Nanoprobes Based On The Nanostructure Of Nucleic Acids And Their Application In Live-Cell Detection

Cancer seriously threaten human life and health due to its high mortality,high metastasis rate and high recurrence rate.With the in-depth study of cancer,researchers have discovered a variety of cancer biomarkers,such as lipids,proteins,DNA,RNA,metabolites,etc.Their detection can quantify the specific state of cancer and help to develop adaptive treatment strategies for patients of different clinical stages.Due to the heterogeneity of tumors and the difficulty of sample concentration,the sensitive detection of specific protein signals and other low-abundance substances is still challenging.Biosensors based on nucleic acid structures are usually composed of DNA or RNA molecules.The variability and specificity of the sequence of such sensors enables them to efficiently identify target molecules and provide new detection tools for the field of bioanalysis.In order to more effectively realize the monitoring and prevention of early cancer and improve the detection sensitivity of biomarkers in living cells,a series of biosensors based on nucleic acid structure design have been constructed in this paper.The specific work carried out is as follows:1.According to the catalytic properties of multi-component nucleases（MNAzymes）,we have developed a new nuclease-encoded multicolor framework nucleic acid probe,which realizes the simultaneous imaging of two miRNAs in living cells with high sensitivity and high selectivity.The framework nucleic acid probes are synthesized through one-step thermal annealing.Two fluorescent groups（FAM and ROX）are labeled in the structure as signal indicators for the targets miRNA-21and miRNA-155.In the intracellular environment,the framework nucleic acid probe can enter the cell through endocytosis,specifically bind to the target miRNA and activate the MNAzyme structure,cleavage the active site,trigger the release of the target miRNA and amplify the circular fluorescent signal,and realize the effect on different cell lines.The miRNA is highly sensitive to diagnosis.At the same time,we loaded the framework nucleic acid probe with AS1411 aptamer that specifically binds to the nucleolin overexpressed on the surface of cancer cells to improve the precise targeting ability of the probe.In addition,the framework nucleic acid probe shows good distinguishing ability between cancer cells and normal cells,can realize the simultaneous detection of multiple intracellular miRNAs,realize double signal amplification,and the detection limit is as low as 0-100 f M.This strategy provides a new detection tool for understanding the biological functions of miRNA in cancer pathogenesis and therapeutic applications.2.According to the catalytic activity of ATP aptamer enzyme and the advantages of catalytic hairpin amplification,a composite structure probe based on Mo S₂was constructed to realize the detection and imaging of intracellular low-abundance ATP.Mo S₂ nanosheets can be used as nanocarriers and excellent fluorescence quenchers.DNA structure probes can be transferred to cells by endocytosis without adding additional transfection reagents.The DNA structure probe is composed of an aptamer enzyme structure that specifically recognizes ATP and three hairpins labeled with FAM fluorescent groups.The presence of ATP will activate the aptamer enzyme to cleave specific RNA sites,thereby releasing the trigger chain that triggers the catalytic hairpin assembly（CHA）reaction.The hairpins form a three-way connection of CHA（Y）nanostructures through base complementary pairing structure.Due to the low adsorption force of the double-stranded Mo S₂nanosheets,the"Y"structure can be released from the surface of the Mo S₂ nanosheets to enhance the fluorescence in the system.In the DNA structure probe,the dual-cycle strategy of the cleavage cycle reaction and the multi-site fluorescent modified CHA cascade catalyzed the fluorescence recovery significantly,which can effectively improve the detection sensitivity.The detection limit of this probe for ATP is 0.21μM,which is 84 times lower than that of the detection method based on a simple aptamer enzyme cycling strategy（17.62μM）.This method can realize the sensitive detection of ATP in living cells,which helps to understand the in the concentration of ATP in tumors,and of great significance for the prevention and detection of early cancers.