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^5,6. The presence of AFB₁ in maize not only affects crop yield and quality but also poses a serious threat to both human and animal health through dietary exposure^7,8.

Currently, aflatoxin management predominantly relies on post-harvest strategies, such as proper drying, sorting damaged kernels, chemical treatments (e.g., ammonia or ozone), irradiation, and hermetic storage to prevent fungal growth during storage⁹. Strategies include good crop management, biocontrol agents (such as lime), and improved storage facilities¹⁰. Physical and chemical treatments, such as microwave, ultraviolet (UV), pulsed light, electrolyzed water, cold plasma, ozone, electron beam, and gamma irradiation, have also been tested¹¹. However, these post-harvest methods are often costly, energy-intensive, or impractical for widespread use, and they do not prevent aflatoxin translocation within the plant during growth.

Pre-harvest approaches that are being used include agronomic practices like resistant cultivars, timely planting, irrigation to reduce drought stress, and biocontrol with aflatoxigenic A. flavus strains (e.g., Aflasafe), which competitively exclude toxigenic strains but require repeated applications and may not fully address soil-borne translocation^11,12,13. These methods often face limitations: post-harvest interventions cannot reverse pre-existing contamination, incur high costs for sorting/storage infrastructure in resource-limited settings, risk nutrient loss or residues from chemicals, and biocontrol efficacy varies with environmental conditions¹⁴.

In contrast, green-synthesized CuO-ZnO hybrid nanoparticles offer a novel pre-harvest soil amendment strategy. Unlike conventional chemical NPs, green synthesis using plant extracts (e.g., mushroom substrate) avoids toxic solvents, reduces costs, and enhances biocompatibility, making them environmentally sustainable^15,16. The CuO-ZnO hybridization synergistically enhances antifungal activity by generating reactive oxygen species (ROS), disrupting fungal membranes, and providing a high surface area for AFB₁ adsorption, thereby preventing uptake and translocation into edible plant parts like grains. This differs from existing strategies by enabling early intervention at the soil-plant interface, achieving up to 80-99% AFB₁ reduction at low doses (e.g., 125 mg/kg), without compromising crop yield or leaving harmful residues¹⁷. Key advantages include eco-friendliness compared to chemical fungicides, superior efficacy compared to single-metal NPs or biocontrol under variable field conditions, and potential for scalable integration into farming practices in aflatoxin-prone regions such as sub-Saharan Africa.

Metal oxide NPs, specifically zinc oxide (ZnO) and copper oxide (CuO), have attracted attention among these materials because of their antifungal characteristics and capacity to absorb toxins^18,19. For example, it was reported that Cu NPs have the ability to create ROS, which can harm bacterial and fungal biological macromolecules²⁰. Also, in another experiment, it was demonstrated that fungal strains have a notable shift with a clear tendency toward an order increase when F. Solani was treated with chitosan-based CuO NPs, resulting in the largest ordering effect²¹. Green synthesis of NPs has gained attention as a safer and sustainable alternative to conventional chemical methods, thereby minimizing toxic byproducts and environmental impact^15,22.

The hybridization of CuO and ZnO NPs combines the beneficial properties of both metals, potentially enhancing their efficacy in degrading or adsorbing AFB₁^23,24. Presently, these green-synthesized NPs are mainly used to control AF production in fungal strains in in vitro experiments; for example, Sheik and Awad studied the impact of AgNPs, AuNPs, and their mixture on the growth and aflatoxin B₁ accumulation of the aflatoxigenic A. flavus strain²⁵. However, the concentration required to control A. flavus growth is unknown. Thus, there is a critical need to evaluate green CuO-ZnO hybrids under field-relevant conditions for their impact on AFB₁ translocation from soil-amended sources into maize tissues, compared to conventional NPs. Hence, the objectives of this study were to determine the effect of different concentrations of CuO/ZnO green hybrid NPs on AFB₁/A. flavus concentration in the maize plant tissues and to assess the risk of AFB₁ in the maize after treating the maize with CuO/ZnO green hybrid NPs. Research hypotheses were to provide a mechanistic basis for the observed efficacy of green-synthesized CuO-ZnO hybrid NPs. This study tests the following measurable hypotheses: 1) The antifungal inhibition hypothesis, whereby hybrid NPs were to significantly reduce A. flavus sporulation and subsequent de novo AFB₁ production in soil compared to single-metal NPs, identified by lower fungal load and toxin levels at the Molelwane site. 2) The adsorption-translocation hypothesis: in which the presence of pre-existing toxins (Campus site), the NPs were to demonstrate a high binding affinity for AFB₁, significantly decreasing the rate of toxin translocation from the soil to the maize root system. 3) The biocompatibility (phytotoxicity) hypothesis: whereby green-synthesized hybrid NPs were to result in lower residual metallic accumulation and reduced oxidative stress markers in maize tissues compared to conventional chemical benchmarks, thereby enhancing crop biosafety.

