Construction Of Novel Fluorescent Nanoprobes For Detection Of Intracellular Biological Sulfur And H₂S

The progress of life is an integrated result of many complicated chemicalreactions with the participation of various biological-active substances. With thedevelopment of chemical biology and biosensing technology, a series of biochemicalreactions in organism are gradually clarified and the deep awareness for life activitiesor life phenomenon is achieved. Bio-active substances possess special physiologicalproperties, play the important biological functions and have a close association withmany kinds of diseases and cancers. Therefore, realizing the detection of thesebio-active molecules is of significance. Fluorescent probes have a series of advantagessuch as high sensitivity, excellent selectivity and easy to operation. More importantly,the fluorescent probes can deeply penetrate into cells without any damage to the tissueand organism. After reaction with targets, the fluoresce of the probe will occur. Withthe help of the scanning confocal microscopy, visual and real-time measurement ofbiological substances can be triumphantly realized. Thence, more attention is paid tothe application of these useful tools in bioassay and biomedical field. In recent years,the development of nanotechnology is rapid. Owing to the nano-size effects （forinstance, surface effect, quantum size effect, macroscopic quantum tunneling effectand dielectric confinement effect）, nanomatericals have many unique properties, suchas superior electrical and optical property, excellent biological affinity and goodbiocompatibility. So, nanomaterials have been widely used for analytical detection andbiosensors construction. Considering the importance of detecting bio-active moleculesby combining the organic fluorescent probes with the nanomaterials, this thesis wasfocused on the following three aspects works:（1） A gold nanocarrier and DNA-metal ligation-based sensing ensemble forfluorescent assay of thiol-containing amino acids and peptides. Based on the strategiesof thymine-Hg²⁺-thymine (T-Hg²⁺-T) and fluorescence resonance energy transfer（FRET）, we designed a novel fluorescent sensing ensemble by combininghairpin-structured DNA segment with gold nanoparticle for the sensitive detectionbiological of thiol-containing amino acids and peptides. The T-Hg²⁺-T-structured stemwas first formed by the addtion of Hg²⁺and fluorescence emission of the sensingensemble can not be observed due to the quenching of AuNPs to the fluorophore In thepresence of thiols, the fluorophore/quencher distance will increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg²⁺-T due to theextraction of Hg²⁺ions, then resulting in switching the sensing ensemble to the “on”state and the sensitive detection and cell imaging of targets can be reached.（2） Fabrication of novel two-photon fluorescent nanoprobe based oncyclodextrin-functionalized graphene oxide and fluorescenece molecule. Based on thecharacteristic of reactive nucleophile and undergoing nucleophilic reaction twice, wereport a two-photon fluorescent nanoprobe containing α,β-unsaturated phenyl ketoneby engineering the two-photon fluorescence dye and graphene oxide to detecthydrogen sulfide. Due to the carbon-carbon double bond, the back ground fluorescenceis very low. Through the host-guest interaction between adamantane and cyclodextrin,two-photon fluorescent molecules are immobilized on the grapheme oxide’s surface.After reaction with H₂S, the fluorophore will be released from the graphene oxide’ssurface and the fluorescence emission of two-photon dyes will be restored. Due to thegood water-soluble property of cyclodextrin-functionalized graphene oxide and itshigh membrane penetration ability, the proposed two-photo fluorescent nanoprobe willbe successfully used to real-time monitoring and imaging of H₂S in living cells.（3） Metal nanoparticle’s ion release-triggered hydrolysis cascade of rhodaminehydrazide for fluorescence signal-amplified detection of proteins. Based on theorganometallic-catalyzed reactions to promote hydrolysis of rhodamine hydrazide toamplify fluorescence signal, we developed a novel fluorescence enhancement method.In the presence of IgG, which is used as a model target protein, the sandwichedimmunocomplexes will be formed between two antibodies, one of which isimmobilized on the magnetic bead to simple the separation process and the second islabeled with CuO nanoparticles to realize the signal amplification. After aciddissolution, Cu²⁺will be released from the CuO nanoparticles and promotes thehydrolysis reaction of rhodamine hydrazide resulting in enhancement of thefluorescence emission.