Microfluidic mimicry of the Golgi-linked <i>N</i>-glycosylation machinery

The complexity of the eukaryotic glycosylation machinery hinders the development of cell-free protein glycosylation since <i>in vitro</i> methods struggle to simulate the natural environment of the glycosylation machinery. Microfluidic technologies have the potential to address this limitation due to their ability to control glycosylation parameters, such as enzyme/substrate concentrations and fluxes, in a rapid and precise manner. However, due to the complexity and sensitivity of the numerous components of the glycosylation machinery, very few "glycobiology-on-a-chip" systems have been proposed or reported in the literature. Herein, we describe the design, fabrication and proof-of-concept of a droplet-based microfluidic platform able to mimic <i>N</i>-linked glycan processing along the secretory pathway. Within a single microfluidic device, glycoproteins and glycosylation enzymes are encapsulated and incubated in water-in-oil droplets. Additional glycosylation enzymes are subsequently supplied to these droplets <i>via</i> picoinjection, allowing further glycoprotein processing in a user-defined manner. After system validation, the platform is used to perform two spatiotemporally separated consecutive enzymatic <i>N</i>-glycan modifications, mirroring the transition between the endoplasmic reticulum and early Golgi.