Portable electrochemical biosensing platform for rapid, on-site detection of zearalenone mycotoxin in grain corn and animal feed

Nur Azura Mohd Said; Norhafniza Awaludin; Erna Mutiara Masdek; Sahira Akmar Zulkepli; Syah Noor Muhammad Ramli; Mohd Afendy Abdul Talib; Mohd Effendi Mohamed Nor; Halimah Hashim; Nurul Afza Karim; Mohammad Rejab Ismail; Hazana Razali

doi:10.51200/jsffs.v2i2.7498

Authors

Nur Azura Mohd Said Biotechnology & Nanotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI) Headquarter, 43400 Serdang, Selangor, Malaysia.
Norhafniza Awaludin Biotechnology & Nanotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI) Headquarter, 43400 Serdang, Selangor, Malaysia.
Erna Mutiara Masdek Biotechnology & Nanotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI) Headquarter, 43400 Serdang, Selangor, Malaysia.
Sahira Akmar Zulkepli Biotechnology & Nanotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI) Headquarter, 43400 Serdang, Selangor, Malaysia.
Syah Noor Muhammad Ramli Biotechnology & Nanotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI) Headquarter, 43400 Serdang, Selangor, Malaysia.
Mohd Afendy Abdul Talib Biotechnology & Nanotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI) Headquarter, 43400 Serdang, Selangor, Malaysia.
Mohd Effendi Mohamed Nor 2 Industrial Crop Research Centre, MARDI Headquarter, 43400 Serdang, Selangor, Malaysia.
Halimah Hashim 2 Industrial Crop Research Centre, MARDI Headquarter, 43400 Serdang, Selangor, Malaysia.
Nurul Afza Karim Industrial Crop Research Centre, MARDI Bachok, 16310 Bachok, Kelantan, Malaysia.
Mohammad Rejab Ismail MARDI Arau, Lot PT3747 Tambun Tulang, 02600 Arau, Perlis, Malaysia.
Hazana Razali Biogenes Technologies, Jalan Maklumat, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

DOI:

https://doi.org/10.51200/jsffs.v2i2.7498

Keywords:

electrochemical biosensor, grain corn, internet of things (IoT), mycotoxin, portable detection, zearalenone

Abstract

Zearalenone (ZEA) is a heat-stable estrogenic mycotoxin produced by Fusarium species that frequently contaminates cereal grains and animal feed, posing severe threats to food safety, livestock health, and agricultural economies. While laboratory-based detection methods are highly accurate, they are often time-consuming and unsuitable for the real-time, on-site applications required by smart agricultural systems. Here, we present a portable electrochemical biosensing platform designed for rapid, decentralized ZEA quantification in raw grain corn and processed poultry feed. Screen-printed carbon electrodes (SPCEs) were functionalized with a conductive polyaniline/gold nanoparticle (PANi/AuNP) nanocomposite, providing a high-surface-area matrix for stable anti-ZEA polyclonal antibody immobilization and accelerated electron transfer. Using differential pulse voltammetry (DPV) with an optimized 1:5 matrix-to-buffer dilution ratio to minimize matrix effects, the platform achieved a linear detection range of 0-200 µg/kg (ppb) with limits of detection (LOD) of 36.88 ppb in buffer and 57.66 ppb in matrix, aligning with international regulatory thresholds. The biosensor demonstrated high analytical accuracy with spiked recoveries between 80.19% and 93.92%, while functionalized SPCE strips maintained robust storage stability at room temperature for 14 days. The sensor strips were coupled with the handheld MARDIsense reader and a smartphone application for automated data processing and real-time cloud logging. Validation across 52 real-world agricultural samples (n=37 raw grains; n=15 feed/kernel matrices) demonstrated 100% categorical agreement and strong quantitative correlation (r=0.931, p<0.001) relative to reference ELISA. Crucially, epidemiological profiling revealed low contamination in pre-harvest field grains (94.6% <50 ppb) contrasting with severe contamination in processed poultry feed (50% > 100 ppb). This highlights the platform's vital utility for prioritizing post-harvest feed mill surveillance to safeguard the food supply chain.

