ABSTRACT
Pathogens, mycotoxins, or antibiotics may exist in a food sample. Micro- and macromolecular substances must be detected quickly. A rapid and convenient lateral flow immunoassay (LFI) integrated with competitive and sandwich models was developed to detect micro- and macromolecular substances. In this study, aflatoxin M1 (AFM1) and Escherichia coli O157:H7 were selected as the micro- and macromolecular substances, respectively. Two test lines in the LFI test strip were evaluated to detect AFM1 and E. coli O157:H7 by competitive and sandwich models. Results showed that the limits of detection for detecting AFM1 and E. coli O157:H7 were 50 pg·mL-1 and 1.58 × 104 cfu·mL-1, respectively. The whole assay time was 30 min. The recoveries of gold nanoparticle-LFI ranged from 78.0 to 111.6% with coefficients of variation in the range of 3.9 to 8.5% for the detection of AFM1. For the detection of E. coli O157:H7, the range of recoveries was from 70.1 to 89.6% with coefficients of variation ranging from 4.9 to 13.0%. This study not only tested sensitivity and specificity, but also was a systematic study of location of 2 test lines of the LFI test strip integrated with competitive and sandwich models.
Subject(s)
Aflatoxin M1/isolation & purification , Escherichia coli O157/isolation & purification , Immunoassay/methods , Milk/chemistry , Milk/microbiology , Animals , Food Microbiology , Gold , Metal NanoparticlesABSTRACT
Amantadine (AMD), a banned antiviral veterinary drug, is still being abused. This study developed a novel enzyme linked immunosorbent assay for the colorimetric detection of AMD involving DNA hybridization reaction and non-crosslinking gold nanoparticles (AuNPs) aggregation. Accordingly, the Primer 1-AuNPs-anti-AMD monoclonal antibody (mAb) could be captured by AMD artificial antigen on ELISA wells. Primer 2, which was complementary paired to Primer 1, was eventually added into the ELISA wells. After the hybridization reaction, the free Primer 2 in the supernatant was mixed with AuNPs and NaCl and induced a rapid color change of AuNPs. The lack of AMD in the sample resulted in capturing a substantial Primer 1-AuNPs-mAb complex and limited free Primer 2 in the supernatant. After adding NaCl, the color of AuNPs turned blue with limited Primer 2. This simple and visualized novel method had good sensitivity (0.033⯵M) and exhibited a potential application for AMD screening on site.
Subject(s)
Amantadine/analysis , Enzyme-Linked Immunosorbent Assay/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Amantadine/immunology , Antibodies, Monoclonal/immunology , Colorimetry , DNA/chemistry , DNA/metabolism , Nucleic Acid HybridizationABSTRACT
Colloidal gold immunochromatographic assay (ICA) has poor sensitivity when used for Escherichia coli O157:H7 (E. coli O157:H7) detection. Eu (III)-doped polystyrene nanoparticle (EuNP) has a large range of stokes shift, long decay time, and wide excitation spectrum and narrow emission spectra. EuNP has been used as novel probe in ICA to improve sensitivity. In this study, carboxyl-modified EuNPs were prepared with different linkers. ICA based on EuNP, EuNP-6 carbon chain (CC) complex, EuNP-200CC complex, EuNP-1000CC complex, and EuNP-streptavidin (EuNP-SA) complex were systematically compared for the detection of E. coli O157:H7. Under optimized working conditions, the limits of detection (LOD) of EuNP-ICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 9.54 × 102, 1.59 × 102, 3.18 × 102, 2.98 × 102, and 1.08 × 102 colony-forming units (CFU) mL-1, respectively. The linear ranges of EuNP-ICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 6.36 × 102-1.59 × 105, 3.18 × 102-1.59 × 105, 6.36 × 102-1.59 × 105, 6.36 × 102-1.59 × 105, and 8.0 × 101-1.59 × 105 CFU mL-1, respectively. EuNP-SA-ICA exhibited the highest sensitivity and the widest linear range with good specificity, accuracy, and precision. It could be a promising analytical method for detecting E. coli O157:H7 in food samples. EuNP-SA-ICA may be a good model for detecting low concentrations of other food-borne pathogens.
