We report here the design of a novel immunosensor and its application for celiac disease diagnosis, based on an electrogenerated chemiluminescence (ECL) readout, using membrane-templated gold nanoelectrode ensembles (NEEs) as a detection platform. An original sensing strategy is presented by segregating spatially the initial electrochemical reaction and the location of the immobilized biomolecules where ECL is finally emitted. The recognition scaffold is the following: tissue transglutaminase (tTG) is immobilized as a capturing agent on the polycarbonate (PC) surface of the track-etched templating membrane. It captures the target tissue transglutaminase antibody (anti-tTG), and finally allows the immobilization of a streptavidin-modified ruthenium-based ECL label via reaction with a suitable biotinylated secondary antibody. The application of an oxidizing potential in a tri-n-propylamine (TPrA) solution generates an intense and sharp ECL signal, suitable for analytical purposes. Voltammetric and ECL analyses evidenced that the ruthenium complex is not oxidized directly at the surface of the nanoelectrodes; instead ECL is generated following the TPrA oxidation, which produces the TPrA•+ and TPrA• radicals. With NEEs operating under total overlap diffusion conditions, high local fluxes of these reactive radicals are produced by the nanoelectrodes in the immediate vicinity of the ECL labels, so that they efficiently generate the ECL signal. The radicals can diffuse over short distances and react with the Ru(bpy)32+ label. In addition, the ECL emission is obtained by applying a potential of 0.88 V versus Ag/AgCl, which is about 0.3 V lower than when ECL is initiated by the electrochemical oxidation of Ru(bpy)3(2+). The immunosensor provides ECL signals which scale with anti-tTG concentration with a linearity range between 1.5 ng·mL–1 and 10 μg·mL–1 and a detection limit of 0.5 ng·mL–1. The sensor is finally applied to the analysis of anti-tTG in human serum samples, showing to be suitable to discriminate between healthy and celiac patients.

An innovative second generation enzymatic microbiosensor was developed exploiting the properties of nanoelectrode ensembles (NEEs) prepared by electroless gold deposition in track-etched polycarbonate (PC) membrane. As a case study, a miniaturized glucose biosensor was developed and characterized. The micro-NEE glucose biosensor was obtained by immobilizing glucose oxidase (GOx) on the nonconductive PC component of the NEE, while the Au nanoelectrodes were used exclusively as transducers. (Ferrocenylmethyl)trimethylammonium cation (FA+) was used as the redox mediator. The immobilization of GOx on the PC didn’t affect the electrochemical performances of the NEE nor the catalytic activity of the enzyme. The proposed biosensor showed outstanding analytical performances with a detection limit of 36µM. The nanostructured biosensor was miniaturized down to a micro-NEE with overall radius of 400 µm, without any degradation of the analytical performances