Programmable Microfluidic Synthesis of Over One Thousand Uniquely Identifiable Spectral Codes

Abstract

Encoded microparticles have become a powerful tool for a wide array of applications, including high-throughput sample tracking and massively parallel biological multiplexing. Spectral encoding, where particles are encoded with distinct luminescence spectra, provides a particularly appealing encoding strategy because of the ease of reading codes and assay flexibility. To date, spectral encoding has been limited in the number of codes that can be accurately resolved. Here, we demonstrate an automated 5-dimensional spectral encoding scheme using lanthanide nanophosphors that is capable of producing isotropic spherical microparticles with up to 1,100 unique codes, which we term MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding). We further develop a quantitative framework for evaluating global ability to distinguish codes and demonstrate that for six different sets of MRBLEs ranging from 106 to 1,101 codes in size, > 98% of MRBLEs can be assigned to a code with 99.99% confidence. These > 1,000 code sets represent the largest spectral code libraries built to date. We expect that these MRBLEs will enable a wide variety of novel multiplexed assays.

Rising Stars in Science and Technology: Luminescent Lanthanide Materials

“…Several companies are selling rare‐earth‐containing tags for marking batches of various goods, among them ammunition . Codes can be embedded into microparticles, and using 5 different lanthanides (Sm, Eu, Ce/Tb, Dy, Tm) produces up to 1100 different combinations that can be recognized with a 99.99 % confidence …”

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.…”

“…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 ).…”

“…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.…”

“…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.…”