Application of Nanoparticles for the Detection and Sorting of Pathogenic Bacteria by Flow-Cytometry

Abstract

In this paper we will describe a new developed contribution of fluorescence nano-crystal (q-dots) as a fluorescence label for detecting pathogenic bacteria by flow cytometry (FCM) and the use of nano-magnetic particles to improve bacterial sorting by Flow cytometry cell sorting (FACS).FCM or FACS systems are based upon single cell detection by light scatter and Immunofluorescence labeling signals. The common FACS systems are based upon single or dual excitation as excitation source both for light scatter parameters and for several fluorescence detectors. Hence, for multi-labeling detection, there is a need for fluorophores with broad excitation wave length and sharp emission bands. Moreover, such fluorophores should be with high fluorescence efficiency, stable, and available for bio-molecules conjugation. Q-dots benefit from practical features which meet those -criteria. We will describe the use of q-dots as fluorescence labels for specific conjugates against Bacillus anthracis spores and Yersinia pestis bacteria, which enable the specific detection of the different species. A specific and sensitive multiplex analysis procedure for both pathogens was achieved, with high sensitivity down to 10(3) bacteria per ml in the sample.Sorting bacteria by FACS has a tremendous advantage for sensitive and selective analysis and sorting of sub-populations. However it has always been a difficult task due to the fact that bacteria are small particles (usually 1-3 μm). For such small particles, light scatter signal is on the threshold level, and many positive events may be lost. Here we will present the development of a procedure for sorting of the gram negative bacteria Y. pestis from environment samples. We will show that the application of nano-magnetic particles, as a tool for the immunomagnetic labeling and separation of the bacteria, enables fast sorting in high and low bacterial concentration down to 10 (5) cfu/ml. The nano-metric physical size of the immunospecific labeling particles disguises them from the FACS detectors; hence the bacterial population becomes the major population as opposed to being "rare events population" when using standard micro-magnetic beads for pre-enrichment.The procedure of separation and collection of bacteria enables sensitive detection and characterization methods of bacteria from complex samples.

Application of Flow Cytometry to the Detection of Pathogenic Bacteria

“…From such diverse materials, one needs to develop FCM based assays for rapid and specific detection of the harmful pathogens. Such FCM assays should be rapid, sensitive, credible by validation, suitable for simultaneous agent detection (multiplex analysis) and applicable to a variety of complex samples (Zahavy et al, 2012). More recently, because of concerns regarding bioterrorism, accurate analytical techniques for pathogenic agents are needed which can be deployed at public transport hubs or scenes of terrorist incidents.…”

“…simultaneous agent detection (multiplex analysis) and applicable to a variety of complex samples (Zahavy et al, 2012). More recently, because of concerns regarding bioterrorism, accurate analytical techniques for pathogenic agents are needed which can be deployed at public transport hubs or scenes of terrorist incidents.…”

“…Such fluorophores should have high fluorescence efficiency, stability, and be available for conjugation with other bio-molecules. Quantum dots (QDs) provide practical features which meet many of these criteria (Zahavy et al, 2012) as they are inorganic crystalline nanoparticles made of semiconducting materials. QDs used for biological applications typically are composed of three different layers starting with a core, normally cadmium selenide (CdSe) coated with a semiconductor outer shell of zinc sulphide (ZnS).…”

“…QDs are currently being used as a novel fluorophores as their physical and chemical properties confer significant advantages over traditional dyes, including brighter fluorescence and resistance to photobleaching (Ibanez-Peral et al, 2008). In comparison to organic fluorophores, a single excitation source can simultaneously excite several QDs with distinctive emission bands which can be easily separated by optical filters (Zahavy et al, 2012). QDs have applications for bacterial and pathogen detection in combination with FCM as well as for bacteriophages (Edgar et al, 2006).…”

Simplified confocal microscope for counting particles at low concentrations

“…One of the mainstays in cellular and particle detection is flow cytometry, which can be used for measuring pathogens. [4][5][6][7] Flow cytometry combines fluid flow through a confined volume to characterize a large number of particles using fluorescence and light scattering. Hydrodynamic focusing before detection reduces background signal permitting high sensitivity.…”