Periodic Reporting for period 4 - BrightSens (Ultrabright Turn-on Fluorescent Organic Nanoparticles for Amplified Molecular Sensing in Living Cells)
Reporting period: 2019-12-01 to 2020-11-30
Three objectives of BrightSens are to address the three challenges described above: (1) To obtain fluorescent organic nanoparticles with high brightness and collective FRET to a single acceptor by resolving fundamental problems of dye self-quenching and energy transfer on the nanoscale. (2) To develop nanoparticle probes that turn-on >100 fluorescent dyes in response to single molecular targets (membrane receptors and mRNA). (3) To validate the nanoprobes in 2D and 3D cell cultures for ultrasensitive detection of cancer markers at the cell surface (integrin, EGFR and folate receptors) and in the cytosol (mRNA of survivin and Bcl-2).
As an impact of BrightSens project on society, several points should be mentioned. First, we strive to develop biodegradable/biocompatible nanoparticles, which are expected to have minimal danger for human health and ecology. Therefore, we aim to move towards safer nanotechnology. Second, our new probes for detection of cancer markers are expected to become tools for cancer diagnostics and personalised medicine. These probes are expected to simplify and decrease costs of protocols for detection of these markers. Thus, ultimately, BrightSens will have an important impact on human health.
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.
2) We maturated the concept of bulky counterion and extended it cyanines for preparation of ultrabright polymeric NPs (100-fold brighter than quantum dots) of any desired color.
3) Monomolecular protein-sized (~10 nm) fluorescent NPs based on folded amphiphilic polymer were introduced.
4) Giant light-harvesting nanoantenna was developed exhibiting unprecedented FRET efficiency and record-breaking >1000-fold signal amplification, allowing unprecedented single molecule detection in ambient sunlight-like conditions.
5) We introduced a concept of amplified detection of nucleic acids based on DNA-functionalized giant light-harvesting nanoantenna with DNA (Melnychuk & Klymchenko J Amer Chem Soc, 2018), which enables direct detection of nucleic acid cancer markers without PCR.
6) We developed the first example of nanoparticle probe capable to detect single copy of RNA/DNA: it couples single recognition even with response of ~1000 of encapsulated dyes.
7) Fluorogenic dye dimers for imaging natively expressed membrane receptors and intracellular RNA were introduced.