Establishment And Research Of Lead Ion Detection Method Based On DNAzyme

Lead ion is a common contaminant of heavy metal ions,which can accumulate in human and animal bodies and is difficult to be discharged from the body.Lead ions can cause damage to the nervous system,cardiovascular system and reproductive system when they accumulate in human body to a certain extent.In addition,lead ion have a greater impact on children,leading to severe developmental disabilities.Therefore,it is of great significance to establish a biosensor with easy operation,high sensitivity and strong specificity.This paper focuses on the detection of lead ion,as follows:(1)Detection of lead ion by DNAzyme-based lateral chromatographyIn this method,a visual detection method for lead ion was developed based on the cutting activity of DNAzyme in the presence of lead ion combined with a lateral tomographic test paper.Sheep anti-mouse secondary antibody and SAV were fixed on the C line of the lateral chromatographic paper,and the conjugate of latex microspheres and antibodies was fixed on the T line.The DNAzyme solution with lead-free ion reaction was dropped onto the sample mat,and the presence of lead ion in the solution was determined by the color display of T and C line on the lateral chromatography paper.Meanwhile,the gray scale of Image J scanning could be used to conduct a quantitative analysis of the content of lead ion.On the basis of confirming the feasibility of the principle,the sealant,the addition amount of enzyme chain and substrate chain during the formation of DNAzyme,and the addition amount of reaction liquid in the sample loading system were optimized.The best experimental scheme was obtained,and the specificity and sensitivity of the lateral chromatographic test paper to lead ion were tested under the best scheme.Finally,the method used in this chapter can complete the detection of lead ion content within 5 min.The sensitivity of the paper-based analysis device by visual observation is 2 ppb,and the linear range of the standard curve is 0-8 ppb(2)Establishment of lead ion detection method based on HRP catalyzed color methodThis method combines DNAzyme,which can be specifically cleavage by lead ion,with HRP-catalyzed TMB.A conjugate of digoxin antibody and magnetic nanoparticles,digoxin and biotin modified DNAzyme was prepared.DNAzyme was attached to magnetic nanoparticles by the specific binding of digoxin to digoxin antibody,and HRP was attached to DNAzyme by the specific binding of biotin to SAV.After lead ion were cut,the presence of lead ion in the solution was determined by observing the color of the solution with the supernatant catalyzed by TMB colorimetry.In addition,the TMB absorbance of the above solution was determined by ultraviolet spectrophotometer,and the lead ion were quantitatively analyzed by the absorbance value.On the basis of the feasibility study,the addition amount of antibodies,the addition amount of DIG Antibody-MNPs,the addition amount of SAV-HRP and reaction time were optimized.The best experimental scheme was obtained,and the specificity and sensitivity of the experiment were tested.Under the optimal experimental conditions,the naked eye observable sensitivity is 0.1 ppb,and the detection range of the standard curve is 0.1-10 ppb.(3)Establishment of lead ion detection methods using electrochemical sensorsThis method is that will lead ion DNAzyme specificity of cutting and the combination of electrochemical,using the probe on the working electrode,the reaction of the DNAzyme solution drops to the working electrode and probes complementary pairing,using SAV with gold nanoparticles surface coupling,then mark of ferrocene amplification products add to the working electrode.The existence of lead ion in the solution can be judged by observing the electrochemical signals in the electrochemical workstation,and the quantitative analysis of lead ion can also be carried out according to the strength of the electrochemical signals.On the basis of the feasibility study,the concentration of LP1,the sealing time of MCH,the addition amount of SAV,and the reaction time of LP2 and HP amplification products were optimized.Under the best conditions,the detection range of the standard curve is 0.01-10 ppb.