Development of an aptamer-based electrochemical sandwich assay for the detection of a clinical biomarker

Abstract

A disposable electrochemical assay involving magnetic particles and carbon-based screen-printed electrodes (SPEs) is developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein and is among the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay is based on a sandwich format in which a biotinylated RNA aptamer is immobilised onto streptavidin coated magnetic beads and then coupled to the same biotinylated aptamer. A streptavidin-alkaline phosphatase solution is then added to beads and after the recognition biotin-streptavidin, the modified magnetic beads are captured by a magnet on the surface of a graphite working electrode and the electrochemical detection is thus achieved through the addition of the AP substrate (α-naphthyl- phosphate) and α-naphthol produced during the enzymatic reaction is detected using Differential Pulse Voltammetry (DPV). The performance of the assay in terms of sensitivity, reproducibility and selectivity are studied in buffer. The detection limit (LOD) is 2.0.10-2 mg/L and the average coefficient of variation (CV) results 9%. The LOD found is comparable with that reported by ELISA and it was much lower than the clinically useful borderline value (8 mg/L). © 2010 Springer Science+Business Media B.V.