A Metal-Organic Hybrid Composed of Dual Quenching Cofactors as a Nanoquencher for the Fluorescent Determination of Protease Caspase-3.

Nanoquenchers with a single quenching cofactor exhibit limited fluorescence quenching efficiency. In this work, a metal-organic hybrid with dual quenching cofactors (Cu and pyrroloquinoline quinone or PQQ) was prepared by metal-coordinated assembly and used as a nanoquencher for a protease assay with enhanced quenching efficiency. The peptide substrate with an oligohistidine (His) tag was labeled with a fluorophore. Caspase-3 was determined as a protease example. The substrate was attached onto the surface of the Cu-PQQ nanoquencher by a metal coordination interaction between the unsaturated Cu on the nanoparticle surface and the His tag in the peptide. The cleavage of the peptide substrate by enzymatic hydrolysis led to the release of a fluorophore-conjugated segment from the nanoquencher surface, thus turning on the fluorescence. The nanoprobe was used to determine caspase-3 with a linear range of 0.01-5 ng/mL and a detection limit of 7 pg/mL. Furthermore, the method was used to evaluate inhibition efficiency and monitor drug-induced cell apoptosis. In contrast to other means of peptide immobilization, such as physical adsorption and covalent coupling, the strategy based on the metal coordination interaction is simple and powerful, thereby achieving assays of caspase-3 activity in lysates with a satisfactory result. The work should be valuable for the design of nanoquenchers with multiple quenching cofactors and the development of novel biosensors.