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Abstract
In a normal environment, cells are subject to multiple signals such as secretion of proteins from neighboring cells and biochemical interactions with the extracellular matrix (ECM). Specifically, Arg-Gly-Asp-Cys (RGDC) ligands present in ECM has been known to bind to cellular integrins, thereby regulating cell adhesion and signaling pathways. Investigating the link between cell surface interactions and G-protein coupled receptor activation is crucial to understand cancer and various disease mechanisms. The dual electrochemical and optical investigation can provide comprehensive information about these networks as electrochemistry can monitor minute changes in morphologies as well as biochemical and ionic changes while optical microscopy has the potential to monitor specific cell signaling processes as well as morphologic changes. The aim of this work is two fold 1) present RGDC ligands on transparent Indium tin oxide (ITO) electrodes in a controlled manner and 2) monitor the G-protein coupled receptor (GPCR) activation signals on varied cell surface interactions using combined fluorescence microscopy and electrochemical impedance spectroscopy.
To achieve this, 1) the surface were modified with phosphonate based self-assembled monolayers (SAMs) so that adhesion and electron transfer properties of the electrode is well controlled, 2) a mixture of cell adhesive GRGDC ligands and antifouling molecules were covalently coupled on top of the monolayer to control cell adhesion, 3) finally, simultaneous live cell fluorescence imaging and electrochemical impedance spectroscopy was conducted on the prepared surfaces to analyze GPCR activation (calcium signals) and their link with cell adhesion.
The results showed stable and well packed phosphonohexadecanoic acid SAMs formed only on smooth amorphous ITO surfaces. Stochastic optical reconstruction (STORM) single molecular fluorescence imaging of ITO surfaces showed well distributed RGDC-Alexa Fluor 647 molecules on modified ITO surfaces. The cell adhesion and spreading was found maximum on 1:10^3 (GRGDC:antifouling) surface and minimum on 1:10^9 surfaces. The dual optical electrochemical investigations showed that GPCR activation of cells attached on 1:10^6 and 1:10^9 surfaces showed large increase in impedance (morphological changes) whereas 1:10^3 and fibronectin coated surfaces showed maximum calcium signals. This dual investigations allow comprehensive understanding of cell signaling processes, fluorescence microscopy provides minute changes in calcium flux, while impedance spectroscopy providing information about minute changes in morphologies.