Construction Of Aptamer-based Pesticide Residue Detection Sensors And Their Research In Detection Performance

Agricultural productivity has been greatly enhanced via effective pesticide application,expanded acreage,excellent breeding techniques and good water management.It is estimated that the use of pesticides almost guarantees one-third of the world’s crop production.The use of pesticides has been recognized as a major factor in the high growth of wheat and maize production in the United Kingdom and the United States,as well as a factor in crop yield growth in large population countries such as China and India.However,the pollution caused by the uncontrolled use of pesticides has become one of the most worrying challenges for humanity to pursue sustainable development.Although pesticides are applied directly to plants or soil,only about 1% of the pesticides are sprayed to the intended target.Due to the long half-life of pesticides,pesticide leakage,storage and water washing may result in accidental release of pesticides and long-term presence in the environment.Foods with pesticide residues may enter the food industry or the catering industry via various sources,posing serious risks to human and animal health.In order to properly manage and use pesticides,people need to accurately assess their contamination in the environment,especially in food.Therefore,the need for accurate and sensitive detection of pesticides in food has become the key to achieving public health protection and safety.The modern pesticide detection methods basically adopt non-specific detection methods such as high performance liquid chromatography(HPLC)and gas chromatography(GC),which were difficult to meet the high sensitivity and selectivity detection of pesticides.In view of this,aptamers have been widely used in sensor research as an emerging specific sensitive element in recent years.An aptamer is a small segment of an oligonucleic acid strand that has been specifically screened for binding to a target.Compared with sensing elements such as antibodies,aptamers have the advantages of in vitro synthesis,thermal stability,reproducibility,easy storage and easy modification.Aptamer technology has been used in the detection of many types of substances,such as metal ions,drug molecules,proteins,viruses and cells.In this study,we used aptamers as specific response receptors for pesticides,and constructed a series of promising aptamer biosensor with strong specificity and high sensitivity in food safety testing by nano-material signal amplification technology,array sensing technology and electrochemical technology.From the synthesis and characterization of nanomaterials,the design of sensing mechanism and the enhancement of sensing signals,to the repetitive use of sensors,a series of pesticide aptamer biosensors were developed in this study,and their application were explored and studied.It opens up new ideas for the design of pesticide detection sensors in food.The specific work and research results of this paper are as follows:(1)Detection of different pesticides based on aptamer-nanogold constructed colorimetric arrayBased on aptamer protection,gold nanoparticle(AuNP)could produce different color reactions for different pesticides.Using three aptamers as sensitive components of colorimetric sensing arrays,fingerprint response patterns of eight pesticides were obtained.We performed TEM and EDS characterization and particle size calculations for the dispersion and aggregation of nanogold before and after the reaction.The UV-Vis absorption spectra of the aptamers and pesticides were collected and analyzed.The absorbance of the two ultraviolet characteristic peaks and their ratios were used as model variable parameters,and different pesticides were identified by means of multivariate analysis methods.When the Euclidean distance was 20-25,the hierarchical clustering analysis model(HCA)could distinguish 8 pesticides.Principal Component Analysis(PCA)results showed that only the first two principal components accounted for 88.8% of the total variance information,which could distinguish 8 pesticides well.Pearson model showed that the main factors affecting the binding between aptamers and small molecule analytes were the conformation of the aptamer and the number of small loops,while the secondary factor was the similarity or difference in analyte atoms or groups.Linear discriminant(LDA)results showed that the first two dimensions of the factor explained 92.2% of the total variance information,which could clearly distinguish 8 pesticides.The constructed array sensor had an identification rate of 93.8% for unknown samples.The method was simple,label-free and accurate,and required only a limited number of sensitive components to achieve high discrimination of pesticides.This work broadens the field of application of bioreceptors(aptamers)and plasmonic nanoparticle sensors,providing new directions for the development of sensitive array sensing systems.