Gold nanoparticles-based lateral flow immunoassay with silver staining for simultaneous detection of fumonisin B1 and deoxynivalenol

A lateral flow immunoassay with silver staining for the simultaneous detection of fumonisin B₁ (FB₁) and deoxynivalenol (DON) in maize samples was reported. The assay was based on the competition between target mycotoxins and corresponding coating antigens immobilized on test lines for binding to limited gold nanoparticles (AuNPs)-labeled antibodies. The detection signal was further amplified by employment of silver staining on AuNPs. In the process, silver ions were catalyzed by AuNPs into metal silver that deposited on the surface of AuNPs, allowing not only the enlargement of particle dimensions of AuNPs but also a more distinguishable black coloration on the test zone. Under optimized conditions, the cut-off values of silver staining lateral flow immunoassay were 2.0 ng mL⁻¹ for FB₁ and 40 ng mL⁻¹ for DON in buffer, which was improved at least 2 times in comparison to those of AuNPs-based method. The assay was further applied to detect FB₁ and DON in naturally contaminated maize samples and a good agreement was found with the data obtained from HPLC-MS/MS.     

A modified lateral flow immunoassay for the detection of trace aflatoxin M1 based on immunomagnetic nanobeads with different antibody concentrations

A modified lateral flow immunoassay (two-step assay) was developed to detect trace aflatoxin M1 (AFM1) in raw milk. In contrast to conventional LFIA, two kinds of immunomagnetic nanobeads (IMNBs) were used. One IMNB with high antibody concentration was used to capture AFM1 in the test sample, whereas the other IMNB with low antibody concentration was used to elucidate the results of the test. Critical factors, such as antibody concentration of IMNBs and size of IMNBs, were investigated. The two-step assay exhibited an ideal sensitivity to screen trace AFM1 in milk samples without extra sample pretreatment. The cutoff value of the naked eye was 0.02 μg/L and satisfied the European Union's maximum limit of AFM1 in raw milk, heat-treated milk, and milk used to manufacture milk-based products and even in baby foods. With the same antibody, sensitivity was enhanced approximately 25 and 50 times when compared with conventional IMNB-based LFIA and gold-based LFIA, respectively. Corresponding results of 13 raw milk samples were obtained between this two-step assay and referenced enzyme-linked immunosorbent assay.     

Synchronous fluorescence and multivariate classification analysis as a screening tool for determining Sudan I dye in culinary spices

Spices are a globally traded commodity which has been found to be adulterated with forbidden Sudan dyes. This work proposes a screening method for determining the adulteration of paprika varieties (mild, hot and smoked) with Sudan I dye, based on constant-wavelength synchronous fluorescence spectroscopy with multivariate classification. Different wavelength-intervals (Δλ) were evaluated. Classification models were built with Partial Least Squares-Discriminant Analysis (PLS-DA) at two Sudan I dye concentration levels (1 and 5 mg L⁻¹) and they were tested with samples at a lower level (0.5 mg L⁻¹). Classification results were quite satisfactory when a strategy based on first-derivative spectra was used for improving classification results. Δλ = 60 nm was chosen as the optimum wavelength interval giving a 100% of sensitivity and specificity. These results are promising because the risk of assigning adulterated samples as safe to be consumed is highly minimized. The proposed method is feasible, rapid and simple taking advantage of Sudan I fluorescence phenomena in a direct way.     

Performance evaluation of lateral flow immuno assay test kits for quantification of deoxynivalenol in wheat

Lateral flow immuno assay test kits for determining the presence of deoxynivalenol (DON) in grain provide relatively simple, cheap and quick detection means, but are only useful if they provide reliable and accurate results. This study aimed to evaluate the performance of four different lateral flow immune assay test kits, being Reveal Q+ DON (Neogen), Donsensor (Unisensor), RidaQuick DON (R-Biopharm AG), and RosaFast DON (Charm Sciences Inc.). The performance was evaluated based on accuracy, repeatability, and toxin recovery of the test kits using three types of wheat sample material. These included samples spiked at three different DON concentrations, reference material with a DON concentration of 900 μg/kg, and naturally contaminated samples with a wide range of DON concentrations.Results revealed that, in general, Reveal Q+ and Donsensor were the most accurate methods, whereas the performances of RidaQuick and RosaFast were much lower. Mean toxin recovery and repeatability of Reveal Q+ and Donsensor complied with EC requirements for analytical methods used for official control of DON in foodstuffs. Reveal Q+ and Donsensor also showed high accuracy. RidaQuick and RosaFast showed lower performance, in particular recovery of RidaQuick was lowest. However, laboratory procedures for the latter two test kits were easier to follow. In particular, RidaQuick has the easiest testing procedure and can be applied on-site without technical skills. Future research could focus on a more in-depth evaluation of the performance of Reveal Q+ and Donsensor, also including evaluation of between laboratory reproducibility.     

