Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 mu m in diameter) droplets for bacterial encapsulation followed by in situ permeabilization, hybridization, and signal detection. Employing locked nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based hybridization buffer, the assay was characterized with Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. The assay performed with single-cell sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at similar to 3 x 10(3) bacteria/mL to an upper limit of quantification (ULOQ) at similar to 3 x 10(7) bacteria/mL, anda linearity R-2 = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.