Design And Application Of Fluorescence Sensing Probe Based On DNA Base Mismatch

Isothermal nucleic acid amplification is a kind of nucleic acid amplification technology,and is a nucleic acid amplification method for amplifying single-stranded primers in vitro at a fixed temperature.Isothermal nucleic acid amplification completely eliminates the requirements of thermal cycling devices,thus significantly simplify live real-time usage.Isothermal nucleic acid amplification technology is more economical,easier to use than polymerase chain reaction(PCR).Terminal deoxynucleotidyl transferase(TdT)is a template-free DNA polymerase that can add deoxynucleotide triphosphate(dNTP)to the 3’-OH end of single-stranded DNA.TdT is often used in isothermal nucleic acid amplification to add nucleotides to the 3’-OH end of a single primer.TdT isothermal nucleic acid amplification is more convenient than PCR which requires high purity primers,complex thermal cycling denaturation and annealing.With the advancement of science and technology,isothermal nucleic acid amplification has been widely used in various research fields,including analysis of disease markers,small organic molecules,amino acids,peptides,proteins,nucleic acid,metal ions,etc.It shows great application prospects in analytical science.In this paper,we carried out the following works:1.Taking the advantage of TdT isothermal nucleic acid amplification,a label-free fluorescence analysis method for Hg2+ detection has been developed based on the specific binding of thymine to Hg2+forms a T-Hg2+-T mismatched double helix structure.TdT recognized the 3’-OH end of primers and prolonged subsequently the single strand poly-T at 3’-OH with the assistance of deoxythymidine triphosphate(dTTP).The generated products coordinated with copper ions to form DNA-templated CuNCs resulting remarkable fluorescence.The introduction of Hg2+ make the primers form T-Hg2+-T mismatch duplex,which prevent the capture of 3’-OH end by TdT.The poly-T single strands prolong reaction was suspended,leading to fluorescence quenching.Hg2+could be detected as low as 76 pM with a great linear range from 200.0 pM to 500.0 nM.This method performed in one tube,one step was not required washing and separation,which provides a new sight for ultra-sensitive detection of toxic analyte in water quality.2.Using a competitive reaction of thymidine deoxyribonucleotides(poly-T)and L-cysteine to mercury ions,a lable-free fluorescence probe was constructed for L-cysteine.The single-stranded DNA of polythymidine can form a T-Hg2+-T mismatched double helix with mercury ions,while Sybr Green I(SG)can be inserted into the T-Hg2+-T mismatched double helix structure,which showed strong fluorescence under correct the excitation.When L-cysteine is added,L-cysteine will capture mercury ions in the T-Hg2+-T mismatched double helix structure,causing SG to fall off from the T-Hg2+-T mismatched double helix structure,and it is extinguished with fluorescence.The detection limit for L-cysteine is 7.4 nM and the linear range is 10 nM to 500 nM.The method is simple in operation and good in sensitivity,and has certain feasibility for detecting L-cysteine in actual samples.3.Melamine(Me)can form a triple hydrogen bond(T-Me-T)with thymidine deoxyribonucleotides in DNA.A fluorescence enhanced method for melamine detection was constructed based on T-Me-T.L-cysteine can capture mercury ions in the T-Hg2+-T mismatched double helix structure,resulting in the disintegration of the T-Hg2+-T mismatched double helix to form free poly-T single-stranded DNA.When melamine is added,melamine forms a triple hydrogen bond(T-Me-T)with the free poly-T single-stranded DNA.As SG is added,the SG can be embedded in the T-Me-T structure to generate a strong fluorescent signal.In this paper,L-cysteine is used to link the T-Hg2+-T mismatched double helix structure with the T-Me-T triple hydrogen bond to achieve specific detection of melamine.In this method,melamine could be detected as low as 14 nM with a great linear range 20 nM to 500 nM,which is lower than the national standard.The method is lable free and simple,which provide a reference for the detection of melamine in food.