Construction Of A Fluorescent Probe For Hypochlorous Acid And Its Regulatory Factors And Its Imaging In Vivo

Hypochlorous acid(HClO)is highly oxidizing,plays an important role in the body's immune system,and helps to eliminate invading bacteria and pathogens.The increase in the concentration of HClO will cause severe stress response and induce many diseases.However,due to its low content and high reactivity,it is still a big challenge to track HClO in real time.At the same time,H₂S and CO,as important gas signal molecules in the organism,have a certain mitigation effect on the stress response caused by HClO.Therefore,the realization of real-time detection of hypochlorous acid and its regulatory factors is of great significance.Here,we have designed and constructed a variety of fluorescent probes for HClO and its regulatory factors,which are used in the fluorescence imaging of HClO and its regulatory factors in biological environments.(1)The HClO fluorescent probe(Nil-ClO)based on two-photon excitation near infrared emission of Nile red derivative.Nil-ClO has a large fluorescence response near650 nm,and has good selectivity to HClO in solution.When 300?M of HClO are present,the fluorescence intensity at 650 nm of the probe shows an approximately 8-fold increase in fluorescence within 1 min.The probe is further used for the fluorescence detection of HClO in cells,tissues and mice with acute liver injury.MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)experiment shows that the probe has little cytotoxicity at the concentration required for imaging.Nil-ClO can quickly respond to HClO in HeLa cells and RAW cells.In tissue imaging experiments,Nil-ClO has a significant near-infrared fluorescence response as deep as100?m to HClO.Finally,fluorescence imaging of endogenous HClO in a mouse model of liver injury was achieved.(2)Reasonable design of a powerful ratio fluorescent probe(Cy7-Nil)for measuring the concentration of HClO.Cy7-Nil consists of two near-infrared chromophores with three emission signals(560 nm,650 nm and 780 nm),and Cy7-Nil has HClO concentration-dependent and two-photon excitation characteristics.These superior characteristics of Cy7-Nil enable it to perform proportional detection of HClO concentration range in dual ratio channels(I₆₅₀/I₅₆₀ and I₆₅₀/I₇₈₀),and for the first time to perform proportional detection of endogenous HClO concentration in living animals.This work provides a universal method to create a new type of ratiometric probe to monitor the level of reactive small molecules in living animals.(3)A unique palladium coordination polymer(Pd-NA)based on naphthalene derivatives was constructed and used for fluorescence imaging of CO.The morphology and coordination structure of Pd-NA were studied by SEM,XPS and XRD.Spectral experiments show that Pd-NA can have obvious fluorescence changes to CO.Due to the heavy metal effect of Pd,the fluorescence of Pd-NA is negligible,but with the addition of CO,Pd-NA produces a significant increase in fluorescence at 510 nm.In biological experiments,Pd-NA was used to image the exogenous and endogenous CO produced in HeLa,and the cells showed up to 10-fold fluorescence enhancement.In addition,Pd-NA exhibits excellent single-photon and two-photon fluorescence imaging capabilities for detecting CO in zebrafish.(4)A new cell-capturing H₂S fluorescent probe Rho630-AM-H₂S with red light emission.Researched its application in the fluorescence detection of H₂S in Hela cells and zebrafish.As expected,Rho630-AM-H₂S showed a huge fluorescence turn-on response of about 20-fold at 630 nm and good selectivity toward H₂S in solution.An MTT assay demonstrated that the probe showed negligible cytotoxicity in the concentrations typically used in fluorescence imaging experiments.Cell imaging experiments revealed that compared with compound 4 without cell-trappable unit modification,Rho630-AM-H₂S exhibited remarkably enhanced cell penetration ability,as an enormous fluorescence signal increase was observed at the red channel within 5min after Rho630-AM-H₂S was incubated with HeLa cells.Finally,Rho630-AM-H₂S was used to detect H₂S in zebrafish.