Sensing Research Of Disease Markers Based On Signal Amplification Strategy

Disease markers such as proteins and nucleic acids have seriously threatened the health of human being,which caused great influence on vital processes including hereditation,aberrance and metabolism.Therefore,it would have important significance on food security,environment monitoring,drug delivery and disease diagnosis to devote to working on the research of biomolecules.With the rapid development of biomedicine and analytical chemistry,scientists have established a series of effective methods to monitor these disease markers.However,the main emphasis of modern bioanalysis has shifted to establish more sensitive,simple,accurate and real-time analysis methods due to the limitations of interference and low concentrations.In recent years,fluorescence signal amplification technology has attracted wide attention due to its high sensitivity,good selectivity,fast response speed and low detection cost.Based on a variety of signal amplification techniques,this dissertation constructed three fluorescent biosensors combined with molecular biology techniques such as rolling circle amplification,DNA self-assembly and live cell imaging for real-time monitoring the target of uracil glycosylase?UDG?,thrombin and microRNA-21.Part 1.A label-free fluorescence biosensor assisted by rolling circle amplification strategy for sensitive detection of uracil DNA glycosylase.As a significant DNA repair enzyme,uracil-DNA glycosylase?UDG?is responsible for recognizing and eliminating uracil base from duplex DNA to pioneer the base excision repair?BER?process,which plays a pivotal role in the maintenance of genome integrity.Therefore,it is essential to carry out the research of UDG activity in fundamental bioanalysis and clinical application.Here,we have proposed a label-free fluorescent approach based on G-quadruplex for monitoring UDG activity by using rolling circle amplification?RCA?.In this work,a hybridized three-stranded DNA complex modified with uracil bases is designed for UDG recognition.Under the action of UDG,the uracil bases can be specifically recognized and excised from the deoxyriboses phosphate backbone to create abasic sites,which successfully result in the disassembly of the hybridized three-stranded DNA substrate and the formation of annular precursor.The annular precursor ligated by T4 ligase could subsequently trigger RCA reaction and generate a prolonged massive repeated G-quadruplex unit to interact with protoporphyrin IX?PPIX?to produce an enhanced fluorescence response for amplified detection of UDG activity.Not only can it successfully distinguish UDG from other nonspecific biomolecules,but also could be applied for screening the inhibitors of UDG.This proposed strategy is highly sensitive and selective with a detection limit as low as 0.00014 U/mL.These results indicate that the simple,sensitive and economic strategy holds great potential applications in UDG associated researches and related clinical diagnosis studies.Part 2.Aptamer proximity recognition-dependent strand translocation for enzyme-free and amplified fluorescent detection of thrombin via catalytic hairpin assembly.By coupling a new aptamer proximity recognition-dependent strand translocation strategy with catalytic hairpin assembly?CHA?signal amplification,we have developed a simple and sensitive method for detecting thrombin in human serums.Simultaneous binding of two engineered aptamer probes to the thrombin target significantly increases the local concentrations of the two probes and facilitates the translocation of a ssDNA strand from one of the probes to the other through toehold mediated strand displacement.Such strand translocation leads to the generation of a ssDNA tail in the aptamer sequence for subsequent initiation of the assembly of two fluorescently quenched hairpin into many DNA duplexes via CHA.The formation of the DNA duplexes thus results in significant fluorescence recovery for amplified detection of thrombin down to 8.3 pmol/L.The developed method is highly selective to the thrombin target against other interference proteins due to the dual recognition mode,and can be employed to monitor thrombin in human serum samples.With the advantage of simplicity,sensitivity and selectivity,this method can be a universal enzyme-and nanomaterial-free amplified sensing platform for detecting different protein molecules.Part 3.Biodegradable MnO₂ Nanosheet-Mediated Signal Amplification in Living Cells Enables Sensitive Detection of Down-Regulated Intracellular MicroRNA.The monitoring of intracellular microRNAs plays important roles in elucidating the biogenesis and biological function of miRNAs in living cells.However,because of their small size,low abundance and sequence similarity,it is of great challenge to detect intracellular miRNAs,especially for those with much lower expression levels.To address this issue,we have developed an in cell signal amplification approach for monitoring down-regulated miRNAs in living cells based on biodegradable MnO₂ nanosheet-mediated and target-triggered assembly of hairpins.The MnO₂ nanosheets can adsorb and exhibit excellent quenching effect to the dye labeled hairpin probes.Besides,due to their biodegradability,the MnO₂nanosheets feature with highly reduced cytotoxicity to the target cells.Upon entering cells,the surface-adsorbed FAM-and Tamra?TMR?-conjugated hairpins can be released due to the displacement reactions by other proteins or nucleic acids and the degradation of the MnO₂ nanosheets by cellular GSH.Subsequently,the down-regulated target miRNA-21 triggers cascaded assembly of the two hairpins into long dsDNA polymers,which brings the fluorescence resonance energy transfer?FRET?pair,FAM?donor?and TMR?acceptor?,into close proximity to generate significantly amplified FRET signals for highly sensitive detection of trace miRNA-21 in living cells.By carefully tailoring the sequences of the hairpins,the developed method can offer new opportunities for monitoring various trace intracellular miRNA targets with low expression levels in living cells.