Study Of Fluorescent MiRNA Detection Based On Two-dimensional Hybrid Polydopamine Nanoquencher

Micro RNAs(miRNAs)are a class of endogenous non-coding RNA molecules(?22nt),whose accurate detection is of great significance for clinical diagnosis,prognosis and treatment of tumors.However,miRNAs'unique characteristics,including their small size,high homology of sequences,low abundance in cells,and easy degradation,present a tremendous challenge for specific,sensitive and rapid miRNA detection.Nano/biosensing interfaces constructed from fluorescent nucleic acid probes and nanomaterials with excellent optical,electrical and thermal properties provide new design ideas for miRNA quantitative detection.Nevertheless,most nanomaterials still have some disadvantages,including complex preparation process,low fluorescence quenching efficiency,and high fluorescence background,which limit their value in practical applications.Therefore,the construction of excellent biosensors by rational design and optimization of nanoquenchers has become a fundamental problem to be solved.Polydopamine(PDA)is a class of novel?-conjugated polymer,which has the advantages of simple preparation,diverse molecular structure,rich and adjustable physicochemical properties.Hence,PDA has a unique potential to mediate conjugated polymer interfacial recombination.As a?-electron-rich system,PDA can strongly interact with?-conjugated bases of single-stranded DNA(ss DNA)probes via?-?accumulation.In addition,PDA can carry out efficient photogenerated electron transfer(PET)with fluorescent molecules,which provides a new possibility for the construction of fluorescence sensing systems.Based on the unique advantages of PDA,DNA and polypyrrole(PPy)with rich?-electrons,two-dimensional(2D)conjugated polymer nanoquenchers with unique structures and excellent properties were prepared and applied to the miRNA quantitative analysis.The main research contents are as follows:(1)Construction of?-electron-rich biosensor interface based on DNA-PDA composite nanomaterials.Based on herring sperm DNA(hs DNA)as the structural guidance agent,the two-dimensional DNA-PDA?-conjugated nanomaterials with lateral?-?stacking and vertical staggered arrangement were synthesized,and their lateral sizes could reach more than 10?m.By adjusting the mass ratios of hs DNA/dopamine(0.75,1,1.5,2),PDA nanosheets(NS)with different thicknesses(12.5-56 nm)and roughness(4-9 nm)were constructed.Subsequently,it was found that 1-NS with a higher specific surface area had a stronger quenching ability to ss DNA fluorescent probes at low concentrations(60?g m L^-1),which was quite to or even lower than solid and mesoporous PDA nanospheres.Finally,miRNA sensors were constructed based on the affinity difference of ss DNA and double-stranded DNA(ds DNA)toward nanosheets.The results demonstrated that the sensor has a low detection limit(LOD,168.97 p M)and good selectivity.The high loading capacity of ss DNA probes toward PDA nanosheets,as well as high affinity difference for ss DNA and ds DNA,demonstrated the crucial functions of the system design in molecular recognition and signal transduction of sensing platform and showed the great potential of the platform in miRNA detection.(2)Construction of?-electron-rich biosensor interface based on PDA-PPy composite nanomaterials.The polydopamine-polypyrrole composite nanosheets(PDA-PPy-NS)with thicknesses of 12.4-21.5 nm were successfully prepared by in-situ polymerization of pyrrole(Py)monomers on PDA nanosheets(PDA-NS)via?-?stacking and oxidant(Fe³⁺)induction.With varying doping ratios of Py/PDA-NS(w/w,0.39,0.68,0.97),PDA-PPy-0.97-NS exhibited remarkable photophysical and electrochemical properties:excellent optical absorption performance(?,10.55 L g^-1cm^-1),narrow band gap(0.29e V),?electron delocalization,high conductivity and PET efficiency.Afterward,femtosecond transient absorption spectroscopy(TAS)and fluorescence quenching properties were used to demonstrate the PET fluorescence quenching mechanism and high fluorescence quenching ability toward fluorescence-labeled DNA probes.More importantly,the quenching efficiency(QE)of PDA-PPy-0.97-NS was up to 99.22%,which was 35.08%higher than PDA-NS(64.14%).The quenching ability of PDA-PPy-0.97-NS was higher than that of previously reported PPy and PDA nanomaterials,even comparable to that of graphene oxide(GO).Furthermore,the miRNA-21 detection performance of PDA-PPy-NS nanoquencher was also investigated,and it was found that PDA-PPy-NS had a lower detection limit(23.1 p M)and higher selectivity.The above results proved that the conceptual confirmatory design of?-electron-rich biosensor interface could significantly enhance the PET efficiency of the sensing system,which provided a theoretical and practical basis for the design of high-performance sensors.In this thesis,all results showed that based on PDA,double-stranded nucleic acid,PPy and other?-electron-rich molecules via interface?-?accumulation,as well as the heterocomposites of conjugated polymer products driven by?-cation,two-dimensional?conjugated hetero-nanosheets had been successfully prepared.Its unique?-electron coupling and?-plane packing structures provided a new idea and a theoretical basis for the controllable preparation of 2D heterostructures with adjustable band gaps,high PET efficiency and excellent water solubility.Meanwhile,the PET fluorescence quenching mechanism of PDA-PPy-NS was verified by femtosecond transient absorption spectroscopy.This discovery provided a conceptual verification platform for fundamentally exploring the fluorescence quenching mechanism and improving fluorescent biosensors'detection performance.