Electrochemical Immunosensor for Detection of Celiac Disease Toxic Gliadin in Foodstuff

摘要

Celiac disease is a gluten-sensitive enteropathy that affects as much as 1percent of the population. Patients with celiac disease should maintain a lifelong gluten-free diet, in order to avoid serious complications and consequences. It is essential to have methods of analysis to reliably control the contents of gluten-free foods, and there is a definitive need for an assay that is easy to use, and can be used on site, to facilitate the rapid testing of incoming raw materials or monitoring for gluten contamination, by industries generating gluten-free foods. Here, we report on the development of an electrochemical immunosensor exploiting an antibody raised against the putative immunodominant celiac disease epitope, for the measurement of gliadin content and potential celiac toxicity of a foodstuff. To develop the gliadin immunosensor, we explored the use of two surface chemistries, based on the use of dithiols, 22-(3,5-bis((6-mercaptohexyl)oxy)phenyl)-3,6,9,12,15,18,21-heptaoxadocosanoic acid (1) and 1,2-dithiolane-3-pentanoic acid (thioctic acid) (2), for anchoring of the capture antibody. The different surface chemistries were evaluated in terms of time required for formation of self-assembled monolayers, stability, susceptibility to nonspecific binding, reproducibility, and sensitivity. The thioctic acid self-assembled monolayer took more than 100 h to attain a stable surface and rapidly destabilized following functionalization with capture antibody, while the heptaoxadocosanoic acid surface rapidly formed (less than 3 h) and was stable for at least 5 days, stored at room temperature, following antibody immobilization. Both surface chemistries gave rise to highly sensitive immunosensors, with detection limits of 5.5 and 11.6 ng/mL being obtained for 1 and 2, respectively, with nonspecific binding of just 2.7percent of the specific signal attained. The immunosensors were extremely reproducible, with RSD of 5.2 and 6.75percent obtained for 1 and 2 (n velence 5, 30 ng/mL), respectively. Finally, the immunosensor was applied to the analysis of commercial gluten-free and gluten-containing raw and processed foodstuffs, and excellent correlation achieved when its performance compared to that of an ELISA.