Laboratoire d'Electrochimie Physique et Analytique, EPFL Valais Wallis, École Polytechnique Fédérale de Lausanne , CH-1951 Sion, Switzerland.
Laboratory of the Physics of Living Matter, École Polytechnique Fédérale de Lausanne , CH-1015 Lausanne, Switzerland.
Anal Chem. 2016 Dec 6;88(23):11436-11443. doi: 10.1021/acs.analchem.6b02379. Epub 2016 Nov 16.
Scanning electrochemical microscopy (SECM) has been widely used for the electrochemical imaging of dynamic topographical and metabolic changes in alive adherent mammalian cells. However, extracting intracellular information by SECM is challenging, since it requires redox species to travel in and out the lipid cell membrane. Herein, we present cell fixation and permeabilization approaches as an alternative tool for visualizing cell properties by SECM. With this aim, adherent cells were analyzed in the SECM feedback mode in three different conditions: (i) alive; (ii) fixed, and (iii) fixed and permeabilized. The fixation was carried out with formaldehyde and does not damage lipid membranes. Therefore, this strategy can be used for the SECM investigation of cell topography or the passive transport of the redox mediator into the cells. Additional permeabilization of the cell membrane after fixation enables the analysis of the intracellular content through the coupling of SECM with immunoassay strategies for the detection of specific biomarkers. The latter was successfully applied as an easy and fast screening approach to detect the expression of the melanoma-associated marker tyrosinase in adherent melanoma cell lines corresponding to different cancer progression stages using the SECM substrate generation-tip collection mode. The present approach is simple, fast, and reliable and can open new ways to analyze cell cultures with electrochemically based scanning probe techniques.
扫描电化学显微镜（SECM）已广泛用于电化学成像活贴壁哺乳动物细胞中的动态形貌和代谢变化。然而，通过 SECM 提取细胞内信息具有挑战性，因为它需要氧化还原物质进出脂质细胞膜。在此，我们提出了细胞固定和通透的方法，作为通过 SECM 可视化细胞特性的替代工具。为此，以三种不同的条件（i）活细胞，（ii）固定细胞，和（iii）固定和通透细胞的方式，在 SECM 反馈模式下分析贴壁细胞。固定采用甲醛进行，不会破坏脂质膜。因此，该策略可用于 SECM 研究细胞形貌或氧化还原介体被动进入细胞的运输。固定后对细胞膜进行额外的通透处理，可以通过 SECM 与免疫测定策略相结合来分析细胞内内容物，以检测特定生物标志物。后者成功地应用于通过 SECM 基底物生成-收集模式检测黑色素瘤相关标志物酪氨酸酶在不同癌症进展阶段的贴壁黑色素瘤细胞系中的表达，作为一种简单、快速和可靠的筛选方法。本方法简单、快速、可靠，可为使用基于电化学的扫描探针技术分析细胞培养物开辟新途径。
