In biological systems, many processes in cell signaling function by the release and uptake of chemical species, such as neurotransmitters, ions or reactive oxygen species. The qualitative and quantitative study of these processes give new insights on how biological systems work. Nano electrochemistry offers powerful tools in the fields of biophysics, electrophysiology as well as neurochemistry where either electric currents or chemical species can be detected. Additionally, the study of cell morphology is possible in ion conductance measurements.

Commonly, carbon microelectrodes are used due to good chemical stability under physiological conditions. Chronoamperometric measurements or fast-scan cyclic voltammetry (FSCV) can be used for the detection and quantification of chemical species from single cells. These methods offer temporal resolution of the processes under investigation.

Scanning electrochemical microscopy (SECM) enables the exact positioning of the microelectrode and adds lateral resolution by scanning of the microelectrode. With this, 2D and 3D concentration profiles of chemical species after release can be mapped.

Scanning ion conductance microscopy (SICM) can be used for contact-less topography imaging of sensitive biological samples. The high resolution and performance under physiological conditions allows the imaging of single live cells.

Scanning Techniques

The redox state of cardiomyocytes are investigated in different mechanical microenvironments. The microenvironment has an influence on the regulation of the phenotype and function of cardiac cells, which are strongly associated with the intracellular redox mechanism of cardiomyocytes.

SECM depth scans and extracted approach curves can be used to determine the GSH level outside a single cell which is a direct measure of the cells redox state. With this study it was shown that a stiff microenvironment results in a more oxidative state of the cardiomyocyte compared to a softer microenvironment. It shows the ability of SECM to elucidate redox mechanisms within cells via a label-free in situ technique.

redox-state-cells
Depth scan above a single cardiomyocyte (left) and extracted approach curves along vertical lines above cells on substrates with different stiffness and a control on an empty petri dish (right).
MDR_Cancer
Cells with overexpressed MRP1 have a big efflux of GSH which reduces the redox mediator and a large current is recorded (a) and cells with normal MRP1 which have smaller efflux of GSH and result in smaller currents (b).

Multi-drug resistance in cancer cells can be a result of chemotherapeutic treatment and has its microstructural origin in the overexpression of membrane proteins, e.g. MRP1 or P-pg. The increased efflux of these transmembrane pumps flushes out drugs before they can be effective.

The increased MRP1-mediated efflux can be monitored by scanning electrochemical microscopy (SECM) with the non-toxic redox mediator ferrocenemethanol (FcMeOH). The quantification of MRP1 activity is quantified via the electrochemical reaction between this redox mediator and glutathione. Glutathione is a peptide molecule involved in MRP1-related transport and therefore a direct measure of its activity.

Tracking the evolution of MRP1 activity and expression is important because direct clinical implications make MRP1 a relatively unique molecular marker in comparison with other prognostic variables identified for several cancer types.

Kuss, D. Polcari, M. Geissler, D. Brassard, J. Mauzeroll (2013). “Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy.” Proceedings of the National Academy of Sciences 110(23):9249-9254.

The study of morphological features of cells can help to understand disease. Scanning ion conductance microscopy (SICM) uses an ionic current between a quasi-reference electrode within a micropipette and a quasi-reference electrode in the bulk solution to map the topography of sensitive biological samples with lateral resolution greater than 100 nm. SICM can be easily combined seamlessly with fluorescence imaging.

SICM topography cell imaging
Schematic of SICM above a cell.
SICM imaging cell topography
SICM topography image above a single live cell.Image by courtesy of Prof. Dr. L. F. Santana, Physiology and Membrane Biology Department, UC Davis, USA.
SECM ROS biological cells
Detection of hydrogen peroxide at a single cell upon stimulation.

In living systems, free radicals and reactive oxygen / nitrogen species (ROS/RNS) play important roles, because they can cause oxidative damage and tissue dysfunction and serve as molecular signals activating stress responses that are beneficial to the organism. Scanning electrochemical microscopy (SECM) and spatially-resolved electrochemical methods were effectively applied to image and quantify extracellular local concentrations of ROS and RNS, as well as other redox active signaling molecules. Stable long-term (>3 h) quantification measurements with high temporal (>1 Hz) and spatial (<1 µm) resolutions have been achieved from individual live cell in the ROS production and degradation processes.

M. Bozem, P. Knapp, V. Mirceski, E.J. Slowik, I. Bogeski, R. Kappl, C. Heinemann, M. Hoth (2018). “Electrochemical Quantification of Extracellular Local H2O2 Kinetics Originating from Single Cells.”  Antioxidants & Redox Signaling.

respiratory_burst
SECM imaging of cells during cellular respiration (left) and during a respiratory burst (right). Image by H. Kikuchi and A. Prasad in Front. Physiol. 7:25..

The cellular respiration of cells can be monitored by scanning electrochemical microscopy (SECM). The microelectrode is held at -0.5 V vs. Ag/AgCl to detect oxygen in the oxygen reduction reaction. In the bulk and above the petri dish a constant oxygen level is detected which gives rise to a steady state current which is directly proportional to the oxygen concentration. The microelectrode is then scanned above the cells. In the vicinity of the cell the current at the microelectrode is reduced due to lower oxygen levels. During a  PMA-induced respiratory burst the current at the microelectrode records even higher consumption of oxygen at the cells.

Kikuchi H, Prasad A, Matsuoka R,Aoyagi S, Matsue T and Kasai S (2016) Scanning Electrochemical Microscopy Imaging during Respiratory Burst in Human Cell. Front. Physiol. 7:25.

Stationary Techniques
SECM Fast Scan CV neurotransmitter
Release of neurotransmitters from a vesicle and detection at the microelectrode.

The release of many neurotransmitters (e.g. dopamine, epinephrine or serotonin) can be investigated electrochemically by oxidation at a microelectrode upon application of a suitable potential. Usually the technique Fast-Scan Cyclic Voltammetry (FSCV) is used for high temporal resolution. The FSCVs can analyzed to quantify the released neurotransmitters. Using a scanning probe platform, such as ElProScan can facilitate positioning of the microelectrode and FSCV can be combined with other techniques (see examples below).

color_plot_only
False color plot of an FSCV for the detection of dopamine.