On the Effect of Downscaling in Inkjet Printed Life-Inspired Compartments

The fabrication of size-scalable liquid compartments is a topic of fundamental importance in synthetic biology, aiming to mimic the structures and the functions of cellular compartments. Here, inkjet printing is demonstrated as a customizable approach to fabricate aqueous compartments at different size regimes (from nanoliter to femtoliter scale) revealing the crucial role of size in governing the emerging of new properties. At first, inkjet printing is shown to produce homogenous aqueous compartments stabilized by oil-confinement with mild surfactants down to the hundreds of picoliter scale [1]. Raster Image Correlation Spectroscopy allows to monitor few intermolecular events by the involvement of protein-binding assays within these compartments [2]. Subsequently, in order to reduce droplet size at values below the nozzle size, a theoretical model from Eggers et al. [3] is experimentally reproduced permitting to obtain femtoliter-scale aqueous droplets from picoliter-scale microchannels [4]. As a remarkable difference to picoliter scale droplets, downscaling at the femtoliter-size triggers the spontaneous formation of molecularly crowded shell structures at the water/oil interface stabilized by a mixture of biocompatible surfactants. The shells have typical thickness in order of hundreds of nanometers, in accordance with theoretical models [5]. Molecular crowding effects in these systems are tested by using fluorescence lifetime imaging under the phasor plot approach [6], revealing different characteristic lifetimes of specific probe molecules in the confined volumes with respect to bulk solutions. The femtoliter-scale compartments autonomously trigger the formation of unique features (e.g., up-concentration, spatial heterogeneity, molecular proximity) that are mediated by the intermolecular interactions in these novel environments, ultimately permitting to mimic the native conditions of sub-cellular scale compartments. The crowding conditions in femtoliter-scale droplets do not to affect the conformation variation of a model DNA hairpin in presence of molecular triggers and of a CYP2E1-catalyzed enzymatic reaction. Our results can be a first step towards the fabrication of size-scalable lab-on-a-chip compartments mimicking sub-cellular environments. References 1. G. Arrabito, F. Cavaleri, V. Montalbano, V. Vetri, M. Leone, B. Pignataro, Lab on Chip, 2016, 16, 4666. 2. M.A. Digman, C. M. Brown, A. R. Horwitz,W.W. Mantulin, and E. Gratton, Biophysical Journal, 2016, 94, 2819. 3. J. Eggers, Phys. Rev. Lett. 1993, 71, 3458. 4. G. Arrabito, F. Cavaleri, A. Porchetta, F. Ricci, V. Vetri, M. Leone, B. Pignataro, Adv. Biosys. 2019, 1900023. 5. M. Staszak, J. Surfactants Deterg., 2016, 19, 297. 6. C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P.J. Donovan, and E. Gratton, Proc. Natl. Acad. Sci. USA 2011, 108, 13582.