Comparison study of three rapid test kits for histamine in fish: BiooScientific MaxSignal enzymatic assay, Neogen Veratox ELISA, and the Neogen Reveal Histamine Screening test

Two new test kits for quantifying histamine in fish are investigated and compared to the Neogen Veratox ELISA. One of the kits is based on selective enzymatic conversion of histamine by histamine dehydrogenase (MaxSignal, BIOO Scientific, Austin, Texas) and the other is dipstick based on lateral flow immuno-chromatography (LFIC). These new kits are compared to Veratox using spiked and naturally contaminated fresh and frozen yellowfin tuna and mahi mahi, as well as canned mackerel in broth and canned tuna in oil and in broth. Histamine levels determined using the MaxSignal enzymatic kit in spiked and naturally contaminated fish were strongly correlated with levels detected with the Veratox ELISA. Comparing both new kits and also using the AOAC performance tested Veratox, spiking at 0 and 50 mg/kg, no false negatives or false positives were found over a wide range of concentrations of histamine. In contrast to competitive ELISA kits, which give a sigmoidal (logistics curve) color response that decreases with increasing analyte, the enzymatic kit gives a linear response, increasing with histamine concentration. Linearity is excellent and samples can be analyzed without further dilution using the included standards to quantify up to 70 mg/kg histamine free base. The 450 nm endpoint color develops rapidly, and using a reaction time of 30 min, recoveries in canned tuna and mackerel were greater than 90% and apparent histamine levels reach a plateau versus time. In contrast, recoveries using the manufacturer’s recommended reaction time of 10 min were low, and further study supported the presence of naturally occurring inhibitors of histamine dehydrogenase in fish. Using an extended (30 min) reaction time, the enzymatic kit performed well and was still rapid. The simplicity of the procedure results in a kit more easily mastered and potentially more rugged than competitive ELISA assays, which require multiple incubations, a wash step, additional dilution steps, and twice as many pipetting steps. The LFIC kit Reveal was also found to be simpler and easier to use than ELISA kits, having only one critical pipetting step, requiring no acylation, and only minimal sample manipulation. If accelerated sample removal and grinding could be implemented and validated, the LFIC kit Reveal appears to have excellent potential for onsite testing.     

Development of a lateral flow colloidal gold immunoassay strip for the simultaneous detection of Shigella boydii and Escherichia coli O157:H7 in bread,milk and jelly samples

A colloidal gold immunochromatographic strip (ICS) was proposed with double monoclonal antibodies (McAbs) for rapid detections of multiple foodborne pathogens, and we successfully developed a model strip for simultaneous detection of Shigella boydii (S. boydii) and Escherichia coli O157:H7 (E. coli O157:H7) in this study. The sensitivity of the strip test was determined to be 10⁶ CFU/mL for both S. boydii and E. coli O157:H7. There was no cross-reaction with other related bacteria. Parallel analysis of pathogen detection from bread, milk and jelly showed consistent results between the strip test and enzyme-linked immunosorbent assay (ELISA). The detection limit was substantially improved to 4 CFU/mL of the original bacterial content after pre-incubation of the bread, milk and jelly samples in broth for 10, 10 and 8 h respectively. This ICS was able to finish test within 5–10 min and has advantages in high throughput and easy operation without requiring sophisticated equipment and specialized skills. Overall, to our knowledge, this is the first report of semi-quantitative ICS for simultaneously detecting two pathogens. This protocol might provide insight into the development of ICS technology for simultaneously examining multiple food-borne pathogens from foods.