Printing Life‐Inspired Subcellular Scale Compartments with Autonomous Molecularly Crowded Confinement

Abstract

A simple, rapid, and highly controlled platform to prepare life‐inspired subcellular scale compartments by inkjet printing has been developed. These compartments consist of fL‐scale aqueous droplets (few µm in diameter) incorporating biologically relevant molecular entities with programmed composition and concentration. These droplets are ink‐jetted in nL mineral oil drop arrays allowing for lab‐on‐chip studies by fluorescence microscopy and fluorescence life time imaging. Once formed, fL‐droplets are stable for several hours, thus giving the possibility of readily analyze molecular reactions and their kinetics and to verify molecular behavior and intermolecular interactions. Here, this platform is exploited to unravel the behavior of different molecular probes and biomolecular systems (DNA hairpins, enzymatic cascades, protein‐ligand couples) within the compartments. The fL‐scale size induces the formation of molecularly crowded confined shell structures (hundreds of nanometers in thickness) at the droplet surface, allowing discovery of specific features (e.g., heterogeneity, responsivity to molecular triggers) that are mediated by the intermolecular interactions in these peculiar environments. The presented results indicate the possibility of using this platform for designing nature‐inspired confined reactors allowing for a deepened understanding of molecular confinement effects in living subcellular compartments.

Artificial Biosystems by Printing Biology

“…[31,33] Differently from previously developed set-ups such as satellites printing or droplet generation in liquid environments [82,83] which have not been demonstrated to be suitable for biomolecular systems, our group has recently designed a printing approach based on piezoelectric IJP for the fabrication of artificial compartments at fL scale. [85] In particular, by following the theoretical model from Eggers and coworkers, [195] in which the droplet size can be finely tuned by minimizing the actuation time of the transducer, we were able to produce Figure 7C). In turn, the downscaling at the fL-size triggered the formation of molecularly crowded shells at the water/oil interface, with a typical thickness in the order of hundreds of nanometers, in accordance with models.…”

“…Note that several reports have demonstrated the possibility to tune droplet formation dynamics by using a set of different boundary conditions that involve hydrodynamic droplet dispensing under electrical field guiding, [81] droplets production within liquid environments, [82] satellite droplets printing, [83] breaking up in a double-orifice system, [84] reducing the impulse duration time. [85] Other approaches that can reach sub-cellular scale resolution include electrohydrodynamic, [70] or pyroelectrodynamic dispensing. [71] One hurdle of these approaches is that they are associated with significant shear/compression stresses that can ultimately lead to the alteration of the biomolecular structures and functions.…”

“…1,2-dioleoyl-sn-glycero-3phosphocholine Polysorbate 20; cholesterol 10 1 µm Single-cell Raman calibration standard [193] Proteins polyoxyethylene (20) sorbitan monolaurate 10 1 -10 2 µm pL-to nL-scale aquoeus compartments by inkjet printing [95] DNA hairpin, Proteins polyoxyethylene (20) sorbitan monolaurate and poly(ethylene glycol)-blockpoly(propylene glycol)-blockpoly(ethylene glycol) 10 0 -10 1 µm fL-scale aquoeus compartments by inkjet printing [85] Egg phosphatidylcholine and poly(2-vinyl-pyridineb-ethyleneglycol) Ethanol 10 -1 µm Direct formation of lipid and polymer vesicles . [200] Phospholipids in aqueous phase 7.5 w/v% ficoll 400; 300 mM glucose solution [2][3] µm Unilamellar lipid vesicles by inkjet printing [201] Alkaline phosphatase, Urease, β-lactamase Glycerol (5 w/w%) in 10 mM PBS 10 2 µm Protein immobilization in printed hydrogels [190] Lipidoids Aqueous sodium acetate buffer 10 2-3 µm Surface-mediated molecular delivery into arrays of cells [191] Table 3.…”

Printing ZnO Inks: From Principles to Devices

“…Notably, it is possible to integrate the hardware with automated translation stages, allowing precise pattern placement and registration to prepare multilayered patterns with different materials. Inkjet printing has found many applications in the field of direct patterning of many different materials, such as colloidal silver nanoparticles [58], oil droplets [8], biomolecular systems [59][60][61]. Notably, the rheological properties of the ink play a fundamental role in the printing process.…”

“…Notably, the droplet diameter is usually similar or slightly larger than the size of the nozzle. However, some reports have shown that it is possible to reduce its size by adapting waveform [61] or by employing satellite printing [63].…”