Evaluation of quantum dot immunofluorescence and a digital CMOS imaging system as an alternative to conventional organic fluorescence dyes and laser scanning for quantifying protein microarrays.

Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile, and hard to deploy widely. Quantum dots (Qdot) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here, we have evaluated a small benchtop "personal" optical imager (ArrayCAM) developed for quantification of protein arrays probed by Qdot-based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labeled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA, and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R > 0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust, and deployable alternative to conventional laser array scanners.

Cell-Sized Lipid Vesicles as Artificial Antigen-Presenting Cells for Antigen-Specific T Cell Activation.

In this study, efficient T cell activation is demonstrated using cell-sized artificial antigen-presenting cells (aAPCs) with protein-conjugated bilayer lipid membranes that mimic biological cell membranes. The highly uniform aAPCs are generated by a facile method based on standard droplet microfluidic devices. These aAPCs are able to activate the T cells in peripheral blood mononuclear cells, showing a 28-fold increase in interferon gamma (IFNγ) secretion, a 233-fold increase in antigen-specific CD8 T cells expansion, and a 16-fold increase of CD4 T cell expansion. The aAPCs do not require repetitive boosting or additional stimulants and can function at a relatively low aAPC-to-T cell ratio (1:17). The research presents strong evidence that the surface fluidity and size of the aAPCs are critical to the effective formation of immune synapses essential for T cell activation. The findings demonstrate that the microfluidic-generated aAPCs can be instrumental in investigating the physiological conditions and mechanisms for T cell activation. Finally, this method demonstrates the feasibility of customizable aAPCs for a cost-effective off-the-shelf approach to immunotherapy.

Directed evolution of custom polymerases using droplet microfluidics.

DNA polymerases are critical tools for a large number of emerging applications in biotechnology, but oftentimes polymerases with desired functions are not readily available. Directed evolution provides a possible solution to this problem by enabling the creation of engineered polymerases that are better equipped to recognize a given unnatural substrate. Here we report a microfluidic-based method for evolving new polymerase functions that involves ultrahigh throughput sorting of fluorescent water-in-oil (w/o) microdroplets. The workflow entails the expression of a diverse population of polymerase variants in E. coli, production of microfluidic droplets containing one or less E. coli, bacteria lysis to release the polymerase and encoding plasmid into the surrounding droplet, a fluorescence-based activity assay to identify variants with a desired activity, isolation of fluorescent droplets using a fluorescence activated droplet sorting (FADS) device, and plasmid recovery with DNA sequencing to determine the identity of the functional variants. This technique is amenable to any type of unnatural nucleic acid and/or polymerase function, including DNA-templated synthesis, reverse transcription, and replication.

Programmed Allelic Mutagenesis of a DNA Polymerase with Single Amino Acid Resolution.

Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.

Elucidating the Determinants of Polymerase Specificity by Microfluidic-Based Deep Mutational Scanning.

Engineering polymerases to synthesize artificial genetic polymers with unique backbone structures is limited by a general lack of understanding about the structural determinants that govern substrate specificity. Here, we report a high-throughput microfluidic-based approach for mapping sequence-function relationships that combines droplet-based optical polymerase sorting with deep mutational scanning. We applied this strategy to map the finger subdomain of a replicative DNA polymerase isolated from Thermococcus kodakarensis (Kod). The enrichment profile provides an unbiased view of the ability of each mutant to synthesize threose nucleic acid, which was used as a model non-natural genetic polymer. From a single round of sorting, we discovered two cases of positive epistasis and demonstrate the near inversion of substrate specificity from a double mutant variant. This effort indicates that polymerase specificity may be governed by a small number of highly specific residues that can be elucidated by deep mutational scanning without the need for iterative rounds of directed evolution.

Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution.

Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ∼2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.

Functionalized Vesicles by Microfluidic Device.

In recent years, lipid vesicles have become popular vehicles for the creation of biosensors. Vesicles can hold reaction components within a selective permeable membrane that provides an ideal environment for membrane protein biosensing elements. The lipid bilayer allows a protein to retain its native structure and function, and the membrane fluidity can allow for conformational changes and physiological interactions with target analytes. Here, we present two methods for the production of giant unilamellar vesicles (GUVs) within a microfluidic device that can be used as the basis for a biosensor. The vesicles are produced from water-in-oil-in-water (W/O/W) double emulsion templates using a nonvolatile oil phase. To create the GUVs, the oil can be removed via extraction with ethanol, or by altering the interfacial tension between the oil and carrier solution causing the oil to retract into a cap on one side of the structure, leaving behind an exposed lipid bilayer. Methods to integrate sensing elements and membrane protein pores onto the vesicles are also introduced in this work.