Construction And Application Of Biosensor For Heavy Metal Ions Detection

Heavy metal ions are difficult to degrade,seriously endangering environmental quality,food safety and human health,so the detection of heavy metal ions is of great significance.Traditional detection methods mainly rely on large-scale instrument analysis such as mass spectrometry and chromatography,which makes it difficult to achieve rapid detection.The detection method based on biosensor has the advantages of simple operation,low cost,fast response and so on,which has been developed into a class of detection methods with a wide range of application prospects.In this paper,specific nucleic acid sequences were used as molecular recognition elements to construct Hg²⁺,Cd²⁺,and As³⁺detection biosensors based on signal amplification.With high sensitivity and good selection,it can be used for rapid detection of real samples.（1）Based on the fact that Hg²⁺can combine with thymidine base T to form T-Hg²⁺-T complex,Hg²⁺biosensor was constructed by using T-rich nucleic acid sequence as molecular recognition element.In the presence of Hg²⁺,the T-rich sequence combined with the 3’end of stem loop of hairpin A to form a blunt 3’terminus,which initiated the cleavage of exonuclease III（Exo III）and released the G-quadruplex nucleic acid sequence.On the one hand,G-quadruplex can combine with N-Methyl mesoporphyrin IX（NMM）to transmit fluorescence signal;On the other hand,G-quadruplex bound to the 3’end of the stem loop of hairpin B to form a blunt 3’terminus to trigger the hydrolysis of Exo III.A large number of G-quadruplex were released.The biosensor exhibited excellent high sensitivity for Hg²⁺with a detection limit as low as 10 f M.（2）Based on the catalytic activity of G-quadruplex,using toehold-mediated DNA strand displacement reaction to amplify signal,a Cd²⁺biosensor was constructed.In the presence of cadmium,the combination of Cd²⁺with its aptamer brought domains a and b into close-enough proximity to bind to hairpin 1.Then branch migration was initiated to open hairpin 2 and hairpin 3.The G-quadruplex was resynthesized and the catalytic activity was activated,which can effectively catalyze the H₂O₂-mediated oxidation of TMB to generate a colored signal readout.The detection limit of this sensor is 0.5 p M and the recovery is 96.5%-105.3%.This sensor can be used for the determination of Cd²⁺in environmental samples.（3）Based on the combination of 2-amino-5,6,7-trimethyl-1,8-naphthidine（ATMND）and base C,the As³⁺biosensor was constructed by using hairpin probes rich in base C as signal amplification element.In the presence of As³⁺,the stem loop sequence of triplex DNA structure could specifically bind to As³⁺and release STP.The combination of STP and HP1 triggered the hydrolysis of Exo III,resulting the releasing of ATMND and STP analogues.The free ATMND emited fluorescent signal.The STP analogues bound to HP1 for a new round of hydrolysis to amply signal.The detection limit of the sensor is 5ng/L.This sensor can be used for the determination of As³⁺in environmental water samples.