Nanostructured Electrochemical immunosensors for the Identification of Egg Proteins: Immunoglobulin Y and Ovalbumin - Application to Cultural heritage and Food Analysis

Despite biosensors are widely used in many research fields and offer many advantages, as high selectivity and sensitivity, low costs and fast analytical procedures, they have been introduced in the cultural heritage field only recently. In this PhD project, two electrochemical immunosensors were developed, with Immunoglobulin Y (IgY) and Ovalbumin (OVA) as the target proteins. IgYin an antibody present in high concentration in egg yolk, while OVA is the main protein in egg white. To this aim, the development of sensors capable of identify these proteins provides complete information about samples from tempera paintings with egg as the organic binder. The electrochemical transducer used for these sensors is an ensemble of gold nanoelectrodes, prepared by electroless deposition of gold in track-etched nanoporous polycarbonate membranes. Thanks to the specific conformation of the NEEs, it is possible to exploit the superficial polycarbonate present in large amount to capture the target proteins and the chemical modification of the surface is not needed. In a first phase of this project, some parameters of the IgY-immunosensing procedure were optimised, to improve the applicability of the sensor in the cultural heritage field. Indeed, low amount of samples is a key role parameter in this research field. To this aim, the geometric area of the sensor was reduced to sub-millimetre dimensions, and it has been proved that this miniaturization does not affect the analytical performance of the sensors. Afterwards, the OVA immunosensor was developedex novo. The analytical procedure was optimised, focusing in particular on reducing aspecific interactions possibly cause by other proteins. The performances of the OVA-immunosensors were evaluated in terms on sensibility and selectivity. Both the IgY- and OVA-electrochemical immunosensors were applied in the identification of egg yolk and egg white in complex mock-up samples, preparedas cross-sections, that simulate multilayer painting. In particular the layers were prepared with whole egg or egg components separately, in a mixture with pigments and other organic binders, as linseed oil or animal glues. IgY-immunosensor was also applied in the study of food supplements that undergone an industrial process such as freeze-drying. Also in this case, the identification of the protein was successful. Measurements carried out with on extracts obtained by direct incubation of the cross-sections, successfully demonstrated the ability of the electrochemical immunosensors to detect the two target proteins (IgY and/or OVA) in most of the samples. With the aim of extending the applicability of the sensors to other research fields, finally, the IgY-immunosensors was exploited for the identification of the protein in food and food supplements based on egg yolk. The results obtained with the electrochemical immunosensor on these samples were compared with those obtained by electrophoretic and western blot analysis, indicating moreover a higher sensitivity of the sensor when samples with small amount of egg yolk were analysed.