Construction And Performance Study Of Dnazyme-based Highly Sensitive Fluorescence Sensing System For Lead Ion(?) Detection

With the advancement of the industrialization process of human society,industrial production has led to a large amount of pollution into the environment in which humans depend for a living.Pollution aggravates in environmental and poses a serious threat to human health.Lead pollution has become a study of hot topics.However,traditional methods for detecting lead ions have been difficult to meet the practical needs of monitoring environment and food safety.Therefore,it is of great theoretical and practical significance to construct a new type of highly sensitive lead ion???sensor.Deoxyribozymes?DNAzymes?are functional oligonucleotide fragments that have been artificially screened and synthesized by vitro molecular evolution techniques.They have high catalytic property,good selection specificity,and biostability.Moreover,they are synthesis and modification easy.They have become an important identification component for constructing highly sensitive and highly selective heavy metal ion biosensors.At present,bioprobes based on DNAzymes are usually designed by fluorescence,colorimetry and electrochemical methods.Among them,the fluorescent DNAzyme-based probes have the advantages of high sensitivity,good specificity,simple operation,portability,and good biocompatibility,thus they can realize practical advantages such as high-resolution imaging in living cells under a relatively low degree of damage.It is widely studied that probe construction.At present,DNAzyme-based fluorescent probes for detecting lead ion???have made great progress,but constructing a fluorescent probe with high sensitivity,high selectivity,rapid detection,and environmental friendliness is still a major challenge.In this study,two novel fluorescent probes were constructed based on DNAzymes to achieve high sensitivity detection of Pb²⁺using two different signal amplification ideas.The main research contents are as follows:?1?A kind of algae fluorescent protein R-phycoerythrin with high quantum yield extracted from natural seaweed was selected as the fluorescent group,and its fluorescence intensity was 30 to 100 times that of fluorescein at the same concentration.At the same time,GR-5 DNAzyme,DNAzyme with a specific recognition ability for Pb²⁺,was used as the recognition element of the fluorescent probe.Based on the principle of fluorescence resonance energy transfer,a highly sensitive,highly selective and environmentally friendly fluorescent probe DNAzyme-R-PE for trace lead ion???detection was constructed.The probe has a linear detection range of 0.5 to 75 nM and a minimum detection limit of 0.16 nM.At the same time,the DNAzyme-R-PE fluorescent probe developed in this paper is expected to be a powerful tool for the assessment of drinking water quality because the green material of the probe is environmentally friendly.Moreover,this fluorescent probe has good resistance to enzymatic cleavage and exhibits good biostability in 10%of FBS in which this fluorescent probe substantially maintains the detection performance of Pb²⁺.Therefore,the fluorescent probe DNAzyme-R-PE is expected to be used to detect whether the lead content in living tissues and organs exceeds the standard in the future.?2?Based on the specific recognition ability of GR-5 DNAzyme for Pb²⁺,a high sensitivity and high selectivity method for rapid detection of trace lead ion???was established.In the presence of Pb²⁺,a large number of specific product chains are synthesized by isothermal amplification under the joint action of DNA polymerase BSM and DNA cleavage enzyme Nt.BbvCI.Then,a structurally stable G-quadruplex is formed,and can be combined with zinc.The combination of porphyrins causes a fluorescence signal change,and as the concentration of lead ion???increases,the fluorescence intensity gradually increases.The combination of strand displacement amplification?SDA?and exponential amplification?EXPAR?enables rapid and highly sensitive fluorescence detection of Pb²⁺by double-cycle amplification.The sensor has a linear detection range of 0.1 to 50 nM with a minimum detection limit of 0.03 nM.The method has been successfully applied to the rapid detection of Pb²⁺in complex water samples and expected to play an important role in the detection of trace lead ion???in drinking water.The two fluorescent probes constructed in this study have high sensitivity,excellent specificity,superior fluorescence probe compared with the same type fluorescent probes.The two fluorescent probes have wide linear detection range,and do not cause secondary pollution,and have been successfully applied in sample testing.