Abstract
Bioactive microcapsules containing Bacillus thuringiensis (BT) spores were generated by a combination of a hydro gel, microfluidic device and chemical polymerization method. As a proof-of-principle, we used BT spores displaying enhanced green fluorescent protein (EGFP) on the spore surface to spatially direct the EGFP-presenting spores within microcapsules. BT spore-encapsulated microdroplets of uniform size and shape are prepared through a flow-focusing method in a microfluidic device and converted into microcapsules through hydrogel polymerization. The size of microdroplets can be controlled by changing both the dispersion and continuous flow rate. Poly(N-isoproplyacrylamide) (PNIPAM), known as a hydrogel material, was employed as a biocompatible material for the encapsulation of BT spores and long-term storage and outstanding stability. Due to these unique properties of PNIPAM, the nutrients from Luria-Bertani complex medium diffused into the microcapsules and the microencapsulated spores germinated into vegetative cells under adequate environmental conditions. These results suggest that there is no limitation of transferring low-molecular-weight-substrates through the PNIPAM structures, and the viability of microencapsulated spores was confirmed by the culture of vegetative cells after the germinations. This microfluidic-based microencapsulation methodology provides a unique way of synthesizing bioactive microcapsules in a one-step process. This microfluidic-based strategy would be potentially suitable to produce microcapsules of various microbial spores for on-site biosensor analysis.
Highlights
The generation of spatially well-defined, three dimensional (3D) microstructures for whole-cell sensing system have attracted interest in the development of portable bacterial whole-cell biosensing systems, high-throughput cellular analysis as well as in fundamental studies of cell biology [1,2,3]
As increasing the continuous phase (CP) flow rate under the same dispersive phase (DP) flow rate, the size of produced microdroplets is decreased. These results indicate that high-flow rate of CP strengthens the shearing force and accelerate the detachment of the droplets from the DP flow at the orifice in the microfluidic device
We developed a novel method for producing bioactive microcapsules which encapsulated a biological species, Bacillus thuringiensis (BT) spores, in a one-step process using a microdroplet-based microfluidic system
Summary
The generation of spatially well-defined, three dimensional (3D) microstructures for whole-cell sensing system have attracted interest in the development of portable bacterial whole-cell biosensing systems, high-throughput cellular analysis as well as in fundamental studies of cell biology [1,2,3] To achieve this goal, three major aspects should be considered: (a) selection of biocompatible materials to construct 3D microstructures; (b) fabrication methods to control the size and uniform shape of the 3D microstructures; (c) polymerization methods to produce hydrogels [4,5,6]. We have developed a new method to produce bioactive monodisperse PNIPAM-based microcapsules by using a combination of a microfluidic device and a chemical polymerization method. To further demonstrate the ability of the synthesized PNIPAM-based microcapsules to act as bioactive containers, BT spores were employed, and encapsulated inside the microcapsules and subsequently cultivated inside the microcapsules for shuttling to the vegetative cells
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