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.

基于珠子的多重生物测定的广泛采用需要能够轻松合成编码微球并将感兴趣的分析物结合到其表面。在这里，我们提出了一种简单的方法 (MRBLEs 2.0)，用于高效高通量生成具有比率条形码镧系元素编码 (MRBLEs) 的微球，该微球带有用于下游表面生物共轭的官能团。 MRBLE 2.0 中的珠子生产依赖于手动混合镧系元素/聚合物混合物（每种混合物都包含一个独特的光谱代码），然后使用单层并行流动聚焦装置生成液滴，并将液滴在片外批量聚合成珠子。为了简化下游分析物耦合，MRBLEs 2.0 在微珠生成过程中交联微珠表面带有官能团的共聚物。使用 MRBLE 2.0 管道，我们生成单分散 MRBLE，其中包含 48 个不同的高分辨率光谱代码，具有高吞吐量（>150,000/分钟，可提升至 450,000/分钟）。我们进一步证明了寡核苷酸和整个蛋白质与羧基 MRBLE 以及生物素与氨基 MRBLE 的有效缀合。最后，我们表明，MRBLE 也可以通过同时掺入磁性纳米粒子而被磁化，而潜在的代码空间仅略有减少。 MRBLEs 2.0 具有显着简化设备制造、无需定制设备以及能够以高通量生产具有直接表面功能化的光谱和磁性编码珠子的优点，可直接被许多实验室应用于各种领域。下游检测，从基础生物学到诊断和其他转化研究。