MRBLES 2.0: High-throughput generation of chemically functionalized spectrally and magnetically encoded hydrogel beads using a simple single-layer microfluidic device.
MRBLES 2.0: High-throughput generation of chemically functionalized spectrally and magnetically encoded hydrogel beads using a simple single-layer microfluidic device.
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DOI：
10.1038/s41378-020-00220-3
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发表时间：
2020
影响因子：
7.9
通讯作者：
Fordyce PM
中科院分区：
文献类型：
--
作者：
Feng Y;White AK;Hein JB;Appel EA;Fordyce PM
基于微珠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缀合到其表面。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，该微球具有下游表面生物缀合的官能团。MRBLEs 2.0中的珠粒生产依赖于镧系元素/聚合物混合物的手动混合（其中每种混合物包含唯一的光谱代码），然后使用单层平行流动聚焦装置产生液滴，并将液滴在芯片外分批聚合成珠粒。为了简化下游分析物偶联，MRBLEs 2.0在微珠生成期间交联微珠表面上带有官能团的共聚物。使用MRBLEs 2.0流水线，我们生成了包含48个不同的高分辨率光谱代码的单分散MRBLEs，具有高吞吐量（> 150，000/min，可以提升到450，000/min）。我们进一步证明了寡核苷酸和整个蛋白质与羧基MRBLES和生物素与氨基MRBLES的有效缀合。最后，我们表明，MRBLEs也可以磁化通过同时纳入磁性纳米粒子，只有一个微小的减少，在潜在的代码空间。凭借显著简化的设备制造，消除对定制设备的需求，以及通过直接表面功能化以高通量生产光谱和磁性编码珠的能力，MRBLES 2.0可以直接应用于许多实验室的各种下游检测，从基础生物学到诊断和其他转化研究。光谱编码的微珠为多重生物测定提供了一个方便的平台，提供了快速的结合动力学和每次测定的多次重复。最近开发的技术MRBLES通过镧系元素纳米磷光体的比例掺入对水凝胶珠进行光谱编码。在本论文中，由Polly福代斯领导的斯坦福大学的研究小组报告了一种显着简化的方法，用于生产带有各种官能团的MRBLE珠，用于下游化学偶联或珠上合成。使用“跨接电缆”管道策略，他们在3D中路由微流体通道，而不需要复杂的制造技术来创建多喷嘴液滴发生器。使用这些简单的单层微流控装置，他们创建了具有48个独特光谱代码的珠子，这些代码带有羧基和胺基，用于下游耦合，吞吐量增加了1000倍以上。最后，他们证明了MRBLE可以同时进行光谱和磁性编码。
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. Spectrally encoded beads provide a convenient platform for multiplexed bioassays, offering fast binding kinetics and many replicates per assay. A recently developed technology, MRBLEs, spectrally encodes hydrogel beads via the ratiometric incorporation of lanthanide nanophosphors. In the present paper, a team from Stanford University led by Polly Fordyce reports a dramatically simplified method for producing MRBLEs beads bearing various functional groups for downstream chemical coupling or on-bead synthesis. Using a ‘jumper cable’ tubing strategy, they route microfluidic channels in 3D without a need for complex fabrication techniques to create multi-nozzle droplet generators. Using these simple single-layer microfluidic devices, they create beads with 48 unique spectral codes bearing carboxyl and amine groups for downstream coupling with over 1000-fold increase in throughput. Finally, they demonstrate that MRBLEs can be simultaneously spectrally and magnetically encoded.
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