Peiwu Li

Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts

An ultrasensitive immunochromatographic (IC) assay for simultaneous detection of total aflatoxins (AFB1, AFB2, AFG1, and AFG2) was developed to meet the requirement for rapidly monitoring aflatoxins in agro-products. The assay was based on a competitive format and its sensitivity was improved by using a novel criterion to screen the optimal amount of monoclonal antibody (MAb) labeled to nanogold particles. The visual detection limits (VDLs) for aflatoxins B1, B2, G1, and G2 in peanut matrix were 0.03, 0.06, 0.12, and 0.25 ng mL(-1), respectively, which were lower than those of published literatures. The results of IC assay were in good agreement with those of high performance liquid chromatography (HPLC) in the analysis of aflatoxins in peanuts, demonstrating the practical applicability of the developed assay in real samples. This qualitative test based on the visual evaluation of results did not require any equipment. Overall, to our knowledge, this is the first report of qualitative detection for total aflatoxins by immunochromatographic assay.

Multi-component immunochromatographic assay for simultaneous detection of aflatoxin B1, ochratoxin A and zearalenone in agro-food

Mycotoxins are highly toxic contaminants and have induced health threat to human and animals. Aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEA) commonly occur in food and feed. A multi-component immunochromatographic assay (ICA) was developed for rapid and simultaneous determination of these three mycotoxins in agro-food. The strategy was performed based on the competitive immunoreactions between antibody-colloidal gold nanoparticle conjugate probes and mycotoxins or mycotixin antigens. Each monoclonal antibody specially recognize its corresponding mycotoxin and antigen, and there was no cross reactivity in the assay. Three mycotixin antigens were immobilized as three test lines in the nitrocellulose membrane reaction zone, which enable the simultaneous detection in one single test. The visible ICA results were obtained in 20 min. The visual detection limits of this strip test for the AFB1, OTA and ZEA were 0.25 ng/mL, 0.5 ng/mL and 1 ng/mL, respectively. The assay was evaluated using spiked and naturally contaminated peanuts, maize and rice samples. The results were in accordance with those obtained using enzyme-linked immunosorbent assay. In summary, this developed ICA could provide an effective and rapid approach for onsite detection of multi-mycotoxin in agro-food samples without any expensive instrument.

Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure.

Monoclonal antibodies (McAbs) cross-reactive with four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30min, aflatoxin B(1), B(2), G(1) and G(2) were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2-3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to four aflatoxins. Five McAbs were chosen for further analysis. Of the five, two antibodies had similar reaction efficiency with aflatoxin B(1), B(2) and G(1) but showed a weak cross-reaction to G(2). Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxin B(1), B(2), G(1) and G(2) at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0pgmL(-1) respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had superiority and these antibodies could be used for simultaneous analysis of total aflatoxins.

A high selective immunochromatographic assay for rapid detection of aflatoxin B1

To solve the problem of low selectivity of current immunochromatographic assay (ICA) for aflatoxin B(1) (AFB(1)) alone detection, a novel selective ICA was developed here. With very high selectivity, a new AFB(1) monoclonal antibody (MAb) 3G1 was prepared by immunizing Balb/c mice with aflatoxin B(2a)-BSA (AFB(2a)-BSA) rather than AFB(1)-BSA used in other reports and 3G1 possessed the highest selectivity than those used in published ICAs. The ICA with visual detection limit (VDL) of 1 ng mL(-1) showed no cross-reactivity with other aflatoxins. Comparing with previous reports, the ICA here provided the most powerful guarantee for avoiding false positive results leaded by coexistence of other aflatoxins in samples. For validation, naturally contaminated samples including peanut, puer-tea, vegetable oil and feedstuff were respectively assayed by ICA and a standard high performance liquid chromatography (HPLC), and good agreement of results was obtained between two methods. Therefore, the developed ICA could well meet the selective detection of AFB(1) in agro-products.

Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol.

A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB1) conjugated with bovine serum albumin (AFB1-BSA). Two AFB1-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 °C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB1, exhibiting an IC50 value of 0.754 ng/mL (2.4 μM), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination.

Monoclonal antibody-quantum dots CdTe conjugate-based fluoroimmunoassay for the determination of aflatoxin B1 in peanuts

A fluoroimmunoassay towards aflatoxin B1 (AFB1) was presented using quantum dots as the fluorescent label. The CdTe QDs were successfully linked to the monoclonal antibody against AFB1. Based on the conjugated complexes, a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) was developed for AFB1 detection. The 50% inhibition value (IC50) of the cFLISA was 0.149ng/mL in peanuts matrix. The method performance included the limit of detection (LOD) of 0.016ng/mL and considerable recoveries of 85-117% at three fortification levels (0.075, 0.15, and 0.3ng/g) from spiked AFB1 blank peanuts samples, along with coefficients of variation (CVs) below 10%. The cFLISA provided an alternative of rapid and sensitive detection for AFB1 and, moreover provided great potential for multiplexed mycotoxins determination simultaneously.

