Rapid preconcentration of viable bacteria using magnetic ionic liquids for PCR amplification and culture-based diagnostics

Kevin D. Clark, Jeffrey A. Purslow, Stephen A. Pierson, Omprakash Nacham, Jared L. Anderson

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 ([P66614(+)][Ni(hfacac)3(-)]) 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 F) 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 [P66614(+)][Co(hfacac)3(-)], [P66614(+)][Ni(hfacac)3(-)], [P66614(+)][Dy(hfacac)4(-)], and [P66614(+)][Nd(hfacac)4(-)] MILs. While direct sampling of an aqueous cell suspension at a concentration of 1.68 × 10(4) colony-forming units (CFUs) mL(-1) yielded no amplicon when subjected to PCR, extraction of the sample with the [P66614(+)][Ni(hfacac)3(-)] 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(2) CFUs mL(-1). 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.