The dissociation constant (Kd) is a crucial parameter for characterizing binding affinity in molecular recognition, including antigen-antibody, DNA-protein, and receptor-ligand interactions. However, conventional methods for Kd characterization usually involve a multi-step process and time-consuming operations for incubation, washing, and detection, thus causing problems, such as time delays, microbead loss, degradation of sensitive molecules, and personal errors. Here we demonstrate an automated ligand binding affinity evaluation platform (Auto-affitech) using digital microfluidics (DMF), with individual droplets at the microliter level, programmed to rapidly perform the incubation and separation of target-beads and binding ligands. Because the loss of the beads influences the detection results, we propose a new strategy for magnetic bead separation using DMF, termed the bidirectional separation method. By splitting one droplet into two asymmetric droplets, high bead retention efficiency (89.57% ± 0.05%) and high washing efficiency (99.59% ± 0.17%, with four washings) were obtained. We demonstrate the determination of Kd of an aptamer-protein system (EpCAM and its corresponding aptamer SYL3C) and an antigen-antibody system (H5N1 antigen and antibody), proving the capability and universality of Auto-affitech in various receptor-ligand systems. Integrating all the sample processing procedures, the Auto-affitech not only saves manual labor and minimizes personal errors, but also conserves samples and shortens analysis time. Overall, this platform successfully demonstrates to be an automated approach for dissociation constant evaluation and exhibits great potential for highly efficient screening of ligands.

中文翻译：

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Auto-affitech：一种自动配体结合亲和力评估平台，使用数字微流控技术和双向磁分离方法。

解离常数（Kd）是表征分子识别中的结合亲和力的关键参数，包括抗原-抗体，DNA-蛋白质和受体-配体相互作用。然而，用于Kd表征的常规方法通常涉及多步过程以及温育，洗涤和检测的耗时操作，因此会引起问题，例如时间延迟，微珠损失，敏感分子降解以及人为错误。在这里，我们展示了使用数字微流控（DMF）的自动配体结合亲和力评估平台（Auto-affitech），其中微滴级的单个液滴经过编程可快速进行目标珠子和结合配体的孵育和分离。由于珠子的丢失会影响检测结果，我们提出了一种使用DMF进行磁珠分离的新策略，称为双向分离方法。通过将一个液滴分成两个不对称液滴，可以获得高的珠保留效率（89.57％±0.05％）和高清洗效率（99.59％±0.17％，四次清洗）。我们证明了适体蛋白系统（EpCAM及其相应的适体SYL3C）和抗原-抗体系统（H5N1抗原和抗体）的Kd测定，证明了Auto-affitech在各种受体-配体系统中的功能和通用性。Auto-affitech集成了所有样品处理程序，不仅节省了人工，并且最大程度地减少了人为错误，还节省了样品并缩短了分析时间。总体，