Quantitative Super-Resolution Microscopy to Assess Adhesion of Neuronal Cells on Single-Layer Graphene Substrates.

Agnieszka Pierzynska-Mach,Francesca Pennacchietti,Silvia Scalisi,Silvia Dante,Dario Pisignano,Nathan D Derr,Francesca Cella Zanacchi,Sandeep Keshavan,Alberto Diaspro

doi:10.3390/membranes11110878

Abstract

Single Layer Graphene (SLG) has emerged as a critically important nanomaterial due to its unique optical and electrical properties and has become a potential candidate for biomedical applications, biosensors, and tissue engineering. Due to its intrinsic 2D nature, SLG is an ideal surface for the development of large-area biosensors and, due to its biocompatibility, can be easily exploited as a substrate for cell growth. The cellular response to SLG has been addressed in different studies with high cellular affinity for graphene often detected. Still, little is known about the molecular mechanism that drives/regulates the cellular adhesion and migration on SLG and SLG-coated interfaces with respect to other substrates. Within this scenario, we used quantitative super-resolution microscopy based on single-molecule localization to study the molecular distribution of adhesion proteins at the nanoscale level in cells growing on SLG and glass. In order to reveal the molecular mechanisms underlying the higher affinity of biological samples on SLG, we exploited stochastic optical reconstruction microscopy (STORM) imaging and cluster analysis, quantifying the super-resolution localization of the adhesion protein vinculin in neurons and clearly highlighting substrate-related correlations. Additionally, a comparison with an epithelial cell line (Chinese Hamster Ovary) revealed a cell dependent mechanism of interaction with SLG.

Highlights

In recent years, the rise of a new family of carbon-based nanomaterials has attracted increasing attention in the scientific community
Among the different types of graphene [3], Single Layer Graphene (SLG) grown by chemical vapor deposition is the most suitable for the development of biosensing architectures given the ease with which it can be used to functionalize other surfaces and given the possibility of its being processed by microfabrication methods [25,26]
Most individual vinculin clusters exhibit a lower density on the graphene substrate, we found a larger amount of the clusters per unit area

Summary

Introduction

The rise of a new family of carbon-based nanomaterials has attracted increasing attention in the scientific community. The effects of graphene on neurons have been extensively studied, highlighting excellent compatibility with neuronal cells, as well as enhanced cellular growth and vitality compared to conventional culture substrates [21,22,23,24]. Among the different types of graphene [3], SLG grown by chemical vapor deposition is the most suitable for the development of biosensing architectures given the ease with which it can be used to functionalize other surfaces and given the possibility of its being processed by microfabrication methods [25,26]. Recent studies propose SLG as a substrate for growing large-area patterned neuronal networks [27,28]. Patterned surfaces of SLG are shown to promote ordered neuronal growth and preferential adhesion [28]

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Results

Conclusion