Research On DNA Fluorescence Sensor Array Based On Molecular Beacon

Fluorescent biosensors have the advantages of simple structure,high sensitivity,good selectivity and fast response time,and are widely used in biological analysis such as genetic diagnosis.The fluorescence sensor array composed of fluorescent biosensor not only has all the above advantages,but also greatly enhances the feasibility of multi-target recognition.Combined with principal component analysis(PCA),the fluorescence sensor array can be used to identify complex analytes.However,in the actual bioanalytical detection,its inadequacies such as fluorescent probes with high background noise,complicated synthesis and low stability are also exposed.This paper uses the synergistic toehold to mediate the low cost and high stability of the DNA strand displacement reaction,and uses it as a new type of signal switch for the design of fluorescent biosensors.The synergistic toehold mediated DNA strand displacement mechanism performs strand displacement reaction by base stacking,and provides additional stability by base stacking between identical bases of two adjacent independent oligonucleotide strands.The feasibility of this strategy was verified by non-denaturing polyacrylamide gel electrophoresis and fluorescence spectroscopy.In addition,due to the high sensitivity and specificity of molecular beacons,this paper will use the advantages of molecular beacons to construct fluorescent biosensors and use them in the study of base mismatches.The specific work content is as follows:(1)Based on the proposed "cooperative toehold-mediated DNA strand displacement mechanism",a fluorescent sensor array 1 based on allosteric DNA molecular beacons was constructed to identify single base mismatches and perfectly matched sets of 9 target DNAs.The principle is based on the DNA hybridization reaction that occurs between the loop of the molecular beacon and the target.Non-denaturing polyacrylamide gel electrophoresis,real-time fluorescence monitoring and fluorescence spectroscopy experiments,combined with principal component analysis(PCA)technology,verified that our designed fluorescence sensor arrays are versatile and can identify each group with high sensitivity.The method has a response range of 10-100 nM and is expected to be used for the diagnosis of DNA-related diseases in clinical diagnosis.(2)Based on the principle of "cooperative toehold-mediated DNA strand replacement mechanism",we designed and constructed a fluorescence sensor array 2 based on allosteric DNA molecular beacons.The principle is based on the DNA hybridization reaction between the molecular beacon and the short-stranded DNA complementary to its loop,forming a DNA double-stranded hybrid,and the presence of the target is easy to produce the strand displacement reaction,and the molecular beacon strand is displaced from the DNA duplex.The sensor array 2 can detect four sets of target miRNAs with high similarity.The method has a response range of 10-200 nM and can be used for quantitative detection and identification of miRNAs in complex environments.(3)In the allosteric DNA molecular beacon fluorescent sensor array 3,the mismatch type with the target is cluster mismatch and the mismatched sites are located at the toehold,so the same molecular beacon chain can be used when designing different fluorescent biosensors.The array can be used not only to visually detect and distinguish 6 groups of highly similar toehold mismatched resistance genes(YMDD,YIDD,YVDD,YSDD,YLDD,YADD),but also to quantify and identify two different amounts of targets have a good value.Further research shows that the array method is suitable for complex systems and has high sensitivity and good anti-interference ability in the presence of interfering material fish sperm DNA.It is expected to provide a simple and rapid method for detecting and identifying highly similar targets in complex systems,and has great application value in clinical diagnosis.