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

Abstract

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 µm 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 ~3 × 103 bacteria/mL to an upper limit of quantification (ULOQ) at ~3 × 107 bacteria/mL, anda linearity R2 = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.

Keywords

• fluorescence <i>in situ</i> hybridization
• locked nucleic acid
• molecular beacons
• droplet microfluidics
• amplification-free detection
• digital assays