Quantitative Real‐Time Monitoring of Antibody‐Induced Internalization of Epidermal Growth Factor Receptor on Single Living Mammalian Cells Using Scanning Electrochemical Microscopy

Abstract

An antibody-induced internalization of epidermal growth factor receptor (EGFR) was quantitatively monitored using scanning electrochemical microscopy (SECM) at the single-cell level. The antibody Cetuximab is used for cancer treatment because of its capability to bind and internalize EGFR, which is one of the key membrane proteins associated with many cancers. To monitor antibody-induced EGFR internalization, Cetuximab was simply labelled with enzymes and the resulting enzymatic products were then electrochemically detected by using a microelectrode. By optimizing experimental parameters, such as immunostaining condition, enzyme type, and buffer solution, we were able to successfully monitor the EGFR internalization by using SECM, from which we found a decrease in current responses from a single living cell by 20 % in the first 60 min, suggesting different internalization pathways from inherent ligand-induced internalization.

Electrochemical Biosensing System for Single Cells, Cellular Aggregates and Microenvironments

“…5). [77][78][79] This research clarified that we could judge from the SECM response whether the reaction occurs inside or at the surface of the targeted cell.…”

High-Content Label-Free Single-Cell Analysis with a Microfluidic Device Using Programmable Scanning Electrochemical Microscopy

“…In response to these challenges, scanning electrochemical microscopy (SECM), as a label-free and non-invasive analytical technique, has been extensively applied for the in situ investigation at the single-cell level. − By scanning a microelectrode over a surface and recording the Faradaic current response, it is possible to simultaneously characterize the morphology and electrochemical reactivity of the underlying sample with extremely high spatial and temporal resolutions. , In terms of the application of SECM for single-cell analysis, cell morphology, and membrane properties, , the cellular expression of proteins, − the activity of transmembrane proteins, , respiration activity, cellular response to environmental stimuli, , and cell-released species − have been thoroughly explored. Despite the intense efforts on single-cell analysis, it remains a challenge for SECM cell imaging because the measurement conditions are considerably different from the ideal physiological environment for cell culture, including the composition of the medium, levels of humidity and CO 2 , and the temperature as well .…”

Local electrochemical impedance spectroscopy: A window into heterogeneous interfaces

“…The study of the electrochemical interface by means of local measurements is a subject that remains in constant evolution, and is accelerated by the numerous technical developments involving probe size reduction (used to sense the interface reactivity) or the development of more advanced associated electronics for data acquisition. In fact, the versatility offered by local electrochemical techniques benefits a large panel of research topics as different as the local catalytic or photocatalytic activity of materials [1][2][3], the production of redox species in living cells [4,5], corrosion processes on various interfaces [6][7][8][9] or complex kinetics mechanism [10][11][12].…”