In Situ Measurement of ATP in Single Cells by an Amphiphilic Aptamer-Assisted Electrochemical Nano-Biosensor

Abstract

In situ and quantitative measurements of adenosine 5′-triphosphate (ATP) in single living cells are highly desired for understanding several sorts of necessary physiological and pathological processes. Due to its small size and high sensitivity, an ultra-microelectrode can be used for single-cell analysis. However, ATP is difficult to detect in single cells because it is nonelectroactive and low in content. Herein, we introduced an electrochemical nano-biosensor based on an amphiphilic aptamer-assisted carbon fiber nanoelectrode (aptCFNE) with high signal-to-noise ratio. The low current (e.g., 60 pA) and the tiny diameter of the tip (ca. 400 nm) of the nanosensor made it noninvasive to living cells. The amphiphilic aptamer has good biocompatibility and can be stably modified to the surface of functionalized electrodes. CFNE, which was modified with ferrocene-labeled aptamer, could quickly and selectively detect ATP content in the nucleus, cytoplasm, and extracellular space of single HeLa cells. The results showed that the ATP contents in the nucleus, cytoplasm, and extracellular space were 568 ± 9, 461 ± 20, and 312 ± 4 μM, respectively. The anticancer drug treatment effects on the cellular level were further recorded, which was of great significance for understanding ATP-related biological processes and drug screenings. This strategy is universally applicable to detect other targets by changing the aptamer sequence, which will greatly improve our understanding of cell heterogeneity and provide a more reliable scientific basis for exploring major diseases at the single-cell level.

Recent Developments in Single-Entity Electrochemistry

“…Moreover, the nanopipette-based nanoelectrode configuration offers a small tip opening size combined with a large inner electrochemically active area, which aids in minimizing damage to the cell while retaining a high detection sensitivity. Recent progress includes functionalization of the nanoelectrode surface with aptamers, , ligands, and tannic acid to analyze a range of biomolecules from living cells. Another approach that facilitates intracellular delivery and detection is the use of a double barrel pipet where one barrel is filled with carbon for electrochemical detection and the other barrel is filled with biomolecules for delivery .…”

Recent Advances in Electrochemical Detection of Cell Energy Metabolism

“…ATP is the main energy carrier in biological organisms, playing an essential part in cellular metabolism pathways and several biochemical reaction processes [105]; thus, intracellular ATP serves as a reliable biomarker for the presence and level of biological activity in living organisms [106]. To this end, considerable work has been carried out to detect ATP at the subcellular scale in the context of cellular energy metabolism using electrochemical method on various modified electrodes [88,89]. Specifically, to enhance their sensitivity, aptamers were used for ATP detection.…”

Perspective—Assessing Electrochemical, Aptamer-Based Sensors for Dynamic Monitoring of Cellular Signaling

“…Carbon fiber electrodes have also been modified to act as E-AB sensors for ATP in and around cells. 48 Although the response time could be as low as 1 minute, it took about 5 min for the response to stabilize at 25 μ M ATP. The sensor could be inserted into the cytoplasm for 20 min and the nucleus for 10 min without resulting in cell death.…”

“… 50 Given these considerations, the relevant biological concentration ranges of some of the common targets for real-time cell measurements must be considered when developing sensing platforms. These can range from ∼4 mM 54 to hundreds of μ M 40 , 47 , 48 for extracellular ATP and ∼10–100 pg ml −1 55 – 58 for IFN- γ , two common targets (see Current Status section).…”