Droplet digital microfluidic system for screening filamentous fungi based on enzymatic activity

Abstract

Fungal cell-wall-degrading enzymes have great utility in the agricultural and food industries. These cell-wall-degrading enzymes are known to have functions that can help defend against pathogenic organisms. The existing methods used to discover these enzymes are not well adapted to fungi culture and morphology, which prevents the proper evaluation of these enzymes. We report the first droplet-based microfluidic method capable of long-term incubation and low-voltage conditions to sort filamentous fungi inside nanoliter-sized droplets. The new method was characterized and validated in solid-phase media based on colloidal chitin such that the incubation of single spores in droplets was possible over multiple days (2–4 days) and could be sorted without droplet breakage. With long-term culture, we examined the activity of cell-wall-degrading enzymes produced by fungi during solid-state droplet fermentation using three highly sensitive fluorescein-based substrates. We also used the low-voltage droplet sorter to select clones with highly active cell-wall-degrading enzymes, such as chitinases, β-glucanases, and β-N-acetylgalactosaminidases, from a filamentous fungi droplet library that had been incubated for >4 days. The new system is portable, affordable for any laboratory, and user-friendly compared to classical droplet-based microfluidic systems. We propose that this system will be useful for the growing number of scientists interested in fungal microbiology who are seeking high-throughput methods to incubate and sort a large library of fungal cells.

Integrating microfluidics and synthetic biology: advancements and diverse applications across organisms

“…The use of droplets have opened the discovery of new therapeutics and has created a lens for visualizing mammalian cells at the single cell level. 100,[131][132][133][134] Two most popular applications that use such tools are antibody discovery for therapeutic purposes 113 and single cell sequencing for disease pathology. 134,135 The extensive application of antibodies in therapeutic contexts has rendered them a significant focus for highthroughput screening.…”

“…The "mother-machine", initially developed by Wang et al 37 in 2010, serves as a foundational platform for bacterial time-lapse studies and exemplifies this adaptability. Recent innovations in droplet sorting devices by Isozaki et al, 163,164 Caen et al, 165 and Samlali et al, 100 offer promising prospects for standardizing sorter devices in highthroughput screenings across various organisms. These devices possess the capacity to sort different droplet sizes, catering to bacterial, yeast cells, single mammalian cells in the picolitre range, as well as spheroids, organoids, and filamentous fungi in the nanoliter range.…”

High-throughput screening of microbial strains in large-scale microfluidic droplets

“…Furthermore, the long droplet incubation times needed for protein expression and the different individual growth rates contribute to droplet volume polydispersity post-incubation, which complicates the sorting procedure. Last but not least, droplets containing mycelium are more susceptible to high-voltage electric fields, which deform and break the droplets (Samlali et al, 2022). Therefore, diverse solutions for optimal droplet morphology and material systems need to be developed to match categories of strains.…”

An Artificial Intelligence-Assisted Digital Microfluidic System for Multistate Droplet Control

“…[4][5][6][7] This technique has advantages such as miniaturization, programmability, parallelization, and low power consumption. 8,9 Due to these bene ts, DMF devices have been used in a wide range of applications, including point-of-care testing, 10,11 cell research, 12,13 biomedicine, 14,15 and environmental monitoring. 16 Typically, DMF devices accept a standard set of basic instructions to perform droplet manipulations.…”