The Research Of Detection For Zearalenone By Two Colloidal Gold Immunochromatography

Zearalenone(ZEN)is an estrogenic fungal toxin produced by fusarium.In warm and humid environments,ZEN can contaminate crops such as maize,wheat,and barley,of which the most common pollution in maize.As one of the fungal toxins,ZEN is highly toxic.There are some residues and accumulations in animals,and the toxins are not easy to get out of the body,which poses a serious threat to food safety and human health.Lateral Flow Immunoassay(LFIA)has been widely used in the field of vitro diagnosis,food safety and environmental monitoring due to its simplicity,rapidity and low cost.The signal label is a key factor that affects the LFIA performance,especially sensitivity.Gold Nanoparticles(AuNPs),as a label with red color,good biocompatibility and high molar extinction coefficient,have been widely used in immunological detection in recent years.However,the low color intensity and poor colloidal stability of traditional AuNPs result in the low sensitivity of colloidal gold immunoassay(CGIA).Therefore,it is of great significance to establish a rapid and sensitive method for detecting trace amounts of ZEN in food and feed.In Chapter 1,we first gave a brief overview of the target ZEN,including the physical and chemical properties,hazards and limit standards of ZEN,and currently used methods for detecting ZEN residue.Secondly,we introduced LFIA in general,and discusses strategies to improve its detection sensitivity.Finally,we briefly introduced the new material PDA,including its properties and applications.In Chapter 2,a CGIA to detect ZEN in maize was established.After optimized the pH of AuNPs labeled antibody,the concentration of complete antigen(ZEN-BSA)in test line(T line),and the concentration of anti-ZEN monoclonal antibody(mAb),the standard curve was established by plotting the signal intensities of the T line against the ZEN concentrations.The results showed that the cut-off value of naked-eye was 2.5 ng/mL,and the limit of detection(LOD)and linear range were 76.1 pg/mL and 0.1-10 ng/mL,respectively.In chapter 3,PDA-based LFIA for the sensitive detection of ZEN in maize was established.Dopamine(DA)was oxidized and polymerized on the surface of AuNPs to form PDA-coated AuNPs(Au@PDA).The synthetic conditions of Au@PDA,including the pH of synthetic solution,the volume of 3%H2O2 and DA were optimized.The characterization of UV-vis spectrum,Zeta potential,and transmission electron microscope showed that the molar extinction coefficient of Au@PDA-10 was 1.68 times higher than that of AuNPs.Besides,Au@PDA-10 has high antibody coupling efficiency,good stability,and strong resistance to high-salt and strong-alkali environment,which could effectively improve the detection sensitivity of traditional CGIA.Therefore,Au@PDA-10 was choosed as signal label,and then 6 important experimental parameters were optimized.The optimal working conditions was obtained:blocking agent was 1%casein,antibody labeling pH was 7,mAb concentration was 2.5 μg/mL,the concentration of ZEN-BSA was 2.5 mg/mL,the amount of probe was 2.5 μL,and the immune reaction time was 30 min.The mAb amount used in this method was 1 times less than that of the conventional CGIA,which could significantly reduce the detection cost.The standard curve was established under the optimal conditions,and the results showed that the cut-off value of naked eye was 1.0 ng/mL,the LOD and linear range were 7.4 pg/mL and 0.01-5 ng/mL,respectively.The sensitivity of quantitative detection was 10 times higher than that of traditional CGIA.The recoveries of this method were 93.80%-111.82%,and coefficient of variation(CV)value was 1.08%-9.04%.There was no cross-reaction with 6 mycotoxins.High performance liquid chromatography and this method were used to simultaneously detect 20 real maize samples with CV values 2.91%-6.08%,indicating that this method has great potential in the real sample detection.In chapter 4,we summarizes and prospects the full work.The two LFIAs for the detection of ZEN established in this work are systematically studied,which proves from multiple performance that the two LFIAs established have certain guiding significance for the detection of ZEN and other mycotoxins.It also proposes new ideas and methods for improving the sensitivity and other performance of LFIA.