Fluorescence signal amplification assay for the detection of B. melitensis 16M, based on peptide-mediated magnetic separation technology and a AuNP-mediated bio-barcode assembled by quantum dot technology.

Members of the Brucella spp. are facultative intracellular bacteria that can cause global brucellosis, a zoonotic disease. Herein, a novel fluorescence signal amplification (FSA) method for the rapid detection of B. melitensis 16M was developed based on peptide-mediated magnetic separation (PMS) technology and Au nanoparticle (AuNP)-mediated bio-barcode assay technology assembled by quantum dots (QDs). The PMS technology was used to specifically capture and isolate B. melitensis 16M from food. The immunomagnetic bead-B. melitensis 16M bioconjugates (IMBs-B. melitensis 16M) were then identified by IgY on the surface of AuNPs and the oligonucleotide chains on the surface of the gold nanoparticles were hybridized with bio-barcodes assembled by quantum dots (QD-probe2). The IMB/B. melitensis 16M/IgY-AuNP-probe1/QD-probe2 bioconjugates were concentrated by magnetic separation. Therefore, as the concentration of B. melitensis 16M in the sample increased, the unbound QD-probe2 in the supernatant reduced, and the B. melitensis 16M in the sample could be indirectly measured by detecting the fluorescence in the supernatant. This FSA method can detect B. melitensis 16M concentration in the range of 10 to 106 cfu ml-1 without pre-enrichment, and the limit of detection (LOD) is as low as 10 cfu ml-1 with high specificity. Furthermore, the proposed method for the detection of B. melitensis 16M has a LOD of 1.07 × 102 cfu ml-1 and a linear range from 102 to 107 cfu ml-1 in milk, and a LOD of 1.72 × 102 cfu ml-1, and a linear range from 102 to 106 cfu ml-1 in lamb leach. In addition, this method takes less than 3 h to perform. Thus, the assay that was developed in this study shows promise for rapid, sensitive, and specific detection of B. melitensis 16M.

Lateral Flow Immunoassay for Visible Detection of Human Brucellosis Based on Blue Silica Nanoparticles.

Brucellosis is a highly contagious zoonosis chronic infectious disease with a strong latent capability to endanger human health and economic development via direct or indirect ways. However, the existing methods for brucellosis diagnosis are time-consuming and expensive as they require a tedious experimental procedure and a sophisticated experimental device and performance. To overcome these defects, it is truly necessary to establish a real-time, on-site, and rapid detection method for human brucellosis. Here, a lateral flow immunoassay (LFIA) with a rapid, sensitive, and alternative diagnostic procedure for human brucellosis with a high degree of accuracy was developed based on blue silica nanoparticles (SiNPs), Staphylococcal protein A (SPA), and surface Lipopolysaccharide of Brucella spp. (LPS), which can be applied for rapid and feasible detection of human brucellosis. To our knowledge, this is the first report that uses blue SiNPs as a signal probe of LFIA for the rapid diagnosis of human brucellosis. The precursor of blue SiNPs@SPA such as colorless SiNPs and blue SiNPs was synthesized at first and then coupled with SPA onto the surface of blue SiNPs by covalent bond to prepare blue SiNPs@SPA as a capture signal to catch the antibody in the brucellosis-positive serum. When SPA was combined with the antibodies in the brucellosis-positive serum, it was captured by LPS on the test line, forming an antigen-antibody sandwich structure, resulting in the T line turning blue. Finally, the results showed that it is acceptable to use blue SiNPs as visible labels of LFIA, and standard brucellosis serum (containing Brucella spp. antibody at 1,000 IU/ml) could be detected at a dilution of 10-5 and the detection limit of this method was 0.01 IU/ml. Moreover, it also demonstrated good specificity and accuracy for the detection of real human serum samples. Above all, the blue SiNPs-based LFIA that we developed provides a rapid, highly accurate, and inexpensive on-site diagnosis of human brucellosis, and shows great promise in clinical diagnostics for other diseases.

A detection method of Escherichia coli O157:H7 based on immunomagnetic separation and aptamers-gold nanoparticle probe quenching Rhodamine B's fluorescence: Escherichia coli O157:H7 detection method based on IMS and Apt-AuNPs probe quenching Rho B' s fluorescence.

This research aimed to detect Escherichia coli O157:H7 in milk based on immunomagnetic probe separation technology and quenching effect of gold nanoparticles to Rhodamine B. Streptavidin-modified magnetic beads (MBs) were combined with biotin-modified antibodies to capture E. coli O157:H7 specifically. Gold nanoparticle (AuNPs) was incubated with sulfhydryl-modified aptamers (SH-Aptamers) to obtain the Aptamers-AuNPs probe. After magnetic beads captured target bacteria and formed a sandwich structure with the gold nanoprobe, Rhodamine B was added into complex to obtain fluorescent signal changes. Our results demonstrated that the established method could detect E. coli O157:H7 in the range of 101-107 CFU/mL, and the limit of detection (LOD) was 0.35 CFU/mL in TBST buffer (pH = 7.4). In milk simulation samples, the LOD of this method was 1.03 CFU/mL. Our research provides a promising approach on the detection of E. coli O157:H7.

Simultaneous Detection of Three Foodborne Pathogens Based on Immunomagnetic Nanoparticles and Fluorescent Quantum Dots.

This paper presents a peptide-mediated immunomagnetic separation technique and an immunofluorescence quantum dot technique for simultaneous and rapid detection of Escherichia coli O157:H7, Staphylococcus aureus, and Vibrio parahaemolyticus. First, three peptides that can specifically recognize the three foodborne pathogens were combined with magnetic nanoparticles to form an immunomagnetic nanoparticle probe for capturing three kinds of target bacteria and then added three quantum dot probes (quantum dots-aptamer), which formed a sandwich composite structure. When the three quantum dot probes specifically combined with the three pathogenic bacteria, the remaining fluorescent signal in the supernatant will be reduced by magnetic separation. Therefore, the remaining fluorescent signal in the supernatant can be measured with a fluorescence spectrophotometer to indirectly determine the three pathogens in the sample. The linear range of the method was 10-107 cfu/mL, and in the buffer, the detection limits of E. coli O157:H7, S. aureus, and V. parahaemolyticus were 2.460, 5.407, and 3.770 cfu/mL, respectively. In the tap water simulation, the detection limits of E. coli O157:H7, S. aureus, and V. parahaemolyticus were 2.730, 1.990 × 101, and 4.480 cfu/mL, respectively. In the milk simulation sample, the detection limits of E. coli O157:H7, S. aureus, and V. parahaemolyticus were 6.660, 1.070 × 101, and 2.236 × 101 cfu/mL, respectively. The method we presented can detect three kinds of foodborne pathogens at the same time, and the entire experimental process did not exceed 4 h. It has high sensitivity and low detection limit and may be used in the sample detection of other pathogens.