Development Of Bio-receptors And Rapid Detection Methods For Typical Mycotoxins In Agricultural Products

Mycotoxins are harmful to the quality of agricultural products,and difficult to prevent.To prevent mycotoxins poluting food chains,it is vital important to study the rapid analysis methods.However,it is challenging for the design of on-site detection methods due to the lack of high affinity antibodies,difficulty in simultaneous assay of multiple risk factors,poor sensitivity and the shortage of anti-interference etc.Therefore,three bio-receptors were developed for the detection of mycotoxins in this thesis via the development of highly sensitive monoclonal antibody,the design of nanobody and the the modification of the aptamers,respectively.Based on the three bio-receptors,novel rapid detection methods were established,which have high sensitivity in the detection of typical mycotoxins in grains,oil,milk,and fruits,etc.The bio-receptors were analyzed and compared accoringly,including their potentials in being applied to the development of rapid detection products.The research contents are present as the follows:（1）A series of monoclonal antibodies（mAbs）,including FB₁,and diacetoxyscirpenol（DAS）,etc.were developed.Based on the novel mAbs,lateral flow chromatography assays for single and multiple different kinds of targets（mycotoxins and pesticide residues）,also a competitive-type pressure-dependent immunosensor were developed.（1）The mAbs 7A11 of fumonisin was developed.The IC₅₀（concentration of target produced 50%inhibition）of 7A11 was 0.66 ng/ml.A gold-naonoparticles-based gray imaging quantification immunoassay was developed.It could be easily observed by naked eyes,or be quantified using gray-imaging method with dynamic range of 0.24～15 ng/ml.Compared with HPLC analysis,the gray-imaging method indicated that it could be used for further detection in rice,corn,peanut and wheat samples,so it provided an economical,convenient and quantitative solution for the rapid screening of fumonisin in grains and oil.（2）Two novel mAbs against carbaryl and carbofuran（1D2 and G11）were developed,the IC₅₀values of 1D2 and G11 were 0.8 ng/mL and 217.6 ng/mL,respectively.In view of the toxic hazard factors existence in the agricultural products,carbamates and aflatoxins were selected in this study to establish the multi-TRFICA-sensor.The calculated LODs of aflatoxin,carbaryl and carbofuran were0.03,0.02,and 60.2 ng/mL in corn samples,respectively.Multi-TRFICA-sensor overcomed the shortages of poor sensitivity,and difficulty in simultaneous assay for mycotoxins and pesticides.（3）A sensitive and specific mAbs 5E7 against DAS was developed.Then,the antibody was applied to develop a competitive-type pressure-dependent immunosensor.The K_a of 5E7 was 5.4×10⁸,and the IC₅₀ was 3.08 ng/ml.The Au@PtNPs and Au@PtNPs-IgG probe were synthesized and characterized.The immunosensor for DAS was established with a LOD of 0.46 ng/mL.The method provided solutions for the lack of sensitive,accurate and on-site rapid screening technologies due to lack of specificity of DAS antibodies.（2）A phage display nanobody library was constructed and aflatoxin M₁（AFM₁）anti-idiotypic nanobody was developed.Based on substitute antigen,the electrochemical detection method for AFM₁in milk and TRFICA method for dual mycotoxins were developed.（1）A novel anti-idiotypic nanobody for AFM₁ named VHH 4-1-1(IC₅₀=8.54 ng/mL)was developed.The screen-printed carbon electrode was modified via electrografting of the diazonium salt for label nanobody,then a competitive immunosensor was established by chromatography spectrometry.The immunosensor exhibited good specificity with LOD of 0.18 ng/ml and the linear range of 0.25～5 ng/ml.The method provided a new green detection in the field of food safety.（2）In order to study the feasibility of utilizing nanobody as substitute antigen in TRFICA method,two patterns of competitive detection:AIdnb–TRFICA（immobilization the AIdnb）and mAb–TRFICA（immobilization the mAb）were established and compared.The IC₅₀ of AIdnb–TRFICA were 18.3-and 20.3-fold more sensitive than that of mAb-TRFICA for AFB₁ and ZEN,respectively.AIdnb–TRFICA for the simultaneous detect dual mycotoxins were established and provided with LODs of 0.13 ng/mL and 0.20 ng/mL,respectively.AIdnb–TRFICA was applied to detect dual mycotoxin in maize real samples,the results exhibited good correlation coefficients with the results of HPLC-MS/MS for AFB₁ and ZEN（0.9931 and 0.9952）,respectively.The AIdnb–TRFICA could be used for the multiple mycotoxins detection,providing new way for non-toxic on-site detection in food rapidly and accurately.（3）Based on the aptamer probe,a rapid detection method for lateral flow chromatography of patulin in fruit juice was established.The"digoxin（DIG）antibody-capture probe-recognition probe-AuNPs signal factor"was constructed using the complementary strand of patulin aptamer labeled with DIG as capture probe and biotin-labeled aptamer as recognition probe.The biosensor exhibited limit of detection（LOD）of 2.3 ng/mL,good dynamic range of 2.7～139.8 ng/mL.The detection method resolved the low sensitivity issue within the rapid detection due to the lack of high-affinity bio-receptor for patulin.（4）The sensitivity and specificity of bio-receptors,including aptamers,monoclonal antibodies,nanobodies were compared with the previous report.It was found that the sensitivity of 7A11 and the specificity of 5E7 was the highest till now,and the VHH 4-1-1 was the first reported nanobody for AFM₁.Development of high quality bio-receptor is the key factor for detection technology.Accuracy and sensitivity were obtained from the aptamer-based analysis method.However,the aptamer structure result in long incubation time with 40 min,which needed to be further optimized.Aptamers could be utilized as the alternative bio-receptors and the surrogate of high-affinity antibody.The mAbs against FB₁,DAS,carbaryl and carbofuran exhibited high affinity and specificity.All of the gray-imaging method,TRFICA and pressure-immunosensor showed good sensitivity and accuracy.Therefore,mAbs are still widely used for the development of rapid bio-receptor analytic method.Nanobody is a new bio-recepor which developed rapidly in recent years.The anti-idiotype nanobody VHH 4-1-1 exhibited good affinity with 2C9.The screen-printed carbon electrode was modified with nanobody exhibited accpetable stability.In addition,nanobody could be used as the antigen in TRFICA,realizing non-toxic rapid detection with satisfied sensitivity.For the development of the rapid detection method in agricultural products,nanobody not only showed high affinity,specificity,high stability and modifiable.The main advantage was nanobody could be used as surrogate antigen to develop non-toxic detection method.Nanobody could be further improve affinity through screening in vitro evolution and fictionalization,expanding new detection methods and formats,which presents a broader application prospect for the rapid detection of mycotoxins in agriculture products.In summary,the bio-receptors developed in this thesis,e.g.,mAbs,nanobodies,can resolve the lack of high-affinity antibodies in the on-site rapid assay for mycotoxins in agricultural products.It has been demonstrated that the developed materials could be used for establishing biosensors for mycotoxins.The established Multi-TRFICA-sensor overcome the challenge in the simultaneous assay of mycotoxins and pesticides.The non-toxic on-site detection method could resolve the low anti-interference issue in the existing approach.Therefore,the biosensors developed in this thesis provide benchmarks for future research works studying the challenges in agricultural products.