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Accurate and sensitive quantification of a specific class of mycotoxins at trace levels in complex matrices with greener approaches is of significant importance. In this study, a green and economical protocol of magnetic microspheres-based cytometric bead array (CBA) assay on indirect competitive principle was developed for sensitive and rapid detection of ochratoxin A (OTA) in malts with a small number of standard and sample solutions. The protocol included the competition of OTA in malt samples and that covalently coupled on the surface of microspheres with its monoclonal antibodies, the separation and aggregation of the magnetic microspheres, and the fluorescence detection of fluorescein isothiocyanate labeled goat anti-mouse immunoglobulin G probes. The magnetic microspheres-based CBA assay allowed for ultralow limit of detection (0.025 μg kg −1 ) for OTA and showed higher sensitivity compared with the common polystyrene beads-based CBA method. This is the first report on the magnetic microspheres-based CBA assay by using a simple and easy-to-operate magnetic separator for highly sensitive and rapid detection of OTA in complex malt samples. By consuming less solvent, time and cost, as well as fewer standard and samples, the developed green protocol expressed high potential for one-site real-time detection of trace components in complex matrices. [ABSTRACT FROM AUTHOR]

An aptamer based lateral flow strip based on competitive format was developed for on-site rapid detection of ochratoxin A (OTA) in Astragalus membranaceus . Some crucial parameters that might influence the sensitive detection, such as the characterization of the colloidal gold, size and shape of gold nanoparticles (AuNPs), amount of AuNPs-aptamer conjugate, migration rate and the addition amount of methanol, were investigated to provide the optimum assay performance. To perform the test, 1 g sample was extracted with 2.5 mL of methanol-water (80:20, v/v ) and diluted by 4-fold running buffer to eliminate the matrix and methanol interferences. Under optimized conditions, the aptamer-based assay showed a visual limit of detection (LOD) of 1 ng mL −1 , and with no significant cross-reactivity with several homologous toxins. The whole detection could be completed within 15 min without special equipment because of available visual results. One out of nine A. membranaceus samples was found to be positive of OTA, which was in a good agreement with those obtained from LC–MS/MS analysis. The results demonstrated that the aptamer-based lateral flow assay could be used as a rapid, reliable, cost-effective and robust on-site screening technique for mycotoxins at trace level in complex matrices without special instrumentation. [ABSTRACT FROM AUTHOR]

A rapid, selective, and sensitive ultra-fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity ( r2 ≥ 0.9993), low limit of detection (0.5-0.8 μg/kg), and satisfactory recovery (83.54-94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer-affinity column, prepared in-house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2→358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 μg/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to optimize and validate a powerful method for the simultaneous analysis of aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and ochratoxin A (OTA) in ginger and related products collected from local markets in Beijing, China. The optimized analytical procedure was based on immunoaffinity column (IAC) clean-up, followed by ultra-high performance liquid chromatography with fluorescence (UHPLC-FLR) detection. Limits of detection (LOD) and quantification (LOQ) for the five mycotoxins were 0.005–0.2 and 0.0125–0.5 μg kg−1, respectively. The average recoveries ranged from 84.2 to 97.3% with relative standard deviations (RSDs) from 0.63 to 7.86% at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all higher than 0.9995. The established method was applied to 30 samples of 10 different species of ginger and related products, and all positive samples were confirmed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The results showed that 5 samples of ginger products were contaminated with AFB1 at 0.13–1.38 μg kg−1, while 3 samples of ginger and 2 samples of ginger products were contaminated with OTA at 0.31–5.17 μg kg−1. All the contamination levels were below the legally allowable limits. [Copyright &y& Elsevier]

Aptamers are single-stranded oligonucleotides with high affinity and specificity and are widely used in targets separation and enrichment. Here, an aptamer-affinity column (AAC) was firstly prepared in-house through a covalent immobilization strategy. Then, ochratoxin A (OTA) in ginger powder was absorbed and enriched using the new aptamer-based clean-up technology for the first time, and was further analyzed by ultra high performance liquid chromatography with fluorescence detection. After optimization, the average recoveries for blank samples spiked with OTA at 5, 15, and 45 μg/kg ranged from 85.36 to 96.83%. Furthermore, the AAC exhibited a similar accuracy as an immunoaffinity column to clean up OTA in ginger powder. Above all, it exhibited better reusability, twice that of the immunoaffinity column, had lower toxicity and cost, and took less time. Of 25 contaminated ginger powder samples, OTA contamination levels ranged from 1.51 to 4.31 μg/kg, which were lower than the European Union (EU) regulatory limits. All the positive samples were further confirmed by ultra-fast LC with MS/MS. In conclusion, the method of clean-up based on the AAC coupled to ultra-HPLC with fluorescence detection was rapid, specific, and sensitive for the quantitative analysis of OTA in a complex matrix. [ABSTRACT FROM AUTHOR]

