The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and, more recently, luminescent nanoparticles, such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular, for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared to GNPs and QDs using the same immunoreagents, except for the labels, allowing for their analytical characteristics to be objectively compared. The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection of ACNP-LFAs in maize was as low as 20 μg/kg for deoxynivalenol, 13 μg/kg for T-2 toxin, and 1 μg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6–109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from high-performance liquid chromatography–tandem mass spectrometry, indicating that ACNPs were more sensitive labels than and a promising alternative to GNPs used in LFAs for detecting mycotoxins in cereals.Keywords: amorphous carbon nanoparticles; gold nanoparticles; multiplex lateral flow immunoassay; mycotoxins; quantum dots;

We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL–1, which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants.Keywords: chemical contaminant; chemiluminescence resonance energy transfer; hapten functionalization; immunoassay; quantum dots;

•Three carefully designed fluorescein-tracers were synthesized and purified by preparative HPLC.•A FPIA for detecting microcystins and nodularin-R in water was developed and validated by UPLC–MS/MS.•The developed FPIA was easy-to-operate and could be completed within 10 min with a simple pretreatment procedure.This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for detection of microcystins (MCs) and nodularin-R (NOD) in water. MCs and NOD, the most widespread cyanobacterial toxin are hepatotoxins and tumor promoters, and their acute exposure may result in severe health problems in animals and humans. The fluorescein-based tracers were synthesized, and for the first time preparative high performance liquid chromatography (HPLC) was employed for their purification. Optimal tracers for the analysis were selected by evaluating the immunochemical activity. Under the optimal conditions, the achieved limits of detection (LODs) for MC-LR and NOD were 0.86 and 0.95 μg L−1, respectively, providing a sufficient sensitivity to meet the provisional guideline value recommended by the World Health Organization (WHO). An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to confirm the accuracy and precision of the FPIA, and no obvious difference in recovery between these two methods was found. The correlation coefficients (R2) were higher than 0.968. The developed FPIA was easy-to-operate and could be completed within 10 min after simple filtration and adjustment of pH for water samples. The method can be easily extended for screening of other cyanotoxins, representing a versatile strategy for environmental sample analysis.Download high-res image (235KB)Download full-size image

•An MWFPIA was firstly proposed for multiplexed detection of mycotoxins.•The time needed for the detection was less than 30 min including sample treatment.•The MWFPIA shows promise as a versatile strategy for food safety analysis.Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0 μg kg−1 for DON, 17.8 μg kg−1 for T-2 toxin and 331.5 μg kg−1 for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5–106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC–MS/MS, with correlation coefficients (R2) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis.

This paper reports the development of a screening fluorescence polarization immunoassay (FPIA) for the simultaneous detection of fumonisins B1 (FB1) and B2 (FB2) in maize. Three FB1 tracers including FB1-fluorescein isothiocyanate isomer I (FB1-FITC), FB1-5-([4,6-dichlorotriazine-2-yl]amino)-fluorescein (FB1-5-DTAF), and FB1-Texas Red-X succinimidyl ester (FB1-TRX) were synthesized and studied to select appropriate tracer–antibody pairs using seven previously produced monoclonal antibodies (mAbs). An FPIA employing the pair of FB1-FITC and mAb 4B9 showing 98.9% cross-reactivity (CR) toward FB2 was used to simultaneously detect FB1 and FB2. Maize flour samples were extracted with methanol/water (2:3, v/v). After optimization, the FPIA revealed a limit of detection (LOD) of 157.4 μg/kg for FB1 and an LOD of 290.6 μg/kg for FB2, respectively. Recoveries were measured for spiked samples of FB1 or FB2 separately, ranging from 84.7 to 93.6%, with a coefficient of variation (CV) of <9.9%. Total time needed for FPIA including sample pretreatment was <30 min. The FPIA was used to screen naturally contaminated maize samples. Results detected by FPIA showed good agreement with that of HPLC-MS/MS with a fit of R2 = 0.99 for the simultaneous detection of FB1 and FB2. The established method offered a rapid, simple, sensitive, and high-throughput screening tool for the detection of fumonisins in maize.

