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Nano-etched fused-silica capillary used for on-line preconcentration and electrophoretic separation of bacteriophages from large blood sample volumes with off-line MALDI-TOF mass spectrometry identification



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Časopis / Zdroj Microchimica Acta
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Klíčová slova Capillary electrophoresis; MALDI-TOF mass spectrometry; Nano-etched fused-silica capillary; Phage propagation; Staphylococcal bacteriophages; Supercritical water
Přiložené soubory
Popis The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10 mu L of the prepared sample at the optimized flow rate of 6.5 mu L min(-1). The limit of detection was determined to be 10(4) phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R-2 = 0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (+/-) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract
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