基于氧化石墨烯荧光生物传感器用于CAPN-2活性监测和实时成像

Abstract

Calpain-2 (CAPN-2) or m-calpain is a major calpain isoform that exerts its regulatory functions via limited proteolysis in a calcium flux-dependent manner, and dynamic changes of CAPN-2 activity participate in physiological and pathological events including cell migration, apoptosis, and synaptic plasticity. Accordingly, development of highly efficient sensing and imaging systems for CAPN-2 is crucial for both fundamental research and clinical diagnostics. This study presents an energy-transfer-based fluorescent biosensing platform by assembling FITC-labeled substrate peptide (FSP) probes on graphene oxide (GO) nanosheets for specific detection and real-time monitoring of CAPN-2 activity. The sensing mechanism relies on the CAPN-2-specific cleavage of the peptide substrate, which triggers the release of the FITC-labeled fragment from the GO surface and subsequently leads to the recovery of fluorescence. The FSP-GO assay demonstrated high sensitivity for CAPN-2 quantification, showing a strong linear correlation between the fluorescence signal and CAPN-2 concentration with a low detection limit of 0.33 U/mL. Additionally, the system exhibited high specificity in vitro, successfully discriminating between the highly homologous CAPN-1 and CAPN-2 isoforms. This biosensor was further applied for real-time monitoring and in-situ visualization of CAPN-2 activity in living cells with calcium flux modulation, CAPN-2-specific siRNA interference and calpastatin (CAST) deletion. These attributes of simplicity, cost-effectiveness, high specificity, and excellent selectivity underscore the potential of the FITC-Peptide-GO system as a versatile tool for probing CAPN-2-related biological processes and advancing therapeutic discovery.