Localization of proteins in paint cross-sections by scanning electrochemical microscopy as an alternative immunochemical detection technique

• Advanced immuno-electrochemical detection of proteins in paint samples by SECM.
• Analysis performed directly on cross-section with high spatial resolution.
• Identification of HRP catalytic activity for a selective location of analyte.
• Satisfactory results were obtained for aged real samples.
• The way forward for an extensive application of SECM in conservation science is shown.

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.

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