Continuous Recirculation of Microdroplets in a Closed Loop Tailored for Screening of Bacteria Cultures

Abstract

Emerging microfluidic technology has introduced new precision controls over reaction conditions. Owing to the small amount of reagents, microfluidics significantly lowers the cost of carrying a single reaction. Moreover, in two-phase systems, each part of a dispersed fluid can be treated as an independent chemical reactor with a volume from femtoliters to microliters, increasing the throughput. In this work, we propose a microfluidic device that provides continuous recirculation of droplets in a closed loop, maintaining low consumption of oil phase, no cross-contamination, stabilized temperature, a constant condition of gas exchange, dynamic feedback control on droplet volume, and a real-time optical characterization of bacterial growth in a droplet. The channels (tubing) and junction cubes are made of Teflon fluorinated ethylene propylene (FEP) to ensure non-wetting conditions and to prevent the formation of biofilm, which is particularly crucial for biological experiments. We show the design and operation of a novel microfluidic loop with the circular motion of microdroplet reactors monitored with optical sensors and precision temperature controls. We have employed the proposed system for long term monitoring of bacterial growth during the antibiotic chloramphenicol treatment. The proposed system can find applications in a broad field of biomedical diagnostics and therapy.

Droplet-Based Screening for the Investigation of Microbial Nonlinear Dose–Response Characteristics System, Background and Examples

“…The modular system has the advantage of flexible assembly related to the desired fluidic protocols and readout wavelengths. The concept of modular microfluidic assemblies has been proven to be utterly practical in numerous publications before [36][37][38][39].…”

Microfluidics for antibiotic susceptibility testing

“…The first system designed for this was called "death galaxy" by Austin et al and comprised a set of interconnected microfluidic chambers to which antibiotics were introduced through diffusion through a series of slits that stopped bacteria from infecting the fresh media source. 44 Microdroplet chemostats were presented in different forms 126,127,213,214 that allowed for observation of antibiotic resistance changes in hundreds of separated bacteria populations in large microliter dropletsthe media used by growing bacteria would be partially removed in an automated fashion, and portions of fresh media with a higher than before antibiotic concentration would be introduced instead. We believe that evolution studies would be improved if a microdroplet chemostat could operate on single cells, i.e., after each exchange of the medium, a single cell would remain per droplet: this would allow for analysis of rates of emergence of resistance from a large number of clones and couple with the research of individual bacteria scMIC would inform about whether clonally identical but more resistant cells gain resistance faster than their isogenic yet less resistant counterparts.…”

Fixing the direction of droplets in a bifurcating microfluidic junction

“…The sequence of consecutive snapshots for: left column-type J1 and right column-type J2. For both cases, the output flow is the same and Q OUT = 1 ml/h of droplet operations (Debski et al 2018;Abolhasani and Jensen 2016).…”

“…The peculiar character of this micro-world has sparked the curiosity of many researchers, which efforts within the last decades opened the way for the development of various strategies for droplet manipulation such as generation (van Steijn et al 2013), sorting (Tan et al 2007), trapping (Huebner et al 2009), splitting (Park et al 2018) or merging (Bremond et al 2008). Moreover, droplets can be considered as tiny biochemical reactors (Debski et al 2018).…”