MRBLES 2.0: High-throughput generation of chemically functionalized spectrally and magnetically-encoded hydrogel beads using a simple single-layer microfluidic device

Abstract

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 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.

MRBLE-pep measurements reveal accurate binding affinities for B56, a PP2A regulatory subunit

“…We speculate that these systematic shifts may result from differences in the density of displayed peptides after on-bead synthesis. In recent work, we described a dramatically simplified method for encoded bead production and functionalization (MRBLEs 2.0) (Feng et al 2020), and we anticipate that peptide synthesis on beads produced via this new pipeline will yield more consistent density coverage. Furthermore, by simplifying the technology we hope that these improvements will allow researchers to more broadly adopt MRBLE-pep to systematically characterize SLiM binding determinants and to validate candidate interactions identified by high-throughput phage-display, yeast-two-hybrid or in silico screens.…”

Magnetic Particles for CTC Enrichment

“…Feng et al used PAA as a ligand and made hydrophilic magnetic nanoparticles through the co-precipitation method to enhance the potential encoding capacity of their beads called MRBLEs [46].…”

“…For this purpose, the particle surface is often coated to prevent the aggregation of the bare MNPs. Commonly used coatings are PEG [49], polyacrylic acid (PAA) [46,102], polyvinylpyrrolidone (PVP) [103], polydopamine (PDA) [104], dextran [105] and BSA [106,107]. The electrostatic repulsion or steric hindrance between these coated surfaces can prevent the agglomeration of nanoparticles, while at the same time non-specific binding is reduced due to the exposure of bare nanoparticles being avoided.…”