Study Of Convenient And Sensitive Detection Method Based On Enzyme-free Amplification To Detect MiRNA And Telomerase

Since the 21st century,Analytical Chemistry for Life Science has become a research hotspot and focus as a deeply interdisciplinary new discipline such as analytical chemistry,biology,physics,and clinical medicine.Biomacromolecules(nucleic acids,proteins,enzymes,etc.)have a significant relationship with human cell functions,gene regulation,and tumor cancer.Therefore,the construction of sensitive,fast,simple,reliable,and specific biosensors for the research of biomacromolecules preventing systemic lesions is extremely important.In this paper,the strategy of quantitative analysis and detection of telomerase and miRNA was mainly applied by using a strategy combining enzyme-free amplification signal amplification technology with fluorescence analysis technology and PGM.Enzyme-free amplification avoids complicated procedures and high experimental costs.More importantly,in the detection process use a portable quantitative detection device PGM bypasses the need for complex instruments and the corresponding complicated operations,so that our method can be easily and flexibly applied to instant detection.Therefore,we have designed a variety of enzyme-free signal amplification strategies using DNA nanoprobes and nanomagnetic spheres as materials to achieve accurate,sensitive and instant detection of biomacromolecules.Thus,the proposed point-of-care assay has potential applications for the detection of telomerase activity with good sensitivity and specificity,which can provide a convenient approach for telomerase-related cancer diagnosis and anti-cancer drug development.The main researches are presented as following:1.Label-Free Fluorescent DNA Dendrimers for micro RNA Detection Based On Nonlinear Hybridization Chain Reaction-Mediated Multiple G?Quadruplex with Low Background Signal.In this chapter,a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear HCR-mediated multiple G-quadruplex has been developed for simple,sensitive,and selective detection of miRNA with low background signal.In the strategy,by taking advantage of the high target-dependent switch of the hairpin probe and Zn PPIX high selectivity for the G-quadruplex,the strategy exhibits splendid selectivity toward the target miRNA,and even can perfectly discriminate the single base mismatch miRNA from the miRNA target.Benefiting from excellent performance of nonlinear HCR and low background signal,as well as high sensitivity,the LOD is 0.7 f M.Thus,this proposed strategy will become an alternative approach for simple,sensitive,and selective miRNA quantification.2.Multi-code magnetic beads based on DNAzyme-mediated double-cycling amplification for a point-of-care assay of telomerase activityIn this chapter,we have developed a point-of-care assay of telomerase activity based on multi-code magnetic beads initiated by DNAzyme-mediated double-cycling amplification coupling with a glucometer as the signal transducer.By exploiting the high efficiency of double-cycling amplification,a high signal-to-background ratio was achieved,and the point-of-care assay realizes sensitive telomerase activity detection down to 5 He La cells.Additionally,the designed DNAzyme-based telomerase substrate prime(DTSP)probe and magnetic separation help to realize highly specific and reliable detection,even in a complex cell lysate.The combination of the enzyme-free signal amplification strategy and PGM not only overcomes the shortcomings of the high detection limit of the blood glucose meter,but also avoids complicated operation processes and high experimental costs,so that our method can be simply and flexibly applied to instant detection.These results prove that our method provides a simple,sensitive and robust platform for the detection of telomerase activity.3.Digital quantitative detection of serum circulating miRNAs using dual-enhanced magnetobiosensors based on cascaded nucleic acid circuitsIn this chapter,we have successfully developed cascaded nucleic acid circuit-based dual-enhanced magnetosensors for the digital quantitative detection of serum circulating miRNAs by PGM.Benefiting from the dual enrichment from the target and signal,cascaded nucleic acid circuit amplification and magnetic separation,this magnetosensor effectively amplified the detectable signal,and decreased the irrelative interference.A high signal to noise(S/N)and a high signal-to-background ratio were achieved,and the magnetobiosensors realized sensitive miRNA detection down to 0.7 f M.The use of a PGM device bypasses the requirement of laboratory-based instruments,making the magnetobiosensors simple,digital and feasible for on-site detection.The results showed that the magnetobiosensors have the potential for convenient and quantitative determination of circulating miRNAs in the peripheral blood with.