Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~10⁷ enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.

Optofluidic techniques could evolve to perform a series of single-cell analysis processes, including manipulation, treatment, and property detection.

Localized surface plasmon resonance (LSPR) nanoplasmonic effects allow for label-free, real-time detection of biomolecule binding events on a nanostructured metallic surface with simple optics and sensing tunability. Despite numerous reports on LSPR bionanosensing in the past, no study thus far has applied the technique for a cytokine secretion assay using clinically relevant immune cells from human blood. Cytokine secretion assays, a technique to quantify intercellular-signaling proteins secreted by blood immune cells, allow determination of the functional response of the donor’s immune cells, thus providing valuable information about the immune status of the donor. However, implementation of LSPR bionanosensing in cellular functional immunoanalysis based on a cytokine secretion assay poses major challenges primarily owing to its limited sensitivity and a lack of sufficient sample handling capability. In this paper, we have developed a label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-α cytokines in clinical blood samples. Our approach integrates LSPR bionanosensors in an optofluidic platform that permits trapping and stimulation of target immune cells in a microfluidic chamber with optical access for subsequent cytokine detection. The on-chip spatial confinement of the cells is the key to rapidly increasing a cytokine concentration high enough for detection by the LSPR setup, thereby allowing the assay time and sample volume to be significantly reduced. We have successfully applied this approach first to THP-1 cells and then later to CD45 cells isolated directly from human blood. Our LSPR optofluidics device allows for detection of TNF-α secreted from cells as few as 1000, which translates into a nearly 100 times decrease in sample volume than conventional cytokine secretion assay techniques require. We achieved cellular functional immunoanalysis with a minimal blood sample volume (3 μL) and a total assay time 3 times shorter than that of the conventional enzyme-linked immunosorbent assay (ELISA).

Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli. The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.