Mycotoxins are secondary metabolites produced by filamentous fungi, such as Aspergillus, Penicillium, Fusarium, etc., that are toxic to animals and humans upon consumption. The most common mycotoxins that are found to contaminate feed are aflatoxins, ochratoxins, fumonisins, patulin, zearalenone, and trichothecenes, including deoxynivalenol and T-2 toxin 1. Ochratoxin (A, B, and C) are common contaminants of agricultural products, such as barley, beans, coffee, groundnut, corn, oats, rice, wheat, and also percolate into animal products, such as eggs, milk, and meat, including human milk 1,2 .OTA is nephrotoxic, genotoxic, neurotoxic, immunotoxic, and embryotoxic with teratogenic and carcinogenic effects 3. According to the International Agency for Research on Cancer (IARC), OTA has been classified as a group 2B carcinogen 4,5. It was observed that there was an occurrence of renal tumors when the dietary intake was more than 70 µg/kg of OTA per day in humans 6. In poultry farms, if consumption of OTA contaminated feed is around 0.025 g/kg for a prolonged period, it may lead to tumor formation in the liver, kidneys, ureters, or spleen 7 .
Several researchers have attempted to develop strategies for rapid and sensitive detection of food-based toxic elements, including mycotoxins using different matrices, nanomaterials, and fluorescence quenchers, including quantum dots, Cu2+, Co2+, gold nanomaterials, and silica nanoparticles 8–12. However, only a few reports have been published on sensing devices showing improved performance on real food samples that may further lead to the development of portable commercialized sensing kits for easy conceptualization and their application in the detection of food toxicants in corn and groundnut samples. Luan et al. developed a label-free aptamer-based colorimetric sensing technique for the detection of OTA 13. Majdinasab et al. demonstrated the different colorimetric-based techniques, such as enzyme-linked assays, lateral flow-assays, microfluidic devices, and homogenous in-solution strategies 14. Moreover, to the best of our knowledge, there are very few reports available where comparative detection capabilities of the developed sensing technique have been compared with conventional detection methods in real food samples (corn and groundnut).
The conventional methods based on Thin Layer Chromatography (TLC), Gas Chromatography (GC), Mass Spectrometry (MS), High-Performance Liquid Chromatography (HPLC) require extensive sample preparation, highly skilled personnel, and are time-consuming 15,16, which demands the development of a rapid and sensitive method for detection of ochratoxin (OTA). Biosensors are analytical tools that can be used for on-site detection of narcotic drugs, herbicides, pesticides, cancer, preeclampsia, and arthritis 17–20. Aptamer-based assays are emerging as a promising alternative tool for quick detection of aflatoxins and ochratoxins based on an aggregation of gold nanoparticles (AuNPs) or microfluidic devices 16 ]. In addition, aptamer-based microfluidic devices can be used for the rapid detection of toxins and have gained ample significance due to their cost effectiveness in recent years.
The microfluidic device was fabricated in-house and combined with an aptamer switching technique to detect OTA in corn and groundnut samples. To the best of our knowledge, for the first time, we have demonstrated a colorimetric sensing method based on the AuNPs aggregation and aptamer switching technique in combination with a microfluidic device for detection of OTA in corn and groundnut. Along with ease of fabrication, this device was also rapid and cost-effective. Here, the aim of the present study was to quantify the detectable limit of OTA in corn and groundnut samples. For this, we have developed a microfluidic device for rapid colorimetric detection with specific aptamers coupled with gold nanoparticles (AuNPs). The specific aptamers physically adsorbed on the surface of AuNPs, and displacement occurred in the presence of OTA analyte due to its specificity. The biophysical characterization of OTA-AuNPs complex was achieved by UV-vis spectroscopy, DLS (dynamic light scattering), and TEM (transmission electron microscopy). The AuNPs were incubated with 36-mer aptamer specific against OTA, and detection was done in spiked corn and groundnut samples. For the quantitative analysis of the assay, the absorbance ratio of A630 and A520, which corresponded to the wavelength of aggregated (grey color) and dispersed (red color) AuNPs, was spectrophotometrically measured. The A630/A520 ratio indicated the transition of the dispersed state of AuNPs to the aggregated state in the presence or absence of the analyte. The OTA was prepared in the concentration range of 60 to 2 µg/mL with 545.45 and 95.69 ng/mL limit of detection in spiked corn and groundnut samples via AuNPs based rapid aggregation assay. The developed OTA-aptamer-AuNPs based assay was further tested by a microfluidic device validated by HPLC, can be used for rapid and cost-effective detection of ochratoxin without the requirement of highly sophisticated techniques and skilled labor.