Synthesis of eco-friendly multifunctional dextran microbeads for multiplexed assays.

There has been an increasing demand for simultaneous detection of multiple analytes in one sample. Microbead-based platforms have been developed for multiplexed assays. However, most of the microbeads are made of non-biodegradable synthetic polymers, leading to environmental and human health concerns. In this study, we developed an environmentally friendly dextran microbeads as a new type of multi-analyte assay platform. Biodegradable dextran was utilized as the primary material. Highly uniform magnetic dextran microspheres were successfully synthesized using the Shirasu porous glass (SPG) membrane emulsification technique. To enhance the amount of surface functional groups for ligand conjugation, we coated the dextran microbeads with a layer of dendrimers via a simple electrostatic adsorption process. Subsequently, a unique and efficient click chemistry coupling technique was developed for the fluorescence encoding of the microspheres, enabling multiplexed detection. The dextran microbeads were tested for 3-plex cytokine analysis, and exhibited excellent biocompatibility, stable coding signals, low background noise and high sensitivity.

A Scalable Microfluidic Platform for Nanoparticle Formulation: For Exploratory- and Industrial-Level Scales.

Nanoparticle synthesis on microfluidic platforms provides excellent reproducibility and control over bulk synthesis. While there have been plenty of platforms for producing nanoparticles (NPs) with controlled physicochemical properties, such platforms often operate in a narrow range of predefined flow rates. The flow rate limitation restricts either up-scalability for industrial production or down-scalability for exploratory research use. Here, we present a universal flow rate platform that operates over a wide range of flow rates (0.1-75 mL/min) for small-scale exploratory research and industrial-level synthesis of NPs without compromising the mixing capabilities. The wide range of flow rate is obtained by using a coaxial flow with a triangular microstructure to create a vortex regardless of the flow regime (Reynolds number). The chip synthesizes several types of NPs for gene and protein delivery, including polyplex, lipid NPs, and solid polymer NPs via self-assembly and precipitation, and successfully expresses GFP plasmid DNA in human T cells.

Fully integrated sample-in-answer-out platform for viral detection using digital reverse transcription recombinase polymerase amplification (dRT-RPA).

Recombinase polymerase amplification (RPA) is one of the most promising diagnostic methods for pathogen detection, owing to the simplified isothermal amplification technique. Using one-step digital reverse transcription RPA (dRT-RPA) to detect viral RNA provides a fast diagnosis and absolute quantification. Here, we present a chip that purifies, digitalizes, and detects viral RNA of SARS-CoV-2 in a fully automated and sensitive manner. The chip purifies the RNA using the surface charge concept of magnet bead-RNA binding, then mixes the RNA with the amplification reagents, digitalizes the amplification mixture, and performs dRT-RPA. RNA-bead complex is transported among purification buffers that are separated by an oil phase. For reagent manipulation and mixing, a magnetic valve system is integrated on the chip, where an external magnet controls the reagent direction and time of addition. Besides, a novel vacuum system is suggested to drive and regulate the reagents into two fluid systems simultaneously in ∼2 min. We also developed a cost-effective way to perform fluorescent detection for dRT-RPA on chip by using EvaGreen® dye. With integrated heating and optical detection system, the on-chip dRT-RPA presents a sample-to-answer detection platform for absolute viral RNA quantitation in 37 min and a sensitivity as low as 10 RNA copies/µL. Hence, this platform is expected to be a useful tool for accurate and automated diagnosis of infectious diseases.

Metabolic engineering of Deinococcus radiodurans for pinene production from glycerol.

BACKGROUND: The objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries. RESULTS: To enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-D-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli. CONCLUSIONS: In this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules.