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

Yinnian Feng,Polly M Fordyce,Eric A Appel,Adam K White,Jamin B Hein

doi:10.1038/s41378-020-00220-3

Abstract

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.

Highlights

The recently developed ability to identify molecules across a cell’s genome, transcriptome, and proteome has dramatically increased the need for technologies capable of detecting interactions between biological macromolecules at scale to understand interactome networks
Spatial arrays suffer from relatively slow kinetics and typically require relatively large amounts of sample[1]
In the first stage (“polymer mixing”), ratiometric mixtures of polyacrylic acid (PAA)wrapped lanthanide nanophosphors (Lns) are combined with a polymer solution off-chip via manual or robotic pipetting and deposited into a standard multiwell plate

Summary

Introduction

The recently developed ability to identify molecules across a cell’s genome, transcriptome, and proteome has dramatically increased the need for technologies capable of detecting interactions between biological macromolecules at scale to understand interactome networks. Multiplexed bead-based assays provide an appealing alternative, with near fluid-phase interfacial kinetics, many replicates per experiment, opportunities for quality control, and the ability to flexibly couple different probes and targets across experiments[3,4,5]. Feng et al Microsystems & Nanoengineering (2020)6:109 combinations of fluorescent (e.g., organic dyes[6,7,8], quantum dots (QDs)9–12) or luminescent materials (e.g., lanthanide nanoparticles13–15), provide a convenient format for multiplexed assays and have already been used for a wide variety of applications[15,16,17,18,19,20,21,22,23,24,25]. Luminex multianalyte profiling (xMAP) technology represents the most widely used spectrally encoded bead-based technology. Luminex xMAP beads are commercially available, are compatible with flow cytometry[26] and are widely used for a variety of bioassays[27]

Methods

Results

Conclusion