Amplified biosensors for metabolites based on fluorescent nanoantennas – AmpliSens

Detection of metabolites in biological fluids and their profiling at the point of care enable early diagnostics of diseases. However, current fluorescence assays are limited by the modest specificity to small organic molecules and insufficient brightness of fluorescent molecular probes. To address these challenges, we propose to couple engineered/evolved biosensors based on fluorogen-activating proteins and nucleic acid aptamers with light-harvesting nanoantenna based on dye-loaded polymeric nanoparticles. In these amplified biosensors (AmpliSens), the metabolite induces a conformational change of the biosensor leading to fluorogen binding and fluorescence light up. The fluorescence signal is then amplified >100-fold by the nanoantenna through (i) efficient Forster resonance energy transfer (FRET) from ~1000 encapsulated donor dyes to the fluorogen or (ii) immobilization of nanoantennas at the surface driven by affinity between biosensor and fluorogen in the presence of the target metabolite. The work plan is composed of four work packages (WPs). WP1 is the synthesis and characterization of nanoantenna amplifiers based on specially designed dye-loaded polymeric NPs conjugated with a fluorogenic modules of proteins and aptamers. Special focus will be made to achieve efficient energy transfer (FRET) from nanoparticles (NPs) to the fluorogenic module, required for signal amplification, and to preserve activity of fluorogenic module at the particle surface. WP2 and WP3 deal with preparation of biosensors for metabolites based on fluorescence-activating and absorption-shifting tag (FAST) protein and RNA aptamers, respectively. Protein-based biosensors will be obtained by fusion and further evolution of FAST protein with protein domains sensitive to enzyme cofactors. Aptamer-based biosensors will combine riboswitch-derived sequences, sensitive to tryptophan metabolites, with a fluorogenic module Gemini/Coral developed in collaboration of the partners. Finally, WP4 will combine nanoantenna amplifiers of WP1 with biosensors of WP2 and 3 to obtain nano-biosensors for amplified detection of metabolites. These nano-biosensors will exploit two amplification mechanisms: (i) direct signal amplification of the fluorogen by FRET from the nanoantenna, which will provide ratiometric response to the recognition of the target metabolite in solution; (ii) affinity between the fluorogen and the biosensor, where the metabolite will trigger the specific interaction of ultrabright NPs with fluorogen-functionalized surfaces, allowing detection on surfaces. The AmpliSens probes will be applied to high-sensitivity detection of kynurenine, serotonin, phenylalanine and glucose in serum (blood) and urine. The consortium is composed of three partners with complementary expertise: fluorescent probes and nanoparticles (P1), fluorogen-activating proteins (P2) and RNA light-up aptamers and microfluidics (P3). The project has a strong potential for further valorization, because the proposed AmpliSens nano-biosensors will be applied for detection of medically relevant metabolites in point-of care diagnostics of diseases.