Research ArticleChemistry

Combinatorial microfluidic droplet engineering for biomimetic material synthesis

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Science Advances  07 Oct 2016:
Vol. 2, no. 10, e1600567
DOI: 10.1126/sciadv.1600567
  • Fig. 1 Overview of the droplet screening platform.

    (A) Schematic summary of the microfluidic screening platform, which includes 24 PDMS droplet-based microfluidic devices, a modified 96-well microtiter plate to hold water and oil source solutions and interface PDMS devices with a vacuum manifold, and a commercial vacuum manifold including a 24-well capture plate in its interior. See photograph of the system in fig. S1. (B) Schematic top plan view of the microfluidic device design, indicating the position of the device’s T-junction, inlets, and outlet relative to four adjacent 96-well plate wells (dotted lines) below the device. (C) Schematic image showing the direction of fluid flows through each microfluidic device. For illustration, an approximate microfluidic channel pattern is projected on the bottom surface of the device [channels actually reside at the interface between two PDMS slabs, and accurate microfluidic pattern dimensions are shown in (B) and in Fig. 2A]. (D) Magnified schematic view of one microfluidic device within the platform, highlighting relevant platform components. (E) Fluid flow through each microfluidic chip is motivated by a pressure differential across an exit tube connected to the chip’s outlet (at pressure Po) that opens into a pressure-regulated vacuum environment, over a receiving well (at Pressure Pv) that collects the emulsion (or separated biphasic solution).

  • Fig. 2 Overview of the combinatorial droplet mineralization screening assay.

    (A) Combinatorial oils are tested in parallel for their ability to form stable water droplets in a segmented flow microfluidic chip (triangle, circle, and star represent unique surfactant species in different initial stock oils). (B) Schematic of interface mineralization reaction that leads to droplet stability via formation of shell-like mineral elements. Diverse surfactants can act in concert at the oil-water interface to catalyze mineralization and template the mineral phase at droplet boundaries through reaction with a titanium salt that is included as a precursor in the water phase. (C) Optical stereomicrograph images of the PDMS device outlets show intact, packed water droplets (left) or merged droplets (right), according to the type of combinatorial oil used as the carrier fluid in parallel devices. (D) Images of solutions received in capture wells, with a red dye included in the water phase, showing examples of droplets that have remained intact (left) or of oil-water separation (right). (E) After winning combinatorial oils are selected from a given screening round, constituent oil components are randomly recombined as determined by a genetic algorithm (see Materials and Methods), producing a new generation of combinatorial oils for an additional screening.

  • Fig. 3 Droplets produced with oil r1A (see Table 1 for oil composition) yield anatase TiO2 nanoparticles.

    (A) Bright-field (left) and polarized light (right) optical micrographs reveal birefringence at droplet boundaries, indicating the presence of crystalline materials. (B) XRD of washed precipitates recovered from TiBALDH/oil r1A emulsions confirms the presence of anatase TiO2 products. (C) Transmission electron microscopy reveals that the precipitates were structured as solid films, suggestive of interface mineralization. (D) EDX from the material shown in (C) confirms the presence of Ti, originating from the mineral titania phases, and Si, from a silicone surfactant (ABIL EM90; see Table 1 and table S1). (E) Selected area diffraction from the area indicated by the box in (C) corroborates XRD data, showing the presence of anatase TiO2, as indicated by diffraction from the (101) plane. (F) High-magnification TEM image shows that anatase is present in the form of nanocrystals embedded in an amorphous film; the indicated 0.35-nm spacing within the nanocrystal is characteristic of anatase (101).

  • Fig. 4 Combinatorial oils are optimized to support off-chip droplet stability and monodispersity after three screening rounds.

    (Screening in each round was conducted as summarized in Figs. 1 and 2.) Between each screening round, successful oil combinations from the previous round were diversified using a genetic algorithm. (A) Optical stereomicrographs of example winning emulsions selected from each screening round. (B) Histograms of droplet sizes measured from emulsions shown in (A) highlight the fact that droplet sizes become increasingly monodisperse over successive screening rounds. (C) The percentage of screened combinatorial oils that produce stable droplets increased in successive rounds. In each round, 45 to 55 oils were screened.

  • Fig. 5 W/O emulsions generated using oil r3 (Table 1) yield droplet-stabilizing TiC minerals.

    (A) TEM images of solid films comprising washed precipitates recovered from TiBALDH/oil r3 emulsions, with associated electron diffraction pattern revealing the presence of TiC as a mineral product (top right); the selected area for electron diffraction is indicated by a red box in the top left image; high-magnification images reveal lattice fringes (bottom left image) and atomic ordering (bottom right image). (B) Droplet generation with or without TiBALDH shows that interface mineralization is important in achieving stable droplets. A gradual reduction in droplet volume suggests that either water or lactate released from hydrolyzed TiBALDH is partially soluble in the oil phase.

