Green CuO/ZnO Nanoparticles: Reducing AFB1 Translocation and Accumulation in Maize

Summary

This study investigated the efficacy of green-synthesized CuO/ZnO hybrid nanoparticles in reducing aflatoxin B₁ (AFB₁) accumulation and translocation in maize, demonstrating that a 125 mg/kg treatment significantly lowered AFB₁ levels and minimized human and animal dietary exposure risks.

Abstract

Aflatoxin B₁ (AFB₁) is a potent carcinogen produced by Aspergillus species that contaminates maize crops, posing severe health risks to humans and animals through dietary exposure. Traditional post-harvest management and chemical interventions are often costly or ineffective at preventing the translocation of soil-borne toxins into the plant during growth. The primary goal of this protocol was to evaluate green-synthesized copper oxide/zinc oxide (CuO/ZnO) hybrid nanoparticles (NPs) as a sustainable pre-harvest soil amendment. The study aimed to determine if these NPs could effectively inhibit the translocation and accumulation of AFB₁ within maize tissues, thereby reducing toxin accumulation in edible grains. Using a randomized complete block design across two distinct field sites, maize was grown in soil inoculated with AFB₁ or Aspergillus flavus spores and treated with varying concentrations of NPs (0–125 mg/kg). The protocol employed green synthesis with Pleurotus ostreatus substrate to ensure eco-friendliness. Plant tissues were analyzed at different physiological stages using High-Performance Liquid Chromatography (HPLC) to measure AFB₁ levels and calculate human and livestock risk assessments. Treatment with 125 mg/kg of green hybrid NPs significantly (p < 0.05) decreased AFB₁ concentrations across all growth stages. At the on-campus site, AFB₁ was reduced by 68% at the vegetative stage, 82% at flowering, and 76% at maturity compared to the positive control. Crucially, grain AFB₁ levels dropped to 6.91 ppb, significantly lowering the estimated daily intake for humans. However, further research is required to evaluate potential nanoparticle residues and long-term toxicity. This method demonstrates that green CuO/ZnO hybrid NPs act as an effective bioremediation agent, preventing the transport of toxins to edible parts of the plant. This approach offers a scalable, environmentally safe strategy to enhance food security and mitigate AFB₁ exposure in aflatoxin-prone regions.

Introduction

Aflatoxins, particularly Aflatoxin B₁ (AFB₁), are among the most significant mycotoxins due to their prevalence, toxicity, and impact on humans. They are produced by certain fungi, primarily those belonging to the Aspergillus species^1,2,3. AFB₁ is linked to hepatocellular cancer and is the most hazardous of all the aflatoxins⁴. These powerful carcinogens infect a variety of agricultural crops, including maize (Zea mays L.), leading to serious food safety concerns^5,

Protocol

Experimental sites
The trials were conducted at two sites located 7 km apart; the sites were inoculated with different treatments. In the first trial, a net-house facility with 40% shade was set up at the Molelwane, North-West University (NWU) Research Farm (25.810°S, 25.630°E, 1276 m) in the North-West Province of South Africa. The red sandy loam soil was categorized as Hutton form and contained 75% sand, 4% silt, and 21% clay²⁵. The second experiment was conducted at NWU Mafikeng campus’s (25. 8278 °S, 25. 6079 °E, 1276 m) net-house facility. The soils in the second site had sandy loam with 82% sand, 3% silt, 15% clay con....

Results

Aflatoxin concentration in growth components
The results showed that aflatoxin concentrations in the growth components and in maize grain treated with green-synthesized CuO/ZnO NPs were detected, indicating the presence of all the major aflatoxin types (AFG₂, AFG₁, AFB₂, and AFB₁). The calibration curves used to calculate aflatoxin concentrations showed good linearity across the different aflatoxins, with r² valu.......

Discussion

The success of this protocol in reducing AFB₁ translocation and accumulation in maize depends on several critical steps that ensure both biological efficacy and analytical reliability^16,27. The results demonstrate that 125 mg/kg of green-synthesized CuO-ZnO hybrid NPs significantly reduced AFB₁ concentrations in both experimental models, albeit via potentially distinct mechanisms¹⁶. Adsorption Dynamics (Campus Site): .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Copper(II) acetate [Cu(CO2CH3)2]	Sigma-Aldrich Pty, St Louis, MO, USA	10,53,753	Dark blue-green crystalline solid (98% purity)
Glass microfiber filter	Sigma-Aldrich Pty, St Louis, MO, USA	1703965	Borosilicate glass
High-performance liquid chromatography (HPLC) system	Shimadzu	20H2	For seperating and quantification of analytes
HPLC grade Ethanol	Sigma-Aldrich Pty, St Louis, MO, USA	34923	High-purity colourless solvent ( ≥ 99 %)
HPLC grade Methanol	Merck KGaA, Darmstadt, Germany	322415	High-purity colourless solvent ( ≥ 99 %)
Immune affinity column	Shimadzu	KJ524	For selective purification of target analytes
Maize seeds of PAN 5R-590R	NWK	N/A	Small, hard kernels with white coluor
Phosphate buffered saline (PBS)	Merck KGaA, Darmstadt, Germany	3526580	An isotonic buffer solution
Plastic bags	Eco-Agro Enterprise (Pty) Ltd	N/A	Perforated
Purified AFB1, AFB2, AFG1, AFG2 and AFs-total in situ	Trilogy Analytical Laboratory, Inc (Vossbrink Drive, WA, USA)	TAS-M12DA1-10	White to off-white crystalline solid
Sodium chloride (NaCl)	Sigma-Aldrich Pty, St Louis, MO, USA	N/A	White crystalline solid
Zinc acetate [Zn(CH3COO2)2H2O]	VWR chemicals	2331 210 2	White crystalline solid (99 % purity)