Department of Chemistry, ‡Department of Biomedical Engineering, and §Institute for Quantitative Health Science and Engineering, Michigan State University , 775 Woodlot Dr., East Lansing, Michigan 48824, United States.
Anal Chem. 2017 Jul 18;89(14):7302-7306. doi: 10.1021/acs.analchem.7b01848. Epub 2017 Jul 6.
Equilibrium dialysis is a simple and effective technique used for investigating the binding of small molecules and ions to proteins. A three-dimensional (3D) printer was used to create a device capable of measuring binding constants between a protein and a small ion based on equilibrium dialysis. Specifically, the technology described here enables the user to customize an equilibrium dialysis device to fit their own experiments by choosing membranes of various material and molecular-weight cutoff values. The device has dimensions similar to that of a standard 96-well plate, thus being amenable to automated sample handlers and multichannel pipettes. The device consists of a printed base that hosts multiple windows containing a porous regenerated-cellulose membrane with a molecular-weight cutoff of ∼3500 Da. A key step in the fabrication process is a print-pause-print approach for integrating membranes directly into the windows subsequently inserted into the base. The integrated membranes display no leaking upon placement into the base. After characterizing the system's requirements for reaching equilibrium, the device was used to successfully measure an equilibrium dissociation constant for Zn and human serum albumin (K = (5.62 ± 0.93) × 10 M) under physiological conditions that is statistically equal to the constants reported in the literature.
平衡透析是一种简单而有效的技术，用于研究小分子和离子与蛋白质的结合。本文使用 3D 打印机创建了一种设备，能够基于平衡透析测量蛋白质与小离子之间的结合常数。具体来说，这里描述的技术使用户能够通过选择不同材料和分子量截止值的膜来自定义适合自己实验的平衡透析设备。该设备的尺寸与标准 96 孔板相似，因此适用于自动化样品处理和多通道移液器。该设备由一个印刷底座组成，底座上有多个窗口，其中包含一个分子量截止值约为 3500 Da 的多孔再生纤维素膜。在制造过程中的一个关键步骤是打印-暂停-打印方法，该方法可将膜直接集成到随后插入底座的窗口中。集成的膜在放入底座时不会泄漏。在对达到平衡的系统要求进行特征描述后，该设备成功地在生理条件下测量了 Zn 和人血清白蛋白的平衡离解常数（K =（5.62 ± 0.93）× 10 M），该常数与文献中报道的常数在统计学上相等。
