Using fluorescent nanoparticles and SYBR Green I based two-color flow cytometry to determine Mycobacterium tuberculosis avoiding false positives

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) cells ml(-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 2 h. This proposed FSiNP@SG-FCM method will be promising for rapid detection of M. tuberculosis or other pathogenic bacteria in clinical samples. (C) 2008 Elsevier B.V. All rights reserved.