An ultra-high temperature flow-through capillary device for bacterial spore lysis.
Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.
Hukari
Kyle W
KW
Microfluidics Research Group, Sandia National Laboratories, Livermore, CA, USA.
Patel
Kamlesh D
KD
Renzi
Ronald F
RF
West
Jay A A
JA
D001407:Bacillus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification / Q000502:physiology; D001408:Bacillus anthracis / Q000235:genetics / Q000502:physiology; D001409:Bacillus cereus / Q000235:genetics / Q000502:physiology; D001412:Bacillus subtilis / Q000235:genetics / Q000502:physiology; D001433:Bacteriolysis / Q000502:physiology*; D002197:Capillary Action; D002455:Cell Division; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D005456:Fluorescent Dyes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012995:Solubility; D013171:Spores, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D013816:Thermodynamics
D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes