Search

International audience; Detection and imaging of RNA at the single-cell level is of outmost importance for fundamental research and clinical diagnostics. Current techniques of RNA analysis, including fluorescence in situ hybridization (FISH), are long, complex and expensive. Here, we report a methodology of amplified FISH (AmpliFISH) that enables simpler and faster RNA imaging using small and ultrabright dyeloaded polymeric nanoparticles (NPs) functionalized with DNA. We found that the small size of NPs (below 20 nm) was essential for their access to the intracellular mRNA targets in fixed permeabilized cells. Moreover, proper selection of the polymer matrix of DNA-NPs minimized nonspecific intracellular interactions. Optimized DNA-NPs enabled sequence-specific imaging of different mRNA targets (survivin, actin and polyA tails), using a simple 1h staining protocol. Encapsulation of cyanine and rhodamine dyes with bulky counterions yielded green, red and far-red emitting NPs that were 2-100-fold brighter than corresponding quantum dots. These NPs enabled multiplexed detection of three mRNA targets simultaneously, showing distinctive mRNA expression profiles in three cancer cell lines. Image analysis confirmed the single-particle nature of the intracellular signal, suggesting single-molecule sensitivity of the method. AmpliFISH was found to be semi-quantitative, correlating with RT-qPCR. In comparison with the commercial LNA-based FISH technique, AmpliFISH provides 8-200-fold stronger signal (dependent on the NP color) and requires only 3 steps vs. ~20 steps together with much shorter time. Thus, combination of bright fluorescent polymeric NPs with FISH yields a fast and sensitive single-cell transcriptomic analysis method for RNA research and clinical diagnostics.

Polymeric nanoparticles (NPs) are highly attractive for biomedical applications due to their potential biodegradability and capacity to encapsulate different loads, notably drugs and contrast agents. For in vivo optical bioimaging, NPs should operate in the near-infrared region (NIR) and exhibit stealth properties. In the present work, we applied the approach of ionic dye insulation with bulky hydrophobic counterions for encapsulation of near-infrared cyanine dyes (Cy5.5 and Cy7 bearing two octadecyl chains) into biodegradable polymer (PLGA) NPs. We found that at high dye loading (20–50 mM with respect to the polymer), the bulkiest fluorinated tetraphenylborate counterion minimized best the aggregation-caused quenching and improved fluorescence quantum yields of both NIR dyes, especially of Cy5.5. In addition, bulky counterions also enabled formation of small 40 nm polymeric NPs in contrast to smaller counterions. To provide them stealth properties, we prepared 40 nm dye-loaded PEGylated NPs through nanoprecipitation of synthetic PLGA–PEG block copolymer with the dye/counterion salt. The obtained NIR NPs loaded with Cy5.5 dye salt allowed in vivo imaging of wild-type mice with a good contrast after IV injection. Compared to the bare PLGA NPs, PLGA–PEG NPs exhibited significantly slower accumulation in the liver. Biodistribution studies confirmed the preferential accumulation in the liver, although PLGA and PLGA–PEG NPs could also be distributed in other organs, with the following tendency: liver > spleen > lungs > kidney > heart > testis > brain. Overall, the present work validated the counterion approach for encapsulation of NIR cyanine dyes into biodegradable polymer NPs bearing covalently attached PEG shell. Thus, we propose a simple and robust methodology for preparation of NIR fluorescent biodegradable polymer NPs, which could further improve the existing optical imaging for biomedical applications., Bulky hydrophobic counterions were applied for encapsulation of near-infrared cyanine dyes into biodegradable polymer matrix with minimized self-quenching, yielding fluorescent nanoparticles with stealth PEG shell for in vivo imaging.

International audience; Whispering gallery mode (WGM) microcavities are emerging as potential candidates in the field of biosensing applications, as their resonance wavelengths shift with changes in the refractive index in the region of their evanescent field. Their high-quality resonance modes and accessible surface functionalities make them promising for molecular assays but their high sensitivity makes them inherently unstable. Here we demonstrate that WGM resonances also strongly enhance fluorescence energy transfer between donors placed inside the microcavity and acceptors placed outside. We load colloidal quantum dots (QDs) into polymeric microspheres, to provide WGMs that benefit from the QD optical features when used as energy transfer donors. Spectroscopic analysis of the emission from the microcavities shows that the high quality of WGMs enables a very efficient energy transfer to dyeloaded polymer nanoparticle (dyeNP) acceptors placed in their vicinity. Compared to Förster Resonance Energy Transfer, WGM-enabled energy transfer (WGET) occurs over a much more extended volume, thanks to the delocalization of the mode over a typically 10 5 times larger surface and to the extension of the WGM electromagnetic field to larger distances (> 100 nm vs. a few nm) from the surface of the microcavity. The resulting sensing scheme combines the sensitivity of WGM spectroscopy with the specificity and simple detection schemes of fluorescence energy transfer, thus providing a potentially powerful class of biosensors.