Also in the Article



Microfluidic chip construction. The capillary microfluidic device was constructed by coaxially assembling two round capillaries (World Precision Instruments Inc.) on a glass slide. The inner capillary was tapered with a laboratory portable Bunsen burner (Honest MicroTorch) to reach an orifice inner diameter of ~250 μm. The outer diameter and inner diameter of the outer capillary were 1000 and 580 μm, respectively. The inner wall of the outer capillary was immersed in the OTS for 20 s to wet the inner wall completely and then incubated for 30 min for further hydrophobic treatment. After this, the solution was blown out with nitrogen. Then, the capillaries were coaxially assembled and a transparent epoxy resin (Devcon 5 Minute Epoxy) was used to seal the tubes where required.

Fabrication of template silica colloidal crystal beads. The silica colloidal crystal beads were generated using the droplet template method that we reported. Briefly, the nanoparticles and silicon oil were injected into the microfluidic chip through two different channels, as inner phase and outer phase. The concentration of the nanoparticles was 13 weight % (wt %), and the flow rates of the oil and water phases were 3 and 0.5 ml/hour, respectively. When the system was running, the inner phase was cut into droplets by the outer phase when they met in the microfluidic channel. A box with high-viscosity silicon oil was used to collect the droplets. Then, the box was transferred to oven at 75°C overnight to dry the droplets and the silica nanoparticles self-assembled into ordered lattices during the evaporation of water. After that, the silica colloidal crystal beads were gently washed with n-hexane to remove the residual silicon oil. Last, the silica colloidal crystal beads were calcined at 800°C for 4 hours to enhance their mechanical strength. The reflection spectra of the silica colloidal crystal beads were measured using a metalloscope (Olympus BX51) with a fiber-optic spectrometer (QE65000, Ocean Optics), and the photographs of the silica colloidal crystal beads were taken using a metalloscope (Olympus BX51) with a charge-coupled device camera (Media Cybernetics Evolution MP 5.0). SEM (Hitachi, S-300N) was used to characterize the microstructures of the silica colloidal crystal beads.

Fabrication of inverse opal hydrogel particles. AAm and Bis were dissolved in deionized water (mass ratio, 29:1) and then mixed with HMPP (volume ratio, 99:1) to obtain the pregel. The template colloidal crystal beads were immersed in the pregel solution so that the voids of the beads could be filled with the pregel. After polymerization by UV light irradiation, the beads with gel were stripped from the bulk and the silica template were removed by HF. The microstructures of the inverse opal hydrogel particles were also characterized by SEM. To bind with enzymes, the inverse opal particles were hydrolyzed with 10% TEMED and 0.1 M NaOH. After activation by EDC and NHS, the enzymes (in PBS) were cross-linked with the inverse opal hydrogel particles. Fluorescein isothiocyanate–bovine serum albumin was used to mimic the enzyme distribution in the inverse opal particles.

Fabrication of the multienzyme microcapsule system. The multienzyme microcapsule system was prepared using a coaxial capillary microfluidic chip with three-bore capillary injection channels integrated with the electrospray collection device. The inverse opal particles were dispersed in 2 wt % sodium carboxymethylcellulose solution and used as inner phase, and 2 wt % Na-Alg solution was used as outer phase. Voltages (5.2 kV) from a voltage generator were used to provide electric field. Because of the hydrodynamic focusing effect, the outer phase formed sheath flow stream around the inner phase and then was broken up into microdrops by the electric field. The microdrops were collected using a container with 2 wt % calcium chloride solution, and the alginate shell was gelled. Thus, the inverse opal particles were packaged in the microcapsules as position-free cores.

Reaction of the microcapsules with HRP-immobilized single core. The mixture of ODS (0.5 g/liter) and 0.3% H2O2 was used as the substrate of the microcapsules with HRP-immobilized single core, and about 150 microcapsules treated with trypsin were used for biocatalysis. To monitor the reaction process, the absorbance of the substrate was monitored with a microplate reader (Synergy|HTX) every 15 min at 460 nm. About 150 free particles immobilized with HRP were used as control.

Reaction of the microcapsules immobilized with β-G, GOD, and HRP. The mixture of ODS (0.5 g/liter) and octyl β-d-glucopyranoside (25 g/liter) was used as the substrate of the microcapsules immobilized with β-G, GOD, and HRP, and about 150 microcapsules were used for biocatalysis. To monitor the reaction process, the absorbance of the substrate was monitored with a microplate reader every 15 min at 460 nm. Free particles immobilized with β-G, GOD, and HRP were used as control.

Reaction of the microcapsules immobilized with AOx and Cat. The mixture of DMEM (high level of glucose in medium, with 10% FBS) and 3% alcohol was used as the substrate of the microcapsules immobilized with Cat and AOx. The 3T3 cells with the microcapsules were cultured in the substrate for 1 hour. To monitor cell viability, the cells were treated with DMEM containing MTT (0.5 g/liter) for 3 hours. Then, DMEM was replaced with dimethyl sulfoxide. After gentle shaking, the solution was monitored with a microplate reader at 490 nm. To observe changes in cell morphology, the cocultured cells were dyed with calcein. As controls, cells cocultured without alcohol, with microcapsules but without enzymes, or with microcapsules containing only AOx were monitored simultaneously.

Reaction of the microcapsules immobilized with AOx, Cat, and ALD. The mixture of 3% alcohol and 10 mM NAD was used as the substrate of the microcapsules immobilized with AOx, Cat, and ALD, and about 3000 microcapsules were used for biocatalysis. The ALD could transform NAD to NADH, and NADH has an absorption peak at 340 nm. Thus, the cascade reaction was detected by monitoring the change of the optical density of the solution at 340 nm every 15 min.

Note: The content above has been extracted from a research article, so it may not display correctly.



Also in the Article

Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.