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.

中文翻译：

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使用微流体富集装置实现基于流动的动力学解离速率选择。

现代基因组测序工作正在识别潜在的诊断和治疗靶标，其速度比现有方法更快，无法生成研究它们所需的基于肽和蛋白质的配体。为了解决这个问题，我们开发了一种微流体富集装置（MFED），能够在不使用外源竞争剂的情况下进行动力学解离速率选择。我们调整了设备的条件（床体积、流速、固定靶标），使得流速的适度、容易实现的变化有利于基于亲和力的靶标-配体复合物的形成或解离。简单的动力学方程可用于描述 MFED 中配体结合的行为，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度提高了 4 倍来证明 MFED 的实用性。这里描述的 MFED 提供了一种同时将池偏向高亲和力配体的途径，同时将每次选择对靶蛋白的需求减少到小于纳摩尔。