Programmable microfluidic synthesis of spectrally encoded microspheres

Abstract

Spectrally encoded fluorescent beads are an attractive platform for assay miniaturization and multiplexing in the biological sciences. Here, we synthesize hydrophilic PEG-acrylate polymer beads encoded with lanthanide nanophosphors using a fully automated microfluidic synthesis device. These beads are encoded by including varying amounts of two lanthanide nanophosphors relative to a third reference nanophosphor to generate 24 distinct ratios. These codes differ by less than 3% from their target values and can be distinguished from each other with an error rate of <0.1%. The encoded bead synthesis strategy we have used is readily extensible to larger numbers of codes, potentially up to millions, providing a new platform technology for assay multiplexing.

Microfluidic Synthesis of Barcode Particles for Multiplex Assays

“…To simplify this procedure, Gerver et al developed an automated microfl uidic device with a staggered herringbone mixer channel for the generation of barcode particles. [ 72 ] Different lanthanide nanophosphors of poly(ethylene glycol) diacrylate solutions fl owed into their device and were mixed in the mixer channel. During this process, the relative fl ow rates from the different nanophosphor inputs determined the relative abundance of each nanophosphor in a barcode.…”

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

“…We developed a new high-throughput protein-peptide interaction assay based on spectrally encoded hydrogel beads containing unique ratios of lanthanide nanophosphors (MRBLEs) 16,17 . MRBLEs containing a given code are output to a particular filtered-tip in a 96-well format or two 48-2mL reaction tubes (Fig.…”

“…Droplets were then photopolymerized with UV light from a full-spectrum 200W Xenon arc lamp (Dymax, Torrington, CT, USA). With this setup, ~3,000 beads containing each spectral code were synthesized in 70 seconds (as described previously 16,17 ); for the 48-plex MRBLE library used here, each code was produced 10 times to yield ~30,000 beads per code.…”

“…MRBLEs were synthesized using a previously published microfluidic device 16,17,23 with each code collected into wells of a 96-well plate using an open-source in-house fraction collector. Briefly, all lanthanide input mixtures contained double-distilled water (ddH 2 O), 42.8% v/v 700 MW PEG-diacrylate (PEG-DA) (Sigma-Aldrich), 19.6 mg mL-1 lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and 5.0% v/v YVO4:Eu (at 25 mg mL-1 ).…”

“…Peptides are synthesized directly on spectrally encoded beads (MRBLEs, for Microspheres with Ratiometric Barcode Lanthanide Encoding) 16,17 with a unique linkage between each peptide sequence and a given spectral code. MRBLE-pep libraries can then be pooled and assayed for protein binding in a single small volume before being imaged to identify the peptide sequence associated with each bead and quantify the amount of protein bound.…”