em Nat

em Nat. attempt to mimic the exceptional material properties observed in natural silk1,3,4,5,6,7. Many components of fibrillar silk can be chemically re-solubilized; in particular reconstituted silk fibroin (RSF)5,6, obtained by dissolving spun cocoons, has been used in a wide range of applications.5,6,8,9,10,11,12,13,14,15 The widespread TUG-891 use of reconstituted silk feedstocks is largely enabled by the relative ease in which this material can be prepared and stored16. NSF feedstocks can be obtained directly from the gland of the silkworm where it is TUG-891 stored in a spatially distinct location from sericin3. However, such feedstocks are renowned for their extreme sensitivity TUG-891 to shear and high propensity to aggregate once isolated17, in marked contrast to RSF, which is significantly more stable in solution1,17,18,19. Indeed, the inherent difficulties in handling NSF feedstocks significantly limit the potential of this material for use in biotechnological applications. To address the fundamental challenges in processing NSF, we have explored a microfluidic platform that enables the investigation of the artificial spinning of native silk as well as providing routes towards its long-term storage in an active state ready for a range of possible uses. We report the generation of a wide range of micron-scale shapes, herein referred to as micrococoons or micron-scale capsules20,21, and demonstrate that morphological diversity can be generated through fine tuning of the shear conditions, and through variation in the flow rates used, and by modulating the surface tension and viscosity of the feedstock. Importantly for potential applications, we find that micrococoons exhibit a distinct core-shell structure with an internal environment apparently ideal for the storage of sensitive and aggregation-prone materials. Results Micron scale silk capsule synthesis We have applied a microfluidic strategy to control the level of shear applied to an NSF solution22,23 to induce the transition of NSF from its initial, native state, into highly aggregated silkworm gland3, fluorinert FC-70 (Sigma-Aldrich, UK) and N,N bis ( em n /em -propyl)polyethylene oxide-bis(2-trifluoromethyl polyperfluoroethylene oxide) amide surfactant47. Droplet microfluidics The single and double T-junction droplet makers were fabricated from PDMS (polydimethylsiloxane, ca. 50,000Mw, Sylgard 184, Dow Corning, USA) as chips by using standard soft lithography methods48, 49, 50, 51. The synthesis of the NSF micrococoons was performed on a specially designed microfluidic system with 20?m diameter channels. 1?ml of aqueous NSF at pH7 and 1?ml of fluorinert oil containing 2% w/v of N,Nbis(n-propyl)polyethylene oxide-bis(2-trifluoromethyl polyperfluoroethylene oxide) amide surfactant, were mixed at the T-junctions of microfluidic channels by using flow control through syringe pumps. The initial concentration of NSF WNT4 varied from 1 to 10?mg?ml?1. The multi-shell micron-scale capsules were formed using a double T-junction device in which the NSF and oil solutions were mixed at the first T-junction to form their initial shape and then passed through the second T-junction with NSF dope as a continuous phase. The capsules were then washed with doubly distilled water (DDW) at pH7 to remove the surfactant and any unreacted protein. Confocal and light microscopy Samples were deposited as aqueous dispersions, without further purification, onto a glass slide. The NSF micrococoons were analysed by confocal microscopy (Laser Scan Confocal, Zeiss Microscope 5,100), using a laser 405?nm at 25?mW for violet excitation. Because of the intrinsic native fluorescent signal emitted from the aggregated NSF protein, the NSF micrococoons were analysed by confocal microscopy without labelling; the emission maxima, in the blue region of the fluorescence spectrum, originated from the aggregated component of the gelled NSF micrococoons, while for the single shell structures the aggregated NSF content was detected at the interface of each micron-scale capsule. The double shell structures exhibited blue emission from the internal as well as the external shells of the NSF micrococoon shapes. 3D images were reconstructed using the Imaris image analysis program (on average 412 em z /em -stack slices per each protein shell). Measurements of loading capacity and release kinetics To calculate the efficiency of the conversion of the NSF into micrococoons, the concentration of unreacted NSF was measured (after washing) by UV absorption by using a NanoDrop 2,000 UV spectrophotometer (Thermo Scientific, UK) and using a bicinchoninic acid (BCA) protein detection kit (ThermoFisherScientific), following absorption at 562?nm; in no case did the difference between the two approaches exceed 3%. In addition, the loading efficiency and release profiles of the C4scFv (ref. 52) antibody domain from NSF micrococoons were probed using an AlexaFluor647 labelled domain43. The.