Daofeng Liu

Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7.

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14×10(3) CFU mL(-1). The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.

Rapid pretreatment and detection of trace aflatoxin B1 in traditional soybean sauce

Soybean sauce, a traditional fermented food in China, has different levels of aflatoxin B1 pollution. Two kinds of direct and indirect immunomagnetic bead methods for the pretreatment of aflatoxin B1 were evaluated in this work. A method was established to detect aflatoxin B1 in soybean sauce using an immunomagnetic bead system for pretreatment and ELISA for quantification. The pretreatment method of immunomagnetic beads performed better compared with the conventional extraction and immunoaffinity column method. ELISA exhibited a good linear relationship at an aflatoxin B1 concentration of 0.05-0.3μg/kg (r(2)=0.9842). The average recoveries across spike levels varied from 0.5 to 7μg/kg were 83.6-104% with a relative standard deviation between 4.2% and 11.7%. With the advantages of rapid detection, easy operation, simple equipment, sensitivity, accuracy, and high recovery; this method can be well applied in the trace determination of aflatoxin B1 in soybean sauce samples.

Rapid Detection of Aflatoxin M1 by Immunochromatography Combined with Enrichment Based on Immunomagnetic Nanobead

Abstract A novel method was developed for detection of aflatoxin M1 by immunochromatography coupled with enrichment on the basis of immunomagnetic nanobeads. The immunomagnetic nanobeads coated anti-aflatoxin M1 antibody was synthesized using EDC/NHS method. Immunochromatographic strip was assembled with conjugate pad, sample pad, absorbent pad, and nitrocellulose membrane. Nitrocellulose membrane sprayed with aflatoxin M1-BSA and donkey anti-mouse antibody served as both test line and control line. For detecting process, immunomagnetic nanobeads were mixed with sample for enriching and separating AFM1. Afterward enrichment solution was detected by immunochromatographic strip. The limit of detection of this assay for aflatoxin M1 in raw milk was 0.1 ng mL−1, which was lower than the legal limit of 0.5 ng mL−1 set by China. No cross reactions were found with other mycotoxins and common illegal additives. The result by the proposed method was in good agreement with that of ELISA. The assay can be applied for rapid detection of on-site aflatoxin M1 in raw milk.

Quantitative detection of β2-adrenergic agonists using fluorescence quenching by immunochromatographic assay

β2-Adrenergic agonists are banned in China and other areas in the world. In this study, a novel method was developed to quantitatively detect β2-adrenergic agonists. The clorprenaline (CLP)–bovine serum albumin (BSA) conjugate is mixed with a BSA–fluorescent microsphere (FM) complex and sprayed on a nitrocellulose membrane as the test line; a goat-anti-mouse antibody is mixed with a BSA–fluorescent microsphere (FM) complex and sprayed on a nitrocellulose membrane as the control line. If the target molecule is absent in the sample, the colloidal gold-monoclonal antibody will bind to the CLP–BSA conjugate coated on the test line, and the colloidal gold quenches the fluorescent microspheres, so that no fluorescent signal develops in the test line, indicating a negative result. The target molecule present in the sample at a cutoff level or higher binds to the colloidal gold-monoclonal antibody in the ELISA well. The colloidal gold-monoclonal antibody (Au-mAb) does not bind to the CLP–BSA conjugate coated on the test line. The fluorescent signal developed in the test line indicates a positive result. The limit of detection (LOD) of the immunochromatographic assay test strip was 0.12 ng mL−1 when the antibody amount was 0.8 μg mL−1 with a detection time of 15 min. The immunochromatographic assay test strip could simultaneously detect five β2-adrenergic agonists, including clorprenaline, bambuterol, terbutaline, clenbuterol, and salbutamol. When spiked swine urine samples (5.0 ng mL−1 and 10.0 ng mL−1) were tested by the novel immunoassay, the recovery was 39.00 ± 3.0 and 32.00 ± 2.0, respectively.

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.xa0coli 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.xa0coli 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 9.54xa0×xa0102, 1.59xa0×xa0102, 3.18xa0×xa0102, 2.98xa0×xa0102, and 1.08xa0×xa0102xa0colony-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 6.36xa0×xa0102-1.59xa0×xa0105, 3.18xa0×xa0102-1.59xa0×xa0105, 6.36xa0×xa0102-1.59xa0×xa0105, 6.36xa0×xa0102-1.59xa0×xa0105, and 8.0xa0×xa0101-1.59xa0×xa0105xa0CFUxa0mL-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.xa0coli 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 Citrobacter freundii 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%.