Design Of Novel Sensing Strategies Based On Function Nucleic Acid And Their Application In Biosensing

Nucleic acid not only functions as the carrier for the storage,transmission and expression of genetic information rn organisms,but also molecular material with multiple functions.Since the discovery of the principle of base complementary pairing and the double helix structure of nucleic acids in 1953,a series of functional nucleic acids have been found and screened,including aptamers,RNAzyme/DNAzyme,G quadruplex,and a series of DNA assemblies.These functional nucleic acids exhibit unique properties in biological recognition,biocatalysis,signal enhancement,and controlable assembly of nanostructures.Due to their good biocompatibility,high-efficiency reaction activity,unique sequence editability and hybridization predictability,functional nucleic acids have shown great application prospects in the field of biosensing.In recent years,the introduction of nucleic acid amplification technology makes the functional nucleic acid-based biosensing strategy have significant improvement in sensitivity.In addition,the integration with micro/nano materials has improved the signal diversity and design flexibility of the biosensing strategy.Through integration with the above technologies and materials,the functional nucleic acid-based biosensing strategy has demonstrated excellent performance in the detection of a variety of disease markers,making it a valued method for disease diagnosis.Due to their flexibility and extensibility,functional nucleic acids still have considerable application potential in the field of biosensing.There is also a continuing need to construct new,stable,accurate,sensitive and high throughput biosensing methods to meet the increasing demands of bioanalytical work for the detection of trace biomarkers.We have developed a series of novel biosensing strategies based on functional nucleic acids for the detection of disease-related bioinolecules.The main contents are as follows:Chapter one is an introduction section,which summarizes several functional nucleic acids,the design and application of the biosensing strategy based on functional nucleic acid-nucleic acid amplification,and the design and application of the biosensing strategy based oil functional nucleic acid-micro/nano materials.This section also briefly introduces the four works carried out in this thesis.In chapter two,a cascade amplification strategy based on primer-template conversion was constructed for sensitive and accurate detection of PNK activity.This strategy integrated rolling circle amplification(RCA)and multiple-repeated-strand displacement amplification(MRSDA)with G-quadruplex based fluorescence lighting-up assay.A delicate dumbbell-shaped DNA probe with 5 '-hydroxyl terminus was designed,in which G-quadruplex and half recognition site of nicking enzyme Nb.BbvCI were encoded in two loops respectively.Under the action of PNK,the 5' terminus on dumbbell probe was firstly phosphorylated,and then the dumbbell was cy-clized with the catalyzation of T4 ligase to become the RCA template.The RCA process produced multiple copies of the prolonged primer.After that,under the assistance of nicking enzyme Nb.BbvCI,a primer-template conversion occurred,which converted the primer and template of RCA into the template and primer of the subsequent MRSDA,respectively.The MRSDA generated multiple repeated ssDNA sequences which possessed G-quadruplexes for outputting signal by lighting-up fluorescence of thioflavin T(ThT).The cascade signal amplification of RCA and MRSDA provided high detection sensitivity,and the target-dependence of template in cascade signal amplification led to a low background.The method showed excellent detection limit of 0.2×10-6 U ?L-1 in buffer and 5 cells in cell lysate sample.Moreover,this method displayed favorable selectivity when interfering proteins were present.The developed strategy has good practical potential for PNK activity detection in clinical diagnosis and medical research.In chapter three,a binding-mediated MNAzyme signal amplification strategy was constructed for enzyme-free and label-free detection of DNA-binding proteins.This strategy relied on the binding-mediated MNAzyme cleavage and G-quadruplex-based light-up fluorescence switch.Three DNA sequences were designed to construct the MNAzyme in which DNA1(including half binding site of the target protein and a toehold sequence)and DNA2(including another half binding site of the target protein and one MNAzyme partzyme)firstly hybridized.The target protein recognized the binding sites on DNA1-DNA2 hybrid to form a stable protein-DNAl-DNA2 conjugates.Then,the MNAzyme was assembled with the presence of DNA3 which contained another MNAzyme partzyme and the complementary sequence of DNA1.The active MNAzyme cleaved DNA4 to release the G-quadruplex that was locked in the stem of DNA4.Finally,N-methyl mesoporphyrin ?(NMM)was inserted into the released G-quadruplex structure and the fluorescence signal was turned on.Taking nuclear factor-?B p50(NF-?B p50)as the model,the limit of detection was low to 0.14 nM.Furthermore,the sequence-specific recognition of NF-kB p50 with DNA displayed excellent selectivity and specificity.The results in present work showed that this strategy will be a promising tool for DNA-binding proteins analysis in biomedical exploration and clinical diagnosis.In chapter four,we constructed a magnetic nanostructure based on dendritic DNA assembly for nucleic acid amplification detection.In this work,dendritic DNA assembly,magnetic separation and silver nanoparticle labeling were integrated to construct a magnetic nanostructure based on dendritic DNA assembly for amplification and detection of nucleic acid.The H5N1 avian influenza virus is a pathogen with high infectious rate and high lethal rate.In this study,the nucleic acid fragment of H5N1 virus was selected as the detection model.First,a magnetic bead-dsDNA composite probe was constructed,in which dsDNA contained the complementary sequence of the target nucleic acid and two blocked Trigger sequences.When that target nucleic acid is present,hybridize with dsDNA in the composite probe.Then,under the action of helper DNA,the beads-dendritic DNA structure was formed through the chain replacement reaction mediated by Toehold.The magnetic bead-dendritic DNA was combined with streptavidin-modified silver nanoparticles(SA-AgNP)by biotin at the end to form magnetic bead-dendritic DNA-AgNP.After magnetic separation,magnetic beads-dendritic DNA-AgNP were collected and subjected to ICP-MS testing,with 107Ag used as the output signal for the detection of the target nucleic acid.In the detection method,as the rnAgnetic beads-dendritic DNA can combine a plurality of sa-AgNPs and each agnp can release a plurality of ag atoms,a single target nucleic acid recognition event is converted into a plurality of 107Ag signals,significant signal amplification is realized,and detection sensitivity is ensured.By magnetic separation,SA-AgNP without binding to the bead-dendritic DNA was removed,which ensured a low background for detection.The method has been successfully applied to the detection of nucleic acids in serum samples,providing a new approach for sensitive,accurate and stable nucleic acid detection,and it will become a promising analytical tool for disease diagnosis and prognosis.In chapter five,a novel metal nanoparticle-labeled magneto-aptasensor was constructed for the simultaneous detection of ochratoxin A and fumonisin B1.In this method,CdSe quantum dots and AgNP were used as elemental lables for the detection of two mycotoxins,respectively.As CdSe quantum dots and AgNP contain a large amount of Cd and Ag elements respectively,and the mass-to-charge ratios of the two elements are high,they can release strong ICP-MS signals,providing high detection sensitivity.In addition,by integrating different kinds of aptamer-element labeled complexes on the magnetic beads,the method can simultaneously identify different mycotoxins and output response signals to realize simultaneous detection.Chapter six is the conclusion section,winch summarizes the innovation and prospect of this thesis.