Detection of Activated Protein C by an ElectrochemicalAptamer-based Sandwich Assay

The goal of this study was to design an aptamer-based sandwich assay with electrochemical detection for activated protein C (APC), a kind of serine protease that plays important roles as anticoagulant, anti-inflammatory and cytoprotective agent. Two sandwich assay formats were tested, one using an anti-APC monoclonal antibody as capturing receptor and a biotinylated aptamer as detection receptor and the other using the same aptamer both for capturing and detection. The assay takes advantage of sandwich binding of two affinity ligands for increased specificity, magnetic beads as carriers of affinity ligands (antibodies or aptamers) for fast magnetic separation, and electrochemical measurement for sensitive detection. Streptavidin-alkaline phosphatase (AP) was fixed to the secondary aptamer through biotin-streptavidin affinity. A magnet placed under the surface of a graphite screenprinted electrode (SPE) captured magnetic beads carrying the affinity complex, and the electrochemical detection was thus achieved through the addition of AP substrate (?-naphthyl phosphate). Differential pulse voltammetry (DPV) was employed to detect the ?-naphthol (the enzymatic reaction product) on the surface of the SPE, which was related to the concentration of the target protein. The conditions for the aptamer immobilization and for the protein binding have been optimized.