Application Of New Technology Of Lead Ion Detection Based On Deoxyribozyme

Heavy metal ions have become a serious threat to human health due to their non-degradable and highly toxic properties,which has attracted extensive attention all over the world.Lead ions(Pb²⁺),mercury ions(Hg²⁺)and silver ions?Ag⁺?are the three most common pollutants,which can cause damage to human health even at low concentrations.Therefore,it is of great theoretical and practical significance to construct a new high sensitive heavy metal ion sensor.Functional nucleic acids have the advantages of good selectivity,high stability,strong binding ability,good biocompatibility,wide range of targets,rapid synthesis and easy modification.They are often used as recognition elements to construct biosensors suitable for complex samples.Deoxyribozyme is an in vitro-selected,artificially synthesized functional oligonucleotide fragment with high catalytic activity and structural recognition ability.The use of deoxyribozymes for the high specific recognition of its cofactor,heavy metal ions,enables the construction of various types of heavy metal ion bio-probes and sensors.Currently,heavy metal ion probes and biosensors based on deoxyribozyme include three types:colorimetry,fluorescence and electrochemistry.The fluorescence and electrochemistry are most studied.Fluorescent nucleic acid probes have many advantages such as high sensitivity,high specificity,simple instrument operation,good biocompatibility,less damage to living cells and capable of realizing resolution imaging,etc.They are mostly used for in vivo imaging and analysis.Electrochemical biosensors have the advantages of low cost,high sensitivity,easy portability,and electrochemical tags that are not easily affected by the detection environment.It has become a research focus in recent years to improve the sensitivity of such sensors through signal amplification.In view of the above-mentioned bottleneck problems in the construction of fluorescent probes and electrochemical biosensors based on deoxyribozymes.This dissertation discusses the chemical modification of GR-5 by performing different chemical modifications?phosphorothioate,2?-O-Methyl?on the 3?and 5?base skeletons of GR-5 deoxyribozymes and their substrates.The effect of ribozymes on catalytic activity,sensitivity,and selectivity;study of biocompatibility,biological stability,and bio-specificity,and detection of heavy metal ions in vivo.In Chapter 2,a highly sensitive and biostable fluorescent probe was constructed by phosphorothioate modification of GR-5 deoxyribozyme.The melting temperature of the probe was 5°C lower than that of the GR-5 deoxyribozyme,which reduced the stability of the enzyme-substrate complex but did not affect the signal to noise ratio of the GR-5deoxyribozyme.Skeleton modification changed the best detection condition,pH changed from 7.26 to 8,and Mg²⁺concentration changed from 5 mM to 1 mM.Under optimized conditions,the fluorescent probe detects lead in a linear range of 1 to 500 nM with a detection limit of 0.3 nM?S/N=3?.It can resist the degradation of DNaseI better,the degradation resistance in the medium can reach 72 hours,and it has the biological stability of long-term follow-up monitoring in vivo.This method was successfully applied to the detection of Pb²⁺in vivo by microinjection into embryonic cells of indigo.2.In chapter 3,a highly stable and highly sensitive fluorescent sensor probe was constructed by modifying the GR-5 deoxyribozyme with 2?-O-Methyl.The melting temperature of the 2?-O-Methyl modified GR-5 deoxyribozyme-substrate complex increased by 13°C,which reduced the signal-to-noise ratio to a certain extent and did not change the optimal conditions for detection.It was not only resistant to DNase I degradation,but also Maintains better biostability in 10%FBS.The fluorescence probe has a linear detection range of 1 to 300 nM and a detection limit of 0.5 nM?S/N=3?.The2?-O-Methyl modified deoxyribozyme probe has strong anti-enzymatic degradation characteristics,and it was successfully applied to the detection of exogenous Pb²⁺in the embryo cells of the medaka.3.In Chapter 4,A highly selective and sensitive lead ion electrochemical biosensor was constructed using Pb^2+-specific deoxyribozyme-substrate complex?GR-5?as the recognition element,6-?ferrocenyl?hexanethiol?FcHT?as an electronic medium.Among them,FcHT activated the entire catalytic cycle of FcHT/HRP/H₂O₂ as an electron mediator,amplifying the electrochemical signal by nearly one order of magnitude,greatly improving the sensitivity of the sensor.The sensor has a high sensitivity,a wide linear range?0.1¹000 nM?,low detection limit?0.03 nM?.The sensor has been successfully applied to the detection of Pb²⁺in tap water samples.The measurement results are consistent with the traditional ICP-MS method.