Work Package 1. Fluorescent organic nanoparticles (NPs).
Polymer NPs. (1) We developed NPs of colors spanning from blue to red, by extending our counterion-based approach to cyanines. (2) We maturated the concept of bulky hydrophobic counterions to obtain bright and stable NPs. (3) We obtained NPs of exceptional brightness (~100-fold brighter than quantum dots of comparable size). (4) Our new copolymers yielded NPs with controlled sizes from 50 to 7 nm (Reisch et al. Adv Funct Mater, 2018) and “stealth” shell (patent application).
Micellar NPs. (1) Protein-sized (7 nm) fluorescent NPs were obtained from calixarene micelles cyanine dye “corona” (Shulov et al. Angew Chem, 2016). (2) We introduced a concept of monomolecular protein-sized (~10 nm) fluorescent NPs based on folded amphiphilic polymer (Collot et al. ACS Nano, 2020).
Photophysics of FONs and collective FRET. We discovered light-harvesting nanoantenna: polymeric NPs containing >10,000 dyes that can efficiently transfer energy to single acceptors (Trofymchuk, et al. Nat Photonics, 2017; patent application). Here, the emission of an acceptor dye is amplified >1,000 (absolute record), which enabled first ever single-molecule detection in ambient sunlight-like conditions.
WP2. Nanoprobe synthesis and evaluation & WP3. Cellular applications of nanoprobes.
Probes for membrane receptors. We developed a series probes based on fluorogenic dimers and dendrimers for imaging membrane receptors (GPCR, integrin and biotin) in live cells and small animals (Fam et al, Chem Sci, 2020).
Nanoprobes for nucleic acids. (1) We introduced a concept of amplified detection of nucleic acids, where a single molecular recognition switches emission of hundreds of dyes inside NPs. We functionalized our giant light-harvesting nanoantenna with DNA (Melnychuk & Klymchenko J Amer Chem Soc, 2018; patent application) and obtained nanoprobes with sequence-specific response to nucleic acids and 0.25-pM detection limit. (2) We improved the nanoantenna to achieve nucleic acids detection with single-molecule sensitivity (Melnychuk, et al. Angew Chem, 2020). (3) Nanoprobes were validated for nucleic acids detection by RGB camera of a smartphone.
Delivery of NPs into the cells. (1) Based on endocytosis of NPs of different color we developed an approach of barcoding of living cells in vitro and in vivo (Andreiuk, et al. Small, 2017). NPs showed almost negligible cytotoxicity. (2) We found that bulky hydrophobic counterions are essential to ensure efficient encapsulation of dyes within minimal leakage in cells and in vivo. (3) We delivered NPs into the cytosol by microinjection and electroporation (Egloff, et al. Small Methods, 2021), showing critical importance of small size of NPs (<20 nm).
Detection of intracellular RNA. Our DNA nanoprobes enabled direct detection of microRNA cancer markers (validated on 4 microRNA) in cell lysates (RNA extracts from 7 healthy and cancer cell lines) without enzymatic amplification (Egloff, et al. Biosens Bioelectron, 2021). We developed a bright fluorogenic dye dimer with its cognate RNA aptamer for intracellular RNA imaging (Bouhedda F, et al. Nat Chem Biol, 2020; patent application). Finally, we found that our DNA-functionalized NPs can be used for fluorescence in situ hybridization (FISH) in fixed cells to detect mRNA cancer markers (in preparation).
In total, ERC project resulted in 57 peer-reviewed articles (including 3 reviews) and 9 patents.
Exploitation of results and knowledge transfer
In connection with the BrightSens project we deposited 9 patents and 1 know-how. The research resulted in several technologies transfer related detection of RNA cancer markers, single cell analysis and virus (SARS-Cov-2) detection. Based on the key technology developments of the BrightSens project on giant light-harvesting nanoantenna for detection of biomolecules, we plan to create a start up at the end of 2021.
