Electrochemical Immunosensor for Detection of Celiac Disease Toxic Gliadin in Foodstuff

Abstract

Celiac disease is a gluten-sensitive enteropathy that affects as much as 1% 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 glutenfree 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 2.7% of the specific signal attained. The immunosensors were extremely reproducible, with RSD of 5.2 and 6.75% obtained for 1 and 2 (n ) 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.Gluten sensitivity, manifesting as celiac disease (CD), affects possibly 1:100 people in Northern Europe and North America. 1,2 Celiac disease is an inflammatory disease of the upper small intestine, results from gluten ingestion in genetically susceptible individuals, and is the only lifelong nutrient-induced enteropathy. 3,4 The small bowel abnormalities are reversed on withdrawal of gluten from the diet. CD is a familial condition with ∼10-15% of first degree relatives being similarly affected. 5 Celiac disease, when untreated or poorly treated, that is with continued ingestion of gluten, leads to a large number of complications, 6,7 which can result in considerable morbidity and repeated hospital visits. In children, symptoms such as growth retardation among others are observed 8,9 and patients with osteoporosis have a higher prevel...

Gliadins in Foods and the Electronic Tongue

“…In recent years, several optical and electrochemical biosensors, including immunosensors and aptasensors, have been developed to detect gliadin in food matrices, namely to evaluate gluten-free foods, since the amount of gluten must be lower than 20 mg/kg (or ppm), according to the legal requirements (Nassef et al, 2008;OJEU, 2009;Zeltner et al, 2009). De Stefano et al (2006 used an optical sensor with a recombinant glutamine-binding protein to detect traces of gluten in food.…”

“…De Stefano et al (2006 used an optical sensor with a recombinant glutamine-binding protein to detect traces of gluten in food. Nassef et al (2008) proposed an electrochemical immunosensor, based on the use of an antibody raised against the putative immunodominant celiac disease epitope, to measure the gliadin content in foods. Detection limits between 5.5 and 11.6 ng/mL (or ppb) could be achieved.…”

“…It should be noticed that the values previously mentioned are only indicative since there is not enough information to make a final decision on them. More recently, foods labeled as "very low gluten" or "gluten-free" must have gluten content lower than 100 and 20 mg/kg, respectively (Nassef et al, 2008;OJEU, 2009;Zeltner et al, 2009). However, commercial foods labeled as gluten-free may be contaminated by gluten in the range of 20-200 ppm (Collin et al, 2004;Scognamiglio et al, 2014).…”

Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic gluten peptide

“…Subsequent assays have improved on some of the important limitations of the techniques based on the Skerritt antibody ELISA. Other antibodies were raised against different epitopes of -gliadin, such as PN3 (residues 31-49) for the toxic 19-mer peptides (Ellis et al, 1998), CDC5 (residues 56-75; Nassef et al, 2008), Abs against T-cell stimulatory peptides present in gluten (Mitea et al, 2008), and R5, which recognized highly repeated peptide sequences present in wheat, barley, and rye grains (Valdés et al, 2003). The sandwich R5 ELISA was endorsed as a type I method by the Codex Committee on Methods of Analysis and Sampling (Codex Alimentarius Commission.…”

Celiac disease diagnosis and gluten-free food analytical control

“…Nassef and colleagues [117] developed an electrochemical immunosensing strategy for the detection of toxic gliadin using an antibody, coined CDC5, which was raised against the putative immunodominant celiac disease toxic epitope of α-gliadin, 56-75. For anchoring the captured antibody, two different surfaces, based on a gold electrode modified with acidic self-assembled monolayers (SAMs), were proposed.…”