Construction Of Aptasensors For Mercury And Lead Ions Based On 2-aminopurine And Exonuclease-assisted Signal Amplification Strategy

In recent years,the pollution of heavy metals to the environment and food has become more serious.Heavy metals are difficult to degrade and easily accumulate in animals and humans through the food chain.Even at low concentrations,heavy metals may cause serious damage to human health.The traditional methods of detecting heavy metals mainly use large instruments,which have limitations and are not suitable for on-site rapid detection.At present,many new sensors have been developed to replace traditional instrument analysis.Fluorescent sensors are widely used because of the advantages of high sensitivity,high accuracy and relatively simple.Aptamer is short single-stranded nucleic acid that can selectively bind to targets and have the advantages of high sensitivity,high binding affinity and high stability.Many aptamer fluorescent sensors have been developed to detect heavy metal ions.In this paper,using heavy metal ion-specific aptamer,the fluorescent characteristics of2-aminopurine and exonuclease-assisted signal amplification technology,two fluorescent sensors were constructed for sensitive and rapid detection of mercury ions(Hg²⁺)and lead ions(Pb²⁺),respectively.The main research contents include:(1)By using base stacking interaction to quench the fluorescence of2-aminopurine,T-Hg²⁺-T mismatch structure and exonuclease III(Exo III)assisted target cyclic signal amplification technology,a fluorescent method was constructed to detect Hg²⁺.Extended T-rich base sequences at the 5'end and 3'end of the hairpin structure,and replaced an adenine in the complementary region of the hairpin structure near the 3'end with 2-aminopurine(2-AP).The extended T-rich base sequence can resist the hydrolysis of Exo III,2-AP was embedded in the double strand and the fluorescence of the system was low.When Hg²⁺was present in the system,Hg²⁺combined with the T-rich sequences to form a blunt-ended hairpin structure which can be hydrolyzed by Exo III.2-AP and Hg²⁺in the T-Hg²⁺-T structures were released to the solution,Hg²⁺continued to combine with T-rich sequences and participated in the reaction cyclically,and the fluorescence of the system increased significantly.Under the optimal reaction conditions,the Hg²⁺concentration in the range of 7.5-200 n M and the fluorescence recovery intensity(F₂-F₁)showed an excellent linear relationship.The detection limit of this fluorescent method was 0.38 n M,which indicates its sensitivity is good.This method has been successfully used for the quantitative detection of Hg²⁺in Songhua River water and grass carp samples,and the results were basically consistent with those obtained by the traditional detection method inductively coupled plasma-mass spectrometry(ICP-MS).The fluorescent method has good accuracy and practicality.(2)By using 2-AP as a fluorescent signal probe,Pb²⁺specific aptamer and exonuclease I(Exo I),a fluorescent sensor was constructed to detect Pb²⁺sensitively.Extended the 3'end sequence of the Pb²⁺aptamer to form a self-complementary blunt-end hairpin structure,and used 2-AP to replace one of the adenines in the extended sequence.Exo I can hardly hydrolyze hairpin nucleic acids,2-AP was embedded in the double strand,and the fluorescence of the system was low.When Pb²⁺was present in the system,Pb²⁺induced the nucleic acid to form a G-quadruplex structure with bases free at 3'end.At this time,2-AP was in a single strand,and the fluorescence of the system increased.Exo I can hydrolyze this structure,2-AP was free,and the fluorescence of the system was further enhanced.Under the optimal experimental conditions,there was a linear correlation between the Pb²⁺concentration in the range of 5-60 n M and the fluorescence recovery intensity(F₂-F₁).The detection limit of this method was 0.049 n M,and the sensitivity is high.This method has been successfully used in the quantitative detection of Pb²⁺in Songhua River water and grass carp samples,and the results were basically consistent with the results obtained by ICP-MS,indicating that this method can be used to detect Pb²⁺in actual samples.In summary,both of the two fluorescence sensors were constructed using 2-AP as the signal molecule,combining with exonuclease and aptamer to detect mercury and lead ions.These methods designed the nucleic acid sequences cleverly,with fast detection speed,simple operation,excellent specificity and anti-interference ability.Therefore,they are expected to be applied to on-site rapid detection.