Electrochemical Aptamer Sensors Based On Signal Amplification Strategy For Detection Of Heavy Metals

Heavy metals are widely distributed,and various ecosystems are affected to varying degrees.Heavy metals have seriously polluted water,soil,plants and animals.Due to their high bioenrichment,high toxicity,single binding site,and non-degradability,heavy metals have attracted wide attention.With the development of industry and social economy,heavy metal pollution of water has become increasingly serious,which further leads to the aggravation of heavy metal pollution in aquatic products,and the accumulation of heavy metals into the human body through the food chain,causing human diseases.In addition to heavy metals,aquatic products are seriously contaminated by pathogenic bacteria,which affects food safety and human health.Conventional detection methods are expensive,difficult to operate,time-consuming,and cumbersome to pre-process,which can no longer meet the needs of trace rapid detection.The electrochemical detection method has a good application prospect in rapid detection due to its high sensitivity and simple pretreatment.Aptamers,as recognition elements,have strong specificity and specificity for target substances.In this thesis,aptamers and electrochemical detection methods were combined to construct a highly sensitive electrochemical aptamer sensor for heavy metal/pathogenic bacteria.The contents of heavy metals and pathogenic bacteria are low but they are seriously harmful,so it is essential to propose effective signal amplification strategies for highly sensitive detection of heavy metals and pathogenic bacteria.In order to solve the above problems,enzyme-assisted and novel nanomaterials signal amplification strategies were used to improve the sensitivity of the sensor to meet the needs of trace detection of heavy metals and pathogenic bacteria.At the same time,the dual-mode visual electrochemical aptamer sensor and the ratiometric electrochemical aptamer sensor were used to realize the visual and accurate detection of heavy metals and pathogenic bacteria in aquatic products,and the feasibility of the detection in actual samples was verified.The main research contents and results of this paper are as follows:（1）Aptamers can specifically recognize specific target substances,Exonuclease I（Exo I）can cleave the single-stranded aptamer,and the combination of aptamer and Exo I can achieve dual-target signal amplification detection.Therefore,based on silver nanoparticles（Ag NPs）and an Exo I-assisted cyclic signal amplification strategy,a novel high-sensitivity dual-target electrochemical biosensor was designed for the detection of Pb（II）or Hg（II）in water.Immobilization of the aptamer chain by thymidine-Hg（II）-thymidine（T-Hg（II）-T）in the presence of Hg（II）resulted in a diminished electrochemical signal.In the presence of Pb（II）,the aptamer dissociates and binds to Pb（II）,automatically triggering Exo I to selectively cleave the single strand of the recognition site,achieving cyclic amplification of the electrochemical signal.Gel electrophoresis analysis was used to verify the interaction between the aptamer and Exo I.Under the optimal conditions,the biosensor showed high sensitivity in the linear range of100 pg/L to 10.0 mg/L,with the lowest limit of detection（LOD）of 17.0 pg/L for Pb（II）and 12.0 pg/L for Hg（II）.The recovery rate of the sensor was 98.3%-107%after standard addition to the actual water sample,which realized the detection of Pb（II）or Hg（II）in water.（2）Due to water pollution,heavy metal pollution in aquatic products is seriously harmful.On the basis of the previous chapter,this part establishes a rapid detection method for heavy metals in aquatic products.As a unique nanomachine,DNA walker can continuously move along a specific track to amplify the signal and realize the detection of heavy metals in complex samples.Therefore,based on the DNA walker and endonuclease assisted signal amplification strategy,a novel dual-mode visual electrochemical aptamer sensor was constructed for the detection of Pb（II）in aquatic products.Cerium oxide@mesoporous carbon（Ce O₂/CS）@Au NPs can not only improve the conductivity of the sensing interface,but also immobilize the aptamer.The DNA walker moves on the surface of the electrode to realize the pairing with the Ag-γFe₂O₃/c DNA probe to form a special base sequence that can be cleaved by the endonuclease Nb.Bbv CI.Under the action of endonuclease Nb.Bbv CI,Ag-γFe₂O₃/c DNA probes were cleaved continuously,and the number of probes on the electrode surface was reduced to achieve signal amplification.In addition,Ag-γFe₂O₃ as a nanoenzyme can catalyze 3’3’5’5’-tetramethylbenzidine（TMB）into blue to realize the visual detection of Pb（II）.The biosensor designed in this paper has a response range of 5.00×10^-2-1.00×10⁶ng/L and a LOD of 7.50×10^-3 ng/L for Pb（II）.Therefore,visual detection is of great significance for the realization of rapid on-site detection in the future,and the detection method of this sensor opens up a new way for the visualization and accurate detection of food contaminants.（3）In addition to heavy metal contamination,food-borne pathogens also exist in aquatic products.Based on the study of heavy metals in aquatic products in the previous chapter,a ratiometric electrochemical aptamer sensor based on a novel nanomal-based signal amplification technology was constructed for the simultaneous and rapid detection of heavy metal Cd（II）and Staphylococcus aureus（S.aureus）.Firstly,zirconia（Zr O₂）nanomaterials were used to improve the conductivity of the sensor.Then,the composite material of nickel-cobalt metal-organic framework combined with gold nanoparticles（Ni/Co-MOFs@Au NPs）is further superimposed to increase the specific surface area of the sensor,thereby increasing the fixation amount of hairpin complementary chains and improving the sensitivity of the sensor.Three signal molecules,anthraquinone-2-carboxylic acid（AQ）,ferrocene（Fc）and methylene blue（MB）,were cited to achieve quantitative detection of Cd（II）and S.aureus.The Fc modified hairpin complementary strand was fixed on the surface of the electrode,the Cd（II）aptamer was modified with MB,and the S.aureus aptamer was modified with AQ.The qualitative and quantitative detection of the target substances was achieved according to the ratio value between AQ,MB and Fc special signal peaks.The sensor realizes the simultaneous and rapid detection of Cd（II）and S.aureus through the signal amplification technology of a new nano-material.The sensor designed in this paper has been successfully used to detect Cd（II）and S.aureus in aquatic products,providing a new strategy for the detection of a variety of small molecular pollutants in aquatic products.