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Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM).

ABSTRACT: Changes in HeLa cell morphology, membrane permeability, and viability caused by the presence of Triton X-100 (TX100), a nonionic surfactant, were studied by scanning electrochemical microscopy (SECM). No change in membrane permeability was found at concentrations of 0.15 mM or lower during an experimental period of 30 to 60 min. Permeability of the cell membrane to the otherwise impermeable, highly charged hydrophilic molecule ferrocyanide was seen starting at concentrations of TX100 of about 0.17 mM. This concentration level of TX100 did not affect cell viability. Based on a simulation model, the membrane permeability for ferrocyanide molecules passing though the live cell membrane was 6.5 ± 2.0 × 10(-6) m/s. Cells underwent irreversible permeabilization of the membrane and structural collapse when the TX100 concentration reached the critical micelle concentration (CMC), in the range of 0.19 to 0.20 mM. The impermeability of ferrocyanide molecules in the absence of surfactant was also used to determine the height and diameter of a single living cell with the aid of the approach curve and probe scan methods in SECM.

SUBMITTER: Koley D

PROVIDER: S-EPMC2947864 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM).

Koley Dipankar D Bard Allen J AJ

Proceedings of the National Academy of Sciences of the United States of America 20100913 39

Changes in HeLa cell morphology, membrane permeability, and viability caused by the presence of Triton X-100 (TX100), a nonionic surfactant, were studied by scanning electrochemical microscopy (SECM). No change in membrane permeability was found at concentrations of 0.15 mM or lower during an experimental period of 30 to 60 min. Permeability of the cell membrane to the otherwise impermeable, highly charged hydrophilic molecule ferrocyanide was seen starting at concentrations of TX100 of about 0. ...[more]

PMID: 20837548

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Project description: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.

Dataset Information

Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM).

Publications

Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM).

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