The Detection Of Mercury Based On The Isothermal Nucleic Acid Amplification Of Biological Sensing

Mercury(Hg²⁺)is a highly toxic properties of heavy metal ions,which can entered the body through food chain enrichment of the kidney,nervous system and other irreparable damage.They can be released from various natural and articially synthesized products,such as oceanic and volcanic emissions,fossil fuel combustion,gold mining,battery manufacturing,and electronic industries.Moreover,it is important to control the leakage of Hg²⁺ ions from amalgamllings in teeth during dental care.Thus,the sensitive and selective detection of Hg?II?is indispensible.Based on the specificity of exonuclease ?,the fluorescence quenching effect of graphene oxide and T-Hg²⁺-T specificity recognition,we carried out the following work to test the mercury ions?1?A label-free,exonuclease ??Exo ??assisted and cost-effective Hg²⁺ fluorometric assay based on a strand displacement amplification strategy was developed with high sensitivity and selectivity.Thymine–thymine?T–T?mismatches between the toehold domains in hairpin DNA and assistant DNA can serve as specific recognition elements for Hg²⁺ binding with the help of T–Hg²⁺–T base pairs to initiate a toeholdmediated strand displacement reaction.Exo ? provides a reduced background signal,and the method is label-free using an intercalator,Sybr green I?SG?.The number of T–T mismatches,the concentration of SG and Exo ?,the incubation temperature and time were all optimized.Under the optimized condition,the limit of detection for Hg²⁺ can be as low as 9.42 pM with a linear range from 0.01 to 1 nM.Moreover,this proposed strategy shows excellent selectivity,and was successfully applied for the detection of Hg²⁺ in laboratory tap water and Yanjing lake water samples in our university.?2?We constructed a fluorometric and sensitive analysis of mercury?II?based on toehold-mediated strand displacement reaction,exonuclease ??Exo ??assisted target recycling,hybridization chain reaction?HCR?,fluorescence intercalator SYBR Green I?SG?and grapheme oxide?GO?.Firstly,toehold-mediated strand displacement reaction was constructed by using thymine-Hg²⁺-thymine(T-Hg²⁺-T)recognition mechanism between the helper hairpin DNA and assisted DNA.And then,the Exo ? disintegrated the double-stranded DNA?dsDNA?by catalyzing the gradual removal of mononucleotides from 3-hydroxyl terminus of dsDNA with a blunt 3? terminus.It resulted in the target and assisted DNA recycling and released atrigger strand DNA at the same time.Finally,the trigger strand DNA initiated the hybridization chain reaction?HCR?,forming long double helices,which achieved a secondary amplification due to the continuous target recycling,trigger strand DNA release and HCR.The number of T-T mismatches,the concentration of SG,Exo ? and GO,the incubation time of Exo ? were all optimized.Under the optimized condition,the limit of detection for Hg²⁺ can be down to 7.37 pM with a linear range from 0 to 1.5 nM.This sensor exhibits high selectivity against other metal ions,and it works well for real samples.?3?Y junction DNA is an alternative secondary structure DNA with three duplex-DNAs which has one fulcrum and two branches.A label-free DNA Y junction sensor for amplified detection of Hg²⁺ has been demonstrated by using thymine-Hg²⁺-thymine(T-Hg²⁺-T)recognition mechanism.In our work,assistant DNA with toehold domains could bond with hairpin DNA?HP1?by the help of thymine-Hg²⁺-thymine(T–Hg²⁺–T base pairs).The exposed part of hairpin DNA?HP2?will then hybridize with the complementary sequences in HP1,again initiating a new toehold domains to open another hairpin DNA?HP3?,triggering a strand displacement reaction.Exonuclease ??Exo ??was added to digest the excess hairpin DNA to reduce the background signal.At the same time,Exo ? is replaced by GO to reduce the background signal because sing-strand DNA could be adsorbed onto the surface of GO.When the strand displacement reaction was completed,GO as an efficient fluorescence quencher for reducing the background signal was added to adsorb the sing-strand DNA.The adding order of GO was tested to compare which is sufficiently sensitive for Hg²⁺ detection.