8:50 AM - *SM07.04.03
Assembling Dyes with Polymers into Small and Bright Fluorescent Nanoparticles for Biosensing and Bioimaging
University of Strasbourg1
Dye-loaded fluorescent polymeric nanoparticles (NPs) appear as an attractive alternative to inorganic NPs, such as quantum dots (QDots). However, preparation of polymeric NPs featuring small size and high fluorescence brightness remains a challenge. To address it, we introduced a concept of charge-controlled nanoprecipitation of hydrophobic polymers in aqueous media. We found that a single charge in polymers, such as poly(methyl methacrylate) (PMMA), poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) can drive self-assembly of polymers into NPs of 20-40 nm size. Further addition of charges into these polymers (up to 10 mol%) can decrease particle size down to 7 nm. On the other hand, to ensure high brightness of polymeric NPs, we proposed to encapsulate charged dyes with bulky hydrophobic counterions. The later serve as spacers between dyes, thus preventing aggregation-cause quenching and, at the same time, favor the co-assembly of the dyes with the polymer into stable NPs without dye leakage. Based on these design concepts, we obtained NPs that are ~100-fold brighter than QDots of similar size. Their small size was found essential for their free diffusion in cytosol of live cells and for their intracellular delivery by electroporation. Their high brightness enabled unprecedented single-particle tracking in the mice brain and visualization of crossing blood-brain barrier. Assembling dyes inside small polymeric NPs revealed also a unique cooperative behavior of dyes through ultrafast dye-dye energy migration, which led to the collective switching of >100 dyes in a single particle.[4, 6] This collective behavior of dyes enabled efficient Förster resonance energy transfer (FRET) from ~10000 encapsulated dyes to a single acceptor. This light-harvesting nanoantenna provided >1000-fold signal amplification (antenna effect), allowing first single-molecule detection in ambient light. Functionalization of these nanoatennas with nucleic acids yielded ultrabright FRET-based nanoprobes for amplified detection of oligonucleotides, opening possibilities for rapid detection of cancer markers using a RGB camera of a smartphone. Nanoantennas of 20 nm size enabled efficient FRET to a single acceptor at their surface and detection of RNA/DNA with a single-molecule sensitivity. The light-harvesting concept was also applied to amplify phosphorescence of porphyrins for ratiometric sensing of oxygen with minimal phototoxicity. The developed small dye-loaded polymeric NPs open the route to ultrabright tools for sensing and tracking biomolecules in biosensing, bioimaging and diagnostics applications.
This work was supported by ERC Consolidator grant BrightSens 648528.
 A. Reisch, A. S. Klymchenko, Small 2016, 12, 1968.
 A. Reisch, A. Runser, Y. Arntz, Y. Mely, A. S. Klymchenko, ACS Nano 2015, 9, 5104.
 A. Reisch, D. Heimburger, P. Ernst, A. Runser, P. Didier, D. Dujardin, A. S. Klymchenko, Adv. Funct. Mater. 2018, 28, 1805157.
 A. Reisch, P. Didier, L. Richert, S. Oncul, Y. Arntz, Y. Mely, A. S. Klymchenko, Nature Commun. 2014, 5, 4089.
 B. Andreiuk, A. Reisch, E. Bernhardt, A. S. Klymchenko, Chem. Asian J. 2019, 14, 836.
 A. Reisch, K. Trofymchuk, A. Runser, G. Fleith, M. Rawiso, A. S. Klymchenko, ACS Appl. Mater. Interfaces 2017, 9, 43030.
 I. Khalin, D. Heimburger, N. Melnychuk, M. Collot, B. Groschup, F. Hellal, A. Reisch, N. Plesnila, A. S. Klymchenko, ACS Nano 2020, 14, 9755.
 K. Trofymchuk, A. Reisch, P. Didier, F. Fras, P. Gilliot, Y. Mely, A. S. Klymchenko, Nature Photonics 2017, 11, 657.
 N. Melnychuk, A. S. Klymchenko, J. Am. Chem. Soc. 2018, 140, 10856.
 C. Severi, N. Melnychuk, A. S. Klymchenko, Biosens. Bioelectron. 2020, 168.
 N. Melnychuk, S. Egloff, A. Runser, A. Reisch, A. S. Klymchenko, Angew. Chem. Int. Ed. 2020, 59, 6811.
 P. Ashokkumar, N. Adarsh, A. S. Klymchenko, Small 2020, 16, 2002494.