The Development Of Parallel Structure Peptide Reagents And Its Application In Benzothiostrobin Immunoassays

Pesticide residue is an important factor for the safety of agricultural products.Immunoassay is being actively promoted because of its simplicity,rapidity,high throughput and low cost for the detection of pesticide residue.However,the sensitivity and specificity for the present immunoassays can’t be satisfied the actual demand for a part of pesticides.Phage-displayed peptides are powerful reagents to improve characteristics of immunoassays by using two formats(1)peptidomimetics,which are used as substitute for hapten or analyte to establish competitive immunoassays,(2)anti-immunocomplex peptides,which can specifically bind with the analyte-antibody immunocomplex to establish noncompetitive immunoassays.In this study,benzothiostrobin was selected as the analyte,the parallel structures of monomer,dimer and tetramer phage-free peptidomimetic and anti-immunocomplex peptides were designed and synthesized.The impacts of affinity,molecular mass for the parallel structure peptides on immunoassay signals and sensitivities were revealed.The internal filtration effect immunoassay(IFE-IA),fluorescence polarization immunoassay(FPIA)and a semi-quantitative multicolor immunochromatographic strip(ICS)was developed using tetramer parallel structure peptides.The related studies provide references for the development and application of high-affinity peptides.The main results are as follows:1.Development of monomer peptides and establishment of competitive and noncompetitive magnetic separation fluorescence immunoassays(FIAs)The monomer peptidomimetic and anti-immunocomplex peptides were synthesized based on phage display peptides.The N-terminal labeled fluorescein isothiocyanate(FITC)peptides were prepared as tracers,because they contained glutamic and aspartic.The competitive and noncompetitive magnetic separation FIAs were developed by using magnetic nanoparticles labeled monoclonal antibody(m Ab-MNPs)and peptide tracers.After optimization,the elution buffer and time were selected as 2% SDS and 10 min;the m Ab-MNPs,tracers,p H,Na+,and methanol content were 200 μL,20 μg m L-1,7.4,0.1 M,2.5% for competitive FIA,while 200 μL,40 μg m L-1,7.4,0.3 M,2.5% for noncompetitive FIA.Under the above conditions,the sensitivity(50% inhibition concentration,IC50)was 16.8 ng m L-1,and the linear detection range was 1.0~759.9 ng m L-1 for competitive FIA,while the 50% signal saturation concentration(SC50)was 93.4 ng m L-1,and the linear detection range was 5.9~788.2 ng m L-1 for noncompetitive FIA.The sensitivities of FIAs were lower than that of ELISAs,however,the FIAs reduced number of steps(from 6 steps down to 2 steps)and time(from more than 5 h to 1.2 h)to complete the analysis,which is simpler and faster.The FIAs didn’t show cross-reactions(CRs)with the structural analogues of benzothiostrobin.In addition,the average recoveries of FIAs in water,soil,cucumber,rice and maize samples were 68.3~98.5% with relative standard deviations(RSD)of 2.1~14.1%.The FIAs detection results for cucumber samples spiked unknown concentrations of benzothiostrobin were consistent with HPLC.This study revealed that the synthetic peptides could be applied in immunoassays.2.Development of parallel structure peptidomimetic and establishment of IFE-IAThe parallel structures of monomer,dimer,and tetramer peptidomimetic were designed and synthesized by using lysine as scaffold.To ensure the activity of tracers and toward outside in a similar way,another lysine was added to the C-terminus to provide active amino groups.The affinity between peptidomimetics and m Ab were measured by isothermal titration calorimetry and the results showed that the tetramer peptidomimetics was 14-fold and 7-fold higher than that of monomer and dimer.The IFE-IA was developed by using upconversion nanoparticles labeled peptidomimetic and gold nanoflowers labeled m Ab as donor and acceptor,respectively.The sensitivity of IFE-IA based on tetramer peptidomimetic was 6-fold and 3-fold higher than monomer and dimer,which because the higher affinity of tetramer peptidomimetic decrease the free antibody and background signal.After optimization,the SC50 and linear detection range of IFE-IA based on tetramer peptidomimetic were 11.8 ng m L-1 and 2.0~106.2 ng m L-1.The IFE-IA didn’t show CRs with the structural analogues of benzothiostrobin.In addition,the average recoveries of IFE-IA for benzothiostrobin in water,soil,cucumber,rice and corn samples were 74.2~105.4% with RSD of 5.2~11.9%.The IFE-IA detection results for cucumber samples spiked unknown