High-throughput Single-cell Screening Research Articles

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.

A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis

The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve’s optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip’s throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

Manipulation of single cells inside nanoliter water droplets using acoustic forces.

Droplet microfluidics enables high-throughput screening of single cells and is particularly valuable for applications, where the secreted compounds are analyzed. Typically, optical methods are employed for analysis, which are limited in their applicability as labeling protocols are required. Alternative label-free methods such as mass spectrometry would broaden the range of assays but are harmful to the cells, which is detrimental for some applications such as directed evolution. In this context, separation of cells from supernatant is beneficial prior to the analysis to retain viable cells. In this work, we propose an in-droplet separation method based on contactless and label-free acoustic particle manipulation. In a microfluidic chip, nanoliter droplets containing particles are produced at a T-junction. The particles are trapped in the tip of the droplet by the interplay of acoustic forces in two dimensions and internal flow fields. The droplets are subsequently split at a second T-junction into two daughter droplets-one containing the supernatant and the other containing the corresponding particles. The separation efficiency is measured in detail for polystyrene (PS) beads as a function of droplet speed, size, split ratio, and particle concentration. Further, single-bead (PS) and single-cell (yeast) experiments were carried out. At a throughput of 114 droplets/min, a separation efficiency of 100% ± 0% was achieved for more than 150 droplets. Finally, mammalian cells and bacteria were introduced into the system to test its versatility. This work demonstrates a robust, non-invasive strategy to perform single yeast cell-supernatant sampling in nanoliter volumes.

A Pichia pastoris single-cell biosensor for detection of enzymatically produced methanol.

We conducted single-cell analyses of the methylotrophic yeast Pichia pastoris to develop a biosensor for the detection of methanol produced by heterologous enzymes. In this biosensor, methanol and its subsequent metabolism induce expression of a gene encoding a fluorescent protein that was placed under the control of a methanol-inducible promoter. Using quantitative analyses of fluorescence microscopy images, a methanol-inducible promoter and a host strain were selected, and preculture and assay conditions were optimized to improve the methanol detection limit. Fluorescence-activated cell sorting (FACS) analysis of the distribution and geometric mean of cellular fluorescence intensity against various concentrations of methanol revealed a detection limit of 2.5μM. Finally, this biosensor was applied to evaluate the activity of a heterologously expressed pectin methylesterase (PME). The cellular fluorescence intensity was proportional to the copy number of the PME expression cassette, the protein level, and the enzyme activity. This biosensor can be used for high-throughput screening of single cells harboring high methanol-producing activity, and thereby, the development of a bioconversion process using methanol-producing enzymes.