| Recently,in the field of life analytical chemistry,the requirement of detection methods of biomolecules is getting higher and higher.Building a sensitive,accurate,economical and convenient detection strategy has become a focus and hotspot in the field of life analysis.The biological signal amplification technology is a kind of advanced analysis technology,which is developed by the use of various kinds of enzymes and nano magnetic spheres,and it has important application in biomolecular detection.Personal glucose meter?PGM?,which is a bedside testing instrument?Point-of care testing POCT?,is a major breakthrough,and it not only can be conveniently purchased for the family,but also broke away from the conditions and instrumentation of laboratory.It has been widely used because of its advantages,such as portable size,cost effective,reliable quantitative detection,simple operation and immediate results.Liposome has good biocompatibility,and its inner cavity is large,which can encapsulate a great quantity of biological molecules.Therefore,we designed a fluorescence signal amplification strategy which based on tool enzyme and nano magnetic ball,and constructed a PGM-multifunctional liposome nano probe detection strategy,achiving accurate,sensitive and economic portable detection of biological macromolecules.In this paper,we took advantage of two kinds of detection techniques of PGM and fluorescence,regarded Dam methyltransferase and phospholipase D 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.Target-controlled gating liposome ‘‘off–on''cascade amplification for sensitive and accurate detection of phospholipase D in breast cancer cells with a low-background signalIn this chapter,we developed a simple,sensitive and accurate PLD detection method based on a target-controlled gating liposome?TCGL?‘‘off–on''cascade amplified strategy and personal glucose meters?PGM?.First,The TCGL provides an unmodified phospholipid natural substrate,avoiding effects on the PLD activity and guaranteeing detection accuracy.Second,the use of a portable quantitative device PGM bypasses the requirement of laboratory-based instruments,making the method simple and feasible for on-site detection.It gave a great sensitivity ang the detection limit is 0.005 U L-1 and well performed PLD activity analysis in breast cancer cells and inhibitor drug screening.2.Magnetic bead-liposome hybrids enable sensitive and portable detection of DNA methyltransferase activity using personal glucose meterIn this chapter,a sensitive,simple,and specific DNA MTase activity assay was developed based on magnetic beads-liposome hybrids combined with personal glucose meter?PGM?for quantitative detection of DNA MTase and inhibitor screening.First,a magnetic beads-liposome hybrid probe is designed by the hybridization of p1DNA-functionalized magnetic bead with p2DNA-functionalized glucoamylase-encapsulated liposome?GEL?.Then,in the presence of Dam MTase,the hybrids probe was methylated,and cleaved by methylation-sensitive restriction endonuclease Dpn I,making liposome separated from magnetic bead by magnetic separation.Finally,the separated liposome was decomposed,liberating the encapsulated glucoamylase to catalyze the hydrolysis of the signal substrate amylose with multiple turnovers,producing a large amount of glucose for quantitative readout by the PGM.It permits detection of as low as 0.002 U/L Dam MTase.In addition,we detecd the activity of Dam methyltransferase in human serum,and got better results.3.Magnetic nanoparticles-cooperated fluorescence sensor for sensitive and accurate detection of DNA methyltransferase activity coupled with exonuclease III-assisted target recyclingA fluorescence magnetic biosensor for the DNA methyltransferase activity was developed based on the cooperative amplification by combining the magnetic nanoparticles synergistic exonuclease III?Exo III?-assisted circular exponential amplification and a supramolecular structure ZnPPIX/G-quadruplex.In the developed sensor,a small amount of target DAM can be converted to a large number of stable DNA triggers,leading to remarkable amplification of the target.Moreover,using MNPs as a vector of the sensor may reduce the interference from the real samples,which increases the anti-interference of the sensing system.Based on this unique amplification strategy,a very low detection limit is 2.0 × 10-4 U m L-1.Furthermore,the sensor could be used to evaluate the DAM activity in different growth stages of E.coli cells and screen Dam MTase inhibitors. |
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