Construction And Application Of Lead Mercury Ion Sensor Based On Pb²⁺ DNAzyme And T-Hg²⁺-T Functional Nucleic Acid

At present,heavy metal pollution is not only the main factor leading to environmental degradation,but also has an impact on food safety and threats human health.In recent years,the emerging DNA sensing technology has provided great feasibility for realizing the rapid on-site analysis of heavy metal ions.In order to build an efficient,sensitive and stable sensing system and promote its application in the field of rapid detection,the design and construction of a lead-mercury ion DNA sensor has specifically carried out the following research work:（1）Detection of Pb²⁺based on hyperbranched hybridization chain reaction mediated by DNA tetrahedrons.Pb²⁺DNAzyme（GR5 DNAzyme）was selected,and tetrahedral DNA nanostructures（TDN）were assembled,and the main reaction conditions（TDN concentration,reaction time,temperature,p H and ionic strength,etc.）were optimized by designing the sequence to characterize the shape of the reaction product.Appearance,detection of FRET efficiency,calculation of reaction kinetic rate and sensitivity,and quantitative detection of Pb²⁺based on the fluorescence signal comparison of different Pb²⁺concentrations.（2）Design and construction of Pb²⁺sensor based on circular DNAzyme.The traditional double-stranded DNAzyme is designed as a single-stranded circular DNAzyme to improve the stability of the sensor.The circular DNAzyme and Td T synergistic amplification reaction are combined to improve the sensitivity of the sensor,and the colorimetric and fluorescent Pb²⁺sensors are designed and constructed,which expands the applicable methods and explores the optimal reaction conditions of the sensor.The sensitivity and stability of the Pb²⁺sensor were studied by studying experimental conditions such as thermodynamic stability,biological stability,and resistance to enzymatic degradation.Pb²⁺was quantitatively detected based on the absorbance or fluorescence signal ratio of different Pb²⁺concentrations.（3）TDN and T-Hg²⁺-T structure mediated hyperbranched HCR to detect Hg²⁺.Replace Pb²⁺DNAzyme with a T-T base that can specifically bind to Hg²⁺.The feasibility of the reaction was verified by designing the reaction sequence,optimizing the reaction conditions,and characterizing the particle size and morphology of the reaction product.Research on the ability of anti-ion interference etc.to study the stability of this Hg²⁺sensor.Based on the fluorescence signal ratio of different Hg²⁺concentrations,a standard curve was established to quantitatively detect Hg²⁺.The main findings are as follows:（1）A Pb²⁺sensor based on Pb²⁺DNAzyme and TDN-mediated hyperbranched hybridization chain reaction was constructed.Under the optimal reaction conditions,the detection limit for Pb²⁺is as low as0.25 p M(5.18×10^﹣8 mg/L),which is lower than most research methods currently reported.The detection process can be completed within 20 minutes,and the reaction rate is greatly improved compared with other detection methods.At the same time,the detection was carried out in actual samples of food and environment,and the detection results were basically consistent with ICP-MS,which showed that this research method can be applied to the detection of actual samples.（2）Constructed a Pb²⁺sensor based on circular DNAzyme.It can maintain stable activity in a wider temperature（4-37°C）,salt concentration（0-500 m M Na⁺）and p H range（5-9）.The detection limits of colorimetric and fluorescent Pb²⁺sensors for Pb²⁺are 0.085 n M(1.76×10^﹣4 mg/L)and 0.0015n M(3.11×10^﹣6 mg/L),respectively.It is also applied to the detection of Pb²⁺in food and environmental samples,and the detection results are consistent with ICP-MS.（3）A Hg²⁺sensor based on T-Hg²⁺-T functional nucleic acid was constructed.Under the optimized reaction conditions,the detection limit of the sensor is 1.29 n M(0.26×10^﹣3 mg/L),which is lower than the Hg²⁺standard in drinking water stipulated by my country.The whole test can be completed within30 minutes.This sensing method is used for the quantitative detection of Hg²⁺in environmental water bodies,and the detection results are basically consistent with ICP-MS,indicating that the sensing method is feasible within a certain range of conditions.