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 content, and were previously classified as Arcadia form²⁵. The CuO/ZnO green hybrid NPs were synthesized in the laboratory of the Department of Chemistry, School of Physical and Chemical Sciences, Faculty of Natural and Agricultural Science, NWU, in South Africa.

Synthesis of CuO/ZnO green hybrid NPs
Ten (10) g of dried Pleurotus ostreatus spent mushroom substrate (SMS) was added to 100 mL of de-ionized water, heated at 70 °C, and stirred for 2 h²⁶. The mixture was then cooled to room temperature, filtered, and 30 mL of the filtrate was added to a solution containing 2.5 g of copper acetate and 2 g of zinc acetate in 30 mL of distilled water. This solution’s pH was adjusted to 8 using sodium hydroxide (NaOH), heated up to 80 °C for 2 h, centrifuged after the reaction, dried overnight in an oven, and calcinated at 650 °C for 2 h²⁶. The synthesized NPs were characterized and confirmed as CuO/ZnO green hybrid NPs.

Soil sampling and analysis
Soil samples were collected from the University Farm at Molelwane and the campus trial site at depths of 0–15 cm and 15–30 cm. They were subsequently categorized and examined for their physical and chemical properties, as it was described by²⁷.

Experimental design
The study employed a factorial experiment arranged in a randomized complete block design (RCBD) with three replicates. To evaluate the efficacy of green-synthesized CuO-ZnO hybrid NPs under diverse contamination scenarios, two independent exposure modes were established across separate sites: Site 1 (North-West University Campus) represented an exogenous contamination model where soil was spiked with extracted AFB₁ at a rate of 160 ppb/kg to evaluate the NPs' direct adsorption and translocation-shielding capabilities. Site 2 (Molelwane Research Farm): Represented a biological infection model where soil was inoculated with A. flavus spores at 160 spores/kg to investigate the NPs' antifungal impact on de novo mycotoxin production and subsequent plant uptake. The powdery NPs were weighed and uniformly mixed with 1 kg of soil in a planting plastic bag after the inoculation (AFB₁ or A. flavus spores). Each treatment was performed separately, and the hands were sanitized between treatments. Then, four maize seeds were initially planted in each pot (9 cm top diameter, 6.5 cm bottom diameter). To keep three healthy seedlings per pot, the seedlings were trimmed a week after they emerged. In accordance with the recommendations for optimal maize production derived from soil analysis, the soil was fertilized. The pots were placed at inter- and intra-row spacings of 0.75 m × 0.3m, for a total of 216 pots. Soil in pots was watered to 60% field capacity after the seeds were sown. Soil and plant samples were collected at three developmental stages: V10 (development), R1 (flowering), and R6 (maturity). Samples were then brought to the lab in an ice-filled cooler box and kept at -20 °C for further examinations. Brown paper bags were used to store and deep-freeze the remaining plant samples for AFB₁ concentrations and other assays. The remaining plant samples for AFB₁ concentrations and other analyses were kept in brown paper bags and deep-frozen until analysis.

