Construction Of Nucleic Acid Probe-mediated Novel Fluorescence Sensing Strategy And Its Biosensing Applications

Nucleic acid has become an excellent biomaterial for the construction of molecular probes due to its easy synthesis and modification,unique hybridization specificity,sequence editability,structural predictability and good biocompatibility.In recent years,a range of nucleic acid probes have been screened and created,such as nucleic acid aptamer,DNAzyme,double-stranded hybrid,G-quadruplexe and dendritic DNA.At present,researchers have constructed a variety of nucleic acid probe-based fluorescence sensing strategies,and have been widely applied in the detection of biomolecules.In particular,the use of nucleic acid probes in combination with some nucleic acid amplification techniques and micro/nanomaterials not only improves the sensitivity of detection,but also broadens its application range.However,there are still some problems to be solved with the deepening of the research and the increasing demand for the detection:(1)How to further improve the detection sensitivity based on cyclic enzyme amplification strategy;(2)How to construct a simple and sensitive universal sensing strategy;(3)How to safely and efficiently deliver the enzyme into the cell,and carry out enzyme-assisted signal amplification strategy in.cells;(4)How to overcome the problem of non-specific amplification caused by additional primers or templates;(5)At present,the detection of biomolecules is mostly single analysis,and the multiple detection of biomolecules is more instructive for the diagnosis and treatment of diseases.To address the above problems,this thesis constructs five novel nucleic acid probe-mediated fluorescence sensing strategies for the detection and imaging of disease-related biomolecules.It mainly includes the following seven chapters:Chapter one is the introduction section,providing an overview of the design and utilization of several common nucleic acid probes.It also discusses the combination of nucleic acid probes with nucleic acid amplification technology,while also highlighting some of the key challenges associated with current strategies.In chapter two,a T7 exonuclease(T7 Exo)-assisted double-cycle cascade signal amplification strategy was constructed for the sensitive detection of circular RNA(circRNA).In the presence of circRNA,the recognition probe specifically hybridized with circRNA,which triggered T7 Exo-assisted target cycle amplification,followed by activation of secondary target cycle amplification,yielding a large number of signal units with G-quadruplex sequences.With the presence of K+,the G-quadruplex sequence bound to thioflavin T to produce enhanced fluorescence for circRNA detection.Benefiting from the high amplification efficiency of the double-cycle cascade signal amplification reaction,the detection limit of this strategy for circRNA was as low as 1.2 × 10-9 M.In addition,the analysis of circRNA in human serum samples and cellular samples was successfully achieved.In chapter three,we constructed a modular universal sensing platform based on dendritic-magnetic sphere superstructures for sensitive detection of biomolecules.First,a 3’-OH primer 1 was generated on the magnetic sphere by the interaction of the target and the recognition probe.Subsequently,the 3’-OH end of primer 1 was extended by terminal transferase(TdT)to produce a long poly-T sequence,which could serve as an anchor template to bind to a large number of primers 2.At the same time,the 3’-OH end of primer 2 was also extended by TdT,generating numerous branched long poly-T sequences to continue binding to primer 2.Finally,the dendritic-magnetic sphere superstructure containing abundant double-stranded structures was obtained after several binding-extensions.Following magnetic separation,SYBR Green I was added and bound to double stranded DNA,producing enhanced fluorescence for biomolecules detection.Benefiting from the high assembly efficiency of the TdT-mediated dendritic-magnetic sphere superstructure,the sensing platform exhibited high sensitivity.The design of magnetic separation could effectively eliminate interferences and ensure low background signal.The modular design improved the versatility of the method as it could be adapted to different biomolecules by simply changing the recognition probes.Based on these,the sensitive detection of microRNA(nucleic acids),hAAG(proteins)and ATP(small molecules)was successfully achieved.In chapter four,an enzyme-embedded ZIF-8/DNA nanocomposite probes were constructed for intracellular double-cycle cascade polymerization nicking reaction to image and detect of ATP.First,the nanocomposite probes were constructed by adsorbing the recognition probe RP and the molecular beacon MB onto the surface of ZIF-8 NPs pre-embedded with enzymes(Bst DP and Nt.BbvCI).After uptake into cells,ZIF-8 NPs were biodegraded in acid lysosome,accompanying by the release of embedded enzymes and surface-adsorbed DNA.The RP specifically recognized and bound to ATP,resulting in its own conformational transformation.Then,with the assistance of embedded enzymes,the 3’-end of the RP-ATP complex acted as primer to trigger the primary polymerization nicking reaction and sequentially activate the secondary reaction.Finally,significantly amplified fluorescence signals were produced for imaging analysis.Based on this,the imaging of ATP in HeLa,A549 and MCF-7 cells was successfully implemented.Furthermore,owing to the high amplification efficiency of the double-cycle cascade polymerization nicking reaction,this strategy achieved the sensitive detection of ATP with a limit of detection of 7.7 nM.These results showed this enzyme-embedded ZIF-8/DNA nanocomposite probe would be a promising tool for ATP-associated biomedical research.In chapter five,a ZIF-8 mediated self-primer and self-template rolling circle amplification(SSRCA)strategy was proposed for living cells imaging of hAAG activity.ZIF-8 NPs were employed as a carrier to deliver the recognition probe RP,substrate probe SP and enzymes into cells.When hAAG specifically removed of damaged base from the RP,the RP was cleaved to generate the primer and template required for RCA.With the assistance of embedding enzymes,the SSRCA reaction was occurred and generated a large amount of DNAzyme to continuously cleave SP,producing enhanced fluorescence signal to imaging hAAG activity.In this work,the primers and templates required for the RCA reaction were generated by the interaction of the target with the recognition probe,which could effectively avoid the occurrence of non-specific amplification reactions and improve the accuracy of imaging.By utilizing this method,the imaging of hAAG activity in NCM 460,MCF-7 and HeLa cells was achieved.In addition,benefiting from the high amplification efficiency of SSRCA,this method displayed high sensitivity with a detection limit of 9.3 × 10-5 U μL-1.This work provided a new idea for sensitive and accurate imaging of hAAG activity.In chapter six,a MnO2 nanosheet-mediated target-binding-induced fluorescence resonance energy transfer(FRET)strategy was developed for multiplexed microRNAs detection and imaging in living cells.In this work,MnO2 nanosheets were served as carriers to deliver the probe pairs into the cells.In the presence of microRNA,the DNA probe pairs specifically recognized and combined with target microRNA to form duplexes,bringing the FRET pairs into close proximity to occur FRET.By utilizing this method,we successfully achieved the simultaneous imaging of microRNA-373 and microRNA-96 in MDA-MB-231 and L02 cells.Furthermore,the ratiometric detections of microRNA-373 and microRNA-96 were successfully implemented by using the DNA probe pairs.This approach had the potential to be applied in the analysis of multiple microRNAs,facilitating accurate diagnosis and treatment of diseases.Chapter seven is the conclusion section,which summarizes the research and prospect of this paper.