Fluorous-based diagnostic platform for multiplexed diagnosis and differentiation of viral infections

Viral infections pose one of the biggest global threats to human populations and agriculture. Successful prevention, monitoring and treatment of viral infections requires the availability of fast and reliable diagnostic methods which can not only sensitively, but rapidly detect a viral infection of interest and differentiate between viral infections. This is particularly important in the winter months where rapid diagnosis of viral infections emerging from SARS-Cov-2 relative to influenza strains is essential in order to assist medical practitioners to suggest the most appropriate interventions and treatment.

At present, methods do not exist which can rapidly detect viral infections in a low-cost, point-of-care device. We propose to develop a biosensing technology which can not only detect viral components, but also has the potential for the platform to be reusable and regeneratable. Central to these developments is the use of fluorous technology as a tool to immobilise elements which detect viral components. Much akin to Teflon, fluorous technology has the dual advantage as a method which can immobilise molecular components which have a complementary fluorous tag, and reduces non-specific binding to non-fluorous biomolecules, thus improving the sensitivity of the approach. Furthermore, the fluorous-directed immobilisation event is inherently reversible by a simple washing step with organic solvent. In this proposal, we will demonstrate the modularity of the strategy to detect viral RNA (by RT-PCR) or protein (by direct detection of intact viral particles). This will provide a powerful new tool for the biosciences which has the potential to be used for any application which requires rapid detection of pathogenic infections.

This TRDF proposal seeks to establish a new method to detect viral infections. Central to the method development is to exploit the 'fluorous effect' as a means to immobilise fluorous-tagged nucleic acids onto a fluorous-coated gold electrode. The advantages of using a fluorous as an immobilisation tool are:

(i) the preparation of fluorous-tagged nucleic acids is facile. This enables the preparation of a suite of fluorous-tagged primers (for RT-PCR) and aptamers by solid phase synthesis.

(ii) fluorous-tagged biomolecules non-covalently bind to fluorous surface.

(iii) fluorous immobilisation is inherently reversible, enabling the regeneration of the biosensing surface by the application of a simple solvent washing step.

We propose a new approach to detect viral infections by developing a biosensor which can detect the presence of viral components. Central to these developments is to exploit the fluorous effect as a means to selectively immobilise fluorous-tagged PCR amplicons from a RT-PCR assay or the direct detection of viral proteins using fluorous-directed immobilisation of aptamers. Finally, we will establish methods to regenerate the surface, thus providing the basic foundation for the development of the approach as a new cost-effective biosensing platform which could be used to detect a wide range of pathogenic infections in a variety of settings.