Ginger, a widely used spice and traditional Chinese medicine, is prone to be contaminated by mycotoxins. A simple, sensitive, and reproducible method based on immunoaffinity column clean-up coupled with HPLC and on-line postcolumn photochemical derivatization with fluorescence detection was developed for the simultaneous determination of aflatoxins ( AFs) B1, B2, G1, G2, and ochratoxin A ( OTA) in 25 batches of gingers and related products marketed in China for the first time. The samples were first extracted by ultrasonication with methanol/water (80:20, v/v) and then cleaned up with immunoaffinity columns for analysis. Under the optimized conditions, the LODs and LOQs for the five mycotoxins were 0.03-0.3 and 0.1-0.9 μg/kg, respectively. The average recoveries ranged from 81.3-100.8% for AFs and from 88.6-99.5% for OTA at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all >0.9995. All moldy gingers were contaminated with at least one kind of the five investigated mycotoxins, while none of them were found in normal gingers. Ginger powder samples were contaminated slightly with the contamination levels below the LOQs, while ginger tea bags were mainly contaminated by OTA at 1.05-1.19 μg/kg and ginger black tea bags were mainly contaminated by AFs at 3.37-5.76 μg/kg. All the contamination levels were below the legally allowable limits. [ABSTRACT FROM AUTHOR]

A simple, reliable, and low-cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A ( OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection ( HPLC- FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC- FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits. [ABSTRACT FROM AUTHOR]

Wolfberry fruit wine (WFW) is widely used as a global functional food to improve the immune system and prevent human disease. A total of 36 bottled WFWs were randomly collected in China between 2005 and 2010. Samples were analysed for the presence of ochratoxin A (OTA) using immunoaffinity column (IAC) clean-up and high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Positive results were confirmed by liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC-ESI-MS/MS). The limit of detection (LOD), based on a signal-to-noise ratio of 3, was 0.05 ng mL–1. Recoveries ranged from 78.3% to 94.7% and relative standard deviations from 1.1% to 4.3% within the spiking range of 0.2–20 ng mL–1. OTA was detected in one sample, below the maximum allowable limit as established by the European community. [ABSTRACT FROM AUTHOR]

Immunoaffinity columns (IACs) are most popularly used for mycotoxin clean-up in complex matrices prior to chromatographic analysis. But, their high cost has limited their wide application and the regeneration of IACs for multiple instances of reuse is important. This study aimed to investigate the feasibility of regeneration and reuse of IACs for purification of ochratoxin A (OTA) in spiked raw malt and dried ginger samples followed by high performance liquid chromatography-fluorescence detection. After each use, the IACs were filled with phosphate buffer saline (PBS) as the preservation solution and stored at 8 °C overnight for regeneration and reuse until the recovery rate was <70%. The results showed that matrix type, preparation procedure, and pH value of sample extraction exhibited major effects on the reuse of IACs for OTA clean-up. While, after modifying the sample preparation procedure using water as the diluent and the solution at a pH of 7 to 8, the IACs could be used eight and three times for the spiked raw malt and dried ginger samples with OTA after regeneration. Regarding the traditional procedure recommended in Chinese Pharmacopoeia (2015 edition), the IACs could be used for three and two times for the spiked raw malt and dried ginger samples with OTA, respectively. Therefore, the corresponding experimental cost could be reduced to one-eighth and one-third of the original cost. This is the first study on the regeneration and reuse of IACs for OTA clean-up in complex Chinese herbal medicines, providing a green and economical tool for a large number of samples analysis with low cost. [ABSTRACT FROM AUTHOR]

Abstract: Ginger, one of the most commonly used spices and additives, is also widely used to prevent and treat human disease as a traditional medicine. However, ginger is prone to contamination by ochratoxin A (OTA), which is toxic, carcinogenic and thermostable. Here, a simple, reliable and low-cost method based on molecularly imprinted polymer (MIP) as selective sorbent of solid-phase extraction (SPE) was proposed for the determination of OTA in ginger by ultra-performance liquid chromatography coupled with fluorescence detection (UPLC-FLR). The samples were firstly extracted and then cleaned up with an AFFINIMIP® SPE OTA column for UPLC-FLR analysis. Under the optimized conditions, the limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.09 and 0.30 ng mL−1, respectively. The recoveries of OTA from ginger spiked at 5, 15 and 25 μg kg−1 ranged from 87.6 to 94.5%. In addition, after a simple regenerated procedure, the MIP-based SPE column could be reused at least forty-one times to obtain more than 80% recoveries of OTA for ginger samples. The developed method was applied to the detection of twenty batches of ginger and six samples were contaminated by OTA with levels below the newest legal limits. [Copyright &y& Elsevier]