(2)Construction of multiple sensor elements array and identification of various pesticidesBased on six sensing elements,we constructed a visual array that combined specific response and cross-response.The six sensing elements contain specific sensing elements such as aptamers and acetylcholinesterase to increase the recognition rate of array detection.This array was successfully applied to the identification of 12 pesticides.Six kinds of sensing elements had obvious optical response to diffrent pesticide molecules by UV-Vis absorption spectrum analysis.The obtained RGB difference spectrum vectors were statistically analyzed by data set,and the accurate identification of 12 pesticides was obtained.After HCA analysis,all pesticides were clearly distinguished and correctly classified.In addition,LDA also obtained similar results as HCA.Based on the total cumulative variance of 70.9% of the first two dimensions of the discriminant factor,the array could accurately and clearly identify 12 pesticides,and could distinguish between organophosphorus pesticides and non-organophosphorus pesticides.By leaving one cross-validation analysis,the array had an accuracy of 100% for all pesticide solution samples.Meanwhile,the PCA results showed that the recognition ability of the array had a high dimension,and seven dimensional principal component factors were required to account for 95% of the total variance information.The analysis results showed that the array had a wide active recognition space,and there were different chemical or physical interaction types between the sensing element and the pesticide analyte.In addition,the constructed array sensor had good reproducibility and stability.The LDA model was used to analyze spiked pesticide samples in cabbage and cucumber samples,and the identification accuracy of 12 pesticides was greater than 92%.(3)Detection of malathion by aptamer sensor based on the formation of metal organic framework and ferroceneA novel electrochemical aptamer sensor platform for the detection of malathion was successfully constructed based on composite nanoprobe.The zirconium-based metal organic framework(MOF)was synthesized by hydrothermal method,and coupled with aptamer complementary probe(CP)and ferrocene(Fc)by covalent bonds to form a CP-MOF-Fc nanocomposite probe.The malathion aptamer was immobilized on the surface of a glassy carbon electrode deposited with gold nanoparticles by a gold-sulfur bond,and then hybridized with CP-MOF-Fc nanoprobe.Successful synthesis of MOF was confirmed by various characterization techniques(SEM,EDS,and XPS).Due to the high specific surface area of MOF,the electroactive substance concentration of CP-MOF-Fc was 1.90 times that of CP-Fc.Combined with the good electrical conductivity of AuNP,the prepared aptamer sensor could detect trace amounts of malathion in the linear range of 25~850 ng/L with LOD of 17.18 ng/L.The aptamer sensor also had high selectivity,repeatability and good stability.The ability of aptamer sensor to analyze and detect malathion in long bean and cucumber samples was studied by sample spike method.The detection results were satisfactory,which proved that the method had certain potential in actual detection.(4)Dual-signal aptamer sensor based on polydopamine-gold nanoparticles and exonuclease I for ultrasensitive malathion detectionA high-sensitivity dual-signal aptamer sensor for malathion detection was prepared using the good biocompatibility of PDA and the enzymatic signal amplification of Exo I.Compared to the method of no signal amplification,Exo I autocatalytic target cyclic amplification could achieve at least 2 times cascade amplification of the current difference of the electrochemically active molecule.Combined with the dual signal design,the prepared biosensor could produce three signal output modes for malathion.By optimizing the concentration of Fc-CP,the hybridization time of aptamer and Fc-CP,the concentration of Exo I and the culture time,the developed sensor had excellent sensitivity,high selectivity and stability.The dual signal design and Exo I’s enzymatic signal amplification allowed the sensor to have up to three linear detection ranges with a low detection limit(0.5 ng/L).In addition,the aptamer sensor was successfully applied to the detection of malathion in cauliflower and cabbage samples with good recovery.(5)A regenerative and selective electrochemical aptasensor based on copper oxide nanoflowers-single walled carbon nanotubes nanocomposite for chlorpyrifos detectionCuO NFs were synthesized by a hydrothermal method and mixed with carboxylated SWCNTs to prepare CuO NFs-SWCNTs hybrid nanocomposites.The synthesized CuO NFs had a three-dimensional flower-like structure and a large specific surface area,which made the CuO NFs-SWCNTs nanocomplex greatly increase the binding sites number of amino probes.Based on the synergistic effect of electrical conductivity of the two nanomaterials,the effective electrochemical active area of the CuO NFs-SWCNTs nanocomposite electrode was 2.06 times that of the bare electrode.Under the optimized conditions,the electrochemical aptamer sensor based on this nanocomposite showed a good linear relationship with chlorpyrifos in the range of 0.1-150 ng/mL,and obtained a detection limit of 70 pg/mL.The sensor was successfully applied to the detection of chlorpyrifos in apple and Chinese cabbage samples,and a satisfactory recovery rate(95.58%~106.72%)was obtained.In addition,the biosensor could be easily regenerated by urea and got repetitive applications,achieving good inter-day repeatability(RSD was 4.87%).The above results indicated that the sensing method proposed in this study provided a new idea of performance improvement and regeneration cycle application for sensor.