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    A Sensitive Electrochemiluminescence Immunosensor for Celiac Disease Diagnosis Based on Nanoelectrode Ensembles
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    † Department of Molecular Sciences and Nanosystems, University Ca’Foscari of Venice, via Torino 155, 30172 Venezia Mestre, Italy
    ‡ Institut des Sciences Moléculaires, CNRS UMR 5255, University of Bordeaux, ENSCBP, 33607 Pessac, France
    § Faculty of Chemistry, University of Belgrade, 11000 Belgrade, Serbia
    ∥ Institute for Maternal and Child Health - IRCCS “Burlo Garofolo”, 34100 Trieste, Italy
    ⊥ University of Trieste, 34127 Trieste, Italy
    *E-mail: [email protected]
    *E-mail: [email protected]
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    Analytical Chemistry

    Cite this: Anal. Chem. 2015, 87, 24, 12080–12087
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    https://doi.org/10.1021/acs.analchem.5b02801
    Published November 10, 2015
    Copyright © 2015 American Chemical Society
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    Abstract

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    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)32+. 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.

    Copyright © 2015 American Chemical Society

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02801.

    • Procedure and scheme to assemble the NEEs. Differential pulse voltammetric data recorded with a bare NEE in solution containing different concentrations of tri-n-propylamine or Ru(bpy)32+. EDX microanalysis of the NEE surface. Dependence of ECL emission on the anti-tTG concentration in the 1–100 ng·mL–1 range (PDF)

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    Analytical Chemistry

    Cite this: Anal. Chem. 2015, 87, 24, 12080–12087
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.analchem.5b02801
    Published November 10, 2015
    Copyright © 2015 American Chemical Society
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