Abstract: Licorice is widely used as additives in food and pharmaceutical industries and Glycyrrhiza uralensis is one of the main sources of licorice in China. G. uralensis may be contaminated by mycotoxins during growth, collection, transportation and, especially, storage. A rapid and highly sensitive method was firstly developed to determine aflatoxins (AFs) and ochratoxin A (OTA) in G. uralensis using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Due to the complexity and high color of the investigated matrices, the proposed method is based on sonication extraction using a methanol/water mixture (80:20, v/v) followed by immunoaffinity column (IAC) cleanup. The limits of quantification (LOQs) of aflatoxins B1, B2, G1, G2 and OTA were 0.020, 0.015, 0.010, 0.015 and 0.010 μg/kg based on a signal-to-noise ratio of 10: 1, respectively. Good linearity was observed for all the analytes of interest, with correlation coefficients all higher than 0.995 within their respective linear ranges. Among the total of 15 G. uralensis samples collected from different areas in China, 6 were moldy due to inappropriate storage and 9 were fine without any visible mold contamination. The results showed that almost all the samples were contaminated with AFs and OTA. The AFs and OTA levels found in the moldy samples were higher than those not visibly moldy and two of the moldy samples exceeded the maximum limit set for OTA in licorice by EU regulation. This is the first report of the co-occurrence of AFs and OTA in G. uralensis in China. [Copyright &y& Elsevier]

[Display omitted] • An electrochemical aptasensor for ultrasensitive detection of OTA was developed. • Nafion-f-MWCNTs as signal enhancers promoted an amplification and robust stability. • Carboxyl groups on f-MWCNTs readily exposed from Nafion to couple with molecules. • The aptasensor demonstrated excellent sensitivity with LOD of 1 pg/mL for OTA. • Practical application in Malt samples confirmed good reliability of the aptasensor. In this study, an ultrasensitive electrochemical (EC) aptasensor with Nafion-stabilized functionalized multi-walled carbon nanotubes (f-MWCNTs) as signal enhancers was established for ochratoxin A (OTA) determination. Herein, f-MWCNTs were prepared through functionalization with nitric acid. The incorporation of Nafion promoted a good dispersion of f-MWCNTs and prevented their leaching on the electrode, making a robust stability of the aptasensor. The Nafion-f-MWCNTs composites were used as the sensing substrates to largely enhance the electroactive surface area and the conductivity of the electrode, realizing a significant signal amplification. Carboxyl groups on the surface of f-MWCNTs readily exposed from Nafion membrane to couple with streptavidin, facilitating the immobilization of biotinylated aptamers to achieve selective recognition towards OTA. When OTA existed, aptamers preferentially combined with it, causing a noticeable decline in the current response. Under optimum conditions, a good linear relationship between the current changes and the logarithm of OTA concentration was observed from 0.005 ng/mL to 10 ng/mL, with a limit of detection low to 1 pg/mL for OTA. The specific, sensitive, and reproducible aptasensor succeeded in application in malt samples, confirming a great promise for more contaminants and providing a universal platform in complex matrices by simply replacing the corresponding aptamers. [ABSTRACT FROM AUTHOR]

• A label-free electrochemical aptasensor was developed for OTA detection. • No extra coupling medium was involved for immobilizing the aptamers. • Nafion-MWCNTs and in-situ generated Au nanopopcorns achieved dual-amplification. • The aptasensor displayed excellent sensitivity and wide linear range for OTA. • Practical application in malt samples confirmed great promise for more contaminants. Trace detection of ochratoxin A (OTA) in foods is essential to mitigate risks to human health. Herein, a label-free electrochemical (EC) aptasensor based on dual-signal amplification of Nafion dispersed multi-walled carbon nanotubes (Nafion-MWCNTs) and Au nanopopcorns was developed for ultrasensitive detection of OTA. Nafion solution prevented the leaching of MWCNTs, and the Nafion-MWCNTs modified screen-printed carbon electrode (SPCE) acted as the sensing substrate which facilitated the uniform distribution of the electrodeposited Au nanopopcorns. The in-situ generated Au nanopopcorns could not only load a large amount of aptamers for specific identification of OTA, but also promote the electron transfer of the sensing platform. The incorporation of Nafion-MWCNTs and Au nanopopcorns realized dual-amplification of the aptasensor due to the enhanced conductivity and the increased electroactive surface area of the electrode. The modified electrodes were characterized through scanning electron microscopy, X-ray photoelectron spectroscopy and EC evaluation. Under optimal conditions, the electrochemical impedance spectroscopy (EIS) was measured for the determination of OTA. The as-fabricated Au nanopopcorns/Nafion-MWCNTs impedimetric aptasensor displayed excellent sensitivity with a detection limit as low as 1 pg/mL and a wide linear range of 1 pg/mL-10 ng/mL for OTA. Practical application of the aptasensor in the spiked malt samples achieved satisfactory recoveries of 89.82–95.65 %, which was also successfully verified to detect OTA in eleven batches of actual malt samples collected from the local market. The creative aptasensor is simple, cost-effective, sensitive, and accurate, showing great promise for on-site monitoring of other trace contaminants in foods by simply replacing the aptamers. [ABSTRACT FROM AUTHOR]

