Aflatoxins and zearalenone (ZEN) are highly common mycotoxins in maize and maize-based products. This study aimed to report a time-resolved fluorescence immunochromatographic assay (TRFICA) developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin B1 (AFB1) and ZEN in maize and its products. A novel Eu/Tb(III) nanosphere with enhanced fluorescence was prepared as a label and conjugated to anti-idiotypic nanobody (AIdnb) and monoclonal antibody (mAb). On the basis of nanosphere–antibody conjugation, two patterns of competitive time-resolved strip methods (AIdnb–TRFICA and mAb–TRFICA) were established and compared. The half inhibition concentration of AIdnb–TRFICA was 0.46 and 0.86 ng·mL–1 for AFB1 and ZEN, which was 18.3- and 20.3-fold more sensitive than that of mAb–TRFICA for AFB1 and ZEN, respectively. Under optimal conditions, AIdnb–TRFICA for dual mycotoxin was established and provided a quantitative relationship ranging from 0.13 to 4.54 ng·mL–1 for AFB1 and 0.20 to 2.77 ng·mL–1 for ZEN, with a detection limit of 0.05 and 0.07 ng·mL–1 in the buffer solution, respectively. AIdnb–TRFICA showed good recoveries (72.6%–106.6%) in samples and was applied to detect dual mycotoxin in maize samples with satisfying results. To the best of our knowledge, it is the first report about a time-resolved strip method based on AIdnbs for dual mycotoxin.

•A time-resolved fluorescent strip was developed based on double-label immunoprobes.•The quantitative TRFICA’s LODs may reach 0.04 mg L−1 in apple extraction solution.•Strip assay sensitivity is increased or similar to the previous GNP strip assays.•The spiked recoveries of carbofuran in the agro-products were 81–103%.•The TRFICA’s results were in good agreement with a standard HPLC method.A rapid and quantitative time-resolved fluorescent immunochromatographic assay (TRFICA) for detecting carbofuran residues in agro-products was reported in this paper. This assay was developed based on double-label immunoprobes, one of which was a carbofuran-specific antibody coupled with europium microbeads for the test (T) line signal while the other was mouse IgG coupled with europium microbeads for the control (C) line signal. Quantitative relationships between carbofuran concentrations and T/C ratios were established to determine the analyte concentration. To increase assay accuracy, four standard curves were established for the agro-products (green bean, cabbage, apple, and pear). The limits of detection (LODs) ranged from 0.04 to 0.76 mg L−1. The spiked recoveries of carbofuran in the agro-products were in the range of 81–103%, which was in good agreement with a standard HPLC method. Therefore, we provided a new and reliable method for determination of N-methylcarbamate pesticide carbofuran residues in agro-products including vegetables and fruits.

•A high sensitive novel monoclonal antibody against fumonisin B1 was achieved.•A indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for fumonisin B1 detection.•A gold nanoparticles-based gray imaging quantification immunoassay (GNPs-GI) was developed for fumonisin B1 detection.•The icELISA and GNPs-GI performed rapidly assay for fumonisin B1 in agricultural products.Fumonisins (FBs) are widely found in rice, maize, peanut, wheat, and other agricultural products. These have been detected using a chromatography technique, whereas the rapid assay by a highly sensitive monoclonal antibody is minimally reported. Herein, a highly sensitive monoclonal antibody (7A11) was successfully developed by hybridoma technique. The 50% inhibition concentration (IC50) of 7A11 monoclonal antibody was 0.32 ng/mL in an optimized buffer. The cross-reactivity between fumonisin B1 and fumonisin B2, B3 was 4.3% and 12.8%, respectively. Based on the newly developed 7A11 antibody, a high sensitivity indirect competitive enzyme-linked immunosorbent assay (icELISA) and gold nanoparticles-based gray imaging quantification immunoassay (GNPs-GI) were established. The analytical range of icELISA was 0.08–1.38 ng/mL and that for GNPs-GI was 0.24–15 ng/mL. Both the methods showed adequate recoveries (80.0–105.8% for icELISA and 78.5–115.2% for GNPs-GI) in spiked samples. Compared to high-performance liquid chromatography (HPLC), icELISA and GNPs-GI indicated reliability that could be used for further detection of fumonisin B1 in agricultural products.

