Final Report Summary - GLUSENS (Reagentless electrochemical & fluorescent biosensor formats for detection of coeliac disease toxic gluten (gliadins and glutenins) in raw and processed foodstuffs) Celiac disease is a gluten-sensitivity problem that affects about 1 % of the population. It is a difficult-to-diagnose digestive disease involving the inability to digest proteins called gluten that occurs in wheat, oats, rye, and barley. 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. The main objective of project GLUSENS is developing a biosensor to detect toxic parts of gluten proteins. The toxic sequences of gliadin is already known and this project aims to develop a recognition element for that toxic parts by using nanotechniques. The biosensor could help millions of people avoid diarrhoea, bloating, and other symptoms that occur when they eat foods containing gluten. In the first six months, an evolutionary procedure called SELEX has been applied for selection of nucleic acid molecules called aptamers which could be important in developing fast and easy-to-use sensors to detect the presence of toxic gluten in foodstuff. The SELEX approach exploited the use of magnetic beads functionalised with gliadin and a short peptide, identified to be the putative immunodominant epitope in the pathology of coeliac disease. Single stranded DNA was generated from a library of of diverse DNA fragments using enzymatic digestion and was incubated with the functionalised magnetic beads and the DNA fragments that interacted with the gliadin target were liberated using affinity purification and amplified for the next round of SELEX. This procedure was followed for 11 cycles and cloning and sequencing subsequently carried out. Aptamer candidates determined will be used to develop biosensors for detection of toxic gluten fragments in foodstuff in future.Current assays available for detection of gluten in food suffer from many drawbacks because of the properties of recognition elements. Aptamer based assays can solve many problems like incompatibility with extraction buffers and long assay times by their inherent properties. The candidate aptamers selected during project should be valuable in developing assays are that are inexpensive, quick and simple. A novel aptamer based ultrasensitive detection system was developed by using thrombin-binding aptamer as proof-of-concept element. The assay system was based on real-time PCR detection combined with aptamer specificity achieving 20 thousand better detection limit which can be crucial in overcoming current limitations of gluten determination assays.