Effect of ozone on aflatoxins detoxification and nutritional quality of peanuts

Aflatoxins are a group of secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus with carcinogenicity, teratogenicity, and mutagenicity. Aflatoxins may be found in a wide range of agri-products, especially in grains, oilseeds, corns, and peanuts. In this study, the conditions for detoxifying peanuts by ozonation were optimised. Aflatoxins in peanuts at moisture content of 5% (w/w) were sensitive to ozone and easily degraded when reacted with 6.0mg/l of ozone for 30min at room temperature. The detoxification rates of the total aflatoxins and aflatoxin B1 (AFB1) were 65.8% and 65.9%, respectively. The quality of peanut samples was also evaluated in this research. No significant differences (P>0.05) were found in the polyphenols, resveratrol, acid value (AV), and peroxide value (PV) between treated and untreated samples. The results suggested that ozonation was a promising method for aflatoxin detoxification in peanuts.

Simultaneous determination of 13 phytohormones in oilseed rape tissues by liquid chromatography‐electrospray tandem mass spectrometry and the evaluation of the matrix effect

In the experiment, a high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry with selected reaction monitoring was used to simultaneously determine various classes of phytohormones, including indole-3-acetic acid, α-naphthaleneacetic acid, 2-chlorobenzoic acid, 4-chlorobenzoic acid, indole-3-butyric acid, gibberellic acid, 2,4-dichlorophenoxyacetic acid, 2-naphthoxyacetic acid, abscisic acid, 2,3,5-triiodobenzoic acid, uniconazole, paclobutrazol and 2,4-epibassinolide in rape tissues. The analyses were separated by an HPLC equipped with a reversed-phase column using a binary solvent system composed of methanol and water, both containing 0.1% of formic acid. The matrix effect was also considered and determined. The technology was applied to analyze rape tissues, including roots, stems, leaves, flowers, immature pods and rape seeds. The rape tissues were subjected to ultrasound-assisted extraction and purified by dispersive solid-phase extraction, and then transferred into the liquid chromatography system. The detection limit for each plant hormone was defined by the ratio of signal/background noise (S/N) of 3. The results showed perfect linearity (R(2) values of 0.9987-1.0000) and reproducibility of elution times (relative standard deviations, RSDs,<1%) and peak areas (RSDs,<7%) for all target compounds.

Classification and Adulteration Detection of Vegetable Oils Based on Fatty Acid Profiles

The detection of adulteration of high priced oils is a particular concern in food quality and safety. Therefore, it is necessary to develop authenticity detection method for protecting the health of customers. In this study, fatty acid profiles of five edible oils were established by gas chromatography coupled with mass spectrometry (GC/MS) in selected ion monitoring mode. Using mass spectral characteristics of selected ions and equivalent chain length (ECL), 28 fatty acids were identified and employed to classify five kinds of edible oils by using unsupervised (principal component analysis and hierarchical clustering analysis), supervised (random forests) multivariate statistical methods. The results indicated that fatty acid profiles of these edible oils could classify five kinds of edible vegetable oils into five groups and are therefore employed to authenticity assessment. Moreover, adulterated oils were simulated by Monte Carlo method to establish simultaneous adulteration detection model for five kinds of edible oils by random forests. As a result, this model could identify five kinds of edible oils and sensitively detect adulteration of edible oil with other vegetable oils about the level of 10%.

Sensitive competitive immunoassay of multiple mycotoxins with non-fouling antigen microarray

Various mycotoxins with strong carcinogenesis and toxicity are fatal threats in food safety, and require highly sensitive and high-throughput detections greatly. Herein a fluorescent competitive immunoassay microarray based on a non-fouling polymer brush, poly[(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-co-GMA) is explored to sensitively detect multiple mycotoxins with aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEN) as template targets. Due to uniformly large protein loading and high resistance to nonspecific protein absorption of the POEGMA-co-GMA brush, the optimal microarray exhibits wide dynamic ranges of three orders of magnitudes and low detection limits of 4, 4 and 3 pg mL(-1), respectively, which is much better than that obtained with an epoxy-functionalized antigen microarray, and is comparable or even better than the conventional ELISA method. This work offers a powerful high-throughput tool to fast screening of toxins in food quality and environmental monitoring.