Rapid And Sensitive Bacteria Detection Using Fluorescent Nanoparticles

Bacterial pathogens pose a significant threat to human, animal, and agricultural health. Rapid and sensitive determination of pathogenic bacteria is extremely important in biotechnology, medical diagnosis, and the current fight against bioterrorism. Containment and proper treatment are contingent upon knowing rapidly, sensitively, and accurately which pathogenic bacteria are present. There has been a critical demand for techniques that can recognize numerous bacterial pathogens simultaneously in a single assay, thus giving an early warning of bacteria contamination. A bioconjugated nanoparticle-based method was developed for the bacteria rapid and sensitive detection. The research details are listed as follows:1. We synthesized RuBpy-doped silica nanoparticles with a modified water-in-oil microemulsion method. The dye molecules were trapped inside a silica matrix to form the dye-doped nanoparticles. The size of the nanoparticles was uniform, with a diameter of50nm. 2. Small and bright TMR-doped nanoparticle was prepared. The size of the nanopartilces were19nm,15nm and13nm respectively. These nanoparticles were monodisperse in solution,16,13and5times brighter than their constituent fluorophore respectively. In this preparation the size was decided by the concentration of the dye in reaction and not related with the time of TEOS hydrolyzing.3. Three types of FRET NPs were prepared. FITC, R6G, and ROX were first covalently linked to the silane coupling agent APTS. The three APTS-dye conjugates were then mixed at desired ratios (1:0.5:1,0.5:1:1,0.5:0.5:3) The spectra of the triple-dye-doped NPs were scanned using the spectrofluorometer, all NPs exhibit three emission peaks due to the encapsulated three types of dyes, while the combinatorial color varies because of the different doping ratio.4. The modification of the nanoparticle. Different antibody labeling strategies were used in the assay to test binding efficiency and specificity: A:directly immobilizing antibody onto COOH-NP surface using carbodiimide interaction; B:linking COOH-NP surface with streptavidin as a bridge molecule then immobilized biotin-antibody onto streptavidin. C:immobilizing NHS-PEG-biotin molecules onto NH2-modified NPs, after incubation with streptavidin, biotin-labeled antibody was anchored onto NP surface. For the bacteria binding the third Strategy is the best. 5. The antibody-conjugated nanoparticles bound to bacteria samples were detected with flow channel, which can precisely detect a single bacterial cell by giving a fluorescent spike when the cell flows through the detection zone. The bacteria number was equivalent to that form plate-counting method.6. Escherichia coli, Salmonella typhimurium and Staphylococcus aureus were used as model pathogens for the multiplex bacteria detection. In the confocal microscope different bacteria with their specific antibody-conjugated nanoparticles emitted different color. Using FRET silica NPs holds great promise to develop multiplexed bacterial detection kits.