  • Fig. 6 Mineral shells formed from [W/O/W] double emulsions support in vitro protein expression.

    (A) Schematic overview of the second, double emulsion–producing, flow-focusing junction of a two-junction flow-focusing microfluidic device used to generate mineralized double emulsions (see fig. S10 and movie S4). (B) Schematic of [W/O/W] droplet mineralization to form an interfacial mineral layer around bacterial extracts in the innermost water phase. Here, “S” is a fluorogenic substrate (CCF2) that changes emission properties when enzymatically converted to product (“P”) by β-lactamase (β-lac) enzyme. (C) Polarized light optical micrograph shows interfacial birefringence. (D) Flow cytometry scatter plots of 50,000 droplets, proving that mineralized droplets support compartmentalized in vitro protein expression.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1600567/DC1

    text S1. Step-by-step screening procedure.

    text S2. Advantages of the new screening platform.

    fig. S1. Photographs of the microtiter vacuum manifold platform.

    fig. S2. Overview of the microfluidic device fabrication.

    fig. S3. Modifying a 96-well plate with metal outlet posts: A schematic summary.

    fig. S4. Procedure for loading source solutions into the 96-well screening plate.

    fig. S5. Interfacing a PDMS microfluidic device with the metal tube–modified 96-well plate.

    fig. S6. Summary of screening trials using syringe pumps.

    fig. S7. Workflows for screening droplet-based microfluidic carrier oils.

    fig. S8. Screened emulsion droplets exhibiting features suggestive of mineralization by optical stereomicroscopy.

    fig. S9. Precipitates recovered from mineralized droplets produced with oil r1B exhibit titania and are morphologically distinct from the precipitates generated from oil r1A.

    fig. S10. Device used for [W/O/W] double emulsion production.

    fig. S11. An alternate approach for preparing double emulsions.

    fig. S12. An evaluation of the structural stability conferred to [W/O/W] double emulsions when mineralized with different metal species.

    table S1. Twenty-five library surfactants.

    table S2. Twenty-two library essential oils.

    movie S1. A collage of four microfluidic devices recorded in operation via stereomicroscopy.

    movie S2. Example of an oil that leads to droplet merging at the PDMS microfluidic chip outlet.

    movie S3. Example of an oil that leads to stable droplets at the PDMS microfluidic chip outlet.

    movie S4. W/O emulsion produced with oil r2 (see Table 1) and 10% TiBALDH as the water phase.

    movie S5. Microfluidic generation of [W/O/W] droplets.

    file S1. A .dwg file of the microfluidic pattern set shown in fig. S2.

    Reference (63)

  • Supplementary Materials

    This PDF file includes:

    • text S1. Step-by-step screening procedure.
    • text S2. Advantages of the new screening platform.
    • fig. S1. Photographs of the microtiter vacuum manifold platform.
    • fig. S2. Overview of the microfluidic device fabrication.
    • fig. S3. Modifying a 96-well plate with metal outlet posts: A schematic summary.
    • fig. S4. Procedure for loading source solutions into the 96-well screening plate.
    • fig. S5. Interfacing a PDMS microfluidic device with the metal tube–modified 96-well plate.
    • fig. S6. Summary of screening trials using syringe pumps.
    • fig. S7. Workflows for screening droplet-based microfluidic carrier oils.
    • fig. S8. Screened emulsion droplets exhibiting features suggestive of mineralization by optical stereomicroscopy.
    • fig. S9. Precipitates recovered from mineralized droplets produced with oil r1B exhibit titania and are morphologically distinct from the precipitates generated from oil r1A.
    • fig. S10. Device used for W/O/W double emulsion production.
    • fig. S11. An alternate approach for preparing double emulsions.
    • fig. S12. An evaluation of the structural stability conferred to W/O/W double emulsions when mineralized with different metal species.
    • table S1. Twenty-five library surfactants.
    • table S2. Twenty-two library essential oils.
    • Legends for movies S1 to S5
    • Legend for data file S1
    • Reference (63)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mov format). A collage of four microfluidic devices recorded in operation via stereomicroscopy.
    • movie S2 (.mov format). Example of an oil that leads to droplet merging at the PDMS microfluidic chip outlet.
    • movie S3 (.mov format). Example of an oil that leads to stable droplets at the PDMS microfluidic chip outlet.
    • movie S4 (.mov format). W/O emulsion produced with oil r2 (see Table 1) and 10% TiBALDH as the water phase.
    • movie S5 (.mov format). Microfluidic generation of W/O/W droplets.
    • file S1. A .dwg file of the microfluidic pattern set shown in fig. S2.

    Files in this Data Supplement:

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