concentrations of benzothiostrobin were consistent with HPLC.This study clarified that the multi-copy numbers of parallel structure peptides can enhance the affinity,and increase the sensitivity of IFE-IA by reducing the background.The results provide references for the development and application of high affinity peptidomimetic in IFE-IA.3.Development of FITC-labeled parallel structure peptides and establishment of competitive and noncompetitive FPIAsThe parallel structures of FITC-labeled monomer,dimer,and tetramer peptidomimetic and anti-immunocomplex peptides were designed and synthesized by using lysine as scaffold.The affinity of the tetramer tracer was 11 and 6 times higher than that of the monomer and dimer peptidomimetic,while 10 and 5 times higher for anti-immunocomplex tracers by using ELISA.The competitive and noncompetitive FPIAs were developed by using FITC-labeled peptidomimetic and anti-immunocomplex peptides as tracers.The theoretical Δm P values will decrease with the increase of peptide copies because of the molecular mass.However,in our experiments,the Δm P values based on tetramer peptidomimetic and anti-immunocomplex peptides tracers higher than that of monomer and dimer,which indicated that the multi-copy tracers could form more tracer-m Ab(or tracer-benzothiostrobin-m Ab)complexes.Comparation with the monomer and dimer tracers,the sensitivity of the tetramer tracers improved approximately 7 and 2 times for the competitive FPIA,while 6 and 2 times for the noncompetitive FPIA.After optimization,the IC50 and linear detection range were 19.7 ng m L-1 and 4.3~129.4 ng m L-1 for the competitive FPIA,while the SC50 and linear detection range were 40.4 ng m L-1 and 9.3~210.6 ng m L-1 for the noncompetitive FPIA.The competitive and noncompetitive FPIAs didn’t show CRs with the structural analogues of benzothiostrobin.In addition,the average recoveries of competitive and noncompetitive FPIAs for benzothiostrobin in paddy water,soil,cucumber,rice,corn,wheat and orange samples were in the range of 78.3~115.53% with RSD of 0.7~15.4%.The FPIAs detection results for rice samples spiked unknown concentrations of benzothiostrobin were consistent with the HPLC.This study clarified that the positive effect of peptide copies was greater than the negative effects of molecular mass for fluorescence polarization signal and revealed that the multi-copy numbers of parallel structure peptides can increase the sensitivity of FPIAs by enhance affinity.The result provide references for the development and application of high affinity peptides in FPIAs.4.Development of biotin-streptavidin mediated parallel structure peptides and application in multicolor immunochromatographic strip ICSThe parallel structure tetramer peptidomimetic and anti-immunocomplex peptides were prepared by using the biotin-labeled peptides and biotin-streptavidin medium.The ratiometric fluorescence ICS(RFICS)based on additive three-primary colors was developed by using quantum dots(QDs)with emission of 525 nm and 623 nm labeled tetramer peptidomimetic and anti-immunocomplex peptides,while the ratiometric colorimetry ICS(RCICS)based on subtractive three-primary colors was developed by using gold nanoparticles and Prussian-blue nanoparticles.The RFICS and RCICS allow intuitive and accurate semi-quantitative detection of analytes by naked eye like a p H test paper.The cutoff points were 1.0 and 10.0 ng m L-1 benzothiostrobin,for which the emission colors of the T-line changed from green to yellow and from yellow to red for RFICS,while the cutoff points were 5.0 and 50 ng m L-1 benzothiostrobin,for which the colorimetric colors changed from red to purple and from purple to blue for RCICS.Combination with a smartphone,the limit of detections of RFICS and RCICS were 0.17 and 0.44 ng m L-1,respectively.The developed RFICS and RCICS are resistant to the interference of visible light.In addition,the average recoveries of RFICS and RCICS for benzothiostrobin in soil,cucumber,rice and wheat samples were 70.7~115.4% with RSD of 2.0~18.6%.The RFICS and RCICS detection results for soil samples spiked with unknown concentrations of benzothiostrobin were consistent with the HPLC.In this study,the parallel structure tetramer peptidomimetic and anti-immunocomplex peptides were prepared by using biotin-streptavidin medium,which overcomes the shortcomings of costly,difficulty and being affected by peptide sequence.The additive and subtractive three-primary colors based multicolor ICS(like a p H test paper)for intuitive and accurate semi-quantitative detection of benzothiostrobin was developed by integration of these two format peptides.