| Immunoassay has been widely in detection of pesticide residue thanks to simplicity,rapidity,low-cost and high throughput.Pesticides are small molecule compounds,so they are often detected using competitive format.Competitor is the key reagents in competitive immunoassay,and its quality directly effects the analytical performance of immunoassay.However,the preparation of chemical hapten as competitor is blindness,high-cost,long-cycle,so that hapten can not meet the actual requirements.Phage display peptide technology can be used to isolate peptidomimetic efficiently and economically,which can specifically react with antibody and be a substitute for chemical hapten as the competitor.Phage enzyme linked immunosorbent assay(P-ELISA)was used in most of the immunoassays based on phage display peptide,but the detection range of P-ELISA was usually narrow.Meanwhile,the phage-displayed peptide suffer the limitation of phage particle,such as large size,poor mobility and safety concerns,which greatly limits the application of peptidomimetic.In this research,imidaclothiz was used as the research object,we isolated the phage-displayed peptidomimetic as competitor to develop P-ELISA for imidaclothiz,which greatly improve the sensitivity of the immunoassay for imidaclohiz.Phage chemiluminescence enzyme immunoassay(P-CLEIA)was developed by introducing chemiluminescence system,which expanded the detection range of P-ELISA.The peptidomimetic was fused with NanoLuc and EmGFP respectively as the recombinant competitors to develop a series of novel immunoassays,which can overcome the limitations of phage partcles,further expand the application of peptidomimetic and improve analytical performance of immunoassay.The main research results are as follows:1.Isolating peptidomimetic of imidaclothiz from phage-displayed peptide librariesAnti-imidaclothiz monoclonal antibody(mAb 1E7)was used to screen three phage-displayed peptide libraries.After three rounds biopanning,six peptidomimetics of imidaclothiz were isolated,and the amino acid sequences are PRIWADS,TYLNSAK,LPPRMIYE,QPWCPPSICGD,TMHLPYCPTNIC and DYHDPSLPTLRK.The standard curves of ELISA for six peptidomimetics were established.The sensitivity(IC50)ranged from 2.97 to 5.74 ng mL-1,in which the sequence TMHLPYCPTNIC(L4-29)showed the optimal sensitivity and its IC50 was 2.97 ng mL-1.2.Phage enzyme immunoassay for the detection of imidaclothizL4-29(TMHLPYCPTNIC)was used to develop two phage enzyme immunoassays:P-ELISA and P-CLEIA for the detection of imidaclothiz in agricultural and environmental samples.By optimizing the reaction buffer,the optimal working buffer of the two methods were obtained:2.5%methanol,pH 8 and 0.14 mol L-1 NaCl.Under optimal case,the IC50,LOD and linear range(IC10-IC90)of P-ELISA were 1.45,0.55 and 0.55～3.82 ng mL-1,respectively,while 0.86,0.13 and 0.13～5.84 ng mL-1 for P-CLEIA.Compared with ELISA based on chemical hapten,the sensitivities of P-ELISA and P-CLEIA to imidaclothiz were increased by 60 times and 101 times,respectively,and were better than the immunoassays have been reported for imidaclothiz.The sensitivity of P-CLEIA was slightly better than P-ELISA,and the linear range was 6.5 times higher than P-ELISA.There was no significant cross-reactivity(CR)of two assays to imidaclothiz-related compounds except imidacloprid.In addition,the average recoveries of imidaclothiz in paddy water,soil,cabbage,rice,apple,pakchoi,pear and tomato samples were 72.3-101.3%by P-ELISA and 73.9-102.6%by P-CLEIA.The P-ELISA and P-CLEIA also showed good correlation with high-performance liquid chromatography(HPLC)for detection of imidaclothiz in authentic samples.The result indicates that phage display peptides could be used as competitor for immunoassay of imidaclothiz,and P-CLEIA is a more sensitive assay with wider detection range.3.Preparation of recombinant peptidomimetic(NanoLuc)and development bioluminescent immunoassay for imidaclothizTwo recombinant peptidomimetic C2-15-NanoLuc(the peptide at the N terminus)and NanoLuc-C2-15(the peptide at the C terminus)were prepared