Abstract
This study presents a novel electrochemical aptasensor for ultrasensitive detection of aflatoxin M1 (AFM1) in milk samples, utilizing a gold electrode modified with chitosan-functionalized multiwalled carbon nanotube/graphene nanocomposite (CS/f-MWCNTs-Gr). The platform was fabricated through covalent immobilization of an amino-modified aptamer onto the nanocomposite surface, enhancing electronic transmission and biorecognition efficiency. Cyclic voltammetry (CV) confirmed stepwise electrode modification, while square wave voltammetry (SWV) quantified AFM1 via suppression of the [Fe(CN)6]3−/4− redox signal upon target binding. Under optimized conditions, the sensor demonstrated a wide linear range (1–1000 nM) covering EU/US regulatory limits, an ultra-low detection limit (0.03 nM, 9.8 ng/L, below EU regulatory limit of 25 ng/kg), and exceptional specificity (> 90% signal suppression against interferents at 10-fold higher concentrations). The aptasensor exhibited high reproducibility (RSD = 5.4%, n = 5) and retained 92% signal after 14-day storage. Validated in commercial milk, it achieved 96–106% recoveries with RSD < 4.9% (n = 5), outperforming reference methods in precision and practicality. This cost-effective platform shows significant potential for on-site monitoring of mycotoxins in dairy products.
Data availability
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
References
Yaacobi-Artzi, S., Kalo, D. & Roth, Z. Effect of the aflatoxins B1 and M1 on bovine oocyte developmental competence and embryo morphokinetics. Reprod. Toxicol. 120, 108437 (2023).
Min, L. et al. An overview of aflatoxin B1 biotransformation and aflatoxin M1 secretion in lactating dairy cows. Anim. Nutr. 7, 42–48 (2021).
Neal, G. E., Eaton, D. L., Judah, D. J. & Verma, A. Metabolism and toxicity of aflatoxins M1 and B1 in human-derived in vitro systems. Toxicol. Appl. Pharmcol. 151, 152–158 (1998).
Zhou, Y. et al. Quantum bead-based fluorescence-linked immunosorbent assay for ultrasensitive detection of aflatoxin M1 in pasteurized milk, yogurt, and milk powder. J. Dairy Sci. 102, 3985–3993 (2019).
Chen, Q. et al. Emerging biosensors to detect aflatoxin M1 in milk and dairy products. Food Chem. 398, 133848 (2023).
Prandini, A. et al. On the occurrence of aflatoxin M1 in milk and dairy products. Food Chem. Toxicol. 47, 984–991 (2009).
Ge, G. et al. A self-assembled DNA double-crossover-based fluorescent aptasensor for highly sensitivity and selectivity in the simultaneous detection of aflatoxin M1 and aflatoxin B1. Talanta, 265: 124908 (2023).
Jalalian, S. H. et al. A novel electrochemical aptasensor for detection of aflatoxin M1 based on target-induced immobilization of gold nanoparticles on the surface of electrode. Biosens. Bioelectron. 117, 487–492 (2018).
Zhao, S. et al. A novel α-Fe2O3 nanocubes-based multiplex immunochromatographic assay for simultaneous detection of Deoxynivalenol and aflatoxin B1 in food samples. Food Control. 123, 107811 (2021).
Singh, H. et al. Development of carbon quantum dot-based lateral flow immunoassay for sensitive detection of aflatoxin M1 in milk. Food Chem. 393, 133374 (2022).
Rabie, M., Movassaghghazani, M. & Mogaddam, M. R. HPLC-FLD determination of aflatoxins M1 and M2 in Raw cow milk samples using in-syringe gas-controlled density tunable solidification of a floating organic droplet-based dispersive liquid–liquid Microextraction method. RSC Adv. 14, 5077–5084 (2024).
Shuib, N. S., Makahleh, A., Salhimi, S. M. & Saad, B. Determination of aflatoxin M1 in milk and dairy products using high performance liquid chromatography-fluorescence with post column photochemical derivatization. J. Chromatogr. A. 1510, 51–56 (2017).
