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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

Abstract

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 μL of the prepared sample at the optimized flow rate of 6.5 μL min−1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 = 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.

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Funding

This work was financially supported by the Grant Agency of the Czech Republic (Grant No. 19-00742S), Ministry of Health of the Czech Republic (Grant No. 16-29916A), Ministry of the Interior of the Czech Republic (Grant VI20172020069), by the Czech Academy of Sciences (Institutional Support RVO:68081715), and by the Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR) under National Sustainability Programme II, project CEITEC 2020 (LQ1601).

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Correspondence to Marie Horká.

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Horká, M., Karásek, P., Šalplachta, J. et al. 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. Microchim Acta 187, 177 (2020). https://doi.org/10.1007/s00604-020-4154-6

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Keywords

  • Capillary electrophoresis
  • MALDI-TOF mass spectrometry
  • Nano-etched fused-silica capillary
  • Phage propagation
  • Staphylococcal bacteriophages
  • Supercritical water