Construction And Application Of Liposomes And Copper Nanoclusters For MiRNA Detection

For a long time,the detection of biomolecules in various fields of society has become increasingly demanding.Therefore,the construction of a simple,economical,sensitive and rapid method for the detection of biological macromolecules has become a hotspot and a focus in many fields.Biomolecule signal amplification technology is a technology developed in recent years.It is a highly sensitive detection method that uses tool enzymes and nanoparticles combined with electrical,optical and other detection methods.It has been widely used in the detection of biological molecules.The home blood glucose meter（PGM）is easy to purchase,and does not require complicated laboratory conditions and instrument operation during the test process.Therefore,home blood glucose meters have been widely used.A liposome is an artificial membrane that is arranged in a bilayer of phospholipids.Its molecular head is hydrophilic and its tail is hydrophobic.Because liposomes have huge internal cavities inside,liposomes can encapsulate drugs in lipid microspheres with submicron or nanometer diameters to detect biomolecules.The copper nanocluster using DNA strand as a template has the advantages of simple preparation method,low preparation cost,good biocompatibility,and environmental protection.Therefore,we have used a variety of nucleic acid tool enzymes,fluorescent nucleic acid probe technology and copper nanoclusters to establish multiple biomolecular signal amplification detection platforms to achieve economical,sensitive and fast detection of miRNAs.In this paper,we took advantage of two kinds of detection techniques of PGM and fluorescence,regarded miRNA as the research object,and constructed a variety of new signal amplification detection technology platform,achieving the quantitative analysis.The main researches are presented as following:1.Liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification for portable and accurate quantification of micro RNAsIn this chapter,we propose a strategy for enzyme encapsulated liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification（P-ERCA）for portable quantification of miRNA in complex biological matrixes with high sensitivity and selectivity by using a glucometer readout.In the sensing system,a small amount of target miRNA-21 can be converted to a large number of glucose signals,leading to a remarkable amplification for the target via P-ERCA and the liposome encoded multiple labeling as well as the enzyme catalytic reaction.The sensor improves the sensitivity of the sensing system with a detection limit as low as 0.1f M.Moreover,the liposome-encoded magnetic beads can circumvent the interference from the complex biological matrixes by magnetic separation.The target-dependent circularization of the padlock probe and the ligation reaction could effffectively improve the specificity.The application of a PGM bypasses the requirement of complicated instruments and sophisticated operations,making the method simple and feasible for on-site detection.2.A dandelion-like liposomes-encoded magnetic bead probe-based toehold-mediated DNA circuit for the amplifification detection of miRNAIn this chapter,,we propose a strategy for a liposome-encoded magnetic bead-based DNA toehold-mediated DNA circuit for the simple and sensitive detection of miRNA based on a toehold mediated circular strand displacement reaction（TCSDR）coupled with a glucometer（PGM）.The strategy realized the quantification of miRNA-21 down to the level of 0.7 f M without enzymatic-assisted amplification and precise instruments.The high-density GELs-MB probe allows the sensitive detection of miRNA-21 to be accomplished within 1.5 h.Moreover,this strategy exhibits the advantages of specificity and simplicity,since a toehold-mediated strand displacement reaction（TSDR）and a portable PGM were used.Importantly,this strategy has been demonstrated to allow the high-confidence quantification of miRNA.3.Construction of dendritic copper nanoclusters for miRNA detectionIn this chapter,a new method for detecting miRNA activity is designed.We use dendritic ds DNA generated by a non-linear HCR reaction to synthesize CuNPs.Quantitative detection of miRNA using a fluorometer.The system dynamically controls the branched growth of DNA dendrimers through the continuous assembly of quenched double-stranded DNA substrates.The generated DNA dendrimer was used as a template for the synthesis of CuNPs.CuNPs were formed after Cu²⁺was added,and the fluorescence signals of CuNPs were detected by a fluorometer.Enzyme-free and label-free amplification detection of miRNA.The detection limit of this method is as low as 5.48×10^-17M.