A versatile monitoring technique for real-time protein activity tracking within cellular and biomimetic scaffold environments

Abstract

High biological noise is intrinsic within biological systems, and this poses critical challenges to the in-situ detection and measurement of biomolecular and protein activities important to advancing approaches to disease and injury treatment. These challenges arise due to possible non-specific binding of other molecules in the analyte’s surrounding medium. Currently the techniques available to characterize protein behaviours in living biological systems are highly elaborate and are generally greatly hindered by the high background noise. Here we present a versatile and straight forward technique for monitoring proteins and protein interactions within cells and other complex environments, based on a novel nano-bio-technology method. Highly sensitive gold edge coated triangular silver nanoplates (AuTSNP), which are highly responsive to molecular interactions on their surfaces, are used to probe protein behaviours within complex cellular and tissue regeneration environments, as well as recognize antibody-antigen (Ab-Ag) interactions within dynamic biological surroundings. The extracellular domains within tissues involve macromolecules vital for the provision of structural support to surrounding cells and signalling cues for the modulation of diverse cellular processes. Tissue scaffolds are designed to mimic the extracellular architecture and functions. In this work, monitoring of the dynamic behaviour of a critical extracellular protein, Fibronectin (Fn), within the presence of bone tissue regeneration scaffolds and living cells is reported. The optical response of Fn functionalised AuTSNP, is used to distinguish between compact and extended conformations of the protein and indicating Fn unfolding and fibril formation on incubation within cells. Moreover, successful detection of native Fn present in isolated Extracellular Matrix (ECM) by using Anti-Fn antibody functionalised AuTSNP is performed. The excellent sensitivity and straight forward application within complex cellular environments, poses AuTSNP as powerful new tools to detect protein interactions and monitor essential protein activity. For this reason, a potential COVID-19 detection platform is explored where SARS-CoV-2 Spike protein is detected through a nanoplate-based system using its corresponding antibody, Anti-Spike. This work is conducted within the presence of horse serum (HS) as complex environment where the dynamic surroundings present a challenge for the straightforward detection of the Ab-Ag complex binding, nonetheless, the research presented is a significant step towards the development of new technologies for medical diagnosis and monitoring