Determination Of Food Allergen By Piezoelectric Immunosensor Detecting Assay

The problem of food allergen is a hidden danger for food safety,shrimp and peanut allergen were listed in the list of eight common allergic foods in the FAO report(1995).However most of the present detecting methods for allergen are time-consuming and cannot satisfy the requirement for a rapid, convenient, and on-line detection of allergen in food samples. The rapid method for determination of food allergen was carried out by piezoelectric immunosensor in this paper, based on antibody antigen reaction and the piezoelectric sensing technology.Shrimp and peanut proteins were prepared by ammonium sulfate fractionated precipitation and isoelectric precipitation, and the main allergic components were analysed by gel chromatography and SDS-PAGE BALB/C mouse were immunized with shrimp and peanut and the titer of anti-shrimp serum was as high as 30000 with an IC50 of 60μg/mL, and anti-peanut serum was 10000 with an IC50 of 150μg/mL.The rapid detecting method of piezoelectric immunosensor was developed on the basis of self-assembly with 1,6-hexanedithiol(HDT)-nanogold. The optimum conditions for detection were as follows: 15mM HDT was coated on the electrode for 90min and nanogold was adsorbed for 110min at room temperature; the antibody was coated for 3 hours under 4℃,and the coating concentration for anti-shrimp antibody was 1:6000, for anti-peanut antibody was 1:4000; the detecting time was only 500s, and the standard curve between 1ug/mL and 300ug/mL of allergen was obtained, in accordance with sauerbrey equation. The detection limit for shrimp was 0.03ug/mL, for peanut was 0.2ug/mL. The specificity was studied with cross reactivities using fish protein and soybean protein respectively: the electrode coated with shrimp antibody had a high cross reactivity with fish protein, while the electrode coated with peanut antibody was low with soybean protein, which were consistent with ELISA results. Shrimp and peanut protein were added to drinking water respectively and the average recoveries were among 90%～110%, the average coefficient of variation of reproducibility test was less than 15%. Regeneration of the electrode was also studied: Piranha and strong base and acid had obvious effect, and the electrode can be reused for more than 13 times; glycine had less regeneration capacity for 5 times, but more conveniently. In general, the rapid detecting method by piezoelectric immunosensor developed in this paper can be used in the food detection, and it showed that this assay had some potential application in practice.