Electrochemiluminescence Imaging for Parallel Single-Cell Analysis of Active Membrane Cholesterol

Abstract

Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 μM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.

Surface‐Enhanced Raman Nanoprobes with Embedded Standards for Quantitative Cholesterol Detection

“…This is also demonstrated by the single Raman band at around 1007 cm −1 in the SERS spectrum at point 2 (Figure b,iv,c,i), indicating the successful oxidization of 4‐MPBA into 4‐HBT. For the NPs+SiO 2 @ChOx group, the map of HBT/BDT signal indicates an intracellular cholesterol concentration range of 3.7–18.3 × 10 −6 m , which is a similar level to the concentration of active membrane cholesterol reported previously . We can also confirm the successful generation of 4‐HBT signals by checking the individual SERS spectra (e.g., see the spectrum at point 3 in Figure c,ii).…”

Essential Role of Electrode Materials in Electrochemiluminescence Applications

“…Amatore and co‐workers combined, for the first time, spatial visualization with ECL in order to observe, with high resolution, concentration profiles of species generated at the microelectrodes or electrode array . Recently, transparent electrodes paved the way for new imaging techniques based on ECL, for example microbeads, fingerprints or cells . The imaging of processes limited near the electrode surface can be easily visualized, avoiding refraction from a thick layer of solution that decreases image resolution.…”

Intracellular Electrochemical Sensing

“…In another study, active membrane cholesterol in a single living cell was imaged via detection of H 2 O 2 generated by a reaction between cholesterol and cholesterol oxidase . Cholesterol in the plasma membrane of single cells can also be detected by using a microcapillary electrode filled with a mixture of cholesterol oxidase and Triton X‐100 .…”