| At present,China’s water resources are being attacked by pollution,water pollution phenomenon is very serious.A class of persistent organic pollutants(POPs)with high toxicity,strong accumulation,difficult to degrade,long migration distance,and can reduce dissolved oxygen in water appeared in polluted water,which seriously harmed human health and the stability of water ecosystem.Therefore,it is particularly important to develop a real-time,rapid,stable,sensitive and simple method for the detection of POPs.At present,the detection methods used in POPs mainly include gas chromatography-mass spectrometry(GC-MS),high performance liquid chromatography-mass spectrometry(HPLC-MS),etc.,but their limitations in time and space hinder their application in field detection.Therefore,a kind of aptamer biosensor detection method with good selectivity and high sensitivity has been developed.Among them,the developed 3,3′,4,4′-tetrachlorobiphenyl(PCB 77)and bis(2-ethyl)hexyl phthalate(DEHP)biosensor detection methods have shortcomings in stability,repeatability and detection limit.In this study,two new biosensor detection methods were constructed for rapid and sensitive detection of the aptamer by taking advantage of the specific recognition advantage of the aptamer and combining with different fluorescence signal amplification methods.The main research work of this subject:(1)An aptamer-coupled catalytic hairpin self-assembly fluorescence detection method(Apt-CHA)was developed for PCB 77 in water environment.The aptamer was used to specifically recognize PCB 77,and the primer chain was replaced at the same time to trigger the CHA reaction and convert it into fluorescence signal output.In order to confirm the accuracy and credibility of the method,this paper evaluated the method through feasibility,condition optimization,quantitative analysis,specificity,reproducibility,repeatability,stability,and actual sample detection.The optimal reaction time(3 h)and temperature(37℃)were optimized.The detection range was0.01 ～ 500 μg/L,and the minimum detection limit was 0.01 μg/L.The fluorescence attenuation of different groups of detection methods was less than 5%,and the actual recovery of water samples was 70 ～ 104.49%.It has the advantages of high sensitivity,good selectivity,simple operation and wide detection range.(2)A method for the fluorescence detection of variable structure adaptor coupled spherical nucleic acids in water environment for DEHP was developed.Molecular probes with different functional domains of aptamer were designed for DEHP recognition and fluorescence signal amplification,and fluorescence signal output was achieved by functional spherical nucleic acid(SNA).In order to confirm the accuracy and reliability of the method,this method was evaluated from the aspects of feasibility,condition optimization,quantitative analysis,specificity and actual sample detection.In this method,the optimal addition amount of variable conformation aptamer(Vs-Apt)was 2.5 n M,the optimal concentration of Nt.Bb VCI was 0.2 U/ μL,the optimal binding time of Vs-Apt and DEHP was 60 mins,and the optimal binding temperature was 37℃.The detection range was 0 ～ 100 μg/L,and the minimum detection limit was 1.008μg/L.In the actual sample analysis,the recovery was 116.4% by standard addition method and 104.02% by HPLC.The biosensor detection method studied in this study has the characteristics of high accuracy,high sensitivity,simple operation and short time consumption,which greatly reduces the time of traditional detection methods and reduces the instrument demand,providing a new idea for the rapid detection of POPs,can be used as a rapid detection method for other pollutants detection. |
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