Weihua Lai

Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays.

Label selection is of vital importance for immunochromatographic assays. In this study, the fluorescent microsphere test strip and colloidal gold immunochromatographic test strip (FM-ICTS and CG-ICTS) were developed for the detection of Escherichia coli O157:H7 on the basis of the sandwich format. Two types of labels, namely, colloidal gold particles (CG) and carboxyl-modified fluorescent microspheres (FMs), were compared while coupling with anti-E. coli O157:H7 monoclonal antibody (mAb). The FM-ICTS and CG-ICTS were also compared. Results show that the coupling rate between FMs and mAb was higher than that between CG and mAb. Under optimum conditions, the sensitivity of FM-ICTS was eight times higher than that of CG-ICTS. Approximately 0.1 μg of mAb was used in every FM-ICTS, whereas 0.4 μg of mAb was used in every CG-ICTS. The coefficient of variation of FM-ICTS and CG-ICTS was 4.8% and 16.7%, respectively. The FM-ICTS and CG-ICTS can be stored at room temperature for 12 months and specific to five E. coli O157:H7 strains. Milk sample inoculated with E. coli O157:H7 were tested by the FM-ICTS and CG-ICTS. The FM-ICTS sensitivity was 10(4) CFU/ml while the CG-ICTS sensitivity was 10(5) CFU/ml. The sensitivity, consumption of antibodies, and coefficient of variation of FM-ICTS were better than those of CG-ICTS for the detection of E. coli O157:H7.

Novel Strategies To Enhance Lateral Flow Immunoassay Sensitivity for Detecting Foodborne Pathogens

Food contaminated by foodborne pathogens causes diseases, affects individuals, and even kills those affected individuals. As such, rapid and sensitive detection methods should be developed to screen pathogens in food. One current detection method is lateral flow immunoassay, an efficient technique because of several advantages, including rapidity, simplicity, stability, portability, and sensitivity. This review presents the format and principle of lateral flow immunoassay strip and the development of conventional lateral flow immunoassay for detecting foodborne pathogens. Furthermore, novel strategies that can be applied to enhance the sensitivity of lateral flow immunoassay to detect foodborne pathogens are presented; these strategies include innovating new label application, designing new formats of lateral flow immunoassay, combining with other methods, and developing signal amplification systems. With these advancements, detection sensitivity and detection time can be greatly improved.

Fluorescent Ru(phen)32+-Doped Silica Nanoparticles-Based ICTS Sensor for Quantitative Detection of Enrofloxacin Residues in Chicken Meat

A Ru(phen)3(2+)-doped silica fluorescent nanoparticle (FN)-based immunochromatographic test strip (ICTS) sensor was developed for rapid, high sensitivity, easy to use, and low cost quantitative detection of enrofloxacin (ENR) residues in chicken meat. The fluorescence signal intensity of the FNs at the test line (FI(T)) and control line (FI(C)) was determined with a prototype of a portable fluorescent strip reader. Unique properties of Ru(phen)3(2+) doped silica nanoparticles (e.g., large Stokes shift, high emission quantum yield, and long fluorescence lifetime) were combined with the advantages of ICTS and an easy to make portable fluorescent strip reader. The signal was based on FI(T)/FI(C) ratio to effectively eliminate strip to strip variation and matrix effects. Various parameters that influenced the strip were investigated and optimized. Quantitative ENR detection with the FNs ICTS sensor using 80 μL sample took only 20 min, which is faster than the commercial ELISA kit (that took 90 min). The linear range of detection in chicken extract was established at 0.025-3.500 ng/mL with a half maximal inhibitory concentration at 0.22 ± 0.02 ng/mL. Using the optimized parameters, the limit of detection (LOD) for ENR using the FNs ICTS sensor was recorded at 0.02 ng/mL in chicken extract. This corresponds to 0.12 μg/kg chicken meat which is two (2) orders of magnitude better that the maximum residue limits (MRLs) imposed in Japan (10 μg/kg) and three (3) orders of magnitude better than those imposed in China. The intra- and inter-assay coefficient of variations (CVs) were 6.04% and 12.96% at 0.5 ng/mL, 6.92% and 12.61% at 1.0 ng/mL, and 6.66% and 11.88% at 2.0 ng/mL in chicken extract, respectively. The recoveries using the new FNs ICTS sensor from fifty (50) ENR-spiked chicken samples showed a highly significant correlation (R(2) = 0.9693) with the commercial enzyme-linked immunosorbent assay (ELISA) kit. The new FNs ICTS sensor is a simple, rapid, sensitive, accurate, and inexpensive quantitative detection of ENR residues in chicken meat and extracts.

Ru(phen)32+ doped silica nanoparticle based immunochromatographic strip for rapid quantitative detection of β-agonist residues in swine urine

A Ru(phen)3(2+) doped silica nanoparticle based immunochromatographic strip was developed for the rapid and quantitative detection of five common β-agonist (salbutamol (SAL), cimbuterol, terbutaline, clenbuterol, and brombuterol) residues in swine urine. The broad spectrum monoclonal antibodies generated by immunizing BALB/c mice with salbutamol conjugated cationic bovine serum albumin. The fluorescence intensities (FIs) of the strip on the test line (FIT) and control line (FIC) were determined using a strip reader. Parameters that influenced the antibody and antigen interaction on the test strip were investigated by recording FIT and FIC values, and the concept of FIT/FIC ratio was used to offset the inherent heterogeneity of the test strips and the effect of the sample matrix. Under optimal conditions, the linear range for the quantitative detection of SAL was 0.6-5.0 ng/ml with a half maximal inhibitory concentration at 1.78 ng/ml. The limit of detection for real swine urine was 0.43 ng/ml. The recovery rates of the intraassay for spiked urine at SAL concentrations of 0.8, 1.5, and 3.5 ng/mL were 88.06%±3.75%, 95.77%±5.33%, and 94.06%±7.43%, whereas those for the interassay were 84.69%±5.0%, 95.06%±9.3%, and 88.34%±7.71%, respectively. The developed quantitative method exhibited excellent agreement with a commercially available competitive enzyme-linked immunosorbent assay kit for SAL-spiked urine samples, with a correlation of coefficient of 0.95 and a slope of 0.99 (n=36). The results indicated that the developed test strip enables sensitive, reproducible, and easily implementable screening for the rapid and quantitative detection of β-agonist residues in swine urine.

