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Synthesis of bioactive microcapsules using a microfluidic device.

Kim BI, Jeong SW, Lee KG, Park TJ, Park JY, Song JJ, Lee SJ, Lee CS - Sensors (Basel) (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanobio Integration & Convergence Engineering (NICE), National Nanofab Center, 291 Daehak-ro, Yuseong-gu, Daejeon 305-806, Korea. kbiset@nnfc.re.kr

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.

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Spore-display of EGFP. (A) Flow cytometry analysis. Black line, BT spores harboring pSD1 as a negative control; green line, BT spores harboring pSD-EGFP; (B) Fluorescence assay. 1, BT spores harboring pSD1 as a negative control; 2, BT spores harboring pSD-EGFP; (C) Confocal microscopy analysis. The inset shows an optical microscopic image. Scale bars represent 5 μm.
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f4-sensors-12-10136: Spore-display of EGFP. (A) Flow cytometry analysis. Black line, BT spores harboring pSD1 as a negative control; green line, BT spores harboring pSD-EGFP; (B) Fluorescence assay. 1, BT spores harboring pSD1 as a negative control; 2, BT spores harboring pSD-EGFP; (C) Confocal microscopy analysis. The inset shows an optical microscopic image. Scale bars represent 5 μm.

Mentions: As a first step towards developing our bioactive microcapsules, we sought to engineer the InhA-mediated spore-surface display of EGFP as a model protein. The EGFP-displayed BT spores were confirmed by flow cytometry (Figure 4(A)), fluorescence assay (Figure 4(B)) and confocal microscopy (Figure 4(C)). Specific fluorescence signals of the spores were observed in EGFP-displayed BT spores, indicating that green fluorescence was observed only with EGFP-displayed BT spores.


Synthesis of bioactive microcapsules using a microfluidic device.

Kim BI, Jeong SW, Lee KG, Park TJ, Park JY, Song JJ, Lee SJ, Lee CS - Sensors (Basel) (2012)

Spore-display of EGFP. (A) Flow cytometry analysis. Black line, BT spores harboring pSD1 as a negative control; green line, BT spores harboring pSD-EGFP; (B) Fluorescence assay. 1, BT spores harboring pSD1 as a negative control; 2, BT spores harboring pSD-EGFP; (C) Confocal microscopy analysis. The inset shows an optical microscopic image. Scale bars represent 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472820&req=5

f4-sensors-12-10136: Spore-display of EGFP. (A) Flow cytometry analysis. Black line, BT spores harboring pSD1 as a negative control; green line, BT spores harboring pSD-EGFP; (B) Fluorescence assay. 1, BT spores harboring pSD1 as a negative control; 2, BT spores harboring pSD-EGFP; (C) Confocal microscopy analysis. The inset shows an optical microscopic image. Scale bars represent 5 μm.
Mentions: As a first step towards developing our bioactive microcapsules, we sought to engineer the InhA-mediated spore-surface display of EGFP as a model protein. The EGFP-displayed BT spores were confirmed by flow cytometry (Figure 4(A)), fluorescence assay (Figure 4(B)) and confocal microscopy (Figure 4(C)). Specific fluorescence signals of the spores were observed in EGFP-displayed BT spores, indicating that green fluorescence was observed only with EGFP-displayed BT spores.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanobio Integration & Convergence Engineering (NICE), National Nanofab Center, 291 Daehak-ro, Yuseong-gu, Daejeon 305-806, Korea. kbiset@nnfc.re.kr

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.

Show MeSH
Related in: MedlinePlus