AuNP And TRFNP Probes Based Immunoassays For Acetochlor

As a chloroacetamide herbicide,acetochlor(ATC)has a wide range of application fields and is applied in corn,soybean and other planting farmland.Some of the sprayed ATC pesticides are absorbed by the targeted parts of plants and play an effect,while others are dissolved,migrated to groundwater or infiltrated and adsorbed in soil,thus damaging the ecological environment and threatening human and animal health.ATC has acute toxicity and chronic cytotoxicity to humans,mammals,amphibians,aquatic organisms and soil microorganisms,showing carcinogenic,teratogenic and mutagenic pathological effects.At present,the conventional detection methods for ATC are high performance liquid(gas)chromatograph based complicated instrument methods.These methods need complex sample pretreatment,high cost and professional technicians.Therefore,it is of great significance to establish fast,sensitive and simple ATC analysis methods for different scenes for veterinary public health.In this study,based on the specific reaction between antigen and antibody and the excellent biocompatibility of nanomaterials,five immunological detection techniques for ATC were established,which are suitable for the circumstances with different detection equipment and needs.The experimental contents and conclusions are as below:1.Indirect competitive enzyme linked immunosorbent assay(ic ELISA)for ATCBased on commercial antigen,antibody and optimized analysis parameters,a conventional ic ELISA for ATC was established.The detection range was 0.27～11.2ng/m L and the limit of detection(LOD)was 0.13 ng/m L,which is lower than the residue limit set by China and the European Union.This method has cross reaction with ATC(100.0%),metolachlor(120.0%)and metolachlor(136.1%),and has no cross reaction with other structural analogues(alachlor,pretilachlor,butachlor,and metalaxine).In the recovery experiment,the average recovery of intra-assay was93.2%～115.6%,and that of inter-assay was 81.0%～112.4%.2.In situ fluorescence ELISA for ATCAscorbic acid(AA),produced by dephosphorylation of ascorbic acid phosphate catalyzed by alkaline phosphatase(ALP),can combine with o-phenylenediamine(OPD)to form blue fluorescent substance in alkaline buffer.Based on this principle,an in situ fluorescence ELISA for ATC was established.The detection range is 3.34～80.87 ng/m L and the LOD is 1.19 ng/m L.This method has cross reaction with ATC(100.0%),metolachlor(85.5%)and metolachlor(48.0%),and has no cross reaction with other structural analogues.In the recovery experiment,the average recovery of intra-assay was 81.0%～98.2%,and that of inter-assay was 85.6%～115.6%.The recovery rates correlated well with the commercial kit(R~2=0.9956),meeting the requirements of the American Association of Analytical Chemists(AOAC).3.Colorimetric and fluorescent dual-mode ELISA for ATCBased on biotin labeled antibody modified gold nanoparticle(Au NP)probe and gold nanostar(Au NS)substrate,a colorimetric and fluorescent dual-mode immunoassay for ATC was constructed.After the formation of immune complex,ALP labeled streptavidin was introduced.The AA,catalyzed by ALP,triggered the deposition of silver ions onto the Au NS surface and subsequently combined with OPD to produce colorimetric and fluorescent signals,respectively.In the colorimetric mode via smartphone,the LOD of ATC is 1.20 ng/m L and the linear range is 1.95～61.66 ng/m L;in fluorescence mode,the LOD of ATC is 0.44 ng/m L and the linear range is 0.63～84.59 ng/m L.In the recovery experiment,the average recovery of colorimetric mode is 91.4%～105.1%,and the average recovery of fluorescence mode is 92.4%～106.2%.The recovery rates of the method correlated well with the commercial kit(R~2=0.9877 for colorimetric mode,R~2=0.9941 for fluorescence mode).This method is not only suitable for convenient,rapid and sensitive on-site detection,but also can be applied to in the laboratory detection scene with high sensitivity requirements.4."Turn on"fluorescence immunoassay for ATCBased on the fluorescent dye labeled nonthiolation DNA modified Au NP(spherical nucleic acid probe)and double strand specific nuclease(DSN),the"turn on"fluorescent immunoassay for ATC was established.After the formation of immune complex,DSN was added to cut DNA to separate the fluorescent label from Au NP,and the fluorescence,quenched by fluorescence resonance energy transfer(FRET)effect,was restored.The LOD of ATC is 0.03 ng/m L,and the linear range is0.07～16.17 ng/m L.In the recovery experiment,the recovery rates of of intra-assay and inter-assay were 93.0%～106.6%and 92.2%～103.1%respectively.The method had good correlation with the commercial kit(R~2=0.9995).The sensitivity of method was greatly improved.5.Dual-mode immunochromatographic strip for ATCBased on HRP modified time-resolved fluorescent nanoparticle(TRFNP),a dual-mode immunochromatographic strip of ATC was established.The fluorescence of TRFNP and the chromogenic results of deposited TMB on the test line constitute fluorescent and colorimetric signals,respectively.In fluorescence mode,the LOD of ATC is 0.08 ng/m L and the linear range is 0.2～50 ng/m L;in colorimetric mode,the LOD of naked eye judgment of ATC is 0.39 ng/m L,and the linear range is 0.39～100ng/m L.In the recovery experiment,the average recovery of fluorescence mode was85.4%～109.3%and that of colorimetric mode was 89.4%～108.3%.The recovery rates correlated well with the commercial kit(R~2=0.9889 of fluorescence mode,R~2=0.9971 of colorimetric mode).This method can not only be applied in the laboratory analysis scenarios with high requirements for LOD,but also be used to detect pesticide residues in field samples by naked eye.In conclusion,five immunoassays were established,which could be used for rapid screening of ATC.The ic ELISA does not need complicated instruments and can be used in general laboratories;After HRP was simply replaced by ALP in fluorescent ELISA,the detection range of ic ELISA was widened to a great extent.The colorimetric and fluorescent dual-mode immunoassay can be used not only for on-site detection with the help of smartphone,but also for laboratory scenes with high sensitivity requirements.The LOD of"turn on"fluorescence immunoassay was the lowest within these five immnuassays.Fluorescence and colorimetric dual-mode immunochromatographic strip has the shortest detection time,which can determine ATC by naked eye and fluorescence quantitative analysis.