Aptamer Screening And Its Sensor Construction And Application Study For Typical Pesticide Residues In Food And Environment

Pesticides,especially organophosphorus pesticides,carbamate pesticides and bipyridine pesticides,are effective ways to improve crop yield and quality for weed and pest control,but the long-term use could lead to their accumulation in soil,crops and wastewater runoff,thus creating significant negative effects of environmental pollution,cross-contamination of crops around the world,and an increase in human and animal diseases.Therefore,the development of rapid detection technology and devices are major strategic demands to ensure food and environmental safety.Aptamers are short single-stranded oligonucleotides(DNA or RNA)obtained by Systematic Evolution of Ligands by Exponential enrichment(SELEX)technique,which can specifically recognize small molecules.Compared with antibodies or enzymes,aptamers have the advantages of better stability,low molecular weight,easy modification and low cost,and are excellent candidates for developing aptamer-based sensors(aptasensors)for pesticide residues detection.Hence,in this paper,the aptamer probes that can specifically capture pesticides were successfully obtained by streptavidin magnetic beads-SELEX technique,which provide novel identification materials for the detection of pesticide residues.In addition,the highly sensitive,rapid and convenient aptasensor for detection of pesticide residues was constructed by combining fluorescence and colorimetric method,respectively.Those methods provide theoretical and experimental basis for rapid on-site detection of pesticide residues in agricultural product quality inspection stations,fruit and vegetable production bases,market and environment supervision administrations,and further development of detection kits for hazardous materials on food and environmental safety.The main contents of this paper are as follows:1.Screening of aptamers for pesticides based on streptavidin magnetic beads-SELEXA technique for screening pesticide aptamer was established,including the design of random libraries and primers,fixation and enrichment of libraries,PCR amplification,preparation and purification of single stranded DNA.In this study,the streptavidin magnetic beads-fixing library method has high efficiency,and the eluent can be quickly obtained by magnetic separation;the PCR bias problem in conventional PCR amplification is reduced by using emulsion PCR to amplify the library;the denatured PAGE electrophoresis is used to separate single-strand DNA and n-butanol is used to concentrate products,which requires low cost,and the single-strand separation effect is better than that by asymmetric PCR amplification.After 7 to 9 rounds of screening,a large capacity aptamer library with good enrichment of carbaryl,carbofuran,chlorpyrifos and paraquat was successfully obtained,respectively.Subsequently,clone sequencing or high-throughput sequencing analysis was conducted.The strongest binding ability to each target pesticide was verified,and the Apta3,CBF-5,Chl-1 and PQ-5 were obtained as the candidate aptamers.Those aptamers provide novel biometric material for the efficient detection of four pesticide residues in food and the environment.2.Construction of turn-on based fluorescence aptasensor and its application in the detection of carbaryl pesticidesIn this study,we constructed a turn-on fluorescence aptasensor to realize the rapid detection of pesticides with small molecules.Firstly,Apta3 S was obtained by clipping and optimization of Apta3,which was used as the recognition probe.Subsequently,the fluorescence signal groups were modified on the Apta3 S aptamer,and the short sequence was designed to modify with quenching groups.Besides,the fluorescence signal of the aptamer was quenched in advance.The fluorescence method has good stability and specificity.It only contains two probes,has the advantages of simple operation and low cost.Moreover,the pre-quenched fluorescence makes a low detection background value and has strong practicability.The developed aptasensor realized the quantitative detection of carbaryl pesticide with the limit of detection of 15.23 nmol/L,meeting the requirements of national Standard [Groundwater Quality Standard(Class III)].3.Construction of DNA-Walker based fluorescence aptasensor and its application in the detection of carbofuran pesticidesIn order to overcome the weakness of turn-on based fluorescence aptasensor in sensitivity,we constructed a DNA-Walker signal amplification technology based fluorescent aptasensor.The aptasensor consists of three parts: swing arm probe,signal probe and aptamer.The aptamer,as a recognition probe,can specifically recognize carbofuran pesticides,after the carbofuran pesticide combined with the aptamer,the swing arm probe immediately binds to the signal probe due to the high specificity of the Watson-Crick base pairing principle.Under the drive of DNA enzyme,the enzyme digestion was started to release the fluorescence signal,and the swinging arm probe was driven to move autonomously along the set oligonucleotide orbit,and then more fluorescence signal was released to achieve the effect of signal amplification.The designed DNA-Walker signal amplification technology drives the swing arm probe to walk through enzymes,avoiding the problem of falling off during the process of carrying multiple DNA to drive the swing arm probe.Besides,the operation is simple and efficient,using magnetic beads as the carrier to recover the solution by magnetic separation.The aptasensor was used for quantitative analysis of carbofuran pesticides,and the sensitivity was as low as72.64 pmol/L,meeting the requirements of national standard [Groundwater quality Standard(CLASS I)] and National Food Safety Standard].4.Construction of label-free Au NPs based colorimetric aptasensor and its application in the detection of chlorpyrifos pesticidesColorimetric analysis is another most commonly used detection method in optical analysis which has the unique advantages of simple production,independent of special equipment,good portability,low cost and strong visual detection ability.In order to simplify the operation steps and study a fast detection method,the label-free Au NPs based colorimetric aptasensor was constructed in this paper.Au NPs have unique optical absorption and scattering properties,whose aggregation can induce interparticle plasmon coupling,resulting in localized surface plasmon resonance shift and visible color change of the nanoparticle solution,which has great potential for colorimetric detection.In this study,aptamer is used as a recognition probe and wrapped on Au NPs surface through electrostatic action,which can act as a stabilizer to protect Au NPs from aggregation in salt solution.In the presence of chlorpyrifos targets,which were specifically captured by aptamers,and then the Au NPs were exposed to high-salt solution.The salt-induced Au NPs aggregated to varying degrees,leading to the solution changing from wine red to blue-purple.The Au NPs used in this study do not require additional modification.Besides,this method has the advantages of few steps,simple operation and low cost,and can be used for quantitative detection of chlorpyrifos pesticides with the detection limit of 14.46 nmol/L,meeting the requirements of national Standard [Groundwater Quality Standard(CLASS II)]and National Food Safety Standard].In addition,the sample tests showed that the colorimetric aptasensor has good stability and specificity in organic solvents or solutions containing matrix components.5.Construction of Au NPs based colorimetric lateral flow aptasensor and its application in the detection of paraquat pesticidesThe lateral flow device is one of the most widely used point-of-care testing(POCT)tools for real-time detection,with the advantages of simplicity,easy operation,fast,miniaturization and low cost.In this study,the Au NPs based colorimetric lateral flow analysis method was developed for rapid detection of pesticides.Firstly,the probes containing aptamer sequences were modified onto Au NPs,then the complementary probes of aptamer were sprayed on the test line.The paraquat pesticides competes with the complementary probes for aptamers.In the absence of paraquat pesticides,the aptamer modified Au NPs combined with the probe on the test line,and a strong signal appeared on the test line.In the presence of paraquat pesticides,the aptamer modified Au NPs combined with the paraquat pesticides,then a weak signal or no signal appeared on the test line.The more paraquat pesticides existed in the tested sample,the weaker the signal on the test line.The Au NPs based colorimetric lateral flow method has good reproducibility and low cost,and the prepared strip can be stored for half a year.The aptasensor can specifically detect paraquat pesticides,and the detection limit was 0.8 μmol/L.In addition,the sample tests showed that this aptasensor could be used as a practical tool for rapid detection of pesticides in agricultural product quality testing stations,fruit and vegetable production bases,and market and environment supervision administrations.