Maize aflatoxins analysis
Maize development components (root, stem, leaf, and grain) at various growth stages were gathered into sterile plastic bags, labeled with care, and transported to the lab. To maintain the integrity of the growth data and ensure independent observations for each developmental stage, a destructive sampling protocol was implemented. To eliminate cross-contamination between nanoparticle concentrations and inoculation types, harvesting tools and gloves were cleaned with 70% ethanol and rinsed with deionized water between every treatment group. Using a mortar and pestle, 10 g of the plant material and 1 g of sodium chloride (NaCl) were pulverized for 10 min in 25 mL of 80% methanol and 20% distilled water. For maize grain and maize core, 25 g of ground sample and 2.5 g of NaCl were blended with 100 mL of 80% methanol:20 distilled water at high speed for 10 min. Then, the homogenized extract was filtered through a glass microfiber filter, followed by washing with 20 mL of 20% Tween-2 in phosphate-buffered saline (PBS). Aflatoxins were extracted from the immune affinity column following elution at a flow rate of one drop per second, and the supernatant (cytoplasm) was collected into an amber vial. Then, AFB₁ and other aflatoxins were measured following the liquid chromatography method²⁸ which involved derivatizing aflatoxins with o-phthaldialdehyde solution and injecting the derivatized compounds into a high-performance liquid chromatography (HPLC) system. The HPLC system was equipped with a Jasco FP-920 fluorescence detector set to 362 nm excitation and 425 nm emission. The AFB₁ was inherently physiologically structured to be detected at an excitation wavelength of 362 nm. A Hichrom column (4.6 mm × 150 mm) with 5 µm particles was used, and the derivatization reactor was a KOBRA Cell program at 100 µA. Separations of chromatographic peaks were done in a Hichrom column to which a pre-column of similar stationary phase had been fitted. The Inertsil ODS-3 and ODS-3 V were used as guard and analytical cartridges, respectively, while the injector consisted of an autosampler with a reodyne valve. The mobile phase was composed of water: methanol (65:35, v/v), potassium bromide (119 mg), and 4 M nitric acid (350 µL) per liter, and was pumped at a 1.0 mL/min flow rate in an isocratic program. Aflatoxin detection was regarded as positive for each peak at a retention time similar to each standard and at a height five times higher than the baseline noise²⁹.

Limits of detection (LOD), limit of quantification (LOQ), and percentage (%) recovery were calculated to confirm the HPLC's analytical approach, using equations 1, 2, and 3, respectively. The LOD is the lowest concentration of aflatoxin that can be reliably distinguished from background noise, while LOQ is the lowest concentration of aflatoxin that can be quantitatively measured with acceptable precision and accuracy³⁰. AFB₁ and its metabolites were recovered by adding 10 µL of total aflatoxin standards (AFB₁, AFB₂, AFG₁, and AFG₂) to the negative maize control samples in triplicate. The samples were then extracted using HPLC utilizing the previously mentioned procedures.

LOD = X + 3s     1

LOQ = X + 10s     2

    3

Whereby: “X” is the mean concentration of fortified sample blank values, and “s” is the sample standard deviation.

AFB1 Exposure estimation
The mean aflatoxin levels in the maize diet, the daily intake of maize, and the average body weight (children, adults, beef cattle, dairy cows, and chicks) were used to calculate the estimated daily intake (EDI). The EDI for mean aflatoxins was calculated according to equation 2 and expressed in (ng/kg body weight/day); for the hazard index (HI) calculation, EDI values were converted to ng/kg body weight/day (1 ppb/kg/day = 1,000 ng/kg body weight/day) to match the units of TD5032³¹.

      4

Risk assessment and characterization
The hazard index (HI) was calculated by dividing the EDI by the TD50 and multiplying by a safety factor of 50,000. Median toxic dose (TD) 50 is the necessary dosage (ng/kg/body weight/day) to cause tumors in 50% of test animals that would not have developed tumors at zero dose^31,32.

Data analysis
Analysis of variance was performed on the collected growth and yield data using the RCBD procedure in SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Multiple comparisons of least squares means were carried out using Tukey’s honestly significant difference (HSD) test³³.

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 va...

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)