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Bioconjugates of mercaptocarboxylic acids functionalized AuNP and superoxide dismutase for superoxide electrochemical monitoring

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Abstract

The use of gold nanoparticles/superoxide dismutase (AuNP/SOD) bioconjugates is described as building blocks in SOD biosensor development for the quantification of superoxide in cell culture media. AuNP functionalization with 11-mercaptoundecanoic acid (MUA) and 4-mercaptobenzoic acid (MBA) (AuNPMUA and AuNPMBA) was used to improve SOD immobilization through EDC/NHS coupling using their –COOH terminus, leading to the formation of more stable bioconjugates. AuNP and AuNP/SOD bioconjugates were characterized by SEM to determine their size and morphology, UV–Vis for optical properties, FT-IR, and Raman spectroscopies for chemical functional group analysis and EDX for elemental analysis. Electrochemical methods were used to characterize the Au/AuNP-modified electrodes. For the optimization of the biosensor architecture, different AuNP/enzyme bioconjugates were prepared by varying the amount of both enzyme and AuNP, as well as their incubation time. Finally, the biosensors incorporating the bioconjugates were characterized by fixed potential amperometry and voltammetric analysis in order to establish the enzymatic mechanism and to elucidate the best biosensor architecture for monitoring superoxide in cell culture media. The best sensitivity value for superoxide detection corresponded to 41.2 nA µM cm−2, achieved by a biosensor based on AuNPMBA/SOD bioconjugates monitored through fixed potential amperometry at 0.3 V vs. Ag/AgCl, with a limit of detection of 1.0 µM, and overall very good operational stability, maintaining 91% of the initial sensitivity after 30 days. Finally, the optimized biosensor was employed for the quantification of successive additions of superoxide in cell culture media, with excellent recovery values.

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Acknowledgements

The authors thank Dr. George E. Stan for the FT-IR measurements.

Funding

Financial support from the Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI), Romania, Project code PN-III-P1-1.1-TE-2019–0387 and PN-III-P1-1.1-PD-2019–0100, and Romanian Ministry of Research and Innovation through Operational Programme Competitiveness 2014–2020, Project NANOBIOSURF-SMIS 103528.

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Authors and Affiliations

  1. National Institute of Materials Physics, Str. Atomistilor 405A, 077125, Măgurele, Romania

    Caroline G. Sanz, Daniel N. Crisan, Ricardo J. B. Leote, Melania Onea & Madalina M. Barsan

  2. Faculty of Physics, University of Bucharest, Str. Atomistilor 405, 077125, Măgurele, Romania

    Ricardo J. B. Leote & Melania Onea

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  1. Caroline G. Sanz
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  2. Daniel N. Crisan
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Correspondence to Madalina M. Barsan.

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Sanz, C.G., Crisan, D.N., Leote, R.J.B. et al. Bioconjugates of mercaptocarboxylic acids functionalized AuNP and superoxide dismutase for superoxide electrochemical monitoring. Microchim Acta 189, 245 (2022). https://doi.org/10.1007/s00604-022-05352-z

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