Department of Chemistry, University of Southern California, 3620 McClintock Avenue, SGM 418, Los Angeles, California 90089, United States.
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, 925 Bloom Walk, HED 216, Los Angeles, California 90089, United States.
Anal Chem. 2020 Aug 4;92(15):10218-10222. doi: 10.1021/acs.analchem.0c01867. Epub 2020 Jul 14.
Modern genomic sequencing efforts are identifying potential diagnostic and therapeutic targets more rapidly than existing methods can generate the peptide- and protein-based ligands required to study them. To address this problem, we have developed a microfluidic enrichment device (MFED) enabling kinetic off-rate selection without the use of exogenous competitor. We tuned the conditions of the device (bed volume, flow rate, immobilized target) such that modest, readily achievable changes in flow rates favor formation or dissociation of target-ligand complexes based on affinity. Simple kinetic equations can be used to describe the behavior of ligand binding in the MFED and the kinetic rate constants observed agree with independent measurements. We demonstrate the utility of the MFED by showing a 4-fold improvement in enrichment compared to standard selection. The MFED described here provides a route to simultaneously bias pools toward high-affinity ligands while reducing the demand for target-protein to less than a nanomole per selection.
现代基因组测序工作正在比现有方法更快地识别潜在的诊断和治疗靶点，而这些靶点需要生成基于肽和蛋白质的配体来进行研究。为了解决这个问题，我们开发了一种微流控富集装置（MFED），无需使用外源性竞争物即可实现动力学解吸率选择。我们调整了装置的条件（床体积、流速、固定化靶标），使得流速的适度、易于实现的变化有利于基于亲和力形成或解离靶标-配体复合物。简单的动力学方程可用于描述 MFED 中的配体结合行为，并且观察到的动力学速率常数与独立测量结果一致。我们通过显示与标准选择相比富集提高了 4 倍来证明 MFED 的实用性。这里描述的 MFED 提供了一种同时偏向高亲和力配体的途径，同时将对靶蛋白的需求降低到每个选择不到纳摩尔。
