Amplification Detection Of MicroRNA And ATP Based On DNA Self-assembly

Important biomolecules,such as enzymes and ATP,are the structural and functional base of organism and life's phenomena.Their contents and activity are directly related to the health of organisms.Among them,microRNA plays an important biological role in the regulation of gene expression.What we should do is to further penetrate the interaction of the biomolecules and to develop rapid,convince,accurate and sensitive assay for them.This project is the forward position and hot point in bioanalytical chemical research.In this paper,we first induced the formation of Gquadruplex-hemin?hemin?complex based on nicking endonuclease?NEase?assisted template enhanced hybridization process?TEHP?and rolling circle amplification?RCA?,then a label-free electrochemical biosensor for ultrasensitive let-7d RNA detection was developed;secondly,we show a DNA self-assembled nanostructures for carrying therapeutic mi RNAs into and abrogating tumor cells,the initial structure of the assembly can be used to detect miRNAs;finally,we design a method to the detect the ATP in the cell cycle of amplification based on exonuclease by the using of graphene oxide nanosheets.The main contents are as follows:1.By using the principle of the DNA self-assembly,a label-free electrochemical biosensor for ultrasensitive let-7d RNA detection was developed based on nicking endonuclease?NEase?assisted template enhanced hybridization process?TEHP?and rolling circle amplification?RCA?induced formation of G-quadruplex-hemin complexes.The hairpin probe?H1?was immobilized on the gold electrode through a strong Au-S bond.With the help of assistant DNA,the introduction of target let-7d RNA forms the nicking site for the NEase,which cleaves the hairpin probe and releases the target RNA.Further addition of another hairpin probe?H2?initiated the next two cascaded recycling process.The cleaved fragment acted as primer to activate RCA reaction,generating product containing hundreds of hemin aptamer and Gquadruplex-hemin complex can be formed with the help of K+ and hemin to give an electrochemical response.The newly designed protocol provides an ultrasensitive electrochemical detection of let-7d RNA down to the 0.42 fM level,and can discriminate target RNA from the let-7 family,holding a great potential for early diagnosis in gene-related diseases.2.We designed an aptamer-carboxyfluorescein?FAM?/graphene oxide nanosheet nanocomplex,combining the cycle amplification of exonuclease,to investigate its ability for molecular probing in living cells.The results showed that the uptake of aptamers-FAM/GO-nanosheets nanocomplex and the intracellular ATP cycle amplification were successfully achieved.The delivery,protection and sensing capabilities of GO-nanosheets in living cells indicate that graphene oxide can be a powerful candidate for many biological fields such as DNA and protein analysis,gene and drug delivery,and intracellular tracking.3.A DNA self-assembly nanostructure was designed to carry therapeutic microRNAs into and abrogate the tumor cells,and the initial structure of DNA selfassembly can be used to detect miRNAs.This system consists of three DNA hairpins modified with FAM and a linear DNA.When the target miRNA was encountered,hybridization chain reaction?HCR?occurs,and the three hairpins were alternately opened to form a stellate DNA self-assembly structure.The fluorescence intensity of unreacted hairpins was quenched by MnO₂ nanosheets,and miRNAs can measured by detecting the stellate DNA self-assembled structure.Linear DNA was ligated into the stellate DNA self-assembly structure under the action of T4 ligase,followed by the action of T7 RNA polymerase.Linear DNA was used as a template to produce a rolling circle transcription?RCT?reaction,and finally formed a DNA self-assembly nanostructures based on satellate DNA nanostructure including multiple copy numbers of CXCR4 hairpin and triple triple-helix structures,and after centrifugation,the DNA nanohydrogels was formed,which carries CXCR4 and triple-helix structures as therapeutic miRNAs entering and abrogating triple negative breast cancer cells.