DNA Nanostructure-Based Biosensors For Mirna Detection And Bioimaging

Cancer is one of the focal points of global health issues,and early diagnosis of cancer can improve patient cure and survival rates.miRNA is a potential cancer marker and therapeutic target,and monitoring intracellular miRNA levels is crucial for early cancer diagnosis.However,due to the small size,low abundance and high inter-family homology of miRNAs,while traditional miRNA detection methods are complicated to operate,have low sensitivity,require proteases to participate in the reaction and demand high reaction conditions,and cannot be used for real-time intracellular miRNA monitoring.Therefore,there is a need to develop accurate,sensitive and rapid methods for miRNA detection.Isothermal enzyme-free signal amplification techniques have attracted a lot of attention in the field of intracellular miRNA analysis,such as branch-mediated strand displacement reaction(STDR),hybridised chain reaction(HCR)and catalytic hairpin reaction(CHA),which have been widely used in the imaging analysis of intracellular miRNA.DNA nanomaterials are nanostructures formed by the self-assembly of DNA molecules.They can form stable double-stranded structures between DNA molecules through the principle of base complementary pairing,with high controllability and precision.Various shapes and sizes of nanostructures have been reported,such as DNA tetrahedra and DNA nanospheres.Based on their good biocompatibility,DNA nanostructures have been successfully applied in molecular diagnostics and bioimaging,etc.In summary,in this paper,three miRNA sensors based on DNA nanostructures were constructed using isothermal enzyme-free amplification strategies and the advantages of DNA nanomaterials,as follows:(1)A strand displacement reaction(STDR)based DNA nanosensor was developed for the sensitive detection of intracellular let-7a.The sensor,consisting of DNA tetrahedra DTN-F and DTN-H,specifically recognises the target upon entry into the cell and generates an amplified FRET signal through the STDR cycle,enabling rapid and sensitive detection of let-7a.The sensor has excellent detection performance,with a detection limit of22.4 p M for the target,and also has a fast response rate,reaching the reaction plateau within 200 s.The sensor also allows cancer cell identification by miRNA expression and dynamic monitoring of intracellular miRNA fluctuations,providing a new tool for bioanalytical and biomedical research.(2)A DNA nanosensor based on three-dimensional hybridisation chain reaction(3D-HCR)was developed for the detection of mi R-21 and mi R-203 in cells.The sensor consists of DNA nanospheres DH-13 and DH-24,which,after entering the cell by cytocytosis,specifically recognise miRNAs and then induce 3D-HCR to generate an amplified fluorescent signal for the detection of the two miRNAs.The sensor uses DNA tetrahedra as a carrier,which can effectively improve the stability of the nucleic acid probe.Meanwhile,the spatial domain-limiting effect of the nanocarrier was utilised to greatly accelerate the detection of mi R-21 and mi R-203,with 13.8-fold and 5.8-fold faster reaction rates compared to the hairpin probe,respectively.Meanwhile,the HCR amplification strategy improved the sensitivity of the two miRNAs detection in buffer solution and under complex conditions,with 1.41 p M and 2.07 p M detection for mi R-21 and mi R-203.In addition,the sensor enables the simultaneous detection of two miRNAs in cells,which is conducive to improving the accuracy of cancer diagnosis and is expected to provide an effective diagnostic and research It is expected to provide an effective tool for disease diagnosis and research.(3)A cascade signal amplification sensor based on the catalytic hairpin assembly reaction(CHA)and the hybridization chain reaction(HCR)was developed for the highly sensitive detection of intracellular mi R-21.The sensor consists of DNA nanospheres DS-13 and DS-24,which specifically recognize mi R-21 upon entry into cells and induce a CHA-HCR cascade reaction between the two DNA nanospheres to achieve highly sensitive analysis of target miRNAs through dual signal amplification of CHA and HCR,with a detection limit of 0.282 p M for mi R-21 in the range of 0.1～2 n M.In addition,a large number of nucleic acid probes can be modified using DNA nanospheres as carriers,which can increase the reaction rate and signal amplification efficiency through the spatial domain-limiting effect.The biological stability of the nucleic acid probes is also improved due to the protective effect of the nanostructures,enabling sensitive imaging of intracellular miRNAs and providing a new idea for molecular diagnosis.