The Study Of Novel Biosensing Strategies Based On Dna Self-assembly And Functional Nucleic Acids

The detection of biomarkers of disease play vital roles in medical research,clinical diagnosis and therapeutic monitoring.Biosensors are devices that use biomolecules activity recognitions to convert measured sensations into output signals and perform detection.They have the advantages of simple operation,high sensitivity and low cost.DNA nanotechnology is programmable and biocompatible,providing a new direction for the development of biosensors.In this paper,to meet the needs of clinical diagnosis,a series of new biosensing strategies have been developed for the detection of nucleic acids and small molecules.Taking advantagies of DNA self-assembly techniques and functional nucleic acids,the developed strategies offer highly effective approaches for biomarkers detection in biomedical research and clinical diagnosis.The study mainly has three parts as follows:1.Surface plasmon resonance biosensing strategy for microRNA detection based on DNA self-assembly and streptavidin signal amplificationMicroRNAs?miRNAs?play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases,making them a promising biomarker.In this work,a novel surface plasmon resonance?SPR?biosensing strategy has been developed for detection of miRNA based on DNA super-sandwich assemblies and biotin-strepavidin signal amplification.The hairpin capture probe specifically hybridizes with the target miRNA and undergoes a conformational change,resulting in the opening of the terminus site for biotin modified auxiliary probe 1hybridization.With the addition of auxiliary probe 1 and auxiliary probe 2,DNA super-sandwich nanostructure is assembled on the chip surface and produces an amplified SPR signal.Furthermore,the added streptavidin attache to the chip surface via binding with biotinylated AP1 on the long DNA super-sandwich assemblies,resulting in a cascading amplification of SPR signal.The method shows very high sensitivity,capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6orders of magnitude(from 1×10^-11 M to 1×10^-6 M)in 30 min,and can successfully discriminate a single base mismatched miRNA sequence.The established method is simple,rapid and stable.It also has the advantage of real-time and enzyme-free.Therefore,it offers a powerful tool for clinical diagnosis,therapeutic monitoring and prognosis evaluation of tumors and other major diseases.2.Surface plasmon resonance biosensing strategy for detection of DNA and small molecule based on nonlinear hybridization chain reactionNonlinear hybridization chain reaction?HCR?is a hairpin-free system in which two kinds of double-stranded DNA monomers could dendritically assemble into highly branched nanostructure upon the introducing of a target sequence.An enzyme-free and label-free surface plasmon resonance?SPR?biosensing strategy has been developed for highly sensitive and specific detection of target DNA by employing the nonlinear HCR amplification.The target DNA partly hybridizes with capture probe on the gold sensing chip and the unpaired fragment of target DNA works as a trigger to initiate the nonlinear HCR,resulting in the formation of chain-branching growth of DNA dendrimer by self-assembly and the amplification of SPR signal.The developed method is capable of detecting target DNA at the concentration down to 0.85 pM in 60 min with a dynamic range from 1 pM to 1000 pM,and can discriminate target DNA from mismatched sequences.Moreover,this biosensing strategy has been successfully applied for detection of target DNA in complex sample matrices.Combined with aptamer technology,this method is extended for the detection of adenosine triphosphate?ATP?with high sensitivity and specificity.The SPR biosensing platform has the advantages of simple,real-time,enzyme-free,and label-free.Moreover,the nonlinear HCR technique is isothermal and flexible,which can be used as an universal signal amplification system for the detection of other biomarkers.Therefore,the developed method provides a new technology platform for the diagnosis of tumors and other major diseases.Forthermore,it has a promising prospect in biomedical research,forensic research,and pathogen detection.3.Homogeneous fluorescent biosensing strategy for the detection of p53gene based on programmed entropy-driven strand displacement reactions and DNAzyme recycling amplificationThe p53 gene is a tumor suppressor gene and plays an extremely important role in the anti-tumor mechanism of cells.The p53 gene mutation is associated with a variety of tumor diseases.Therefore,it is of great significance to establish a new method for simple,rapid and sensitive detection of p53 gene.As a novel nanomaterial,DNA can be utilized to construct promising DNA nanostructures due to the well-characterized nature of base-pairing of nucleic acids,and has been widely used in the detection of biomarkers on biosensing platforms.In this work,an innovative homogeneous fluorescent biosensing strategy has been developed for the detection of p53 gene based on programmed entropy-driven strand displacement reactions?ESDRs?and DNAzyme recycling amplification.Target DNA triggers ESDRs,which brings about the recycle of target and large amounts of displaced DNAzyme sequences in the solution.Subsequently,in the presence of the coenzyme Mg²⁺,DNAzyme circularly cleaves the fluorophore/quencher-modified substrates and leads to the fluorescence as readout signals.The developed strategy shows excellent sensitivity and high selectivity for the detection of p53 gene.The method achieves a linear range of 500 fM to 10 nM with a low detection limit of 220fM,and enables discrimination of target p53 gene from mutation sequences easily.This homogeneous sensing method does not require protein enzyme assistance or separation.Thus,the promising technique can be applied to tumor prediction,early clinical diagnosis,prognosis assessment,and tumor treatment.Furthermore,it promotes the research of clinical laboratory diagnostics.