Analysis of Intracellular Glucose at Single Cells Using Electrochemiluminescence Imaging

Abstract

Here, luminol electrochemiluminescence was first applied to analyze intracellular molecules, such as glucose, at single cells. The individual cells were retained in cell-sized microwells on a gold coated indium tin oxide (ITO) slide, which were treated with luminol, triton X-100, and glucose oxidase simultaneously. The broken cellular membrane in the presence of triton X-100 released intracellular glucose into the microwell and reacted with glucose oxidase to generate hydrogen peroxide, which induced luminol luminescence under positive potential. To achieve fast analysis, the luminescences from 64 individual cells on one ITO slide were imaged in 60 s using a charge-coupled device (CCD). More luminescence was observed at all the microwells after the introduction of triton X-100 and glucose oxidase suggested that intracellular glucose was detected at single cells. The starvation of cells to decrease intracellular glucose produced less luminescence, which confirmed that our luminescence intensity was correlated with the concentration of intracellular glucose. Large deviations in glucose concentration at observed single cells revealed high cellular heterogeneity in intracellular glucose for the first time. This developed electrochemiluminescence assay will be potentially applied for fast analysis of more intracellular molecules in single cells to elucidate cellular heterogeneity.

Nanokit for single-cell electrochemical analyses

“…The relative SD of 63.6% from single-cell analysis was much larger than that of 9.04% from the measurement of aqueous glucose using three electrodes, which indicated the cellular heterogeneity of the intracellular glucose levels. This high cellular heterogeneity of intracellular glucose was similar to our previous results obtained using electrochemiluminescence imaging (26).…”

“…4E shows the charge increase in single HeLa cells, suggesting that intracellular glucose was detected. According to the average charge of 0.44 nC collected from single cells, the intracellular glucose level was estimated to be 2 mM, which was similar to published data and our previous results (25,26). The relative SD of 63.6% from single-cell analysis was much larger than that of 9.04% from the measurement of aqueous glucose using three electrodes, which indicated the cellular heterogeneity of the intracellular glucose levels.…”

“…Electrochemical sensors were developed to analyze the glucose levels in serum and cell populations (24); however, the electrochemical analysis of intracellular glucose levels at the single-cell level was still challenging (25,26). Thus, we attempted to use our nanokit to analyze the glucose levels in single cells.…”

Electrochemiluminescence Single‐Cell Analysis: Intensity‐ and Imaging‐Based Methods

“…To further improve the throughput of single cell analysis, Xu et al fabricated the cell-sized photoresist-based microwell (30 μm in diameter and height) array for simultaneous detection of 64 cells. [59] The microwell could not only retain single cells but also slow down the diffusion of H 2 O 2 away from the ITO region, thus avoiding the cross-interference among single cells. [60] After the cell membrane was removed by triton X-100, the intracellular biomolecules, such as glucose [59][60] and intracellular cholesterol [61] can be determined with the help of corresponding oxidases.…”

Imaging Analysis Based on Electrogenerated Chemiluminescence

“…The addition of detergent prompted the release of the intracellular substance, such as glucose, which could react with glucose oxidase and luminol to generate ECL under a positive voltage [77]. In this format, the ECL image showing 64 individual cells were obtained simultaneously in 60 s, which greatly facilitated the rate of single cell analysis.…”