Premium
Rapid separations of nile blue stained microorganisms as cationic charged species by chip‐ CE with LIF
Author(s) -
Nuchtavorn Nantana,
Bek Fritz,
Macka Mirek,
Suntornsuk Worapot,
Suntornsuk Leena
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100698
Subject(s) - cationic polymerization , nile red , chemistry , microorganism , nile blue , chip , biology , bacteria , physics , organic chemistry , telecommunications , engineering , genetics , quantum mechanics , fluorescence
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 E scherichia coli , S taphylococcus aureus , and C andida albicans were selected as representatives of G ram‐positive bacteria, G ram‐negative bacteria, and fungi. While their cells carry an overall negative charge in neutral to alkaline p H , 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 p H 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 ( R s > 5.3) in an electrolyte consisting of 3.94 m M T ris, 0.56 m M boric acid, 0.013 m M 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.