Construction Of Controlled Aggregation-Induced Emission System And Its Application In Detection Strategy

Fluorecscent probes become powerful tools in biosensing and imaging due to their high resolution and good biocompatibility.Traditional fluorescent bioprobes suffer from aggregation-caused quenching effect,which leads to fluorescent decrease at high concentration or aggregation states,and limits their performance in biosensing.However,molecules with aggregation-induced emission(AIE)characteristics overcome the limitation with low background interference,high signal to noise ratio and good photostability.Due to the complication of real sample environment,developing specific AIE bioprobe with controllable scaffolds is highly demanded in the future.This thesis focuses on design of novel detection methods based on AIE signal transduction with controllable molecular structures such as DNA self-assembly,metal-organic frameworks,and DNA walker.The thesis includes the following four sections:1.DNA Quadruplexes as Molecular Scaffolds for Controlled Assembly of Fluorogens with Aggregation-Induced EmissionAggregation-induced emission(AIE)can be generated due to the restriction of the intramolecular motions.The controllable assembly of fluorogens with AIE properties(AIEgens)is able to provide a new opportunity for precise manipulation of the fluorescent signal transduction.Here,a tetrapod DNA quadruplex(TP-G4)was designed as a molecular scaffold for assembly and precise modulation of light emission of an oligonucleotide-grafted fluorogen with aggregation-induced emission(Oligo-AIEgen).The Oligo-AIEgen was synthesized by attaching the AIEgen to 3'-terminus of the oligonucleotide through a dibenzylcyclooctyne mediated coupling reaction.The AIEgen emitted no detectable fluorescence in the context of a double-stranded structure.When hybridized to a parallel-stranded TP-G4,several AIEgens were located in close proximity to generate fluorescence.The fluorescence intensity has been precisely regulated by manipulation of the spacer length between the core structure of the scaffold and AIEgen,as well as by altering the quartet number of G-quadruplex.Similar control of fluorescence was also demonstrated using tetramolecular and bimolecular i-motif quadruplex structures as the scaffolds.These scaffolds provide a proof of concept on manipulation of molecular interactions,which forms a universal molecular tool for design of new biosensing strategies.2.Quencher-Delocalized Emission Strategy of AIEgen-Based Metal-Organic Framework for Profiling of Subcellular GlutathioneDistinguishing glutathione(GSH)level in different subcellular locations is critical for studying its antioxidant function in the signaling system.However,traditional methods for imaging subcellular GSH were achieved in isolated organelles or fixed cells.In this work,we report a quencher-delocalized emission strategy for in situ profiling of GSH at different subcellular locations in living cells.A non-emissive metal-organic framework(MOF)nanoprobe was designed with AIEgen as the linker and Cu(?)as the node and quencher.The AIEgen in MOF structure was lightened up with green emission at neutral environment due to partial Cu(?)delocalization by competitive binding to GSH.Meanwhile,along with the protonation of AIEgen ligand under acidic environment,the AIEgen-based MOF could be completely dissociated in the presence of GSH to yield yellow emission.The two-channel ratiometric analysis of dual colored emission of AIEgen-based MOF allows visualization of GSH in cytoplasm and lysosome in living cells,which is also able to report the drug effects on different subcellular GSH levels.3.Controlled Assembly of AIEgens Triggered by DNAzyme for Detection of Plasma Membrane ProteinsQuantification of plasma membrane proteins(PMPs)is crucial for understanding the fundamentals of cellular signaling systems and their related diseases.In this work,tetrapod DNA quandruplexes(TP-G4)were reported to regulate assembly of oligonucleotide-grafted AIEgens(Oligo-AIEgen)with super-hairpin based DNAzyme amplification for detection of PMPs.The Oligo-AIEgen was synthesized by attaching the AIEgen to 3'-terminus of the oligonucleotide through click chemistry.The DNAzyme was anchored on the specific PMPs through aptamer-protein recognition.When adding the super-hairpin and cofactor Mn2+ to the pretreated cells,the substrate sequence on the loop of the super-hairpin was cleaved by DNAzyme,resulting in the dehybridization of stem domain and release of the fragments from cell surface.Meanwhile,the DNAzyme continuously cut the next super-hairpin to achieve recycling amplification.The DNA-1 fragment in the cell culture supernatant could induce assembly of Oligo-AIEgen with TP-G4 and HDNA to generate fluorescence.Therefore,this approach demonstrates a simple and sensitive strategy for detection of PMPs.4.Pixel Counting of Fluorescence Spots Triggered by DNA Walkers for Ultrasensitive Quantification of Nucleic AcidA pixel counting strategy is designed based on DNA walker-triggered fluorescence spots for ultrasensitive detection of nucleic acid.The two-dimensional DNA walker was constructed by hybridization of the dye-labeled hairpin structure(hDNA)as track and swing strand(sDNA)as DNAzyme with two types of capture DNAs covalently modified by click chemistry on glass slide.Introduction of target DNA unlocked the sDNA via strand displacement to form the activated DNAzyme,and the latter cut nearby hDNA with Mn2+ as cofactor,resulting in fluorescence recovery of dye-labeled hDNA on the substrate due to the separation from the quencher.Meanwhile,the DNAzyme sequence of sDNA was released to cut the next hDNA,and thus initiated autonomous walking of sDNA for signal amplification.The enhanced fluorescence spots were digitalized as pixels on the basis of DNA walker-built compartments and extracted by home-made program in MATLAB.The concept on the association between fluorescent pixel numbers and DNA concentrations was further proved by mathematical model,and led to an ultrasensitive quantification of nucleic acid with a linear range from 100 fM to 10 pM.The designed pixel counting strategy shows more sensitive and accurate comparing with conventional methods based on fluorescence intensity or spot counts,and provides a new dimension in designing next-type biosensors.