Ratiometric ECL sensor based on Apt-AuNS@Lu nanoprobe for analyzing cell swelling-induced ATP release.

Abstract

A novel ratiometric electrochemiluminescence (ECL) system based on gold nanostars (AuNSs) support was constructed for the determination of hypotonicity-induced ATP release from HepG2 cells. AuNS@Lu nanoprobe was used as anodic luminophore and K2S2O8 as cathodic luminophore as well as anodic co-reactant. AuNS with the large specific surface was adopted to adsorb plentiful luminol to form solid-state probe and as affinity support to immobilize ATP aptamer (Apt). The obtained nanocomposite (Apt-AuNS@Lu) generated astrong ECL signal at + 0.4V (vs. Ag/AgCl) with co-reactant K2S2O8, because of excellent conductivity and catalytic activity of AuNS. Furthermore, graphene oxide was reduced onto indium tin oxide (ITO) electrodes to facilitate the electron transfer. Following, polydopamine (PDA) film was formed via self-polymerization, improving stability and adhesion of the electrode surface. To immobilize ATP capture aptamer (AptC), abounding AuNSs were attached to RGO/PDA surface. When the sensor was incubated in the mixture solution of Apt-AuNS@Lu and target ATP, the ECL signal of Apt-AuNS@Lu increased with the increase of ATP concentration, meanwhile, the signal of K2S2O8 declined. The ratio of the two luminophores was used for the quantitative determination of ATP. The linear range was 5 to 250nM, and the limit of detection was 1.4nM at (3σ)/S. The method was successfully applied to analyze ATP release from HepG2 cells stimulated by 0.45% NaCl hypotonic solution. The results showed that the release kinetics profile of ATP had a sigmoidal shape with rapid release within 10min and then slowed. Compared to the isotonic groups, the intracellular ATP concentration was 3.7 ± 0.3µM (n = 3) decreasing by 40.3% and the extracellular was 23.4 ± 1.2nM (n = 3) increasing by 9.2 times in the hypotonicity for 10min, which showed ATP release from cells and good agreement with commercial ELISA test. The proposed strategy would be beneficial to broadening application of ECL technology in studying cell biological functions.