An intramolecular FRET biosensor for the detection of SARS-CoV-2 in biological fluids

The development of a FRET-based sensor for detecting the Spike surface antigen of SARS-CoV-2 in biological fluids is described here, exploiting the fluorescence properties of Green Fluorescent Protein (GFP). Our design strategy combines experimental and molecular modeling and simulations to build a smart modular architecture, allowing for future optimization and versatile applications. The prototype structure incorporates two reporter elements at the N-terminus and C-terminus, with two interaction elements mediating their separation. This design supports two fluorescence measurement methods: direct measurement and the molecular beacon approach. The former detects changes in GFP fluorescence intensity due to interactions with the Spike protein, while the latter involves an organic quencher that restores GFP fluorescence upon Spike protein binding. In silico design of linkers, using molecular dynamics (MD) simulations, ensured optimal flexibility and stability. The AAASSGGGASGAGG linker was selected for its balance between flexibility and stability, while the LEAPAPA linker was chosen for its minimal structural impact on the interaction elements. Fluorophores' behavior was analyzed, showing stable FRET efficiency, essential for reliable detection. Quenching efficiency calculations, based on Förster energy transfer theory, validated the sensor's sensitivity. Further, MD simulations assessed GFP stability, confirming minimal unfolding tendencies, which explains the sensor functioning mechanism. The sensor was successfully produced in E. coli, and functional validation demonstrated its ability to detect the Spike protein, with fluorescence recovery proportional to protein concentration, while the modular computer aided design allowed for sensitivity optimization. The developed biosensor prototype offers a promising tool for rapid and precise viral detection in clinical settings.