Magnetically-assisted electrochemical immunoplatform for simultaneous detection of active and total prostate-specific...

Simultaneous detection of active and inactive proteases is clinically meaningful for improving diagnostic specificity. In this work, we reported an electrochemical method for simultaneous immunoassays of active and total proteases. Magnetic beads (MBs) were used as the solid supports for immobilization of capture antibodies and enrichment of targets. For the detection of active protease, the proteolytic-reaction-based analysis was carried out by the generation of Cu²⁺-binding peptide, in which a label-free peptide was used as the proteolytic substrate. The redox potential of the resulting peptide-Cu²⁺ complex was intrinsically distinguished from that of free Cu²⁺, thus allowing the "signal-on" detection of active protease. For the immunoassay of total protease in a sandwich-like format, electroactive metal-organic frameworks (Cu-MOFs) were used as the signal tags. The captured Cu-MOFs could directly produce a well-defined electrochemical signal from the reduction of Cu²⁺ ions. The analytical performances of the immunoplatform were evaluated by determining the model analytes of free and total prostate-specific antigen (fPSA and tPSA) in buffer and serum. The detection limits were found to be 0.3 pM for fPSA and 2 pM for tPSA. This work proposed a new strategy for simultaneous detection of active and total proteases, which should be evaluable for clinical diagnosis and treatment of protease-relative diseases.