3D electrochemical and ion current imaging using scanning electrochemical-scanning ion conductance microscopy.

Local cell-membrane permeability and ionic strength are important factors for maintaining the functions of cells. Here, we measured the spatial electrochemical and ion concentration profile near the sample surface with nanoscale resolution using scanning electrochemical microscopy (SECM) combined with scanning ion-conductance microscopy (SICM). The ion current feedback system is an effective way to control probe-sample distance without contact and monitor the kinetic effect of mediator regeneration and the chemical concentration profile. For demonstrating 3D electrochemical and ion concentration mapping, we evaluated the reaction rate of electrochemical mediator regeneration on an unbiased conductor and visualized inhomogeneous permeability and the ion concentration 3D profile on a single fixed adipocyte cell surface.

Nanoscale imaging of an unlabeled secretory protein in living cells using scanning ion conductance microscopy.

Scanning ion conductance microscopy (SICM) was applied to evaluate an unlabeled secretory protein in living cells. The target protein, von Willebrand factor (vWF), was released from human endothelial cells by adding phorbol-12-myristate-13-acetate (PMA). We confirmed that SICM could be used to clearly visualize the complex network of vWF and to detect strings with widths as low as 60 nm without any artifact. By acquiring the sequential SICM images of living cells, the protrusion and strings formation were observed. We also detected the opening and closing motions of a small pore (∼500 nm), which is difficult to visualize with fluorescence methods. The results clearly demonstrate that SICM is a powerful tool to examine the changes in living cells during exocytosis.

Improving the electrochemical imaging sensitivity of scanning electrochemical microscopy-scanning ion conductance microscopy by using electrochemical Pt deposition.

We fabricated a platinum-based double barrel probe for scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM) by electrodepositing platinum onto the carbon nanoelectrode of the double barrel probe. The deposition conditions were optimized to attain highly sensitive electrochemical measurements and imaging. Simultaneous SECM-SICM imaging of electrochemical features and noncontact topography by using the optimized probe afforded high-resolution images of epidermal growth factor receptors (EGFR) on the membrane surface of the A431 cells.

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system.

We evaluated the intracellular NAD(P)H: quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione-ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 µM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 µM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.

Noninvasive measurement of alkaline phosphatase activity in embryoid bodies and coculture spheroids with scanning electrochemical microscopy.

Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.

Evaluation of the differentiation status of single embryonic stem cells using scanning electrochemical microscopy.

The differentiation status of single live embryonic stem (ES) cells was quantitatively evaluated by monitoring the activity of alkaline phosphatase (ALP), an undifferentiation marker of ES cells, using scanning electrochemical microscopy (SECM).