Programmable Microfluidic Synthesis of Over One Thousand Uniquely Identifiable Spectral Codes

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.