References

Ascoli, C. A., & Aggeler, B. (2018). Overlooked benefits of using polyclonal antibodies. Biotechniques, 65(3), 127–136. https://doi.org/10.2144/btn-2018-0065

Aymard, C., Kanso, H., Serrano, M. J., Pagán, R., Noguer, T., & Istamboulie, G. (2022). Development of a new dual electrochemical immunosensor for a rapid and sensitive detection of enrofloxacin in meat samples. Food Chemistry, 370, 131016. https://doi.org/10.1016/j.foodchem.2021.131016

Azri, F. A., Selamat, J., Sukor, R., Yusof, N. A., Raston, N. H. A., Eissa, S., Zourob, M. & Chinnappan, R. (2021). Determination of minimal sequence for zearalenone aptamer by computational docking and application on an indirect competitive electrochemical aptasensor. Analytical and Bioanalytical Chemistry, 413(15), 3861–3872. https://doi.org/10.1007/s00216-021-03336-1

Bianchi, V., Boni, A., Bassoli, M., Giannetto, M., Fortunati, S., Careri, M., & De Munari, I. (2021). IoT and biosensors: A smart portable potentiostat with advanced cloud-enabled features. IEEE Access, 9, 141544–141554. https://doi.org/10.1109/ACCESS.2021.3120022

Curulli, A. (2021). Electrochemical biosensors in food safety: Challenges and perspectives. Molecules, 26(10), 2940. https://doi.org/10.3390/molecules26102940

European Commission. (2006). Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union, L 70, 12–34.

Food and Agriculture Organization of the United Nations (FAO). (2024). Agricultural production statistics. FAO Knowledge Repository. https://openknowledge.fao.org

Forzato, C., Vida, V., & Berti, F. (2020). Biosensors and sensing systems for rapid analysis of phenolic compounds from plants: A comprehensive review. Biosensors, 10(9), 105. https://doi.org/10.3390/bios10090105

Foubert, A., Beloglazova, N. V., Hedström, M., & De Saeger, S. (2019). Antibody immobilization strategy for the development of a capacitive immunosensor detecting zearalenone. Talanta, 191, 202–208. https://doi.org/10.1016/j.talanta.2018.08.062

Fox, P. F., & Brodkorb, A. (2008). The casein micelle: Historical aspects, current concepts and significance. International Dairy Journal, 18(7), 677–684. https://doi.org/10.1016/j.idairyj.2008.03.002

Fumagalli, F., Ottoboni, M., Pinotti, L., & Cheli, F. (2021). Integrated mycotoxin management system in the feed supply chain: Innovative approaches. Toxins, 13(8), 572. https://doi.org/10.3390/toxins13080572

Goud, K. Y., Kumar, V. S., Hayat, A., Gobi, K. V., Song, H., Kim, K. H., & Marty, J. L. (2019). A highly sensitive electrochemical immunosensor for zearalenone using screen-printed disposable electrodes. Journal of Electroanalytical Chemistry, 832, 336–342. https://doi.org/10.1016/j.jelechem.2018.10.058

Gruber-Dorninger, C., Jenkins, T., & Schatzmayr, G. (2019). Global mycotoxin occurrence in feed: A ten-year survey. Toxins, 11(7), 375. https://doi.org/10.3390/toxins11070375

Guo, K. (2024). Design and fabrication of a molecularly imprinted electrochemical sensor with high sensitivity for zearalenone assessment in maize. International Journal of Electrochemical Science, 19(6), 100612. https://doi.org/10.1016/j.ijoes.2024.100612

Jiang, X., Mu, Z., Wang, J., Zhou, J., & Bai, L. (2024). A novel sandwich-type electrochemical immunosensor for sensitive detection of zearalenone using NG/PDDA/HNTs and Ti-MOF-KB composites for signal amplification. Food Chemistry, 436, 137704. https://doi.org/10.1016/j.foodchem.2023.137704

Jusoh, N. S., Said, N. A. M., Salam, F., Awaludin, N., Kadir, A. A. A., & Ismail, M. R. (2022). An integrated handheld biosensing device with IoT system for in-situ and rapid detection of multi-mycotoxins. 2022 4th International Conference on Smart Sensors and Application (ICSSA), 11–16. IEEE. https://doi.org/10.1109/ICSSA54161.2022.9870938

