Ultrasensitive aptamer-based on-off assay for lysozyme using a glassy carbon electrode modified with gold nanoparticles and electrochemically reduced graphene oxide

The authors describe an electrochemical aptamer based assay for the determination of the serine protease lysozyme in very low (pM) concentrations. The method is based on the formation of a complex between anti-lysozyme aptamer fragments and lysozyme, and on electrochemical detection by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The surface of a glassy carbon electrode was modified with a nanocomposite consisting of gold nanoparticles and electrochemically reduced graphene oxide nanosheets (AuNPs/erGO), and the thiolated aptamer was then linked to the AuNPs by self-assembly through Au-S bonds. The interaction of immobilized aptamers with lysozyme leads to the decreased peak current in DPV and increased charge transfer resistance (R-ct) in EIS when using hexacyanoferrate or Methylene Blue as a redox probe. The calibration plot, when applying EIS and working at a typical voltage of -0.22 V (vs. SCE), is linear over 1.0 to 104.3 pM concentration range, with a detection limit of 0.06 pM (at a signal-to-noise ratio of 3). The respective data for DPV are a 9.6-205.5 pM linear range with a detection limit of 0.24 pM. Depending on the redox marker applied, the method works in the signal-off or signal-on mode in DPV and EIS protocols, respectively. The sensing interface is high specific for lysozyme and not affected by other proteins. The method was applied to the determination of lysozyme in spiked diluted human serum, and the results agreed well with data obtained with a standard ELISA.