Microfluidics: high-throughput production of spectrally encoded hydrogel beads for bioassays Spectrally encoded beads provide a convenient platform for multiplexed bioassays, offering fast binding kinetics and many replicates per assay. A recently developed technology, MRBLEs, spectrally encodes hydrogel beads via the ratiometric incorporation of lanthanide nanophosphors. In the present paper, a team from Stanford University led by Polly Fordyce reports a dramatically simplified method for producing MRBLEs beads bearing various functional groups for downstream chemical coupling or on-bead synthesis. Using a 'jumper cable' tubing strategy, they route microfluidic channels in 3D without a need for complex fabrication techniques to create multi-nozzle droplet generators. Using these simple single-layer microfluidic devices, they create beads with 48 unique spectral codes bearing carboxyl and amine groups for downstream coupling with over 1000-fold increase in throughput. Finally, they demonstrate that MRBLEs can be simultaneously spectrally and magnetically encoded.

The widespread 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 the efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding (MRBLEs) that bear functional groups for downstream surface bioconjugation. Bead production in MRBLEs 2.0 relies on the 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 the 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 during bead generation. Using the MRBLEs 2.0 pipeline, we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes with 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 of biotin to amino MRBLEs. Finally, we show that MRBLEs can also be magnetized via the simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space. With the advantages of dramatically simplified device fabrication, elimination of the need for custom-made equipment, and the ability to produce spectrally and magnetically encoded beads with direct surface functionalization with high throughput, MRBLEs 2.0 can be directly applied by many labs towards a wide variety of downstream assays, from basic biology to diagnostics and other translational research.