Li, R., Wen, Y., Wang, F., & He, P. (2021). Recent advances in immunoassays and biosensors for mycotoxins detection in feedstuffs and foods. Journal of Animal Science and Biotechnology, 12(1), 108. https://doi.org/10.1186/s40104-021-00629-4

McDougal, T. (2025, March 9). Persistently high mycotoxin co-contamination levels pose ongoing challenges. https://www.dairyglobal.net/health-and-nutrition/persistently-high-mycotoxin-co-contamination-levels-poses-ongoing-challenges

Meliana, C., Liu, J., Show, P. L., & Low, S. S. (2024). Biosensor in smart food traceability system for food safety and security. Bioengineered, 15(1). https://doi.org/10.1080/21655979.2024.2310908

Nji, Q. N., & Mwanza, M. (2024). Three-year multi-mycotoxin analysis of South African commercial maize from three provinces. Frontiers in Fungal Biology, 5, 1426782. https://doi.org/10.3389/ffunb.2024.1426782

Radi, A. E., Eissa, A., & Wahdan, T. (2020). Molecularly imprinted impedimetric sensor for determination of mycotoxin zearalenone. Electroanalysis, 32(8), 1788–1794. https://doi.org/10.1002/elan.201900528

Rai, A., Das, M., & Tripathi, A. (2020). Occurrence and toxicity of a fusarium mycotoxin, zearalenone. Critical Reviews in Food Science and Nutrition, 60(16), 2710–2729. https://doi.org/10.1080/10408398.2019.1655388

R-Biopharm AG. (2023). RIDASCREEN®FAST Zearalenon: Competitive enzyme immunoassay for the quantitative analysis of zearalenone in cereals and feed (Art. No. R5502) [Product specifications and instructions for use]. R-Biopharm AG. https://food.r-biopharm.com/products/ridascreenfast-zearalenon/

Rizzotto, F., Khalife, M., Hou, Y., Chaix, C., Lagarde, F., Scaramozzino, N., & Vidic, J. (2023). Recent advances in electrochemical biosensors for food control. Micromachines, 14(7), 1412. https://doi.org/10.3390/mi14071412

Robin, P., & Gerber-Lemaire, S. (2023). Design and preparation of sensing surfaces for capacitive biodetection. Biosensors, 13(1), 17. https://doi.org/10.3390/bios13010017

Said, N. A. M., Awaludin, N., Ismail, M. R., Razali, H., Masdek, E. M., Zulkepli, S. A., & Ramli, S. N. M. (2025). Polyclonal Antibodies against Zearalenone: Production, Characterisation, and Application in Food Safety Biosensors. Pertanika Proceedings 1 (7): 133–137 (2025).

Seaton, B. T., Hill, D. F., Cowen, S. L., & Heien, M. L. (2020). Mitigating the effects of electrode biofouling-induced impedance for improved long-term electrochemical measurements in vivo. Analytical Chemistry, 92(9), 6334–6340. https://doi.org/10.1021/acs.analchem.9b05194

Siva Sangu, S., Chandra Bose Gopinath, S., Abdul Shukur, M. F., & Mohamed Saheed, M. S. (2022). An electrochemical approach for ultrasensitive detection of zearalenone in commodity using disposable screen-printed electrode coated with MXene/chitosan film. BioNanoScience, 12(3), 814–823. https://doi.org/10.1007/s12668-022-00984-0

Stejskal, J., & Gilbert, R. G. (2002). Polyaniline. Preparation of a conducting polymer (IUPAC technical report). Pure and Applied Chemistry, 74(5), 857–867. https://doi.org/10.1351/pac200274050857

Thongrussamee, T., Kuzmina, N. S., Shim, W. B., Jiratpong, T., Eremin, S. A., Intrasook, J., & Chung, D. H. (2008). Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals. Food Additives and Contaminants, 25(8), 997–1006. https://doi.org/10.1080/02652030701823159

Umapathi, R., Ghoreishian, S. M., Rani, G. M., Cho, Y., & Huh, Y. S. (2022). Emerging trends in the development of electrochemical devices for the on-site detection of food contaminants. ECS Sensors Plus, 1(4), 044601. https://doi.org/10.1149/2754-2726/ac9d4a

United States Department of Agriculture Foreign Agricultural Service (USDA-FAS). (2024). World Agricultural Production: Grain Markets and Trade. USDA FAS Report. https://www.fas.usda.gov/data/production/0440000

Urraca, J. L., Benito-Peña, E., Pérez-Conde, C., Moreno-Bondi, M. C., & Pestka, J. J. (2005). Analysis of zearalenone in cereal and swine feed samples using an automated flow-through immunosensor. Journal of Agricultural and Food Chemistry, 53(9), 3338–3344. https://doi.org/10.1021/jf048092p

USDA Foreign Agricultural Service. (2023). Malaysia: Grain and Feed Annual. GAIN Report No. MY2023-0001. United States Department of Agriculture.

