“a Schematic of two microfluidic devices used for screening: (i) a droplet generator and (ii) a sorting device containing electrodes. In (i) and (ii), the microfluidic channel is fabricated via soft lithography techniques except that (ii) contains an electrode layer with an SU-8 dielectric layer that is bonded to the channel PDMS layer. Two optical fibers (excitation: 105 μm core, 0.22 NA; emission: 200 μm core, 0.39 NA) are inserted into (ii) and placed orthogonally to the channel to excite and detect droplet fluorescence. b Screening workflow overview. Enzyme screening followed five steps. First, the mutant fungal population is generated through UV mutagenesis. Next, a microfluidic mixer T-junction droplet generator (see device (i)) co-encapsulates the conidial library suspended in colloidal chitin minimal media with a fluorescein-linked enzymatic substrate for glycoside hydrolases. The droplets were then collected and incubated in HFE7500 oil 2% fluorosurfactant at 27 °C for 2–4 days. The droplets contain single spores, minimal media with colloidal chitin, and a cell-wall-degrading specific fluorogenic substrate (FL-GlcNAc, FL-GalNAc, or FD-Glc). During incubation, glycoside hydrolase (GH) activity leads to the cleavage of the fluorescein-based substrate and the release of fluorescent fluorescein units that remain confined within the droplet. The droplet library is reinjected into the microfluidic low-voltage sorter (see device (ii)), which uses a three-electrode system (CE – constant electrode, PE – pulsing electrode, and GE – ground electrode). Mutant populations are sorted when they display high fluorescence intensity and are recovered on PDA plates and grown into clonal colonies, after which enzymatic assays are performed.” Reproduced under a Creative Commons Attribution 4.0 International License from Samlali, K., Alves, C.L., Jezernik, M. et al. Droplet digital microfluidic system for screening filamentous fungi based on enzymatic activity. Microsyst Nanoeng 8, 123 (2022).