Electrochemical immunosensor detection of antigliadin antibodies from real human serum

The determination of antigliadin antibodies from human serum samples is of vital importance for the diagnosis of an autoimmune disease such as celiac disease. An electrochemical immunosensor that mimics traditional ELISA type architecture has been constructed for the detection of antigliadin antibodies with control over the orientation and packing of gliadin antigen molecules on the surface of gold electrodes. The orientation of the antigen on the surface has been achieved using a carboxylic-ended bipodal alkanethiol that is covalently linked with amino groups of the antigen protein. The bipodal thiol presents a long poly(ethyleneglycol)-modified chain that acts as an excellent non-specific adsorption barrier. The bipodal nature of the thiol ensured a good spacing and hence good diffusion properties of electroactive species through the self-assembled monolayer, which is vital for the efficiency of the constructed electrochemical immunosensor. The electrochemical immunosensor was characterized using surface plasmon resonance as well as electrochemical impedance spectroscopy. Amperometric evaluation of the sensor with polyclonal antigliadin antibodies showed stable and reproducible low limits of detection (46 ng/mL; % RSD = 8.2, n = 5). The behaviour and performance of the electrochemical immunosensor with more complex matrixes such as reference serum solutions and real patient samples was evaluated and compared with commercial ELISA kits demonstrating an excellent degree of correlation in thirty minutes total assay time; the electrochemical immunosensor not only delivers a positive or negative result, it allows the estimation of semi-quantitative antibody contents based on the comparison against clinical reference solutions.