Wan Syahidah, H., Suhaimi, D., Hartini, Y., & Nurulhayati, A. B. (2021). Screening of fungal contamination of Aspergillus and Fusarium mycotoxin in grain corn from Peninsular Malaysia. In M. S. Bastami, M. Muhammad, M. A. M. Marzuki, Y. S. Ting, N. H. M. Zairi, M. H. Ahmad, M. M. M. Ajat, A. Q. Sazili, M. A. M. Noor, N. F. M. Sohaimi, & M. A. Azman (Eds.), Proceedings of the 40th Malaysian Society of Animal Production Annual Conference 2021 (pp. 157–159).

Wang, K., Lin, X., Zhang, M., Li, Y., Luo, C., & Wu, J. (2022). Review of electrochemical biosensors for food safety detection. Biosensors, 12(11), 959. https://doi.org/10.3390/bios12110959

Wang, X., Luo, Y., Huang, K., & Cheng, N. (2022). Biosensor for agriculture and food safety: Recent advances and future perspectives. Advanced Agrochem, 1(1), 3–6. https://doi.org/10.1016/j.aac.2022.08.002

Wang, Y., Wang, X., Zhang, H., Jiang, J., & Fotina, H. (2021). Synthesis of zearalenone immunogen and comparative analysis of antibody characteristics. International Journal of Analytical Chemistry, 2021(1), 7109383. https://doi.org/10.1155/2021/7109383

Yagati, A. K., Chavan, S. G., Baek, C., Lee, M. H., & Min, J. (2018). Label-free impedance sensing of aflatoxin B1 with polyaniline nanofibers/Au nanoparticle electrode array. Sensors, 18(5), 1320. https://doi.org/10.3390/s18051320

Yazid, S. N. E., Ng, W. J., Selamat, J., Ismail, S. I., & Samsudin, N. I. P. (2021). Diversity and toxigenicity of mycobiota in grain corn: A case study at pioneer grain corn plantations in Terengganu, Malaysia. Agriculture, 11(3), 237. https://doi.org/10.3390/agriculture11030237

Zambry, N. S., Prathaban, S. N., Ibrahim, F., Thiha, A., Karajacob, A. S., Jamaluddin, N. F., Syed Omar, S.F., Shariffuddin, I.I., Bustam, A. & Tay, S. T. (2025). A label-free electrochemical immunosensor on gold-printed electrode for the electrochemical detection of C-reactive protein from blood samples. Microchimica Acta, 192(7), 437. https://doi.org/10.1007/s00604-025-07252-4

Zeng, Y., Camarada, M. B., Lu, X., Tang, K., Li, W., Qiu, D., Wen, Y., Wu, G, Luo, Q. & Bai, L. (2022). Detection and electrocatalytic mechanism of zearalenone using nanohybrid sensor based on copper-based metal-organic framework/magnetic Fe3O4-graphene oxide modified electrode. Food Chemistry, 370, 131024. https://doi.org/10.1016/j.foodchem.2021.131024

Zhang, J., Wang, B., Xu, B., Cheng, G., & Dong, S. (2000). Amperometric quantification of polar organic solvents based on a tyrosinase biosensor. Analytical Chemistry, 72(15), 3455–3460. https://doi.org/10.1021/ac000373i

Zinedine, A., Soriano, J. M., Moltó, J. C., & Manes, J. (2007). Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food and Chemical Toxicology, 45(1), 1–18. https://doi.org/10.1016/j.fct.2006.07.030

Ziyatdinova, G., Guss, E., & Yakupova, E. (2021). Electrochemical sensors based on the electropolymerized natural phenolic antioxidants and their analytical application. Sensors, 21(24), 8385. https://doi.org/10.3390/s21248385