FRET Sensing Strategies Based On Polydopamine Nanomaterials And Enzyme Assisted Target Cycling Amplification Technology For Biochemical Analysis

It is of great significance to establish highly sensitive and high-throughput tumor marker sensing technologies for the early diagnosis and treatment of cancer.However,many of present methods for the detection of cancer markers are not accurate enough for low-abundance markers,and false positive signals are easy to generate.Therefore,in order to solve these problems,and to make full use of the advantages of fluorescent analysis,a new fluorescent sensing method was developed to reduce background interference,to avoid false positive signal generation,and to improve the accuracy of detection.In addition,signal amplification technology was developed to realize the detection of low-abundance markers and improve the detection sensitivity.In this thesis,the advantages of both fluorescence resonance energy transfer and enzyme assisted cycling signal amplification technology were combined to construct different FRET sensing strategies based on biocompatible polydopamine nanomaterials to achieve sensitive and accurate detection of low-abundance miRNA.These strategies were then applied to the actual sample detection to explore their potential in the diagnosis of cancer.The main research contents are as follows:?1?A novel fluorescence resonance energy transfer?FRET?-based platform using polydopamine nanospheres?PDANSs?as energy acceptors and dual colored Au NCs as energy donors for simultaneous detection of multiple tumorrelated microRNAs with DNase-I-assisted target recycling amplification was developed for the first time.On the basis of monitoring the change of the recovered fluorescence intensity at 445 and 575 nm upon the addition of targets miRNA-21 and let-7a,these two microRNAs?miRNAs?can be simultaneously quantitatively detected,with detection limits of 4.2 and 3.6 pM?3??for miRNA-21 and let-7a,which was almost20 times lower than that without DNase I.Additionally,semiquantitative determination of miRNA-21 and let-7a can also be realized through photovisualization.Most importantly,serums from normal and breast cancer patients can be visually and directly discriminated without any sample pretreatment by confocal microscope experiments,demonstrating promising potential for auxiliary clinical diagnosis.?2?The core-shell nanomaterials of ZnO@PDA were synthesized using biodegradable ZnO as the core and biocompatible polydopamine as the shell.Then,a novel FRET sensing strategy was constructed based on this material.Combined with DNAzymes assisted target recycling amplification technology,the ratio detection of miRNA in cells was realized,and the detection sensitivity was greatly improved.A DNAzymes probe related to Zn²⁺was designed based on the fact that ZnO can be degraded to Zn²⁺under weak acid conditions?pH 5.0-6.0?which could activate DNAzymes and therefore,additional Zn²⁺was no longer required.In this experiment,the highly sensitive detection of in-cell miRNA-21 was realized using a ratio fluorescent probe designed by combining DNAzymes assisted target recycling amplification and the ratio fluorescent method transported into the cell with the nano carrier ZnO@PDA.The detection amplificated signals effectively while reducing the background.Test results of miRNA-21 was in good linear relation a range from 0.01to 2.0 nM,and the detection limit was 3.8 pM.The results proved that the FRET strategy has a broad application prospect in tumor diagnosis and miRNA related biological studies.