Design and optimisation of Photochrome Aptamer Switch Assay (PHASA)

Photochrome Aptamer Switch Assay (PHASA) is our unique and recently developed fluorescent biosensing platform that exploits the combination of aptamer adaptive binding and photoisomerisation kinetics of stilbenes. The PHASA is based on converting any suitable stilbene-ligand-aptamer complex into a reversible biosensor. Binding affinity, aptamer concentration, and stilbene molecular structure determine the PHASA performance criteria. Consequently, understanding of the structure-activity relationship of the stilbene-ligand conjugate is extremely important when predicting and optimising the biosensor sensitivity. In the present manuscript, new analytical methods relating to calculation of the limit of detection (LOD) of the PHASA are proposed and discussed. Using these methods, it is possible to optimise the independent or dependent physicochemical parameters of the stilbene-ligand conjugates and evaluate their general sensing performance. The proposed analytical methods are based on the inter-dependent relationships between the sensitivity and the range of analyte detection and provide starting conditions for known aptamer binding constants in various PHASA applications.