Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform

Abstract

There remains an urgent need for rapid diagnostic methods that can evaluate antibiotic resistance for pathogenic bacteria in order to deliver targeted antibiotic treatments. Toward this end, we present a rapid and integrated single-cell biosensing platform, termed dropFAST, for bacterial growth detection and antimicrobial susceptibility assessment. DropFAST utilizes a rapid resazurin-based fluorescent growth assay coupled with stochastic confinement of bacteria in 20 pL droplets to detect signal from growing bacteria after 1 h incubation, equivalent to 2–3 bacterial replications. Full integration of droplet generation, incubation, and detection into a single, uninterrupted stream also renders this platform uniquely suitable for in-line bacterial phenotypic growth assessment. To illustrate the concept of rapid digital antimicrobial susceptibility assessment, we employ the dropFAST platform to evaluate the antibacterial effect of gentamicin on E. coli growth.

Droplet microfluidics for high‐sensitivity and high‐throughput detection and screening of disease biomarkers

“…Following on-chip incubation, droplets can be detected sequentially in continuous flow by creating a constriction in the delay line where only a single droplet may pass through at a time. Kaushik and Hsieh et al utilized an on-chip delay line with a detection constriction for individually interrogating droplets during continuous-flow operation (Kaushik et al, 2017). In this work, the microfluidic device was directly connected to supporting instrumentation, which consisted of a Peltier heating device, an optical excitation source and a fluorescence detector (Figure 3Cii).…”

“…In that work, a slightly larger droplet volume (4 nL) required 7.5 h off-chip incubation in order to determine the susceptibility and resistance to methicillin. A rapid and integrated approach was presented by Kaushik and Hsieh et al (Kaushik et al, 2017). Here, the authors encapsulated single bacteria in significantly smaller droplets that are 20 pL in volume.…”

“…Optimization between droplet volume and sample volume for each assay may also be important. This is because although smaller droplet volumes can accelerate time-to-detection (Boedicker et al, 2008; Kaushik et al, 2017), they also reduce the volumetric throughput for performing the assay and can consequently lengthen the overall turn-around time if the target biomarker is rare (Rosenfeld, Lin, Derda, & Tang, 2014). …”

“…(Reprinted from Biosensors and Bioelectronics, 97, Kaushik and Hsieh et al, Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform, 260-266, Copyright 2017, with permission from Elsevier.) (Kaushik et al, 2017). …”

“…After co-encapsulation of single cells of S. aureus with blood plasma and ampicillin into 4 nL droplets, the pathogen’s susceptibility/resistance to the drug was determined in 7.5 h. (Reproduced from (Boedicker et al, 2008) with permission of The Royal Society of Chemistry.). (C) An integrated one-step platform was developed for bacterial growth analysis and antibiotic susceptibility testing (Kaushik et al, 2017). Critically, the use of much smaller 20 pL droplets in this platform enabled detection of single-cell E. coli growth and its susceptibility/resistance to gentamicin in as little as one hour, equivalent to 2-3 replications of the bacterial cell.…”