Efficient Electrochemical Coupling of Aptamer to Nanoelectrode for In Situ Detection of ATP in Single Cells.

Nanoelectrodes, renowned for their small size, rapid mass transport, fast response, and high spatiotemporal resolution, have been recognized as a powerful tool in biosensing, especially for single-cell analysis. However, the nanoelectrode itself has no selectivity and cannot respond to nonelectroactive substances, limiting its wide application to some extent. Herein, we propose a simple and efficient electrochemical conjugation strategy to develop an electrochemical aptamer-coupled (E-AC) sensor for detecting adenosine triphosphate (ATP) in single living cells. Through simple electrochemical conjugation, ferrocene-labeled aptamers could be stably and efficiently coupled onto the surface of carbon fiber electrodes within 5 min. The small size (ca. 400 nm) and biocompatibility of the functionalized nanoelectrodes enabled the E-AC sensors to noninvasively and continuously monitor ATP content in single HeLa cells over 20 min, as well as the cellular ATP fluctuations under glucose starvation. Furthermore, the E-AC sensors exhibit superior specificity, sensitivity, and universality in the application of analysis of ATP in single living Hela cells and MCF-7 cells. They were also versatile for sensing other nonelectroactive targets through modification of the corresponding electroactive marker-labeled aptamers, showing great potential in cell-related physiological processes and drug screening.