Abdul-Hay, M. E., Shendy, A. H., Aboalhassen, A. E., Gomaa, A. M. & Ismail, E. H. Development and validation of a simple solid-liquid extraction protocol coupled with LC-ESI-MS/MS for the determination of aflatoxin M1 in products of colostrum-based supplements and Whey protein-based sports food. J. Food Compos. Anal. 126, 105933 (2024).
Radoi, A., Targa, M., Prieto-Simon, B. & Marty, J. L. Enzyme-Linked immunosorbent assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection. Talanta 77, 138–143 (2008).
Liu, B. H., Chu, K. C. & Yu, F. Y. Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk. Food Control. 66, 1–7 (2016).
Ebanks, F., Nasrallah, H., Garant, T. M., McConnell, E. M. & DeRosa, M. C. Colorimetric detection of aflatoxins B1 and M1 using aptamers and gold and silver nanoparticles. Adv. Agrochem. 2, 221–230 (2023).
Aran, G. C. & Bayraç, C. Simultaneous dual-sensing platform based on aptamer-functionalized DNA hydrogels for visual and fluorescence detection of chloramphenicol and aflatoxin M1. Bioconjug. Chem. 34, 922–933 (2023).
Wang, M. et al. A label-free fluorescence strategy for analysis of aflatoxin M1 by self-protected DNAzyme and aptamer recognition triggered DNA walker cascade amplification. Microchem. J. 186, 108356 (2023).
Karczmarczyk, A., Baeumner, A. J. & Feller, K. H. Rapid and sensitive inhibition-based assay for the electrochemical detection of Ochratoxin A and aflatoxin M1 in red wine and milk. Electrochim. Acta. 243, 82–89 (2017).
Chrouda, A., Ayed, D., Zinoubi, K., Majdoub, H. & Jaffrezic-Renault, N. Highly stable and ultra-sensitive amperometric aptasensor based on pectin stabilized gold nanoparticles on graphene oxide modified GCE for the detection of aflatoxin M1. Food Chem. Adv. 1, 100068 (2022).
Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).
Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).
Velasco-Garcia, M. & Missailidis, S. New trends in aptamer-based electrochemical biosensors. Gene Therapy Mol. Biology. 13, 1–10 (2009).
Deiminiat, B., Rounaghi, G. H., Arbab-Zavar, M. H. & Razavipanah, I. A novel electrochemical aptasensor based on f-MWCNTs/AuNPs nanocomposite for label-free detection of bisphenol A. Sens. Actuators B. 242, 158–166 (2017).
Bi, X. et al. Inner filter effect-modulated ratiometric fluorescence aptasensor based on competition strategy for zearalenone detection in cereal crops: Using Mitoxantrone as quencher of CdTe QDs@ SiO2. Food Chem. 349, 129171 (2021).
Wang, L. et al. Colorimetric aptasensor utilizing MOF-235 with exceptional peroxidase-like activity for the detection of oxytetracycline residues in raw milk. Food Chem. 447, 138966 (2024).
Liu, S. et al. A facile dual-mode aptasensor based on AuNPs@ MIL-101 nanohybrids for ultrasensitive fluorescence and surface-enhanced Raman spectroscopy detection of Tetrodotoxin. Biosens. Bioelectron. 201, 113891 (2022).
Dursun, A. D., Borsa, B. A., Bayramoglu, G., Arica, M. Y. & Ozalp, V. C. Surface plasmon resonance aptasensor for Brucella detection in milk. Talanta 239, 123074 (2022).
Deiminiat, B. & Rounaghi, G. H. Fabrication of a novel photoelectrochemical aptasensor using gold nanoparticle-sensitized TiO2 film for quantitative determination of Diazinon in solutions. Electrocatalysis 14, 484–498 (2023).
Zhang, R. et al. Electrochemical aptasensor with signal amplification strategy of covalent organic framework-derived carbon material for ultrasensitive determination of carbendazim. Microchem. J. 200, 110450 (2024).
Jin, Y. et al. A ratiometric electrochemical aptasensor for sensitive detection of Kanamycin in food based on entropy-driven strand displacement reaction. Food Control. 161, 110390 (2024).
Al Fatease, A. et al. A dual-mode electrochemical aptasensor for the detection of Mucin-1 based on AuNPs-magnetic graphene composite. Microchem. J. 180, 107559 (2022).
