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Online Concentration of Bacteria from Tens of 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.
ACS Infectious Diseases ( IF 4.0 ) Pub Date : 2020-01-13 , DOI: 10.1021/acsinfecdis.9b00200 Marie Horká 1 , Jiří Šalplachta 1 , Pavel Karásek 1 , Filip Ru Žička 2 , Michal Roth 1
ACS Infectious Diseases ( IF 4.0 ) Pub Date : 2020-01-13 , DOI: 10.1021/acsinfecdis.9b00200 Marie Horká 1 , Jiří Šalplachta 1 , Pavel Karásek 1 , Filip Ru Žička 2 , Michal Roth 1
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This study presents a 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 a 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 microliter 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 preconcentrated from the rough part of the capillary surface using transient isotachophoretic stacking from a high conductivity model matrix. The charged cells were swep and separated again in micellar electrokinetic chromatography using a nonionogenic surfactant. Static adhesion of the cells onto the roughened part of the capillary is certainly volumetric limited. Dynamic adhesion allows the concentration of bacteria from 100 μL volumes of physiological saline solution, bovine serum, or human blood with the limits of detection at 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 of the sample matrix. A combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 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 online 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.
中文翻译：
在粗糙的熔融石英毛细管中从数十微升样品体积在线收集细菌，随后通过毛细管电泳和基质辅助激光解吸/电离飞行时间质谱分析。
本研究提出了一种基于毛细管电泳与基质辅助激光解吸/电离飞行时间质谱联用的临床样品中病原菌鉴定的及时，可靠和灵敏方法。在这方面，单片熔融石英毛细管的一部分用超临界水蚀刻，目的是将其用于数十微升样品体积的静态或动态细胞表面粘附。优化了该程序的条件。金黄色葡萄球菌（对甲氧西林敏感或耐甲氧西林的细菌）和铜绿假单胞菌的粘附细胞通过从高电导率模型基质中获得的瞬时等速电泳堆叠从毛细血管表面的粗糙部分解吸和预浓缩。扫掠带电的细胞，并使用非离子表面活性剂在胶束电动色谱法中再次分离。细胞在毛细管的粗糙部分上的静态粘附当然在体积上受到限制。动态粘附允许从100μL体积的生理盐水溶液，牛血清或人血中富集细菌，检出限分别为1.8×102、1.7×103和1.0×103细胞mL-1。所有三种检查的细菌菌株的检出限均相同。该方法的回收率约为83％，与样品基质无关。毛细管电泳与基质辅助激光解吸/电离飞行时间质谱的组合至少需要4×103细胞mL-1才能获得可靠的结果。校准曲线是线性的（R2 = 0。99），峰面积的相对标准偏差最大为2.2％。粘附的细菌，无论是单个细菌还是混合物中的细菌，均通过胶束电动色谱法进行在线分析，然后从毛细管中收集并通过基质辅助激光解吸/电离飞行时间质谱进行离线分析，而不会干扰基质成分。
更新日期：2020-01-13
中文翻译：
在粗糙的熔融石英毛细管中从数十微升样品体积在线收集细菌，随后通过毛细管电泳和基质辅助激光解吸/电离飞行时间质谱分析。
本研究提出了一种基于毛细管电泳与基质辅助激光解吸/电离飞行时间质谱联用的临床样品中病原菌鉴定的及时，可靠和灵敏方法。在这方面，单片熔融石英毛细管的一部分用超临界水蚀刻，目的是将其用于数十微升样品体积的静态或动态细胞表面粘附。优化了该程序的条件。金黄色葡萄球菌（对甲氧西林敏感或耐甲氧西林的细菌）和铜绿假单胞菌的粘附细胞通过从高电导率模型基质中获得的瞬时等速电泳堆叠从毛细血管表面的粗糙部分解吸和预浓缩。扫掠带电的细胞，并使用非离子表面活性剂在胶束电动色谱法中再次分离。细胞在毛细管的粗糙部分上的静态粘附当然在体积上受到限制。动态粘附允许从100μL体积的生理盐水溶液，牛血清或人血中富集细菌，检出限分别为1.8×102、1.7×103和1.0×103细胞mL-1。所有三种检查的细菌菌株的检出限均相同。该方法的回收率约为83％，与样品基质无关。毛细管电泳与基质辅助激光解吸/电离飞行时间质谱的组合至少需要4×103细胞mL-1才能获得可靠的结果。校准曲线是线性的（R2 = 0。99），峰面积的相对标准偏差最大为2.2％。粘附的细菌，无论是单个细菌还是混合物中的细菌，均通过胶束电动色谱法进行在线分析，然后从毛细管中收集并通过基质辅助激光解吸/电离飞行时间质谱进行离线分析，而不会干扰基质成分。
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