by fusing C2-15(LPPRMIYE)with NanoLuc.Nanoluc-C2-15 showed the higher affinity to mAb 1E7 and was used to develop two bioluminescent immunoassays for imidaclothiz:bioluminescent enzyme immunoassay(BLEIA)and bioluminescent lateral-flow immunoassay(BLLFIA).The signal of BLLFIA can be detected by smartphone without external excitation light thanks to the high luminous efficiency of NanoLuc.The IC50.LOD and linear range of BLEIA were 3.3,0.2 and 0.2～57.3 ng mL-1,and 6.4,0.3 and 0.3～81.7 ng mL-1 for BLLFIA.The sensitivities of the two bioluminescent immunoassays was similar to C2-15-based P-ELISA(IC50=4.02 ng mL-1).BLEIA and BLLFIA showed 93.6%and 95.1%of CR for imidacloprid,there was no significant CR of two assays to other imidaclothiz-related compounds.Meanwhile,the average recoveries of imidaclothiz by BLEIA and BLLFIA in paddy water.soil,brown rice and orange samples ranged from 79.0 to 104.1%and 78.0 to 110.2%,respectively.The BLEIA and BLLFIA also showed good correlation with HPLC for detection of imidaclothiz in authentic samples.The results showed that the recombinant peptidomimetic(NanoLuc)retained the specific binding between phage display peptide and antibody,and can provide detection signal,which overcame the limitations of phage particle,such as large size,poor mobility and potential biosafety hazard,and avoided the batch difference caused by conjugation.4.Preparation of recombinant peptidomimetic(EmGFP)and development Magnetic-separation fluorescent immunoassay(MAFIA)for imidaclothizTwo fluorescent recombinant peptidomimetic,C2-15-EmGFP(peptide at the N terminal)and EmGFP-C2-15(peptide at the C terminal),were prepared by fusing C2-15 with EmGFP.Although Tthe affinity of C2-15-EmGFP and EmGFP-C2-15 to mAb 1E7 was similar,C2-15-EmGFP was used to develop MSFIA considering the higher yield.The IC50,LOD,and linear ranges of MSFIA were 11.0,1.87,and 1.87～66.0 ng mL-1,respectively.MSFIA shows simple operation,short analysis time,and is suitable for high-throughput detection.The method showed no significant CR with imidaclothiz analogues except imidacloprid.The average recoveries for imidaclothiz in paddy water,soil,brown rice and cucumber samples by MSFIA ranged from 75.3 to 114%.The detection results of imiclothiz residue in real cucumber samples by MSFIA showed good correlation with HPLC.5.A dual signal immunochromatographic strip for imidaclothiz using C2-15-EmGFP and gold nanoparticlesA fluorescence quenching-based immunochromatographic assay(FQICS)was developed using C2-15-EmGFP(containing His tag)as the fluorescent donor and mAb 1 E7 labelled gold nanoparticles as the quencher.Anti-His tag antibody was used as T line to capture C2-15-EmGFP,and anti-mouse IgG antibody was used as C line to capture mAb 1E7.The binding of C2-15-EmGFP and mAb 1E7 can quench fluorescence,and the colorimetric signal appears in T line cause by accumulation of gold nanoparticles accumulation.The presence of imidaclothiz can reduce mAb 1E7 binded with C2-15-EmGFP,which cause the decrease of colorimetric signal and recovery of fluorescent signal.Two signals(colorimetric and fluorescent signals)of FQICS were analyze by visual and ImageJ software respectively.The intensity of fluorescent signal is proportional to the concentration of imidaclothiz.and the colorimetric signal is inversely proportional.The quantitative results of ImageJ showed that the sensitivity of colorimetric signal(IC50=9.62 ng mL-1)is similar to fluorescent signal(SC50=10.1 ng mL-1).The visual results by naked eyes showed that the sensitivity of the fluorescent signal(8.00 ng mL’1)was 8-fold higher than colorimetric signal(64.0 ng mL-1).In addition,the average recoveries of imidaclothiz in soil and wheat samples by FQICS ranged from 80.1 to 97.5%,respectively.The detection results of imiclothiz residue in soil and wheat samples by FQICS showed good correlation with HPLC.These result show the "turn-on" mode of ICS can improves the sensitivity of visual detection and makes the detection results more intuitive. |
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