Identification of lipolytic enzymes using high-throughput single-cell screening and sorting of a metagenomic library.

The demand for novel, robust microbial biocatalysts for use in industrial and pharmaceutical applications continues to increase rapidly. As a result, there is a need to develop advanced tools and technologies to exploit the vast metabolic potential of unculturable microorganisms found in various environments. Single-cell and functional metagenomics studies can explore the enzymatic potential of entire microbial communities in a given environment without the need to culture the microorganisms. This approach has contributed substantially to the discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples, constructing expression libraries comprising the entire microbial genome, and screening of the libraries for the presence of desired phenotypes. In this study, lipolytic enzymes from the Red Sea were targeted. A high-throughput single-cell microfluidic platform combined with a laser-based fluorescent screening bioassay was employed to discover new genes encoding lipolytic enzymes. Analysis of the metagenomic library led to the identification of three microbial genes encoding lipases based on their functional similarity and sequence homology to known lipases. The results demonstrated that microfluidics is a robust technology that can be used for screening in functional metagenomics. The results also indicate that the Red Sea is a promising, under-investigated source of new genes and gene products.

Integration of Droplet Microfluidic Tools for Single-Cell Functional Metagenomics: An Engineering Head Start.

Droplet microfluidic techniques have shown promising outcome to study single cells at high throughput. However, their adoption in laboratories studying "-omics" sciences is still irrelevant due to the complex and multidisciplinary nature of the field. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single cells in droplets at a rate of ∼250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescence-activated droplet sorting systems to integrate the use of 4 independent fluorescence-exciting lasers (i.e., 405, 488, 561, and 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger is also integrated into our pipeline to enable adding new reagents to already-made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput capability (>50,000 cells/day) for mining and bioprospecting metagenomic data.

High-Throughput Incubation and Quantification of Agglutination Assays in a Microfluidic System.

In this paper, we present a two-phase microfluidic system capable of incubating and quantifying microbead-based agglutination assays. The microfluidic system is based on a simple fabrication solution, which requires only laboratory tubing filled with carrier oil, driven by negative pressure using a syringe pump. We provide a user-friendly interface, in which a pipette is used to insert single droplets of a 1.25-µL volume into a system that is continuously running and therefore works entirely on demand without the need for stopping, resetting or washing the system. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5⁻10-fold improvement over traditional agglutination assays. We study system parameters such as channel length, incubation time and flow speed to select optimal assay conditions, using the streptavidin-biotin interaction as a model analyte quantified using optical image processing. We then investigate the effect of changing the concentration of both analyte and microbead concentrations, with a minimum detection limit of 100 ng/mL. The system can be both low- and high-throughput, depending on the rate at which assays are inserted. In our experiments, we were able to easily produce throughputs of 360 assays per hour by simple manual pipetting, which could be increased even further by automation and parallelization. Agglutination assays are a versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as this one is a step towards being able to produce high-throughput microfluidic diagnostic solutions with widespread adoption. The development of analytical techniques in the microfluidic format, such as the one presented in this work, is an important step in being able to continuously monitor the performance and microfluidic outputs of organ-on-chip devices.

Comparison of capacitive and radio frequency resonator sensors for monitoring parallelized droplet microfluidic production.

Scaled-up production of microfluidic droplets, through the parallelization of hundreds of droplet generators, has received a lot of attention to bring novel multiphase microfluidics research to industrial applications. However, apart from droplet generation, other significant challenges relevant to this goal have never been discussed. Examples include monitoring systems, high-throughput processing of droplets and quality control procedures among others. In this paper, we present and compare capacitive and radio frequency (RF) resonator sensors as two candidates that can measure the dielectric properties of emulsions in microfluidic channels. By placing several of these sensors in a parallelization device, the stability of the droplet generation at different locations can be compared, and potential malfunctions can be detected. This strategy enables for the first time the monitoring of scaled-up microfluidic droplet production. Both sensors were prototyped and characterized using emulsions with droplets of 100-150 µm in diameter, which were generated in parallelization devices at water-in-oil volume fractions (φ) between 11.1% and 33.3%.Using these sensors, we were able to measure accurately increments as small as 2.4% in the water volume fraction of the emulsions. Although both methods rely on the dielectric properties of the emulsions, the main advantage of the RF resonator sensors is the fact that they can be designed to resonate at multiple frequencies of the broadband transmission line. Consequently with careful design, two or more sensors can be parallelized and read out by a single signal. Finally, a comparison between these sensors based on their sensitivity, readout cost and simplicity, and design flexibility is also discussed.