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

The goal of this study was to design an aptamer-based sandwich assay withelectrochemical detection for activated protein c (apc), a kind of serine protease that playsimportant roles as anticoagulant, anti-inflammatory and cytoprotective agent. two sandwichassay formats were tested, one using an anti-apc monoclonal antibody as capturing receptorand a biotinylated aptamer as detection receptor and the other using the same aptamer bothfor capturing and detection. the assay takes advantage of sandwich binding of two affinityligands for increased specificity, magnetic beads as carriers of affinity ligands (antibodies oraptamers) for fast magnetic separation, and electrochemical measurement for sensitivedetection. streptavidin-alkaline phosphatase (ap) was fixed to the secondary aptamerthrough biotin-streptavidin affinity. a magnet placed under the surface of a graphite screenprintedelectrode (spe) captured magnetic beads carrying the affinity complex, and theelectrochemical detection was thus achieved through the addition of ap substrate (?-naphthylphosphate). differential pulse voltammetry (dpv) was employed to detect the ?-naphthol(the enzymatic reaction product) on the surface of the spe, which was related to theconcentration of the target protein. the conditions for the aptamer immobilization and for theprotein binding have been optimized.