Chem. SCI.: hydrophilic / hydrophobic environment sensitive dye for cell polarity detection

In the intracellular environment, the distribution, spatial arrangement and composition of parameters such as polarity, viscosity, temperature, redox state and pH are different, which is very important for maintaining the physical and chemical behavior of biomolecules In addition, the intracellular polarity is closely related to various cell processes such as cell proliferation, immune system regulation, cell migration and molecular transcellular transfer, as well as diabetes, nervous system diseases and cancer However, the reported polarity probes can only detect limited polarity in specific intracellular areas, and can not achieve quantitative detection of intracellular polarity Recently, Professor Hwan Myung Kim of the Asian University of Korea designed and synthesized a novel double dye probe, rps-1, which consists of a polar probe dye1 based on the "turn on" of spiropyran intramolecular cyclization ring opening system activated by polar solvent and a benzothiadiazole dye dye3 with large Stokes shift, which only emits light in non-polar solvent （ Scheme 1a ）。 At the same time, the ratio probe was used to quantitatively detect the polar distribution of different cell lines It was found that lysosomes were the most polar organelles in the cells, while lipid droplets showed the most non-polar Relevant achievements were published on Chemical Science (DOI: 10.1039 / c9sc04859f) under the title of "combining hydraulic and hydraulic environment sensitive dyes todetect a wide rang e e of cellular polarity" (source: Chemical Science) firstly, the polar probe dye1 was obtained by condensation of Salicylaldehyde and 1,2,3,3-tetramethyl-3h-indole iodide under the condition of ethanol, and its absorption and emission intensity (Fig 1) were measured with different polar solvents (toluene to water) In nonpolar solvents, due to its closed-loop structure, dye1 limits the charge transfer and generates absorption in the short wave region of 328 nm; in polar solvents, dye1 opens the ring and increases the conjugated structure, so that the charge transfer recovery (scheme 1b) from Diethylaniline to indole salt decreases gradually at 328 nm and increases gradually at 549 nm, and at 573 nm Strong fluorescence (λ ex = 552 nm) was produced at In addition, the cyclization of dye1 was limited by the introduction of methyl to synthesize dye2 It was confirmed that the change of UV and fluorescence of dye1 in different polar solvents was the result of intramolecular cyclization In order to prove that changing the polarity of the solvent can make the cyclization and ring opening process of dye1 reversible, the fluorescence spectrum (Fig 1C, d) of dye1 was tested repeatedly with methanol (MeOH) and tetrahydrofuran (THF) as the solvent, and the constant change of fluorescence was observed (source: Chemical Science) then, the author analyzed 1H NMR spectrum in polar and nonpolar solvents, and further proved the cyclization ring opening system of dye1 (Fig 2) In CDCl3 (nonpolar) solvents, dye1 has a closed-loop structure and an intramolecular chiral center Therefore, the cyclodimethyl in the spiro form has been well separated at 1.14 and 1.32 ppm, respectively In the CD 3od: D 2O = 1:1 (polar) solvent, the spiro ring of dye 1 was opened, the intramolecular structure changed and the chirality disappeared, and the peak of gemodimethyl was only 1.72 ppm At the same time, with the increase of solvent polarity, the amine of dihydroindole was positively charged, and the chemical shift of amine methyl was changed from 2.74 ppm to 3.71 ppm In CDCl3: CD3 od = 1:1 (medium polarity) solvent, closed-loop and open-loop structures coexist (source: Chemical Science) in order to explore the application of dye1 and dye2 in biological imaging, the author used them in HeLa cell imaging Under the excitation of 552 nm light, both dye1 and dye2 show bright fluorescence After incubation with lysotracker green, a commercial lysosomal probe, and mitotracker green, the results showed that dye1 was mainly located in lysosomes with a Pearson coefficient of 0.94 (Fig 3a); dye2 was mainly located in mitochondria with a Pearson coefficient of 0.92 (Fig 3b) The results show that dye1 may be located in lysosomes or distributed in various places, but the polarity of lysosomes is relatively high, so it only shows fluorescence in lysosomes Therefore, in order to quantitatively show the polarity distribution in cells, the design of ratio fluorescent probe is particularly important (source: Chemical Science) the absorption wavelength of dye3, a benzothiadiazole derivative, is similar to that of dye1, but its large Stokes shift makes it produce near-infrared fluorescence and reduces the fluorescence interference of dye3 and dye1 Dye1 and dye3 are connected by piperazine to form ratio polarity probe rps-1 Due to the influence of dye3, rps-1 shows absorption at 510 nm in non-polar solvents (toluene, ether, etc.) and 550 nm in polar solvents (MeOH, water, etc.) due to the influence of dye1 (FIG 4A) In addition, the fluorescence intensity of rps-1 decreased at 650 nm and increased at 580 nm with the increase of solvent polarity (Fig 4b) The two measuring regions of the ratio fluorescence are 565-585 nm (f yellow) and 630-680 nm (f red), respectively The f yellow / F red is highly correlated with the en t value (Fig 4C) In addition, rps-1 showed reversible change of F yellow / F red ratio depending on solvent polarity, and its behavior was similar to dye1 (Fig 4D) (source: Chemical Science) through the ratio images of two channels, f yellow and f red, the author proved that rps-1 can be used to reflect the polarity differences in various regions of the cell HeLa cells incubated with rps-1 showed different fluorescence signals (Fig 5a, 5b) in F yellow and f red channels When ratio imaging (f yellow / F red) was performed with pseudo color image, the polarity distribution of each region of HeLa cells (Fig 5C) could be known at a glance (source: Chemical Science) at the same time, all the polar images presented by rps-1 were verified by the co location experiment of commercial probes located by organelles (Fig 6) In the ratio image, the red region with the largest polarity overlaps the lysosomal probe, the green region with the medium polarity overlaps the mitochondria and endoplasmic reticulum probe (cytoplasm), the blue region with the smallest polarity overlaps the lipid droplet probe, and the lysosome, cytoplasm and lipid droplet are clearly distinguished (source: Chemical Science) finally, in addition to HeLa cells, the author also observed the polarity distribution in Chang, Huh7 and sw837 cells It was found that the polarity of lysosome was the highest (Fig 7) The calculated fluorescence ratio of F yellow / F red of lysosome was between 1.7-1.9, indicating that it was similar to methanol and water (Fig 4C) (source: Chemical Science) in summary, based on two fluorescent dyes with the same absorption but different emission, the author designed and synthesized a ratio probe rps-1 for the detection of organelle polarity It can sensitively and quantitatively detect the change of polarity in a wide range, clearly show the polarity difference of each region in the cell, and provide some help for biomedical research.