Can Single Cell Respiration be Measured by Scanning Electrochemical Microscopy (SECM)?
Can Single Cell Respiration be Measured by Scanning Electrochemical Microscopy (SECM)?
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DOI：
10.1021/acsmeasuresciau.3c00019
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发表时间：
2023-10-18
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影响因子：
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通讯作者：
Unwin, Patrick R
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文献类型：
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作者：
Cremin, Kelsey;Meloni, Gabriel N;Valavanis, Dimitrios;Soyer, Orkun S;Unwin, Patrick R
超微电极（UME），或相当于微电极，探针越来越多地用于单细胞测量细胞特性和过程，包括生理活动，如代谢通量和呼吸速率。这种测量灵敏度的主要挑战包括：（i）细胞和UME通量（表现在电流中）的相对大小;以及（ii）UME响应随时间的稳定性问题。为了探索这些因素在多大程度上影响电化学细胞测量的精度，我们进行了系统的分析测量条件和实验参数确定单细胞呼吸速率通过在单个HeLa细胞的耗氧率（OCR）。使用扫描电化学显微镜（SECM），与铂UME作为探针，我们采用了自参考测量协议，很少采用SECM，其中的UME是反复接近从本体溶液到一个细胞，和短脉冲氧还原反应（ORR）电位附近的细胞和本体溶液中进行。这种方法能够周期性地跟踪体UME响应，与之重复比较（参考）近电池响应，并且还确保仅短暂地执行电池附近的ORR，从而最小化电化学过程对电池的影响。SECM实验与有限元法（FEM）建模框架相结合，以模拟氧气扩散和UME响应。假设单细胞OCR的实际范围为1 × 10-18至1 × 10-16 mol s-1，FEM模拟和自参考SECM测量相结合的结果表明，这些OCR值等于或低于该技术的当前检测灵敏度。我们提供了一组基于模型的建议，以改善这些测量在未来，但强调，如果要实现单细胞OCR测量的稳定性和精度的SECM测量将需要非凡的改进。
Ultramicroelectrode (UME), or, equivalently, microelectrode, probes are increasingly used for single-cell measurements of cellular properties and processes, including physiological activity, such as metabolic fluxes and respiration rates. Major challenges for the sensitivity of such measurements include: (i) the relative magnitude of cellular and UME fluxes (manifested in the current); and (ii) issues around the stability of the UME response over time. To explore the extent to which these factors impact the precision of electrochemical cellular measurements, we undertake a systematic analysis of measurement conditions and experimental parameters for determining single cell respiration rates via the oxygen consumption rate (OCR) in single HeLa cells. Using scanning electrochemical microscopy (SECM), with a platinum UME as the probe, we employ a self-referencing measurement protocol, rarely employed in SECM, whereby the UME is repeatedly approached from bulk solution to a cell, and a short pulse to oxygen reduction reaction (ORR) potential is performed near the cell and in bulk solution. This approach enables the periodic tracking of the bulk UME response to which the near-cell response is repeatedly compared (referenced) and also ensures that the ORR near the cell is performed only briefly, minimizing the effect of the electrochemical process on the cell. SECM experiments are combined with a finite element method (FEM) modeling framework to simulate oxygen diffusion and the UME response. Taking a realistic range of single cell OCR to be 1 × 10–18 to 1 × 10–16 mol s–1, results from the combination of FEM simulations and self-referencing SECM measurements show that these OCR values are at, or below, the present detection sensitivity of the technique. We provide a set of model-based suggestions for improving these measurements in the future but highlight that extraordinary improvements in the stability and precision of SECM measurements will be required if single cell OCR measurements are to be realized.