Microfabricated gold-based aptasensors for label-free electrochemical assay of oxytetracycline residues in milk

Antibiotics are commonly used in veterinary medicine purposes to prevent and treat bacterial infections in food-producing animals [1]. As a result, antibiotic residues may be present in animal-derived products (meat, eggs, milk and dairy products) and eventually consumed by humans, potentially causing resistance to antibiotics and other health issues [2]. Oxytetracycline (OTC) is a commonly used antibiotic in farming animals; in order to protect consumers, a maximum residue limit (MRL) of 100 µg L^-1 has been established by the European Union for OTC residues in milk [3].

Although liquid chromatography is considered the default and most powerful analytical methodology for antibiotics residues [4], biosensors constitute an alternative technology that offers rapidity, low-cost and scope for on-site or field assays. Biosensors are based on a signal arising from the interaction event between the target and a selective bioreceptor [5]. In particular, aptasensors are biosensing devices utilizing aptamers (short single-stranded oligonucleotides with high affinity towards specific targets) as bioreceptors; aptamers exhibit some unique and advantageous properties compared to other bioreceptors and, therefore, different aptasensing strategies have been applied to the assay of antibiotics including OTC [6].

In this work, we describe microfabricated gold-based electrochemical aptasensors for label-free detection of OTC. Thin-film gold electrodes were fabricated by sputtering of gold on a Kapton film. A thiol-modified OTC-specific aptamer was immobilized on the electrode surface exploiting thiol-gold interactions. Then, the sample was incubated with the aptamer-modified electrode to achieve selective capture of OTC. Finally, the binding between the immobilized aptamer and OTC was monitored electrochemically using cyclic voltammetry (CV), differential-pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) using the Fe(CN)₆⁴⁺/Fe(CN)₆³⁺ redox probe. Different experimental variables were studied and the metrological features for OTC were derived. Finally, proof-of-principle applicability of the sensors was demonstrated for the determination of OTC in milk.

This work was supported by European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 101007299.

[1] Virto M., Santamarina-García G, Amores G, Hernández I (2022) Antibiotics in Dairy Production: Where Is the Problem? Dairy 3:541-564, doi:https://doi.org/10.3390/dairy3030039
[2] Landers, T.F.; Cohen, B.; Wittum, T.E., Larson, E.L. A Review of Antibiotic Use in Food Animals: Perspective, Policy, and Potential. Public Health Rep. 2012, 127(1), 4–22. doi: http://www.jstor.org/stable/41639470.
[3] Commission Regulation (EU) No. 37/2010, https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32010R0037
[4] Peris-Vicente, J.; Peris-García, E.; Albiol-Chiva, J.; Durgbanshi, A.; Ochoa-Aranda, E.; Carda-Broch, S.; Bose, D.; Esteve-Romero, J. Liquid chromatography, a valuable tool in the determination of antibiotics in biological, food and environmental samples. Microchem. J. 2022, 177, 107309. doi: https://doi.org/10.1016/j.microc.2022.107309.
[5] Zhou, C.; Zou, H.; Sun, C.; Li. Y. Recent advances in biosensors for antibiotic detection: Selectivity and signal amplification with nanomaterials. Food Chem. 2021, 361, 130109. doi: https://doi.org/10.1016/j.foodchem.2021.130109.
[6] Liang, G.; Song, L.; Gao, Y.; Wu, K.; Guo, R.; Chen, R.; Zhen, J.; Pan, L. Aptamer Sensors for the Detection of Antibiotic Residues— A Mini-Review. Toxics 2023, 11, 513. doi: https://doi.org/10.3390/toxics11060513.