•The IAC column for selective extraction of capsaicinoids from oil was developed.•The major parameters affecting IAC extraction efficiency were optimized.•The IAC column was characterized including binding capacity, accuracy, etc.•Accurate quantification was performed with deuterium-labeled internal standards.•Capsaicinoids in spiked oil was extracted by IAC column and analysed by LC–MS/MS.Capsaicin and dihydrocapsaicin were selected as adulteration markers to authenticate vegetable oils. In this study, a method of immunoaffinity chromatography (IAC) combined with liquid chromatography–tandem mass spectrometry was established for the determination of capsaicin and dihydrocapsaicin in vegetable oils. In this method, immunosorbents were obtained by covalently coupling highly specific capsaicinoid polyclonal antibodieswith CNBr-activated Sepharose 4B, and then packed into a polyethylene column. In this paper, the major parameters affecting IAC extraction efficiency, including loading, washing and eluting conditions, were also investigated. The IAC column displayed high selectivity for capsaicin and dihydrocapsaicin with the maximum capacity of 240 ng. The limit of detection (LOD) and limit of quantification (LOQ) for capsaicin were calculated as 0.02 and 0.08 μg kg−1, and for dihydrocapsaicin were 0.03 and 0.10 μg kg−1. The recoveries of capsaicin and dihydrocapsaicin in oil samples were in the range of 87.3–95.2% with the relative standard deviation (RSD) of less than 6.1%. The results indicated that capsaicinoid compounds could not be found in edible vegetable oils. Therefore, the proposed method is simple, reliable and adequate for routine monitoring of capsaicinoid compounds in vegetable oils and has an excellent potential for detection of adulteration with inedible waste oil.

A highly-sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) was developed to detect aflatoxin M1 (AFM1) in raw milk samples within 6 minutes without any sample pretreatments. This method could meet the requirement for rapid and sensitive milk monitoring in dairy farms and milk industries. Based on a competitive format and the home-made monoclonal antibody 2C9 against AFM1, this assay enhanced the sensitivity from 0.3 ng mL−1 (by using nanogold-strip method previously reported) to 0.03 ng mL−1 (by using this TRFICA method). The improved sensitivity could probably result from the increases in both the higher affinity of monoclonal antibody 2C9 against AFM1 and detection signals of immunoassay probes (with each europium microbead coupled with numbers of 2C9 antibodies). The TRFICA method showed a considerable dynamic range of 0.1–2.0 ng mL−1 and spiked recoveries of 80–110% for AFM1 quantification in raw milk. The results via TRFICA method was found to be high consistency (R2 = 0.988) with results via standard high-performance liquid chromatography (HPLC) method, when detecting AFM1 in 17 blind milk samples.

•Developing a sensitive and quantitative immunochromatographic assay for ochratoxin A.•Using antibody conjugated gold nanoparticles as label for detection.•Using a rapid color intensity portable strip reader for quantifying the method.•The assay was used for ochratoxin A detection in agro-products.•The results were reliable and in accordance with those obtained using HPLC.In most cases of mycotoxin detection, quantitation is critical while immunochromatographic strip tests are qualitative in nature. Moreover, the sensitivity of this technique is questioned. In order to overcome these limitations, an ultrasensitive and quantitative immunochromatographic assay (ICA) for rapid and sensitive quantitation of ochratoxin A (OTA) was developed. The assay was based on a competitive format and its sensitivity was improved by using a sensitive and selective OTA monoclonal antibody (OTA-mAb). The visible ICA results were obtained within 15 min, and in addition to visual examination, they were read by the rapid color intensity portable strip reader. The visual and computational detection limits (vLOD and cLOD, respectively) for ochratoxin A were 0.2 and 0.25 ng mL−1, respectively. These values were lower than those reported by previous studies in a range 5–2500 folds. For validation, contaminated samples including wheat, maize, rice and soybean were assayed by ICA and a standard high performance liquid chromatography (HPLC). The results were in good agreement for both ICA and HPLC methods. The average recoveries of the HPLC were in the range 72–120% while the ICA values were from 76 to 104%, confirming the accuracy and sensitivity of this method.

