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.

Aqueous Processed Biopolymer Interfaces for Single-Cell Microarrays

“…It consists in a short-pulse waveform developed to print aqueous droplets by a conventional 1 pL-ejecting cartridge with nozzle diameters of 10.5 μm, which enabled the formation of a droplet smaller than the nozzle size. As reported in our previous investigations, it is possible to produce subnozzle sized droplets by minimizing the t D . The mechanism for this process is based on the theoretical model described by Eggers, which in turn relies on a singularity of the Navier–Stokes equations describing the droplet size at the initial formation stages .…”

“…The mechanism for this process is based on the theoretical model described by Eggers, which in turn relies on a singularity of the Navier–Stokes equations describing the droplet size at the initial formation stages . This short pulse waveform permitted to obtain droplets which did not show any tail, as a consequence of the reduced volume jetted at the nozzle which, in turn, simply led to almost spherical droplets, in accordance with our previous investigations on aqueous inks jetting at femtoliter-scale volumes . Differently to the chitosan ink, the collagen ink was fairly printable by this waveform at jetting voltages comprised between 30–40 V. It was not possible to print at voltages lower than 30 V. This can be again likely explained by considering the ink viscosity (3 mPa s) and the high surface tension (70 mN/m) which dissipate the droplet kinetic energy during its formation at the nozzles …”

“… 75 This short pulse waveform permitted to obtain droplets which did not show any tail, as a consequence of the reduced volume jetted at the nozzle which, in turn, simply led to almost spherical droplets, in accordance with our previous investigations on aqueous inks jetting at femtoliter-scale volumes. 76 Differently to the chitosan ink, the collagen ink was fairly printable by this waveform at jetting voltages comprised between 30–40 V. It was not possible to print at voltages lower than 30 V. This can be again likely explained by considering the ink viscosity (3 mPa s) and the high surface tension (70 mN/m) which dissipate the droplet kinetic energy during its formation at the nozzles. 62 Figure 4 b shows the stroboscopic images of collagen ink droplets ejected by applying such a waveform at 30 V jetting voltage after droplet formation at the nozzle.…”

“…Moreover, in accordance with previous data, the resulting sessile droplet diameter was found to increase at longer t D , reaching a plateau at t D > 4.0 μs. This effect is likely due to the viscosity of the ink that reduces the further droplet size increase.…”

Peptide–Membrane Interactions Monitored by Fluorescence Lifetime Imaging: A Study Case of Transportan 10

“…The observed shift occurs within the first 5 min after the addition of CF-TP10 to POPC/POPG GVs (see Supporting Information Figure S2), and then, the signal remains stable for at least 24 h. It is well known that changes in fluorescence spectrum shapes and position reflect changes in the fluorophore environment. In particular, fluorescein-based dyes are often used to monitor environmental properties as they are critically sensitive to the pH or the hydrogen-bond character of the environment. , Other factors may also affect the spectral properties of these molecules; among these are solvent relaxation phenomena which are often related to spectral shifts and usually reported as a result of changes in the dielectric constant of the surroundings of the dye. , …”