title: Localization of proteins in paint cross-sections by scanning electrochemical microscopy as an alternative immunochemical detection technique.
abstract: The qualitative identification of proteinaceous substances, as well as their location within a complex paint stratigraphy, is one of the most challenging issues in the characterization of painting materials. Nevertheless, information on paint components represent a crucial task for studies concerning both the ancient painting techniques adopted and the state of conservation, being fundamental investigations for the selection of appropriate conservation actions. The present research was aimed at developing a new detection approach for the immunochemical localization of ovalbumin in paint cross-sections based on the use of scanning electrochemical microscopy (SECM). The immunochemical analyses were performed using an anti-ovalbumin primary antibody and a secondary antibody labelled with horseradish peroxidase (HRP). SECM measurements were performed in feedback mode using benzoquinone (BQ)/hydroquinone (H2Q) redox couple. In presence of hydrogen peroxide (H2O2), HRP catalyzes the re-oxidation of H2Q to BQ and the increment of BQ concentration in correspondence of the target protein was detected by SECM through the electrochemical reduction of the regenerated BQ at the microelectrode. Indeed, the localization of ovalbumin was possible thanks to a clear discrimination of SECM currents, achieved by the comparison of the measurements recorded before and after H2O2 administration, based on the HRP on/off approach. The method was evaluated both on samples from standard mocks-up and on a historical sample, collected from a Renaissance wood painting. The obtained results were promising, foreseeing a wider application of SECM on cultural heritage researches.
authors.affiliation: Microchemistry and Microscopy Art Diagnostic Laboratory, University of Bologna, Via Guaccimanni 42, Ravenna 48121, Italy; Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Sciutto
authors.affiliation: Microchemistry and Microscopy Art Diagnostic Laboratory, University of Bologna, Via Guaccimanni 42, Ravenna 48121, Italy; Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Prati
authors.affiliation: Microchemistry and Microscopy Art Diagnostic Laboratory, University of Bologna, Via Guaccimanni 42, Ravenna 48121, Italy; Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy. Electronic address: rocco.mazzeo@unibo.it.
authors.forename: Rocco
authors.initials: R
authors.lastname: Mazzeo
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Zangheri
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Bardini
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Valenti
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Rapino
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi, Bologna 2 40126, Italy. Electronic address: massimo.marcaccio@unibo.it.
authors.forename: Massimo
authors.initials: M
authors.lastname: Marcaccio
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D016227:Benzoquinones / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D006873:Hydroquinones / Q000737:chemistry; D064347:Microscopy, Electrochemical, Scanning; D010047:Ovalbumin / Q000032:analysis / Q000276:immunology; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis* / Q000276:immunology
chemical_list: D000906:Antibodies; D016227:Benzoquinones; D006873:Hydroquinones; D011506:Proteins; C004532:quinone; D010047:Ovalbumin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C031927:hydroquinone
keywords: Immunoassay; Paint cross-section; Protein binders; Scanning electrochemical microscopy