Competitive immunosensor based on gliadin immobilization on disposable carbon-nanogold screen-printed electrodes for rapid determination of celiotoxic prolamins.

The first competitive disposable amperometric immunosensor based on gliadin-functionalized carbon/nanogold screen-printed electrodes was developed for rapid determination of celiotoxic prolamins. To date, no competitive spectrophotometric or electrochemical immunoassays have yet been successfully applied to gluten detection in processed food samples, which require the use of complex prolamin extraction solutions containing additives with denaturing, reducing and disaggregating functions. Thus, in this work, great effort was put into the optimization and performance evaluation of the immunosensor in terms of suitability as a screening tool for analysis of cereal-based food samples. For this purpose, aqueous ethanol or complex extraction mixtures, as the patented Cocktail Solution®, were proved effective in the extraction of gliadin. Good sensitivity was achieved after optimization of the immunocompetitive assay, giving limit of detection and limit of quantitation of 8 and 22 ng/ml of gliadin, respectively, for ethanol extracts. The immunosensor was proved to be suitable also for samples extracted with Cocktail Solution® after a proper dilution. Analysis of real samples of different flours proved the suitability of the immunosensing device as a powerful tool for safety assessment of raw materials used for the formulation of dietary products for celiac disease patients. This immunosensor combines good analytical performance using a very simplified set-up protocol with suitability for rapid screening analysis performed using inexpensive and portable instrumentation. Graphical abstract Depiction of the development and working principle of the competitive immunosensor.

Multiplex liquid chromatography-tandem mass spectrometry for the detection of wheat, oat, barley and rye prolamins towards the assessment of gluten-free product safety.

Celiac patients should feel confident in the safety of foods labelled or expected to be gluten-free. In this context, a targeted proteomic approach based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was proposed to assess the presence of celiotoxic cereals, namely wheat, oats, barley and rye, in raw and processed food products. To this aim, unique marker peptides were properly selected in order to distinguish between the different cereal types. A revised cocktail solution based on reducing and denaturing agents was exploited for prolamin extraction from raw and processed food; in addition, defatting with hexane was carried out for sample clean-up, allowing to largely reduce problems related to matrix effect. Method validation on fortified rice flour showed good analytical performance in terms of sensitivity (limits of detection in the 2-18 mg kg(-1) range). However, poor trueness was calculated for self-made incurred bread (between 3 and 30% depending on the peptide), probably due to baking processes, which reduce gluten extractability. Thus, it is evident that in the case of processed foods further insights into sample treatment efficiency and reference materials for protein calibration are required to obtain accurate gluten determination. Finally, the developed method was applied for the analysis of market food products, offering the possibility to discriminate among cereals, with good agreement with labelled ingredients for gluten-containing foodstuffs.

An amperometric immunosensor for diagnosis of celiac disease based on covalent immobilization of open conformation tissue transglutaminase for determination of anti-tTG antibodies in human serum.

A new amperometric immunosensor based on the covalent immobilization of tissue transglutaminase enzyme in its open conformation (open-tTG) was developed and optimized for determination of anti-tissue transglutaminase antibodies (anti-tTG) in human serum. Experimental design allowed us to find the optimal conditions for quantification of both IgA and IgG isotypes of anti-tTG in order to assess suitability of the device for diagnostic purposes. The glassy carbon electrodic substrate was electrochemically functionalized with gold nanoparticles and subsequently derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid for the covalent anchoring of the enzyme. This step was performed under carefully controlled conditions in order to keep the open conformation of the tTG. The immunosensor showed good analytical performance with limit of detection levels (1.7 AU mL(-1) for IgA and 2.7 AU mL(-1) for IgG) below the diagnostic threshold value (3.0 AU mL(-1)) and inter-sensor reproducibility giving RSD lower than 10%. The developed sensor was validated in serum samples from pediatric patients for clinical applications, using two ELISA kits specific for the determination of anti-tTG IgA and IgG antibodies as reference methods; good recovery rates ranging from 74% to 117% were calculated.