However, it still requires a high-throughput screening technique and/or compartmentalized environments for cell sorting. Second, a high-throughput screening technology is developed by patterning fluid in which individual Escherichia coli cells can be immobilized and cultured in a cavity array format. In addition, it was successfully demonstrated that the two technologies hold great potential to enable not only high-throughput screening, but also many biological experiments such as detection of cell-secreted products, long-term cell incubation, cell-to-cell communication and detection of target molecules via a whole-cell biosensor .
However, most of these methodological approaches are still crucially limited by their need for high-throughput measurements of target molecule responses or a desired phenotype in a massive, randomly mutated library of microorganisms. When dealing with the artificial and random mutagenic response to a target microbe, the population of the random mutagenic library usually exceeds 105-9 variants [6]. However, the plate reader exhibits low throughput performance because the time-limiting determination step is ultimately limited by the microplate reading speed of the instrument.
In the last decade, microfluidics has made great improvements with the potential to revolutionize high-throughput bioassays [11]. As this microfluidic technique is a suitable platform for fluid sampling with a fully compartmentalized environment and high throughput, many applications have been developed that are a suitable candidate for bioassay, for example, droplet-based microfluidics [12, 13], small bioreactors using complex pneumatic valves [14] and liquid sampling of the aqueous phase with separation of immiscible oil [15, 16]. Due to the above advantages, some researchers have developed new microfluidic devices for liquid sampling, in other words, sample digitization [15], self-priming [16] and droplet sampling [12,13].
Since these liquid modeling technologies fulfill many requirements of directed evolution by the random mutation method, the device can provide a high-throughput screening technique as well as a fully separated environment for the detection of chemical products secreted by microbes.
Patterning Microdroplet
- Fabrication of the microfluidic device
- Experimental procedure and data analysis
- Calibration of microdroplet production rate
- Droplet trapping in a cavity array
- Numerical analysis for agitation of microdroplet
- Single cell level of cell encapsulation in microdroplet
- Biological applications in microdroplet platform
First, each of the two microchannels, indicated as “top” and “bottom”, were specially designed to provide a microfluidic droplet generator in the upper region of the upper device and thousands of microcavities in the lower region of the lower device. At the entrance of the microcavities, flow-focusing shaped microfluidic structures were designed with a width of 50~60 μm, in order to precisely control the droplet production rate as well as the target droplet volume. Second, photomasks provided by microTECH (Ansan, Korea) were used in the photo fabrication process.
Trichloro(3,3,3-trifluoropropyl)silane was used for the silanization process of the channel surface in a vacuum vessel for one hour to minimize the adhesion between the PDMS prepolymer to the substrate. This high-temperature treatment was performed to render the PDMS channel surfaces hydrophobic so that any oil phase easily flowed along the channel. Once the droplet generation was performed on the top layer, while the immobilization of droplets was performed at the bottom layer where the microcavity array was fabricated, the droplets were sequentially trapped in microcavities obtained by the difference in specific gravity between immiscible substrates.
When the condition for the target droplet volume is fixed at approximately 100 μm diameter, which is sufficient for the cell incubation experiment, the droplet production rate depended on the flow rate in the flow focusing structure [ 24 ]. The flow rate in the experiment consists of the continuous flow rate of the oil phase and the discontinuous flow rate of the aqueous phase. The higher flow rate in the droplet generator structure showed the highest droplet production rate (Table 2).
To obtain actual three-dimensional images for the immobilization of droplets, the Olympus MVX-100 confocal microscope was used in Figure 2.3b. The concept of a downstream droplet immobilization process is shown in Figure 2.3 chronologically. When the squeezed droplet reached the microcavities in the bottom device, the droplet tended to be a spherical shape which is an original droplet shape.
Spherical shape of droplets is stuck to the edge of the microcavity, and then sinks down due to the difference of specific gravity mentioned earlier. This is proof positive that the droplet roll occurs not only on the surface, but also inside liquid. It is very clear that faster flow velocity of continuous oil phase drove faster agitation of the droplet inside the trap.
Both Figure 2.4 and Table 3 directly indicate the relationship between optical density and cell encapsulation rate. As can be seen in Figure 2.5a-c, different numbers of individual cells were gradually induced by different concentration of inducer chemical.
Fluid Patterning Array
- A novel parylene to parylene bonding method
- Fluid patterning for a high-throughput screening assay
- Characterization of array using food dye with various oil
- Effect of protein concentration on patterned fluid
- An array for resveratrol over-production cell
- Long-term cell cultivation in a patterned array
- An array for whole-cell biosensor for fatty acid
- Single cell array for Tn5 transposon random mutagenic library
- Discussion and future works
Here, a novel bonding method between both parylene and a parylene-coated surface on a patterned PDMS substrate is presented in an experimental session. The combined parylene and parylene coated device was tested using the hexadecane durability experiments shown in Figure 3.1. As shown in Figure 3.4, FC-40 fluorinated oil and mineral oil were used in the liquid sampling experiments.
In the results, as the protein concentration increased in the array, the stability of the array also increased. Since the new parylene to parylene bonding method was developed in this study as described in session 3.2.3, we achieved more than 12 hours of cell incubation experiment on a single chip. With more than 12 hours of time-lapse images, we speculated on fluorescence intensity, which directly indicates the cell growth in the liquid-patterned arrays.
Even it was possible to see that the heterogeneity of a single colony sperm cell would continue to grow for the long term, as shown in Figure 3.7d. The fatty acid-produced microorganism has long been one of the targets in the field of industrial biology. Its mechanism of action was based on fadR gene specificity for cellular fatty acid production and the fadR is related to the expression of red fluorescent protein [51].
Combining the fatty acid biosensor with liquid pattern device provides a good synergy effect in the form of experiments. We prepared two different E.coli, one with control strain without any manipulation and another strain has fadE gene knock-out region on genomic DNA, which is well understood that the knock-out region on fadE blocks the β-oxidation pathway for fatty acids and induces overproduction of fatty acids by accumulation. The measured fluorescence intensities at the 10 hour time point showed different results as expected with conventional experimental data shown in Figure 3.7d.
Due to accumulation of fatty acid inside cellular membrane, biosensor was induced and clearly expressed more red fluorescence protein than control strain. This experiment proves that the combined parylene-coated device can have great advantages in cell-to-cell communication experiments as well as complete dissection of the individual array. Both contain fatty acid biosensor plasmid, which expresses red fluorescent protein upon accumulation of cellular fatty acid.
Regardless of whether the opportunity to screen 4000 libraries works, we modeled arrays containing a single cell mutagen DH10B Tn5 with a single cell DH10B fatty acid biosensor plasmid. Among the rest of the group that shows the intensity of red fluorescence, we can check the target cell which produces more fatty acid than other mutagenic cells.
Conclusion
Mary, P., et al., Analysis of gene expression at the single-cell level using microdroplet-based microfluidic technology. Huebner, A., et al., Static microdroplet arrays: a microfluidic device for droplet capture, incubation, and release for enzymatic and cell-based assays. Iyer, J., et al., Studies on immediate release drug compounds for microfluidic high-throughput screening applications.
Ollila, O.H., et al., Interfacial tension and surface pressure of high-density lipoprotein, low-density lipoprotein, and related lipid droplets. Katsagonis, A., et al., An HPLC method with UV detection for the determination of trans-resveratrol in plasma. Hoover, S.W., et al., Isolation of improved strains of Escherichia coli overproducing free fatty acids by nile red-based high-throughput screening.
Zhang, F.Z., et al., Enhancement of fatty acid production by expression of the regulatory transcription factor FadR.
Acknowledgements