A novel monoclonal antibody for orbifloxacin (ORB) was produced for the first time and used to develop a homogeneous fluorescence polarization immunoassay (FPIA) for ORB determination in milk. Three coating antigens and six fluorescent tracers were prepared, and evaluated by ELISA and FPIA methods, respectively. Heterogeneous tracer, LOM-BDF, was selected to develop the FPIA, providing high sensitivity and stability. Moreover, a new optimization strategy for tracer and Ab concentration was proposed by integrating Z′ factor into checkerboard titration, and Z′ factor >0.8 was considered as a prerequisite to ensure the robustness of quantitative FPIA. The optimized FPIA provided a detection limit of 3.9 ng mL−1 and IC50 of 24.5 ng mL−1 with an expected Z′ factor of 0.81. Saturated (NH4)2SO4 precipitation was applied for milk sample pre-treatment due to high tolerance to elevated-ionic strength in the FPIA. Mean recoveries of fortified milk ranged from 74.3 to 112% at the adding levels of 10, 20 and 40 ng mL−1. The results indicate that the developed FPIA is efficient and especially suitable for fast screening of ORB in milk.

The optimum conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, detergent, and solvent) were investigated to develop a broad-specific and sensitive immunoassay for detection of sulfonamides in milk samples. Two previously produced broad-specific MAbs, 4D11 and 4C7, and eight structurally different haptens conjugated with bovine serum albumin (BSA) were used as coating antigens in a competitive indirect ELISA (ciELISA). In addition, six hapten-HRP conjugates and the two MAbs were evaluated in a competitive direct ELISA. After optimization, a highly broad-specific and sensitive ciELISA for screening for sulfonamides was obtained based on MAb 4D11 and the BS-BSA heterologous-coating antigen, demonstrating a 50 % specific binding (IC50) for 22 sulfonamides at concentrations below 100 ng mL−1. This is the first report of an immunoassay that is capable of detecting more than 20 sulfonamides based on MAbs. The optimized ciELISA was used to quantify the five sulfonamides, SMZ, SDM, SQX, SMM, and SMX in spiked milk samples. Recoveries of 89–104.6 % and coefficients of variation of 11.9–19.1 % demonstrated the potential of the ciELISA to simultaneously monitor multiple sulfonamides in diluted milk samples without further purification steps.

To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 μg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 μg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL−1 in buffer and 0.11 μg L−1 in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6 % in the inter-batch assay with coefficient of variations of 6.0 to 14.3 %. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

A fluorescence polarization immunoassay (FPIA) was developed for the analysis ofaflatoxins (AFs) using an anti-aflatoxin B1 (AFB1) monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 min. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.

Fumonisin B1 (FB1) is a mycotoxin, mainly produced by Fusarium fungi and present in food and feed. It causes harmful effects on human and animal health. Therefore, it is necessary to develop sensitive and reliable screening methods. In this study, a highly sensitive monoclonal antibody (MAb) against FB1, clone 2D7, was produced, and the 50% inhibition concentration (IC50) of the MAb was 2.2 ng/mL in buffer. The MAb showed high cross-reactivity with fumonisin B2 (FB2), and negligible cross-reactivity with other mycotoxins. A sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this MAb was developed for the determination of FB1 in maize. In spiked samples (100, 200 and 500 μg/kg), the average recoveries ranged from 78 ± 11 to 107 ± 4%, and the coefficient of variation ranged from 3 to 15%. The limit of detection of the icELISA was 5.4 μg/kg. This method was compared to liquid chromatography tandem mass spectrometry (LC–MS/MS) using naturally contaminated samples, and the correlation coefficient was above 0.82. These results show the reliability of the icELISA method for the determination of FB1 in maize.Highlights► A novel anti-fumonisin B1 monoclonal antibody was produced using different immunogens. ► A rapid and sensitive icELISA was developed for the detection of fumonisin B1 in maize. ► The icELISA was preliminary validated by the LC–MS/MS method.

A novel monoclonal antibody for orbifloxacin (ORB) was produced for the first time and used to develop a homogeneous fluorescence polarization immunoassay (FPIA) for ORB determination in milk. Three coating antigens and six fluorescent tracers were prepared, and evaluated by ELISA and FPIA methods, respectively. Heterogeneous tracer, LOM-BDF, was selected to develop the FPIA, providing high sensitivity and stability. Moreover, a new optimization strategy for tracer and Ab concentration was proposed by integrating Z′ factor into checkerboard titration, and Z′ factor >0.8 was considered as a prerequisite to ensure the robustness of quantitative FPIA. The optimized FPIA provided a detection limit of 3.9 ng mL−1 and IC50 of 24.5 ng mL−1 with an expected Z′ factor of 0.81. Saturated (NH4)2SO4 precipitation was applied for milk sample pre-treatment due to high tolerance to elevated-ionic strength in the FPIA. Mean recoveries of fortified milk ranged from 74.3 to 112% at the adding levels of 10, 20 and 40 ng mL−1. The results indicate that the developed FPIA is efficient and especially suitable for fast screening of ORB in milk.