Fluorescent Apta-Sensors Based On DNA-Templated Copper Nanoparticles And Exonuclease-Assisted Signal Amplification Strategy For The Detection Of Lead(?)Ion

Lead ion(Pb²⁺)and other heavy metal ions are the most toxic metal pollutants,which can cause serious harm to the environment and organisms through accumulation in the environment and enrichment in the food chain.Therefore,the development of accurate and sensitive detection method of heavy metal residue is of great significance to ensure the safety of human body and environment.Recent years,the development of biosensing technology based on DNA aptamers provides a new method for the sensitive detection of heavy metals.Aptamers are short strand DNA molecules with high recognition and binding characteristics to specific targets obtained from a large DNA library via in vitro selection,which are stable in nature,biocompatible and easy to modify and synthesize.Biosensors based on aptamers have been widely studied and concerned in the analysis and detection of heavy metal residues by transforming the recognition and binding effects between the aptamers and specific targets into sensitive optical or electrochemical detection signals.In this paper,two fluorescent apta-sensors for the detection of Pb²⁺in the environment and food substrate were constructed based on the optical properties of DNA-Copper Nanoparticles?CuNPs?and exonuclease-assisted signal amplification strategy.The following are the main research content and discoveries:1.By combining Pb²⁺aptamer PS2.M with CuNPs synthesis template poly thymidine?poly T?DNA sequence,a bifunctional DNA template sequence T20-PS2.M-T20?T20 means 20 consecutive thymine?was designed that can combine with target Pb²⁺and induce in situ synthesis of fluorescent DNA-CuNPs.Pb²⁺can insert into PS2.M and induced it to fold into an antiparallel G-quadruplex structure.Subsequently,the fluorescence of DNA-CuNPs synthesized in situ at T20 can be quenched by Pb²⁺in G-quadruplex.By measuring the fluorescence intensity quenching ratio of CuNPs,the rapid,sensitive,selective and low-cost analysis of Pb²⁺can be realized.The detection limit of Pb²⁺in aqueous solution was as low as 0.065nM with a concentration range of 10-60 nM and the analysis time was less than 30minutes.This method and graphite furnace atomic absorption spectrometry?GFAAS?were used to detect the added Pb²⁺in the hairtail samples.The results of these two methods showed no significant difference,which verified the feasibility of this method for the determination of Pb²⁺in the real samples.2.By using the DNA adsorption function of Graphene Oxide?GO?and its fluorescence resonance energy transfer?FRET?with 5-Carboxy Fluorescein?FAM?,a fluorescent sensor based on the exonuclease-assisted target circulating signal amplification strategy was constructed to detect Pb²⁺.Firstly,the 3'end of PS2.M was extended by 8 bases to form a hairpin structure,and FAM was labeled at the 5'end to form a recognition and signal composite probe named PS2.M-4.PS2.M-4 with hairpin configuration is complementary at the end of itself,which can resist the digestion of Exo I.The adsorption of DNA strand by GO leads to the proximity of FAM to GO and the fluorescence of FAM can be quenched through FRET with GO.When the PS2.M part in PS2.M-4 identifies and combines with Pb²⁺,and then PS2.M-4 transformes into a G-quadruplex with 8 bases free at 3'end,Exo I can recognize the convex 3'end and performs enzyme digestion to gradually release Pb²⁺and FAM.The released Pb²⁺can continue to bind PS2.M-4,triggering the cyclic enzyme digestion process,so a small amount of Pb²⁺can induce a large amount of FAM to free from the GO surface,and the FRET efficiency between FAM and GO is reduced,and the fluorescence signal is strongly restored.Under the optimal conditions,there was a good linear relationship between the fluorescence recovery rate of the system and Pb²⁺concentration in the range of 10-100 nM,and the detection limit of the method was0.02 nM.This method was applied to the determination of added Pb²⁺in tap water,Songhua River water and hairtail samples,and the results showed no significant difference with GFAAS,indicating that this method can be used for the determination of Pb²⁺in real samples.