Research And Development Of Immunochromatography Products For T2 Toxin Based On Aggregation-induced Emission Luminogen

Lateral flow immunoassay（LFIA）is widely used in the field of food safety because of its simplicity,rapidity,low cost,and strong specificity.Due to the low molar extinction coefficient and colorimetric intensity of 20 nm～40 nm gold nanoparticles（Au NPs）as the signal probe,the traditional immunochromatography test strip has poor detection sensitivity and cannot meet the requirements of highly sensitive detection.Compared with absorbing materials,emissive fluorescent nanoparticles have better optical performance,which is expected to improve the sensitivity of LFIA.However,traditional fluorescent materials have the phenomenon of fluorescence quenching at aggregation or high concentrations,which severely limits the loading capacity of fluorescent materials.Fluorophores with aggregation-induced emission（AIE）properties provide a new way to solve this problem and are an excellent choice for developing high-performance fluorescent probes,bringing great promise for improving the sensitivity of LFIA.Therefore,this study carried out a series of basic application studies on the preparation of novel AIE fluorescent probes and the development of immunochromatography products using these AIE probes,as follows:（1）The size of the probe is one of the key parameters determining the performance of LFIA.It affects the binding efficiency,convection and diffusion speed of the immune complex on the test strip,which in turn affects the analytical performance of the LFIA.Our previous research had showed that the signal intensity and the detection sensitivity of probes,including Au NPs,colloidal gold microspheres and quantum dot microspheres increased with the increase of particle size.However,the detection sensitivity of probes with too large particle size decreased instead.Due to the density difference among different probes,the optimal particle size is different for each probe.Thus,in this study,four competitive immunochromatographic methods for the detection of T2 toxins（T2T）were established based on 150,250,350 and 450 nm aggregation-induced emission nanoparticles（AIENPs）,and the effect of AIENP size on the analytical performance of competitive immunochromatographic assay was investigated.The results showed that AIENPs with small particle size（150-250 nm）exhibited better analytical performance.Subsequently,using 150 nm AIENPs as signal probes,we successfully developed the immunochromatographic test strips for T2T detection.Benefiting from the ultrahigh optical properties of AIENPs,this strip has a limit of detection of 0.43 ng/m L,IC₅₀ of 2.46 ng/m L,and a linear detection range of0.66 ng/m L～9.17 ng/m L for the detection of T2T in feed.The average recovery rate of T2T in feed was 97.4%～109.0%,with the CV of 4.3%～11.1%.No cross-reaction was observed with other common mycotoxins.Twenty T2T-spiked compound feed samples were tested by using the developed AIE strips and commercially available ELISA kit,and the results showed that the two methods have good consistency（R²=0.93）.The above results showed that the developed fluorescent immunochromatographic test strips based on AIENPs have good specificity,reliability,precision and accuracy.（2）Compared with the single-mode immunochromatography test strips,the colorimetric/fluorescence dual-mode immunochromatography test strips have a flexible reading mode,and different dynamic detection ranges,which are suitable for different application scenarios.In addition,the two signals can corroborate each other and improve the accuracy of detection.Therefore,in this study,the colorimetric/fluorescence dual-mode immunochromatographic test paper products were developed by synthesizing nanomaterials with colorimetric/fluorescent dual functions.Firstly,dendritic mesoporous silica nanoparticles（DMSN）were synthesized by the soft template method,and their surfaces were modified with amino groups.Subsequently,the DMSN were loaded with AIEgen by alkyne-amine click reaction to prepare the DMSN@AIE.Finally,the Fe-tannic acid metallic polyphenol network was modified on the surface of DMSN@AIE（DMSN@AIE@TA）to improve its colloidal stability and biocompatibility.In addition,the modification of metal polyphenol network also provides the possibility for efficient bioconjugation of antibodies.Using the DMSN@AIE@TA as the signal probes,we successfully developed a colorimetric/fluorescence dual-mode immunochromatographic test strip for T2T detection.For the colorimetric mode,this strip has an IC₅₀ of 0.62 ng/m L and a linear range of 0.29 ng/m L～1.34 ng/m L;while for the fluorescence mode,this strip has an IC₅₀ of 0.59 ng/m L and a linear range of 0.26 ng/m L～1.33 ng/m L.The two signal reading modes can corroborate each other,which greatly improves the reliability of detection.The average recovery rate of the developed strip method for T2T in the feed was 92.1%～107.2%,and the CV was 1.9%～8.3%,exhibiting good precision and accuracy.Moreover,no obvious cross-reaction with other common mycotoxins were observed,showing good specificity.The universality of this method was further confirmed by conducting an assay of Staphylococcal enterotoxin A（SEA）on a sandwich immunochromatographic platform.The sensitivity of this method for SEA was 7.81 ng/m L（colorimetric mode）,and 0.43 ng/m L（fluorescent mode）.These results indicated that the colorimetric/fluorescence dual-mode immunochromatographic test strip developed based on DMSN@AIE@TA can not only perform two kinds of signal self-correction,improve the reliability of detection,but also realize multi-end application with only one development,which is expected to play an important role in food safety,infectious diseases prevention,and family self-monitoring.