Construction Of Label-free And G-quadruplex-based Fluorescent Sensors And Application For DNA And MiRNA

Fluorescent biosensors are often used to detect trace substances due to their stability and simplicity.Nucleic acids are closely related to various biological processes.Accurate detection of nucleic acids plays an important role in drug development and medical research.However,the development of simple,fast,low-cost,highly specific and sensitive fluorescence biosensors still remains challenging.Therefore,several simple and label-free fluorescent biosensors were constructed based on G-quadruplex and DNAzyme to detect viral DNA and microRNA(miRNA).The main researches are listed as follows:(1)Efficient Dual-amplification System for G-quadruplex-based Non-enzymatic Fluorescence Detection of MicroRNABased on catalytic hairpin self-assembly amplification(CHA)and hybridization chain reaction(HCR),an efficient dual-amplification system for fluorescence detection of miRNA was successfully developed.H1 recognized the target and released the G-quadruplex sequence,which bound H2 to activate the CHA cycle.The CHA product hybridized with H3 and released the trigger to open HCR circuits,resulting in the cross-opening of H3 and H4 and self-assembling into a long DNA nanowire with abundant G-quadruplexes,which could combine with Thioflavin T(ThT)to produce strong fluorescence.The proposed system could detect miRNA in a wide concentration range with the detection limit as low as 36 fM and exhibit special selectivity in homologous miRNA sequences.Moreover,this method is label-free,enzyme-free,practical and simple,owing to overcoming cumbersome labeling procedures and the use of expensive protein enzymes.(2)Multifunctional and Label-free Fluorescence Probe for Simultaneous Detection of Multiple virus DNAs and MicroRNAsIn our work,with the addition of the targets,the probe with two capitated recognized regions was hybridized with targets.A G-quadruplex/ThT duplex was formed for specific and enhanced fluorescence response.In addition,an increase in the fluorescence intensity of silver nanoclusters(AgNCs)occurred because dark AgNCs became bright nanocluster dimers.Moreover,this single and duplex detection for virus DNAs and microRNAs,which provides a versatile platform for different targets,was sufficiently sensitive for the expected detection limit and specially selective with similar oligonucleotides,and could quickly detect targets in 40 min.Therefore,this strategy might provide new insight into the simultaneous detection of multiple targets.(3)Universal and Label-free Fluorescence Probe for Multiple Oligonucleotides Detection Based on RNA-cleaving DNAzymesIn this study,the method on the basis of Mg2+-dependent DNAzyme and G-quadruplex for fluorescent detection of miRNA and viral DNA was developed.The split DNAzyme could bind with target to produce an active DNAzyme.In the presence of Mg2+,the catalytic core was formed and the active DNAzyme could cleave the substrate strand,resulting in the release of the caged G-quadruplex sequences.Then,these released G-quadruplexes bound with ThT to form G-quadruplex/ThT duplex and produced significant fluorescence.This strategy could detect target as low as 70 pM.Moreover,some other advantages with low cost and simple operation,are also exhibited in the strategy.This method can also detect miRNA and DNA in actual samples and has great potential for application in disease research and clinical diagnostics.