Green Enzyme-Linked Immunosorbent Assay Based on the Single-Stranded Binding Protein-Assisted Aptamer for the Detection of Mycotoxin.

In this work, a green enzyme-linked immunosorbent assay (ELISA) based on the single-stranded binding protein (SSB)-assisted aptamer was designed for biosensing applications. Combined with the biotin-streptavidin (SA) system and the high catalytic activity of horseradish peroxidase (HRP), this SSB-assisted aptamer sensor was applied for the detection of aflatoxin B1, ochratoxin A, and zearalenone. In this novel ELISA, mycotoxin-protein conjugations were replaced by SSB to avoid the hazard of mycotoxin, whereas antibodies were replaced by aptamer to avoid the complex and tedious preparation of antibodies. In the absence of target mycotoxins, SSB can bind the aptamer-biotin specifically. Detection was performed using the strong combination of biotin and SA after adding SA-HRP and substrate/chromogen solution, thereby resulting in a strong yellow color signal. In the presence of target mycotoxins, the aptamer-biotin cannot bind to the SSB, thereby leading to a weak yellow color signal. Under optimal conditions, the designed method was successfully applied for the determination of real sample and exhibited high specificity and low limits of detection in corn (112 ng L-1 for aflatoxin B1, 319 ng L-1 for ochratoxin A, and 377 ng L-1 for zearalenone). The green ELISA may also be extended to the detection of other biohazardous targets by changing the aptamer.

Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M1 and Escherichia coli O157:H7 in milk.

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.

Specific colorimetric ELISA method based on DNA hybridization reaction and non-crosslinking gold nanoparticles aggregation for the detection of amantadine.

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 µM) and exhibited a potential application for AMD screening on site.

Novel immunochromatographic assay based on Eu (III)-doped polystyrene nanoparticle-linker-monoclonal antibody for sensitive detection of Escherichia coli O157:H7.

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.

Strategy for Accurate Detection of Escherichia Coli O157:H7 in Ground Pork Using a Lateral Flow Immunoassay.

Escherichia coli O157:H7 is known to cause serious diseases including hemorrhagic colitis and hemolytic uremic syndrome. A gold nanoparticle lateral flow immunoassay (Au-LFIA) was used to detect Escherichia coli O157:H7 in ground pork samples. False-positive results were detected using Au-LFIA; a Citrobacterfreundii strain was isolated from the ground pork samples and identified by using CHROmagarTM plates, API 20E, and 16S RNA sequencing. Since C.freundii showed cross-reactivity with E. coli O157:H7 when Au-LFIA test strips were used, a novel method combining modified enrichment with a lateral flow immunoassay for accurate and convenient detection of E. coli O157:H7 in ground pork was developed in this study to minimize these false positives. MacConkey broth was optimized for E. coli O157:H7 enrichment and C.freundii inhibition by the addition of 5 mg/L potassium tellurite and 0.10 mg/L cefixime. Using the proposed modified enrichment procedure, the false-positive rate of ground pork samples spiked with 100 CFU/g C.freundii decreased to 5%.

Short communication: A novel method using immunomagnetic separation with a fluorescent nanobeads lateral flow assay for the rapid detection of low-concentration Escherichia coli O157:H7 in raw milk.

Escherichia coli O157:H7 is an important serotype of enterohemorrhagic E. coli that was first identified as a human pathogen in 1982. This pathogen causes several serious diseases. In this study, immunomagnetic separation was coupled with a fluorescent nanobeads lateral flow assay to establish a sensitive and rapid detection method for Escherichia coli O157:H7 in raw milk. The pathogen was captured from raw milk by immunomagnetic separation with immunomagnetic nanobeads and then detected using a fluorescent nanobeads lateral flow assay. A fluorescent line was formed in the test line of the test strip and quantitatively detected using a fluorescent reader. Screening times, which included immunomagnetic separation and the fluorescent nanobeads lateral flow assay, were 8, 7, 6, and 5h when 1, 5, 25, and 125 cfu of E. coli O157:H7, respectively, were inoculated into 25mL of raw milk. The established method could be widely applied to the rapid onsite detection of other pathogens to ensure food safety.

DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA.

This study reported on a novel sandwich enzyme linked immunosorbent assay (ELISA) for the sensitive determination of Escherichia coli O157:H7 (E. coli O157:H7) by using DNA-based hybridization chain reaction (HCR) and biotin-streptavidin signal amplification. The anti-E. coli O157:H7 polyclonal antibody (pAb) was immobilized in the ELISA wells. The anti-E. coli O157:H7 monoclonal antibody (mAb) and initiator strand (DNA1) were labeled on gold nanoparticle (AuNP) to form a mAb-AuNP-DNA1 complex. In the presence of the target E. coli O157:H7, the sandwiched immunocomplex, which is pAb-E. coli O157:H7-mAb-AuNP-DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA (dsDNA) that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase-streptavidin and substrate/chromogen solution. Under optimal conditions, E. coli O157:H7 could be detected in the range of 5×10(2) CFU/mL to 1×10(7) CFU/mL; the limit of detection was 1.08×10(2) CFU/mL in pure culture. The LOD of the novel ELISA was 185 times lower than that of traditional ELISA. The proposed method is considerably specific and can be applied in the detection of whole milk samples inoculated with E. coli O157:H7. The coefficient of variation of in pure culture and in whole milk was 0.99-5.88% and 0.76-5.38%, respectively. This method offers a promising application in the detection of low concentrations of food-borne pathogens.