Aflatoxins are a group of extremely toxic small molecules that have been involved in human hepatic and extrahepatic carcinogenesis as causative agents. Herein, we developed a real-time immuno polymerase chain reaction (IPCR) assay for the accurately quantitative detection of aflatoxins in agri-products base on a M13 phage containing aflatoxin anti-idiotypic nanobody and its encoding DNA which was used to design the specific primers. The limit of detection (LOD) of the assay is 0.02 ng/mL, which exhibits a 4-fold improvement over traditional phage ELISA. The developed method was successfully validated with the samples of corn, rice, peanut, and feedstuff, which are major aflatoxin-contaminated agri-products. And the recoveries were from 77.05 to 122.16%. For further validation, the developed assay was also compared with a reference HPLC method for the analysis of aflatoxins in corn and peanuts, and concordant results (R2 = 0.991) were obtained. In this context, this study provides a novel opportunity to analyze aflatoxins in agri-products.

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.

Anti-idiotypic antibodies recognize the antigenic determinants of an antibody, thus they can be used as surrogate antigens. Single-domain antibodies from camlid heavy-chain antibodies with the benefit features of small size, thermostability, and ease in expression, are leading candidates to produce anti-idiotypic antibodies. In this work, we constructed an antibody phage library from the mRNA of an alpaca immunized with an antiaflatoxin monoclonal antibody (mAb) 1C11. Three anti-idiotypic VHH antibodies were isolated and applied to immunoassay toward aflatoxin as a coating antigen. The best immunoassay developed with one of these VHH antibodies shows an IC50 of 0.16 ng/mL toward aflatoxin B1 and cross-reactivity toward aflatoxin B2, G1, and G2 of 90.4%, 54.4%, and 37.7%, respectively. The VHH-based immunoassay was successfully applied to the analysis of peanuts, corn, and rice, which are the predominant commodities regularly contaminated by aflatoxins. A good correlation (r2 = 0.89) was found between the data obtained from the conventional ELISA and the ELISA based on a VHH coating antigen for the analysis of aflatoxins in peanuts and feedstuff. The use of biotechnology in developing the surrogate, the absence of standard aflatoxin and organic solvents in the synthesis procedures, and the reproducibility of the VHH antibody makes it an ideal strategy for replacing conventional synthesized antigens.

•Graphene oxide (GO) was successfully synthesized and used as an adsorbent for extraction of four aflatoxins.•The aflatoxins extraction efficiency for GO has been investigated.•A method of HPLC-FLD coupled with GO as adsorbent has been developed for the simultaneous determination of four aflatoxins in peanut samples.In this paper, graphene oxide (GO) was synthesized and specifically selected by centrifugation to extract four aflatoxins (B1, B2, G1, and G2) as an effective adsorbent. Then, the amount of aflatoxins was quantitatively measured by high-performance liquid chromatography (HPLC). The GO was characterized by X-ray diffraction (XRD), atomic force microscopy (AFM), and ultraviolet (UV) spectrophotometer. Several parameters that could affect the extraction efficiency, including the GO amount, methanol concentration in the extraction solvent, spiked amount, extraction time, and elution cycle, were also investigated and optimized in this work. Under optimal conditions, good linear relationships were achieved with the correlation coefficient (r) ranging from 0.99217 to 0.99995. The detection limit of this method for the four aflatoxins ranged from 0.08 to 0.65 ng/g. Finally, the proposed method has been successfully applied to determine aflatoxins in peanut samples. The results show that the recoveries of the four aflatoxins range from 85.1% to 100.8% with the relative standard deviations between 2.1% and 7.9%.