Detection of different isotypes of anti-tissue transglutaminase by nanoelectrode ensemble biosensors for celiac disease diagnosis

Abstract

Celiac disease is a gluten-dependent autoimmune disorder with a prevalence of 1% in the general population. Although definitive diagnosis of CD is still based on biopsy, serological test of CD screening based on detection of anti-tissue transglutaminase antibodies (anti-tTG) are less invasive. On this basis, increasing researches are devoted to developing sensitive and selective analytical tests and sensors to detect anti-tTG in human serum. Anti-tTG is indeed present in two different isotypes, IgA and IgG, but the majority of existing methods focus only on the detection of the IgA type. In this thesis we develop electrochemical biosensors based on nanoelectrode ensembles (NEEs) to study the possibility to detect both isotypes. NEEs are prepared in our laboratory by electroless deposition of gold in track-etched polycarbonate membranes. The polycarbonate of the NEEs is functionalized with tissue-transglutaminase as capture antigen. The obtained immunosensor show to be able to capture both the isotypes of anti-tTG in serum samples. The selective detection of isotype IgG is possible by using a secondary anti-IgG antibody labeled with horse radish peroxidase (HRP): in the presence of the target analyte (IgG anti-tTG), the addition of HRP substrate (hydrogen peroxide) and hydroquinone as redox mediator generates an electrocatalytic increase of the cathodic current which scales with the IgG antibody concentration. For the detection of IgA isotype a different secondary antibody is employed, that is anti-IgA labeled with glucose oxidase. In this case with the addition of glucose (the substrate) and ferrocenylmethyl trimethilammonium (the redox mediator), an electrocatalytic increase of the anodic current is now observed. Analytical performances of the two biosensors are presented and discussed together with applicative prospects for molecular diagnosis of the celiac disease.