Fan, L. et al. Design of a facile and label-free electrochemical aptasensor for detection of atrazine. Talanta 201, 156–164 (2019).
Hui, Y. et al. Rapid and label-free electrochemical aptasensor based on a palladium nanoparticles/titanium carbide/polyethyleneimine functionalized nitrogen-doped carbon nanotubes composite for amplified detection of streptomycin. Food Chem. 432, 137271 (2024).
Kolahi-Ahari, S., Rounaghi, G. H. & Deiminiat, B. Simultaneous determination of dopamine and paracetamol using a gold electrode modified with MWCNTs/GO nanocomposite capped Au nanoparticles. Anal. Bioanal. Electrochem. 14, 402–417 (2022).
Cao, S., Zhao, H., Chen, K., Zhou, F. & Lan, M. An electrochemical aptasensor based on multi-walled carbon nanotubes loaded with PtCu nanoparticles as signal label for ultrasensitive detection of adenosine. Anal. Chim. Acta. 1260, 341212 (2023).
Ansari, M. A., Liew, W. J., Kurup, C. P. & Ahmed, M. U. Label-free electrochemical aptasensor for ultrasensitive thrombin detection using graphene nanoplatelets and carbon nano onion-based nanocomposite. J. Electroanal. Chem. 937, 117422 (2023).
Eteya, M. M., Rounaghi, G. H. & Deiminiat, B. Electrochemical determination of tyramine using a carbon ionic liquid paste electrode modified with f-MWCNTs/graphene nanocomposite film. Electrocatalysis 14, 688–696 (2023).
Abedi, R., Raoof, J. B., Mohseni, M. & Hashkavayi, A. B. Sandwich-type electrochemical aptasensor based on hemin-graphite oxide as a signal label and rGO/MWCNTs/chitosan/carbon quantum dot modified electrode for sensitive detection of acinetobacter baumannii bacteria. Anal. Chim. Acta. 1303, 342491 (2024).
Shafiei, F., Saberi, R. S. & Mehrgardi, M. A. A label-free electrochemical aptasensor for breast cancer cell detection based on a reduced graphene oxide-chitosan-gold nanoparticle composite. Bioelectrochemistry 140, 107807 (2021).
Khezrian, S., Salimi, A., Teymourian, H. & Hallaj, R. Label-free electrochemical IgE aptasensor based on covalent attachment of aptamer onto multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite modified electrode. Biosens. Bioelectron. 43, 218–225 (2013).
Yu, Y. et al. Dual-target electrochemical sensor based on 3D MoS2–rGO and aptamer functionalized probes for simultaneous detection of Mycotoxins. Front. Chem. 10, 932954 (2022).
Thurner, F. & AlZahra’a Alatraktchi, F. Recent advances in electrochemical biosensing of aflatoxin M1 in milk—A mini review. Microchem. J. 190, 108649 (2024).
Li, H. et al. Ratiometric electrochemical aptasensor based on split aptamer and Au-RGO for detection of aflatoxin M1. J. Dairy Sci. 107, 2748–2759 (2024).
Dinckaya, E., Kınık, O., Sezgintürk, M. K., Altuğ, Ç. & Akkoca, A. Development of an impedimetric aflatoxin M1 biosensor based on a DNA probe and gold nanoparticles. Biosens. Bioelectron. 26, 3806–3811 (2011).
Nguyen, B. H. et al. Label-free detection of aflatoxin M1 with electrochemical Fe3O4/polyaniline-based aptasensor. Materials Sci. And Engineering: C. 33, 2229–2234 (2013).
Author information
Authors and Affiliations
Contributions
R.V.Z.: Conceptualization, Investigation, Methodology, Formal analysis, Writing - original draft; A.M.S.: Supervision, Project administration, Funding acquisition, Writing - review & editing; V.H.: Validation, Data curation, Visualization; A.F.: Resources, Software, Formal analysis.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Zadeh, R.V., Sani, A.M., Hakimzadeh, V. et al. Ultrasensitive on-site detection of aflatoxin M1 in milk using a chitosan-MWCNT-graphene nanocomposite aptasensor with sub-regulatory limit capability. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38492-w
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-38492-w
