Study On Fluorescent Sensors Using G-quadruplex As Quenching Groups

Nucleic acid aptamers are a class of DNA or RNA molecules with high affinity and specific recognition.g-quadruplex is a unique nucleic acid aptamer secondary structure consisting of a guanine-rich DNA sequence folded into parallel,antiparallel and hybrid structures.In recent years,researchers have developed various sensing platforms based on G-quadruplexes for the analysis and detection of a wide range of substances,including metal ions,toxins,nucleic acids,and other small molecules.The fluorescence of fluorophores can be quenched by nucleotide bases through photoinduced electron transfer(PET),and guanine has the strongest quenching ability among common nucleotide bases.Since G-quadruplex is a structure with a high accumulation of guanine and low oxidation potential,it is the best candidate for electron donor and has the potential to be a quencher of a fluorophore.If the G-quadruplex formed in the molecular recognition process is used as the quenching group to construct the fluorescence aptamer sensor,the detection cost will be greatly reduced.In this study,based on the fluorescence quenching ability of G-quadruplex,a label-free fluorophore and label-free quencher nucleic acid aptamer sensor was designed for biomolecule detection.The main contents and related results are as follows:(1)A fluorescent aptamer sensor was constructed for the detection of ochratoxin A(OTA)using an antiparallel G-quadruplex as a quenching group.A complementary self-hybridizing double strand was designed at both ends of the nucleic acid aptamer,and SYBR Green I(SGI)was embedded in the double-strand to generate fluorescence as the luminescent group,and the G-quadruplex produced during the recognition of OTA was used as the quenching group to achieve the quantitative detection of OTA.By replacing the label-free SGI dye with a 1:1labeled carboxyfluorescein(FAM)molecule and introducing a specific ligand(crystalline violet,CV)for the antiparallel G-quadruplex,it was confirmed that the antiparallel G-quadruplex was a good label-free fluorescent quenching group.Based on the quenching ability of the antiparallel G-quadruplex,a fluorophore-free and quenching group-free labeled fluorescent nucleic acid aptamer sensor scheme was constructed for the detection of OTA.The linear range of the biosensor for the detection of OTA was 0.19 to 15.63 n M,and the lowest detection limit was 0.19 n M.A fluorescent kit based on this detection scheme was developed to achieve rapid detection of OTA in wine.(2)Based on the previous work,a fluorescent aptamer sensor was constructed to detect potassium ions using the parallel G-quadruplex as the quenching group.To further confirm the quenching ability of parallel G-quadruplex,a specific ligand of parallel G-quadruplex(methemoglobin,Hemin)was introduced.The formed G-quadruplex/Hemin complex increased the G-quadruplex electron cloud density,which exacerbated the degree of fluorescence quenching due to the leap of electrons from the complex to the fluorophore HOMO orbitals,which inhibited the return of electrons from the excited state of the fluorophore to the ground state.The biosensor can detect potassium ions in a linear range from 0.24 to 15.63μM with a minimum detection limit of 0.24μM.The fluorescence kit developed based on this assay protocol enables ultra-fast detection of serum potassium and is expected to enable immediate bedside detection(POCT).In this thesis,a fluorophore-free labeling and quenching group-free labeling of fluorescent nucleic acid aptamer sensor scheme was constructed using G-quadruplex as the quenching group to achieve the detection of biological small molecules(OTA and K~+).This biosensor has the advantages of simple operation,low detection cost and high sensitivity.It has wide application prospects in bioanalysis,clinical medicine,food safety and environmental monitoring.