title: Using fluorescent nanoparticles and SYBR Green I based two-color flow cytometry to determine Mycobacterium tuberculosis avoiding false positives.
abstract: A method using an improved two-color flow cytometric analysis by a combination of bioconjugated fluorescent silica nanoparticles and SYBR Green I (FSiNP@SG-FCM) has been developed for detection of pathogenic Mycobacterium tuberculosis. Antibody-conjugated nanoparticles were prepared by oriented immobilization of the anti-M. tuberculosis antibody onto Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate-doped fluorescent silica nanoparticles (RuBpy-doped FSiNPs) through Protein A. M. tuberculosis was specially labeled with antibody-conjugated RuBpy-doped FSiNPs, then stained with a nucleic acid dye SYBR Green I to exclude background detrital particles, followed by multiparameter determination with flow cytometry. With this method, false positives caused by aggregates of nanoparticle-bioconjugates and nonspecific binding of nanoparticle-bioconjugates to background debris could be significantly decreased. This assay allowed for detection of as low as 3.5 x 10(3) and 3.0 x 10(4)cellsml(-1)M. tuberculosis in buffer and spiked urine respectively, with higher sensitivities than the FITC-based conventional flow cytometry. The total assay time including sample pretreatment was within 2h. This proposed FSiNP@SG-FCM method will be promising for rapid detection of M. tuberculosis or other pathogenic bacteria in clinical samples.
authors.affiliation: College of Chemistry and Chemical Engineering, Biomedical Engineering Center, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha, PR China.
authors.forename: Dilan
authors.initials: D
authors.lastname: Qin
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: He
authors.forename: Kemin
authors.initials: K
authors.lastname: Wang
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
mesh_terms: D052160:Benzothiazoles; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D015169:Colony Count, Microbial / Q000295:instrumentation*; D003959:Diamines; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005189:False Positive Reactions; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D009930:Organic Chemicals; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I