase (POD)-like and photoresponsive oxidase (OXD)-like activities, was used as a signal probe, which not only exhibited a PEC signal sensitization effect on the AgInS₂/Ag₂S/magnetic ITO (MITO) photoelectrode but also triggered two-step radical polymerization reactions to form the ZnFe₂O₄@ZrMOF/polydopamine (PDA)/ poly(ferrocenylmethyl methacrylate) (PFcMMA) composite for further enhancing the PEC signal amplification. The proposed PEC biosensing platform achieved ultrasensitive detection of PTP1B activity, demonstrating a wide linear range (10 aM-0.1 μM) and an ultralow detection limit (3.92 aM), along with good reproducibility, satisfactory stability, high selectivity, and promising practical applicability. This work provides a prospective method for protein phosphatase activity assay, facilitating early disease diagnosis and therapeutic development alongside an innovative signal amplification strategy for advancing PEC biosensor sensitivity.