Andrey Klymchenko “Supramolecular approaches in fluorescence biosensing and bioimaging”
November 29 @ 4:30 pm
Laboratory of Bioimaging and Pathologies, UMR CNRS 7021, University of Strasbourg, France
Supramolecular approaches in fluorescence biosensing and bioimaging
Understanding and controlling biological processes require advanced chemical tools ready to co-assemble and interact with biological systems. To this end, we design smart molecules and nanoparticles – fluorescent probes, capable to communicate with biological systems using light. In the field of molecular probes, we develop concepts of biological sensing and imaging based on molecules sensitive to their nanoscale environment – solvatochromic and fluorogenic dyes. They operate by different mechanisms, such as (i) excited-state charge transfer, where the dye changes its emission color as a function of environment polarity and (ii) assembly-disassembly of dyes under environmental control. Introduction of functional groups allow them to specifically target and image membrane receptors, RNA aptamers and lipid membranes of different organelles of the cells and monitor changes in their lipid organization. Alternatively, we exploit dynamic covalent chemistry, where a molecule of interest can be localized at any desired organelle due to reversible covalent bonds, which allows tuning its photodynamic activity.
However, fundamentally limited brightness of organic dyes makes detection and imaging of biomolecules at low concentrations highly challenging. Therefore, we assemble specially designed polymers and dyes into ultrabright dye-loaded fluorescent polymeric nanoparticles. We introduced a concept of charge-controlled nanoprecipitation of hydrophobic polymers in aqueous media. We found that a few charged groups in hydrophobic polymers can drive self-assembly of polymers into NPs of small size ranging from 40 nm down to 7 nm. To ensure high brightness of NPs, we proposed to encapsulate charged dyes with bulky hydrophobic counterions, which serve as spacers between dyes, thus preventing their self-quenching. Based on these concepts, we obtained NPs that are ~100-fold brighter than QDots. Their small size was found essential for their free diffusion in cytosol of live cells, while their high brightness enabled unprecedented single-particle tracking in the mice brain. Assembling dyes inside small polymeric NPs led to the collective behaviour of 100-10000 dyes in a single particle due to ultrafast dye-dye energy migration and further transfer to a single acceptor.[9, 11] The obtained light-harvesting nanoantenna provided >1000-fold signal amplification, allowing first single-molecule detection in ambient light. Functionalization of these nanoantennas with nucleic acids yielded ultrabright FRET-based nanoprobes for amplified detection of RNA/DNA cancer markers, featuring single-copy sensitivity and compatibility with a smartphone RGB camera. The developed small dye-loaded polymeric NPs open the route to ultrabright tools for biosensing, bioimaging and diagnostics applications.
This work was supported by ERC Consolidator grant BrightSens 648528.
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