Clark Kevin D, Purslow Jeffrey A, Pierson Stephen A, Nacham Omprakash, Anderson Jared L
Department of Chemistry, Iowa State University, 1605 Gilman Hall, Ames, IA, 50011, USA.
Anal Bioanal Chem. 2017 Aug;409(21):4983-4991. doi: 10.1007/s00216-017-0439-y. Epub 2017 Jun 20.
In this study, a series of magnetic ionic liquids (MILs) were investigated for the extraction and preconcentration of bacteria from aqueous samples. By dispersing small volumes (e.g., 15 μL) of MIL within an aqueous cell suspension, bacteria were rapidly extracted and isolated using a magnetic field. Of the seven hydrophobic MILs examined, the trihexyl(tetradecyl)phosphonium Ni(II) hexafluoroacetylacetonate ([P][Ni(hfacac)]) MIL exhibited the greatest enrichment of viable Escherichia coli K12 when coupled with microbiological culture as the detection method. The MIL-based strategy was applied for the preconcentration of E. coli from aqueous samples to obtain enrichment factors (E ) as high as 44.6 in less than 10 min. The MIL extraction approach was also interfaced with polymerase chain reaction (PCR) amplification where the positive detection of E. coli was achieved with the [P][Co(hfacac)], [P][Ni(hfacac)], [P][Dy(hfacac)], and [P][Nd(hfacac)] MILs. While direct sampling of an aqueous cell suspension at a concentration of 1.68 × 10 colony-forming units (CFUs) mL yielded no amplicon when subjected to PCR, extraction of the sample with the [P][Ni(hfacac)] MIL under optimized conditions provided sufficient enrichment of E. coli for amplicon detection. Importantly, the enrichment of bacteria using the Ni(II)-, Co(II)-, and Dy(III)-based MILs was compatible with real-time quantitative PCR amplification to dramatically improve sample throughput and lower detection limits to 1.0 × 10 CFUs mL. The MIL-based method is much faster than existing enrichment approaches that typically require 24-h cultivation times prior to detection and could potentially be applied for the preconcentration of a variety of Gram-negative bacteria from aqueous samples. Graphical abstract Magnetic ionic liquid solvents rapidly preconcentrate viable E. coli cells for unambiguous pathogen detection using microbiological culture and qPCR.
在本研究中，对一系列磁性离子液体（MILs）进行了研究，用于从水性样品中提取和预富集细菌。通过在水性细胞悬液中分散少量体积（例如15 μL）的MIL，利用磁场快速提取和分离细菌。在所研究的七种疏水性MIL中，当与微生物培养作为检测方法结合使用时，三己基（十四烷基）鏻六氟乙酰丙酮镍（II）（[P][Ni(hfacac)]）MIL对活的大肠杆菌K12表现出最大程度的富集。基于MIL的策略用于从水性样品中预富集大肠杆菌，在不到10分钟的时间内获得高达44.6的富集因子（E）。MIL提取方法还与聚合酶链反应（PCR）扩增相结合，使用[P][Co(hfacac)]、[P][Ni(hfacac)]、[P][Dy(hfacac)]和[P][Nd(hfacac)] MIL实现了大肠杆菌的阳性检测。当对浓度为1.68×10菌落形成单位（CFUs）/mL的水性细胞悬液进行直接采样并进行PCR时，未产生扩增子，但在优化条件下用[P][Ni(hfacac)] MIL对样品进行提取，可使大肠杆菌充分富集以进行扩增子检测。重要的是，使用基于镍（II）、钴（II）和镝（III）的MIL对细菌进行富集与实时定量PCR扩增兼容，可显著提高样品通量并将检测限降低至1.0×10 CFUs/mL。基于MIL的方法比现有的富集方法快得多，现有方法通常在检测前需要24小时的培养时间，并且有可能用于从水性样品中预富集多种革兰氏阴性细菌。图形摘要 磁性离子液体溶剂可快速预富集活的大肠杆菌细胞，以便使用微生物培养和定量PCR进行明确的病原体检测。
