Research On Important Biological Toxin Reference Materials And On-site Quantitative Detection Methods Of Lanthanide High-sensitivity Fluorescenc

Ricin Toxin（RT）,Abrin Toxin（AT）,Botulinum neurotoxin type A（Bo NT/A）,Clostridium perfringens alpha toxin（CPA）,Clostridium perfringens epsilon toxin（ETX）,fungal T-2 toxin and aflatoxin B₁（AFB₁）have the characteristics of strong toxicity,wide source,and easy preparation.They are potential biological warfare agents and biological terrorist agents,which may be applied in military and have strong social harmfulness.In response to the threat of these important biological toxins,rapid,accurate,sensitive and specific on-site rapid detection methods are urgently needed to deal with biological terrorist incidents,biological warfare sites or major public health events caused by these toxins.Colloidal gold-immunochromatographic assay is the on-site rapid detection method used most extensively.It is time-saving,simple operation and low cost,but low sensitivity and unable to achieve quantitative detection.Time-resolved fluorescence immunoassay（TRFIA）established in this study makes up for the shortcomings of the above method.TRFIA combines time-resolved fluorescence with immunoassay technology.Based on the fluorescence characteristics of lanthanide elements such as large Stokes shift and long fluorescence lifetime,an on-site detection method for biological toxins with high sensitivity,strong specificity,good stability,wide linear range and quantitative detection was established.This method has great potential application value in the field of biosafety.In addition,to improve the accuracy of the biological toxin detection method,we also developed the corresponding reference materials（RM）,which can not only provide more accurate measurement standards for the quantitative results of the detection method,but also serve as an important evaluation tool for the toxin detection method,which can effectively improve and promote the development process of toxin-related detection products.The purpose of this study contains two aspects.One is to prepare seven important biological toxin reference materials.The other is to prepare a kind of polystyrene microspheres based on lanthanide highly sensitive fluorescence,and establish a biological toxin on-site quantitative detection method.The first is the development of five important biological toxin reference materials.Four natural toxins,such as RT,AT,Bo NT/A and ETX,were prepared by affinity chromatography,gel filtration and ion exchange.A total of five macromolecular toxins were obtained including the above four toxins and a CPA recombinant protein toxin.Aiming at the macromolecular toxins（protein toxins）,the specificity,purity,content and activity of proteins were determined by adopting the methods of identification of protein.The purity was determined by SDS-PAGE and HPLC,the activity was determined by in vitro cytotoxicity test or mouse biological test,and the content was determined by ultraviolet method.The natural RT and AT were purified by affinity chromatography,with molecular weights of about 64 k Da and 63 k Da,contents of 0.228 mg/m L and0.317 mg/m L,and purity of 90.59%and 95.96%,respectively.The results of real-time cell analysis（RTCA）showed that the IC₅₀ values were 2.79 ng/m L and 5.41 ng/m L,respectively.Natural Bo NT/A and ETX were obtained by ion exchange method,with molecular weights of about 150 k Da and 34 k Da,and contents of 0.153 mg/m L and0.359 mg/m L,respectively.Bo NT/A was used as the complex to replace it with SDS-PAGE purity index,and its purity was>90%.The LD₅₀ of mouse toxicity test was 1.41ng/m L,the purity of ETX was 96.42%,and the IC₅₀ was 9.9μg/m L.The molecular weight of CPA obtained by recombinant expression and purification was about 43 k Da,the content was 0.182 mg/m L,the purity was 95.87%,and the IC₅₀ was 4.49 ng/m L.As commercial products of T-2 toxin and AFB₁ is more mature,we bought three different brands of toxin for comparison.Aiming at the above small molecule compound toxin,the specificity,purity and content were mainly analyzed by common analytical methods.Their structure and specificity were identified by detecting the hydrocarbon spectrum,and quantified by LC-MS/MS.The molecular weights of AFB₁ and T-2 toxin were about 466.51 Da and 312.27 Da,and the contents were 99.6μg/g and 100.6μg/g,respectively.There were no obvious impurities.The NMR hydrocarbon spectra showed that the structures were correct,which were consistent with the theoretical values.Then,a method for on-site quantitative detection of biological toxins based on lanthanide highly sensitive fluorescence was established.The lanthanide fluorescent microspheres with good homogeneity and dispersion were prepared by the method of seed emulsion polymerization.The characterization results showed that the average particle sizes were 100,185 and 310 nm,which were spherical,uniform and well dispersed.The excitation wavelength was about 412 nm,which guarantee realization of the visible light excitation.Based on this microsphere,the test strips of macromolecular toxins and small molecular compound toxins were designed by using the principle of double-antibody sandwich method and competition method,respectively,according to the different chemical structures of biological toxins.Finally,the detection cards of lanthanide high-sensitivity fluorescence immunochromatography test strips for the above seven biological toxins were successfully developed.The detection card is simple,time-saving,high sensitivity and good specificity.Among them,the LOD of RT,Bo NT/A,ETX,and AFB₁ detection cards was 0.5 ng/m L,and the quantitative range was 0.5～50 ng/m L.The LOD of T-2 toxin detection cards was 10 ng/m L,and the quantitative range was 10～250 ng/m L.The intra-and inter-assay repeatability coefficients of variation were less than 15%.The detection results of orange juice,milk,sewage and serum samples are good.The minimum detection limits of CPA and AT test strips were 1000 and 500 ng/m L,respectively,needed to improve sensitivity by further optimization.Its specificity was good.Finally,the developed lanthanide high-sensitivity fluorescence immunochromatographic test card is used in a small portable detection instrument,which has the advantages of rapid detection,accurate quantification and remote transmission of results.In summary,seven kinds of important biological toxin reference materials were obtained,which had good specificity,purity,activity and other characteristic values.They can be used as reference materials for toxin detection reagents,and provide important measurement tools for the evaluation of biological toxin detection methods.The lanthanide fluorescent microspheres with excellent characters were successfully prepared,and the on-site quantitative detection method of biological toxins based on lanthanide highly sensitive fluorescence was established.The method has good specificity and rapid detection,and has a promising prospect in the field of biological quantitative detection.