Label-Free and Highly Sensitive Detection of Buckwheat 16kD Allergenic Protein with Field Effect Transistor (FET) Sensor and Immunochromatography Strip

Abstract

A field effect transistor (FET) sensor detects the change in charge generated by binding of a specific target molecule to the sensor. This simple mechanism allows label-free rapid detection. However, the sensitivity of detection depends on the net charge of the targeted molecule. Buckwheat 2S albumin (BWp16), which is one of the major allergens of buckwheat, constitutes an analyte of buckwheat in food, but its small net charge limits its sensitivity to a FET sensor. We show here that pretreatment of BWp16 with sodium dodecyl sulfate (SDS) increases the net charge of this analyte and increases sensitivity to 1 ng/ml of BWp16 using a FET sensor. Our results suggest a FET sensor is applicable to food safety management. We also developed an immunochromatography assay strip for convenient and rapid detection of BWp16 to a lower limit of 5 ng/ml. Food Allergen Labeling Regulations in Japan require mandatory labeling of seven allergens in foods at 10 µg/g or more, including buckwheat proteins. Allergenic contamination of food is usually monitored using enzyme-linked-immunosorbent-assay (ELISA), which requires multiple handling steps, an enzyme-labeled secondary antibody, and optical equipment for quantification. In contrast, a FET sensor and an immunochromatography assay strip are potential assay tools without those drawbacks due to their direct sensing mechanism. In this study, we propose a FET sensor and an immunochromatography assay strip be devised for detection of buckwheat protein. Here we attempt to improve the sensitivity of a FET sensor for buckwheat protein BWp16 by increasing the net charge of its molecules. BWp16 was determined specifically using a FET sensor. The response of the FET sensor was improved markedly by pretreatment of recombinant BWp16 (rBWp16) with SDS. The response of the FET sensor was correlated positively with the concentration of rBWp16 down to a detection limit below1 ng/ml. The charge number of BWp16 calculated from its molecular weight (15,106 Da) and isoelectric point (pH 5.25) was small (−3.67/molecule) at the sensing condition of pH 7.4 used with the FET sensor. After the pretreatment with SDS, BWp16 was covered with enough negative charge to increase sensitivity to approximately 10 ng of buckwheat proteins/g of food material. We devised an immunochromatography assay strip and tested the strip on 40 food samples. The assay strip gave a lower limit of detection below 5ng/ml of BWp16. SDS pretreatment of BWp16 protein from buckwheat increased its sensitivity to detection with a FET sensor nearly 1000-fold. This simple charge-amplification technique can increase the sensitivity of a FET sensor to a targeted protein that would ordinarily carry a small net charge. Both a FET sensor and an immunochromatography assay strip achieved highly sensitive and specific detection of buckwheat protein.