The prevalent ochratoxin A (OTA) contamination in food has increased the urgency to develop sensitive and accurate detection methods. This study reported a conveniently fabricated label-free electrochemical (EC) aptasensor for ultrasensitive detection of OTA based on the competitive binding of OTA with its specific aptamer without any signal amplification strategy. A gold electrode (AuE) was modified with a compact layer of double-stranded DNA (dsDNA), obtained via OTA aptamer and complementary DNA (cDNA) hybridization, to act as a barrier for redox probe [Fe(CN) 6 ]3-/4- to AuE access. This led to a high initial electron-transfer resistance (R ct), recorded via electrochemical impedance spectroscopy (EIS). Since the aptamer displayed a higher affinity to OTA than cDNA, the added OTA triggered aptamer dissociation from AuE to form the OTA-aptamer complex, obviously decreasing the R ct. The target-induced variation in the resistance response strongly depended on the OTA concentration. Optimum conditions improved the linear relationship between R ct and the logarithm of OTA concentration in a range of 0.05–10 ng/mL with a limit of detection (LOD) of 0.05 ng/mL for OTA. Recoveries of 74.8–105% with relative standard deviations (RSDs) of 8.86–13.09% were obtained in the spiked malt samples. The Real-world detection of OTA in the actual malt samples confirmed the reliability of the convenient, cost-effective, and highly specific impedimetric aptasensor, exhibiting significant promise for extended application in food control to identify more hazardous contaminants by altering the corresponding aptamers. • A label-free electrochemical impedimetric aptasensor for OTA detection was developed. • No extra coupling medium was involved for immobilizing the aptamers. • The aptasensor achieved high sensitivity for OTA without signal amplification. • The impedimetric aptasensor succeeded in real malt samples with satisfactory recoveries. • The aptasensor can be extended to more contaminants in foods via replacing the corresponding aptamers. [ABSTRACT FROM AUTHOR]

• A self-assembled electrochemical immunosensor was fabricated for determining OTA. • TGA monolayer modified Au electrode could improve electrochemical response. • Some crucial parameters were systematically optimized for improving the sensitivity and reliability. • A ultra-low limit of detection of 0.08 ng/mL was obtained. • Real application in real spiked malt samples with OTA explored high accuracy without matrix interferences. Trace residue of mycotoxins in complex medicinal and edible food matrices has brought huge challenges for the development of ultrasensitive analytical methods. Here, a green electrochemical immunosensor for the ultrasensitive detection of ochratoxin A (OTA) was fabricated by self-assembling a compact 2-mercaptoacetic (TGA) monolayer on the surface of the working Au electrode to form the Au/TGA/bovine serum aibumin (BSA)-OTA/anti-OTA monoclonal antibody composite probes for selective and ultra-sensitive detection of OTA based on indirect competitive principle and differential pulse voltammetry analysis. The electrochemical impedance spectroscopy and cyclic voltammetry methods were introduced to characterize the assemble situation of the TGA-modified Au electrode and optimize some critical parameters for the green electrochemical immunoseonsor. Under the optimal conditions, the developed immunosensor exhibited much lower limit of detection (0.08 ng/mL) in the range of 0.1–1.0 ng/mL for OTA compared with other direct or disposable electrochemical immunosensors. Real application in the spiked malt samples verified high accuracy with no matrix interferences of the proposed immunoseonsor. This is a meaningful study on a self-assembled electrochemical immunoseonsor for ultra-sensitive and rapid detection of OTA in malt samples, which suggested a general simple-to-use sensing platform and prospect as an economical and green tool for ultra-sensitive detection of much more trace-level of toxic small molecules in other complex matrices to ensure their quality and safety. [ABSTRACT FROM AUTHOR]