AbstractWidespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface. Here, we present a simple method (MRBLEs 2.0) for efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding (MRBLEs) bearing functional groups for downstream bioconjugation. Bead production in MRBLEs 2.0 relies on manual mixing of lanthanide/polymer mixtures (each of which comprises a unique spectral code) followed by droplet generation using single-layer, parallel flow-focusing devices and off-chip batch polymerization of droplets into beads. To streamline downstream analyte coupling, MRBLEs 2.0 crosslinks copolymers bearing functional groups on the bead surface simultaneously during bead generation. Using the MRBLEs 2.0 pipeline, we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes in high-throughput (>150,000/min and can be boosted to 450,000/min). We further demonstrate the efficient conjugation of oligonucleotides and entire proteins to carboxyl MRBLEs and biotin to amino MRBLEs. Finally, we show that MRBLEs can also be magnetized via simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space. We anticipate that MRBLEs 2.0 can be directly applied towards a wide variety of downstream assays from basic biology to diagnostics and other translational research.
Synthesis of Size-Tunable Polymeric Nanoparticles Enabled by 3D Hydrodynamic Flow Focusing in Single-Layer Microchannels
