On-line concentration of bacteria from tens-microliter sample volumes in roughened fused silica capillary with subsequent analysis by capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This study presents timely, reliable and sensitive method for identification of pathogenic bacteria in clinical samples based on a combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In this respect, a part of single-piece fused silica capillary was etched with supercritical water with the aim of using it for static or dynamic cell-surface adhesion from tens of L-sample volumes. The conditions for this procedure were optimized. Adhered cells of Staphylococcus aureus (methicillin-susceptible or methicillin-resistant) and of Pseudomonas aeruginosa were desorbed and pre-concentrated from the rough part of the capillary surface using transient isotachophoretic stacking from a high conductivity model matrix. The charged cells were swept and separated again in micellar electrokinetic chromatography using non-ionogenic surfactant. Static adhesion of the cells onto the roughened part of the capillary is certainly volumetric limited. Dynamic adhesion allows concentration of bacteria from 100 L- volumes of physiological saline solution, bovine serum, or human blood with the limits of detection 1.8×102, 1.7×103, and 1.0×103 cells mL-1, respectively. The limits of detection were the same for all three examined bacterial strains. The recovery of the method was about 83 % and it was independent on the sample matrix. Combination of CE with MALDI-TOF MS required at least 4×103 cells mL-1 to obtain reliable results. The calibration plots were linear (R2 = 0.99) and the relative standard deviations of the peak area were at most 2.2 %. The adhered bacteria, either individual or in a mixture, were on-line analyzed by micellar electrokinetic chromatography and then collected from the capillary and off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without interfering matrix components.