Microfluidic isotachophoretic Fluorescence In Situ Hybridization of bacterial cells

Nucleic acid hybridization has been extensively used for diagnostic and biological sample processing [1]because the specificity allowing for the detection of a single sequence from an entire genome. FISH is widely used in clinical analysis to detect whole cells based on their DNA or RNA sequence using fluorescently labelled hybridisation probes. However, the downfall of FISH is that whole cells need to be fixed and incubated with the probe for at least 30 min before analysis, preventing rapid turn around. Recently,ITP has been demonstrated to increase the nucleic acid hybridisation rate of DNA and RNA in free solution, and ionic spacers can be selected to separate the hybridised and free probe. Here, we take a similar approach to the selective labelling of bacterial cells. In order to allow the FISH probe to enter the cells, DMSO was added to the terminating electrolyte to increase the rate at which the probe permeates into the cells. Using a fluorescently labelled hybridisation probe with 27R DNA sequence, ITP was used for on chip FISH of <i>Escherichia coli</i> (<i>E.coli</i>), specifically staining <i>E.coli</i> in less than 4 min.