Study Of Construction And Performance Of Highly Sensitive And Enzyme-free Fluorescence Detection System For Pb²⁺ Based On GR-5 DNAzyme And Isothermal Amplification Strategy

With the stage of social and economic development entering the stage of full industrialization,the problem of heavy metal pollution has attracted more and more attention.Heavy metals are one of the main pollutants in the environment and water resources,especially lead ion(Pb²⁺)pollution seriously endangers human health.Previous studies have shown that lead ions at low concentrations can cause serious damage to many parts of the human body such as the nervous system,digestive system,hematopoietic organs,cardiovascular and kidneys,etc.It can also cause serious pollution to the environment,so it is of great significance to monitor trace amounts of Pb²⁺in the environment.There are often many limitations for traditional detection methods,and it is not possible to detect lead ions quickly,and efficiently,,and at low cost.Therefore,it is important to establish a low-cost,simplesimple,and fast,highly sensitive and selective Pb²⁺detection method.Deoxynuclease（DNAzyme）is a catalytically active DNA molecule obtained by in vitro selection.With its excellent programmability,stability,and catalytic activity,DNAzyme has attracted a lot of attention in therapeutic and diagnostic applications.So far,DNAzyme has been widely used as an important recognition element for biosensors to detect heavy metal Pb²⁺.DNAzyme-based biosensors can be divided into fluorescent,colorimetric,and electrochemical types according to the detection signal.The isothermal amplification of the nucleic acid can rapidly and effectively amplify the sequence of the nucleic acid at a constant temperature.Compared with the traditional Polymerase Chain Reaction（PCR）,the isothermal amplification does not need the thermal cycle of temperature rise and fall,the requirement on the precision of the instrument is reduced,the operation time is shortened,the process is simple,the energy consumption is low,and the method has high sensitivity,can be integrated into portable equipment,is beneficial to on-site real-time detection,and is a technology for efficiently amplifying the nucleic acid instead of a PCR method.The main isothermal amplification methods include loop-mediated isothermal amplification（LAMP）,Helicase-Dependent Amplification（HDA）,Rolling Circle Amplification（RCA）,Strand Displacement Amplification（SDA）,Catalytic Hairpin Assembly（CHA）and Hybridization Chain Reaction（HCR）etc.In recent years,a variety of isothermal amplification techniques have been applied to biosensors and combined with the Pb²⁺specific DNAzyme to detect Pb²⁺in different places quickly,with high sensitivity and high specificity.These sensing strategies have been widely used.In this thesis,GR-5 DNAzyme was used as the recognition element,and combined with the nucleic acid isothermal amplification strategy,two different design ideas were constructed,and an enzyme-free,efficient,low cost and high specific detection system for Pb²⁺was created,and its performance was studied.The specific work includes the following:（1）Enzyme-free and highly sensitive detection of Pb²⁺by fluorescence sensing system based on the amplification strategy of hairpin DNA dynamic self-assembled dendrimerIn this chapter,GR-5 DNAzyme was used as the Pb²⁺specific recognition probe and dynamic self-assembly was used as the amplification signal amplification strategy,to construct a highly sensitive Pb²⁺fluorescence sensing system without enzyme.First,three DNA single chains Y1,Y2 and Y3 are combined to form a three-armed Y skeleton and combined with three hairpins H1,H2 and H3 to form a plurality of hairpin-type trimers.Pb²⁺specifically recognized and triggered the catalytic activity of GR-5DNAzyme,cleaving the substrate chain and releasing the target chain;Then the target opens and binds the hairpin H1 containing the recognition domain of the target chain to the recognition domain;At the same time,the exposed part of H1 opens the hairpin H2 on the other hairpin-type trimer and combines with the recognition domain to form a polymer containing two three arms,and opens H3 on the third hairpin-type trimer and combines with the recognition domain to form a polymer containing three three arms,wherein H3 replaces target from the polymer to catalyze next dynamic self-assembly process to finally form a dendritic polymerization product.In this process,when the hairpin H1 is opened,the above modified fluorophore and the quenching group are separated to generate a fluorescence signal,thereby realizing high sensitivity detection of Pb²⁺.Under the optimal experimental conditions,the detection system could complete the enzyme-free fluorescence detection of Pb²⁺in 40 min,with the linear range of 0.1～10 n M and the detection limit of 18.96 p M.The method was operated in a single tube at a constant temperature without the assistance of protease and was successfully applied to the detection of Pb²⁺in real water samples.（2）Construction and performance study of Pb²⁺highly sensitive and enzyme-free fluorescence detection system based on GR-5 DNAzyme and entropy-driven isothermal cycle amplification systemThis chapter constructed an enzyme-free fluorescent sensing system based on the cascade entropy-driven strand displacement reaction of GR-5 DNAzyme and Molecular Beacon（MB）.The fluorescent group Cy3 was modified at the 5’end of the MB,and the quenching group BHQ2 was modified at the 3’end of the MB.In the presence of Pb²⁺,GR-5 DNAzyme specifically recognizes Pb²⁺,and its catalytic activity is stimulated,thereby cleaving the substrate strand and releasing the target strand（Target）,and then complexing from the prepared triplex through an entropy-driven strand displacement reaction A large number of signal probes（Signal Probes,SP）are replaced in the medium,and through the hybridization of SP and MB,the modified fluorescent groups and quenching groups at both ends of MB are separated,thereby generating fluorescent signals and realizing the specific detection of Pb²⁺.In the absence of Pb²⁺,the substrate chain of GR-5 DNAzyme is not cleaved,and Target cannot be released,so the entropy-driven strand displacement reaction cannot be triggered to release SP,so the detection system does not generate fluorescence.The fluorescence sensing system has strong specificity for Pb²⁺and amplifies the fluorescence signal through entropy-driven strand displacement reaction,thereby realizing the quantitative analysis of Pb²⁺concentration.Its linear detection range is0.01～10 n M with a detection limit of 2.4 p M.The sensor has good selectivity and high sensitivity,can quantitatively detect Pb²⁺in actual water samples,and has good application prospects in the monitoring of heavy metals in the environment and food.