A sensitive impedance biosensor based on immunomagnetic separation and urease catalysis for rapid detection of Listeria monocytogenes using an immobilization-free interdigitated array microelectrode

In this study, we described a novel impedance biosensor combining immunomagnetic separation with urease catalysis for sensitive detection of foodborne bacteria using Listeria monocytogenes as model and an immobilization-free microelectrode as detector. The monoclonal antibodies (MAbs) were immobilized on the surface of the magnetic nanoparticles (MNPs) with the diameter of 180 nm by biotin-streptavidin system for specifically and efficiently separating Listeria cells from sample background. The polyclonal antibodies (PAbs) and the urease were modified onto the surface of the gold nanoparticles (AuNPs) with the diameter of 20 nm and the modified AuNPs were used to react with Listera to form the MNP-MAb-Listeria-PAb-AuNP-urease sandwich complexes. The urease in the complexes could catalyze the hydrolysis of the urea into ammonium carbonate and this led to an increase in the ionic strength of the media, which could be detected by the microelectrode. The magnetic separation efficiencies for L. monocytogenes at the concentrations ranging from 3.0×10(1) to 3.0×10(4) CFU/mL were over 95% for the pure cultures and over 85% for the spiked lettuce samples. The lower detection limit of this biosensor for L. monocytogenes was found to be 300 CFU/mL in both the pure cultures and the spiked lettuce samples. The microelectrode was demonstrated to be reusable for over 50 times with thorough cleaning by deionized water. This biosensor showed its potential to provide a simple, low-cost and sensitive method for rapid screening of foodborne pathogens and could be extended for detection of other biological or chemical targets.

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.

Sulfonated polystyrene magnetic nanobeads coupled with immunochromatographic strip for clenbuterol determination in pork muscle.

A magnetic solid-phase extraction method (MSPE) was developed to pre-concentrate and cleanup clenbuterol (CLE) from pork muscle. Novel sulfonated polystyrene magnetic nanobeads (spMNBs) were synthesized via a one-pot emulsion copolymerization method by using divinylbenzene, styrene, and sodium styrene sulfonate in the presence of oleic acid-modified and 10-undecylenic acid-modified magnetic ferrofluid. The resulting spMNBs exhibited high adsorption efficiency for CLE and for 10 other common beta-adrenergic agonists, namely, brombuterol, ractopamine, tulobuterol, bambuterol, cimbuterol, mabuterol, clorprenaline, penbutolol, salbutamol, and cimaterol. The adsorption behavior of the spMNBs for CLE was described by the Langmuir equation with a maximum adsorption capacity of 0.41 mg/g. Under the optimized parameters, the extraction of CLE from 0.5 g of pork muscle required 25mg of the spMNBs at a shortened adsorption time (0.5 min). The proposed MSPE was coupled with colloidal gold nanoparticle-based immunochromatographic assay (MSPE-AuNPIA) for the quantitative detection of CLE residue in pork muscle. The limit of detection and limit of quantification for the pork muscle were 0.10 and 0.24 ng/g, respectively. The intra-day and inter-day assay recoveries at three CLE spiked concentrations ranged from 92.5% to 98.1%, with relative standard deviations ranging from 3.2% to 13.0%. The results of MSPE-AuNPIA were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CLE values obtained with MSPE-AuNPIA agreed with those obtained with LC-MS/MS.

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.

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.

Advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold as label in lateral flow assays for detection of ractopamine

Label selection is a critical factor for improving the sensitivity of lateral flow assay. Time-resolved fluorescent nanobeads, fluorescent submicrospheres, quantum dots, and colloidal gold-based lateral flow assay (TRFN-LFA, FM-LFA, QD-LFA, and CG-LFA) were first systematically compared for the quantitative detection of ractopamine in swine urine based on competitive format. The limits of detection (LOD) of TRFN-LFA, FM-LFA, QD-LFA, and CG-LFA were 7.2, 14.7, 23.6, and 40.1pg/mL in swine urine samples, respectively. The sensitivity of TRFN-LFA was highest. In the quantitative determination of ractopamine (RAC) in swine urine samples, TRFN-LFA exhibited a wide linear range of 5pg/mL to 2500pg/mL with a reliable coefficient of correlation (R2=0.9803). Relatively narrow linear ranges of 10-500pg/mL (FM-LFA) and 25-2500pg/mL (QD-LFA and CG-LFA) were acquired. Approximately 0.005µg of anti-RAC poly antibody (pAb) was used in each TRFN-LFA test strip, whereas 0.02, 0.054, and 0.15µg of pAb were used in each of the FM-LFA, QD-LFA, and CG-LFA test strips, respectively. In addition, TRFN-LFA required the least RAC-BSA antigens and exhibited the shortest detection time compared with the other lateral flow assays. Analysis of the RAC in swine urine samples showed that the result of TRFN-LFA was consistent with that of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a commercial enzyme-linked immunosorbent assay (ELISA) kit.