Study of a Miniaturizable System for Optical Sensing Application to Human Cells
Emanuele Luigi SciutoGiusy VillaggioMaria Francesca SantangeloSamuele LaudaniConcetta FedericoSalvatore SacconeFulvia SinatraSebania Libertino
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Abstract:
Conventional approaches to human intracellular optical sensing, generally, require dedicated laboratories with bulky detection systems. They are performed by cell labeling procedures based on the use of fluorophores that are, mostly, phototoxic, invasive, bleached in case of prolonged light exposures, which require carriers and/or structural modifications for the cellular uptake. These issues, together with the sensitivity of the eukaryotic cell model, could be problematic towards the development of a robust sensing system suitable for biomedical screening. In this work, we studied a sensing system resulting from the combination of the commercial tris(2,2’bipyridyl)ruthenium(II) fluorophore, for cell labeling, with a potentially miniaturizable optical system composed by a laser source and a photomultiplier tube, for the fluorescence analysis.Keywords:
Optical sensing
Fluorescent molecules have been widely utilized in scientific researches, medical uses a industrial purposes. To develop fluorophores with useful and unpredictable functions, our group have focused on natural products, and identified fluorescent natural products, Amarastelline A and Nigakinone. For the evaluation of those functions as a fluorophore and the development novel fluorescent sensors, we synthesized various derivatives of these common structures, canthin-5,6-dione and 1,5-naphthyridin-2(1H)-one. Among them, novel polarity-sensitive fluorophore, which showed the shift of fluorescence maximum wavelength by the change of solvent polarity, could be obtained. To apply this function to the study of biomolecule interaction, C5 peptide, which could interact calmodulin, labelled with this fluorophore, was synthesized, and its interaction with calmodulin could be detected by the ratio the fluorescence intensities at two fluorescent maximum wavelengths. Our findings indicated the usefulness of natural products for the development of novel fluorescent molecules, as well as the versatilities of canthin-5,6-dione and 1,5-naphthyridin-2(1H)-one.
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In the current paper, three activity-based colorimetric and ratiometric fluorescent probes based on a naphthalimide fluorophore were well designed and synthesized, which can be recognized and hydrolyzed by aminopeptidase N (APN) at both the enzymatic and cellular level by following the fluorescent emission wavelength change from blue to green light. As a result, these molecules were successfully identified as the first ratiometric fluorescent probes for APN cell imaging.
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Abstract Bright fluorescent molecules with long fluorescence lifetimes are important for the development of lifetime‐based fluorescence imaging techniques. Herein, a molecular design is described for simultaneously attaining long fluorescence lifetime ( τ ) and high brightness ( Φ F × ɛ ) in a system that features macrocyclic dimerization of fluorescent π‐conjugated skeletons with flexible linkers. An alkylene‐linked macrocyclic dimer of bis(thienylethynyl)anthracene was found to show excimer emission with a long fluorescence lifetime ( τ ≈19 ns) in solution, while maintaining high brightness. A comparison with various relevant derivatives revealed that the macrocyclic structure and the length of the alkylene chains play crucial roles in attaining these properties. In vitro time‐gated imaging experiments were conducted as a proof‐of‐principle for the superiority of this macrocyclic fluorophore relative to the commercial fluorescent dye Alexa Fluor 488.
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Molecular analogues of the green fluorescent protein (GFP) fluorophore have been designed by these authors (see Figure, X=NC4H9 or O). It is shown that by varying the donor and acceptor groups of the oxazolone (X=O) analogue the fluorescence emission can be tuned from blue to green to orange. The utility of these artificial fluorophores is explored by incorporating them into organic LEDs.
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SUMMARY The quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth‐dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emission.
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1,5‐Naphthyridin‐2(1 H )‐one is structural component of the fluorescent natural products amarastelline A and nigakinone. Herein, we synthesized several 1,5‐naphthyridin‐2(1 H )‐one derivatives and evaluated their fluorescence properties to examine their potential application as fluorescent probes. Among them, compounds that contain a 3‐hydroxyl group have unique solvent‐dependent fluorescence properties, that is, they exhibit stronger fluorescence in polar and protic solvents. The further installation of an 8‐phenyl group resulted in a shifted fluorescence in different solvents. Our results indicate that the 1,5‐naphthyridin‐2(1 H )‐one scaffold would be a valuable and useful fluorophore that displays intense fluorescence relative to known bicyclic fluorophores such as hydroxyquinoline. Our findings suggest that the 1,5‐naphthyridin‐2(1 H )‐one scaffold would be practical as an environment‐polarity‐dependent fluorophore, that is, for the development of sensors to detect structural changes in proteins and biomolecular interactions.
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