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Rapid separations of nile blue stained microorganisms as cationic charged species by chip-CE with LIF


Nuchtavorn, N and Bek, F and Macka, M and Suntornsuk, W and Suntornsuk, L, Rapid separations of nile blue stained microorganisms as cationic charged species by chip-CE with LIF, Electrophoresis, 33, (9-10) pp. 1421-1426. ISSN 0173-0835 (2012) [Refereed Article]

Copyright Statement

Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

DOI: doi:10.1002/elps.201100698


Rapid detection of microorganisms by alternative methods is desirable. Electromigration separation methods have the capability to separate microorganisms according to their charge and size and laser-induced fluorescence (LIF) detection have single-cell detection capability. In this work, a new combined separation and detection scheme was introduced using chip-based capillary electrophoresis (chip-CE) platform with LIF detection. Three microorganisms Escherichia coli, Staphylococcus aureus, and Candida albicans were selected as representatives of Gram-positive bacteria, Gram-negative bacteria, and fungi. While their cells carry an overall negative charge in neutral to alkaline pH, staining them with nile blue (NB) provided highly sensitive LIF detection with excitation and emission wavelengths at 635 nm and 685 nm, respectively, and at the same time, the overall charge was converted to positive. Electrolyte pH and concentration of polyethylene oxide (PEO) significantly affected the resolution of the microorganisms. Their optimal separation in the 14 mm separation channel was achieved in less than 30 s (Rs > 5.3) in an electrolyte consisting of 3.94 mM Tris, 0.56 mM boric acid, 0.013 mM ethylenediaminetetraacetic acid disodium salt dihydrate (pH 10.5), and 0.025% PEO, with injection/separation voltages of +1000/+1000 V. The separation mechanism is likely employing contributions to the overall cationic charge from both the prevalently anionic membrane proteins and the cationic NB. Importantly, the resulting cationic NB-stained cells exhibited excellent separation selectivity and efficiency of ~38000 theoretical plates for rapid separations within 30-40 s. The results indicate the potential of chip-CE for microbial analysis, which offers separations of a wide range of species with high efficiency, sensitivity, and throughput.

Item Details

Item Type:Refereed Article
Keywords:chip-based CE, fluorescent staining, laser-induced fluorescence, microorganisms
Research Division:Chemical Sciences
Research Group:Analytical chemistry
Research Field:Separation science
Objective Division:Expanding Knowledge
Objective Group:Expanding knowledge
Objective Field:Expanding knowledge in the chemical sciences
UTAS Author:Macka, M (Professor Mirek Macka)
ID Code:82981
Year Published:2012
Web of Science® Times Cited:18
Deposited By:Austn Centre for Research in Separation Science
Deposited On:2013-02-26
Last Modified:2017-10-29

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