| In recent years,with the development of the food industry,soy protein has been widely used in food in various forms,making it difficult for consumers to distinguish.Therefore,soy allergies caused by accidental ingestion or unintentional contact with soy protein have been increasing year by year.Soy glycinin is the most abundant protein in soy,and it is also one of the main soy allergens.Due to the complexity of the food matrix,the requirements for the sensitivity of the food allergen detection method have been greatly increased.Traditional detection methods have gradually been unable to meet the needs,so a rapid and sensitive detection method for glycinin is needed.Since the phenomenon that Raman signals can be amplified 106-1015 times on the surface of rough metal particles was discovered,this technology has been widely used in various detection technologies to greatly improve the sensitivity of detection.Therefore,this thesis is based on Raman signal enhancement technology,combining it with immunochromatographic technology,in order to establish a more sensitive detection method for glycinin.And by improving the nanomaterials,the prepared gold nanostars are used to replace colloidal gold,so as to achieve the purpose of further enhancing the Raman signal,so that the sensitivity of the detection means is further improved.The main research of this subject is as follows: The first step is to separate and purify defatted soybean meal to obtain natural soybean glycinin,and use this as an immunogen to immunize mice to prepare poly-antiserum against soybean glycinin.In the second step,a double-antibody sandwich ELISA method was established to detect glycinin,and the performance of this method was tested.The third step is based on the previous two steps to establish a soy globulin-based Raman enhanced detection test strip based on colloidal gold surface.The fourth step is to improve the nanomaterials.The gold nanostar is used instead of colloidal gold to further improve the Raman signal enhancement.A more sensitive detection of soy globulin is prepared based on the gold nanostar Raman enhanced detection test strip.The results of the study are as follows: After alkali-soluble acid precipitation and Sepharose CL-6B column chromatography separation,natural soybean glycinin with a purity of more than 80% was obtained.Then 10 mice were immunized to prepare poly-antiserum.The titers of mice 1,2,8,9,and 10 which were all over 12,800 times are high.The sensitivity test result of polyantiserum showed that the IC50 of No.1 polyantiserum was 602 ng/mL,which was the most sensitive.Cross-reaction experiments show that the polyclonal antibody has strong specificity.The western blot experiment showed that the polyclonal antibody only had a significant reaction with the glycinin A2 subunit,which further proved that the polyclonal antibody has strong specificity.A double-antibody sandwich ELISA detection method for soybean glycinin was established,and its reaction conditions were optimized.After optimization,the actual detection of glycinin was carried out,and the analysis and detection results showed that the detection limit of the detection method was 11.99 ng/mL,and the linear range was 10-2560ng/mL.The recovery rate of the experiment within the batch is in the range of 91-105%,and the range of the CV is in the range of 2.50-7.10%.The recovery rate of the inter-batch experiment is in the range of 87-106%,the CV is in the range of 4.17-10.14%,and the crossreaction rate with each allergic protein is low,which proves that the test kit has good accuracy,precision and specificity.The established detection method for soybean glycinin is based on the colloidal gold Raman enhanced test strip,which not only retains the characteristics of the naked eye detection of the traditional colloidal gold test strip,but also can use the method of detecting the T-line Raman signal to achieve higher sensitivity,and You can create a standard song for quantitative analysis.The visual detection limit of this detection method is 200 ng/mL.Combined with the Raman signal detection method,the detection limit is 4.87 ng/mL,which is an order of magnitude lower than that of the ELISA method.The recovery rate is 91-107% and the CV range is 3-10%.Food allergens have no cross-reactions.This method has high sensitivity,accuracy and specificity.The signal of the immune probe prepared on the basis of gold nanostar is much higher than that of the immune probe prepared on the basis of colloidal gold.The Raman-enhanced test strip prepared by using this immunoprobe has a detection limit of 0.23 ng/mL for glycinin,which is more sensitive.The recovery rate of addition is 91-96%,and the CV is in the range of 1-7%.According to the cross-reaction results,good specificity is also maintained. |
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