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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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BT spore encapsulated in the hydrogel microcapsules. Fluorescent image of BT spore inside of microcapsules before the germination (A) and its optical image (B); Fluorescent image of microcapsules after the germination (C) and its optical image (D). Scale bars represent 100 μm.
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f5-sensors-12-10136: BT spore encapsulated in the hydrogel microcapsules. Fluorescent image of BT spore inside of microcapsules before the germination (A) and its optical image (B); Fluorescent image of microcapsules after the germination (C) and its optical image (D). Scale bars represent 100 μm.

Mentions: For the confirmation of the encapsulation of EGFP-displayed BT spores in the microcapsules BT spore-encapsulated PNIPAM microcapsules were collected and investigated the fluorescent characteristics by confocal microscopy. As shown in Figure 5(A), the strong green fluorescent signal which is derived from encapsulated BT spores was observed from the fluorescent image. The highly transparent and monodisperse microcapsules were also obtained as shown in Figure 5(B). To investigate the viability of the encapsulated spores and subsequently shuttle into vegetative cells, we transfer the BT spores-encapsulated microcapsules were placed in Luria-Bertani (LB) medium for germination and incubated at 37 °C for 24 h. Once germinated, the vegetative cells did not display EGFP on their surface anymore. Nonetheless, it is noteworthy that the fluorescence of vegetative cells became weaker under the germination condition. In particular, the fluorescent and optical property change would be the strong evidence that the BT spores were converted into vegetative cells by the germination in microdroplets. In these reasons, we investigated the fluorescent and optical changes. The BT spores maintained their viability under the microencapsulating conditions and were successfully germinated into vegetative cells. In addition, there were no fluorescent signals in the microcapsules as shown in Figure 5(C). The spatially included microstructures of vegetative cells (live cells) were observed after 24 h of incubation, and some free vegetative cells were observed (Figure 5(D)). In addition, the highly transparent microcapsules were also converted into dark-gray color microcapsules. The darkness of the inside of microcapsule indicates that vegetative cells are agglomerated in the microcapsules. Some microcapsules were covered with vegetative cells as shown in Figure 5(D). Once the vegetative cells were growing and packing inside of the microcapsules, the hydrogels were flexible enough to break out the cells from the microcapsules. These results suggest that the strategy present herein should be useful in generating microstructure of any microbial cells by spatially addressing their spores within microdroplets.


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)

BT spore encapsulated in the hydrogel microcapsules. Fluorescent image of BT spore inside of microcapsules before the germination (A) and its optical image (B); Fluorescent image of microcapsules after the germination (C) and its optical image (D). Scale bars represent 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472820&req=5

f5-sensors-12-10136: BT spore encapsulated in the hydrogel microcapsules. Fluorescent image of BT spore inside of microcapsules before the germination (A) and its optical image (B); Fluorescent image of microcapsules after the germination (C) and its optical image (D). Scale bars represent 100 μm.
Mentions: For the confirmation of the encapsulation of EGFP-displayed BT spores in the microcapsules BT spore-encapsulated PNIPAM microcapsules were collected and investigated the fluorescent characteristics by confocal microscopy. As shown in Figure 5(A), the strong green fluorescent signal which is derived from encapsulated BT spores was observed from the fluorescent image. The highly transparent and monodisperse microcapsules were also obtained as shown in Figure 5(B). To investigate the viability of the encapsulated spores and subsequently shuttle into vegetative cells, we transfer the BT spores-encapsulated microcapsules were placed in Luria-Bertani (LB) medium for germination and incubated at 37 °C for 24 h. Once germinated, the vegetative cells did not display EGFP on their surface anymore. Nonetheless, it is noteworthy that the fluorescence of vegetative cells became weaker under the germination condition. In particular, the fluorescent and optical property change would be the strong evidence that the BT spores were converted into vegetative cells by the germination in microdroplets. In these reasons, we investigated the fluorescent and optical changes. The BT spores maintained their viability under the microencapsulating conditions and were successfully germinated into vegetative cells. In addition, there were no fluorescent signals in the microcapsules as shown in Figure 5(C). The spatially included microstructures of vegetative cells (live cells) were observed after 24 h of incubation, and some free vegetative cells were observed (Figure 5(D)). In addition, the highly transparent microcapsules were also converted into dark-gray color microcapsules. The darkness of the inside of microcapsule indicates that vegetative cells are agglomerated in the microcapsules. Some microcapsules were covered with vegetative cells as shown in Figure 5(D). Once the vegetative cells were growing and packing inside of the microcapsules, the hydrogels were flexible enough to break out the cells from the microcapsules. These results suggest that the strategy present herein should be useful in generating microstructure of any microbial cells by spatially addressing their spores within microdroplets.

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