The Preparation Of Fluorescent Nano-probes Based On The Principle Of Aggregation Induced Luminescence And Theirapplication In Biological Sensing And Imaging

Fluorescent bioprobes are powerful tools for analytical sensing and optical imaging,which allow direct visualization of biological analytes at the molecular level.Most of the traditional dyes decrease or even emit fluorescence at high concentration,which is called aggregation induced quenching effect.Until 2001,Professor Tang Benzhong's research team discovered another phenomenon of dye molecules.In the aggregation state,the fluorescence intensity of the dye molecules will be greatly enhanced,which is called aggregation induced luminescence effect.The simple design and fluorescence turn-on feature of the molecular AIE bioprobes offered direct visualization of specific analytes and biological processes in aqueous media with higher sensitivity and better accuracy than traditional fluorescence turn-off probes.We synthesized three kinds of molecules,which Emit three different colors of fluorescence.AIE molecules connected with DNA by covalent bonding,then developed a new biological sensing probe,and applied the probe in the detection of cancer biomarkers in vitro and in vivo respectively,and the living cell imaging.The main contents of the dissertation were as follows:(1)We designed an ultrasensitive microRNA detection method based on an extremely simple probe with only fluorogens but without quencher groups.It avoided complex and difficult steps to accurately design the relative distance between the fluorogens and quencher groups in the probes.Furthermore,the assay could accomplish various detection limits by tuning the reaction temperature.Specifically,1 pM miR-21 could be detected at 37?,and 10 aM(about 300 molecules in 50?L)miR-21 could be discriminated at 4?.The great specificity of the assay guaranteed that the real 21 urine samples from the bladder cancer patients were successfully detected by our method.(2)We successfully synthesized a facile probe based yellow emission AIEgens.The probe could distinguish the cancer patients' urine samples and the normal person's.Moreover,the probe showed much higher fluorescence intensity in breast cancer cells(MCF-7)(miR-21 in high expression)than that in cervical cancer cells(HeLa)and human lung fibroblast cells(HLF)(miR-21 in low expression),which showed the good performance and super photo-stability for the probe in vivo.As controls,another two probes with FAM/Cy3 and corresponding quenchers respectively,could perform miRNAs detections in vitro,and parts of in vivo tests but not suitable for the long-term cell tracking due to the photo-bleach phenomena.(3)We designed the signal amplification strategy with 1:N2 reaction ratios using exonuclease ?(Exo ?)as a signal amplifier.With the aid of telomerase,repeat units(TTAGGG)n were added into the end of TS primer and formed duplex DNAs with TPE-Py-DNA.Then,Exo ? catalyzed the digestion of duplex DNAs,liberating elongation product and releasing hydrophobic TPE-Py.The released hydrophobic TPE-Py aggregated together and produced a telomerase-activity-related fluorescence signal.The liberated product hybridized with another TPE-Py-DNA probe,starting the second cycle.Finally,we obtained the target-to-signal amplification ratio of 1:N2.This strategy exhibits good performance for detecting clinical urine samples(distinguishing 15 cancer patients'samples from 8 healthy ones)and checking intracellular telomerase activity(differentiating cell lines including HeLa,MDA-MB-231,MCF-7,A375,HLF and MRC-5 from the cells pretreatment with telomerase-related drug),which shows its potential in clinical diagnosis as well as therapeutic monitoring of cancer.(4)We successfully synthesized a red-light AIE molecule,which was then attached to the DNA by the click reaction for intracellular mRNA analysis.The probe showed much higher fluorescence intensity in breast cancer cells(MCF-7)than that in cervical cancer cells(HeLa).Cordycepin and lipopolysaccharide(LPS)were used to downregulate and upregulate the expression level of mRNA in MCF-7 cells,respectively.The probe with AIEgens is a promising approach for the real-time analysis of mRNA expression and processing in live cells for elucidation of biological processes and disease pathogenesis.