Measurement of Protein-Protein Interaction Dynamics Using Microfluidics and Particle Diffusometry.
Measurement of Protein-Protein Interaction Dynamics Using Microfluidics and Particle Diffusometry.
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DOI：
10.1021/acs.analchem.2c02570
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发表时间：
2022-11-15
影响因子：
7.4
通讯作者：
Kinzer-Ursem, Tamara L.
中科院分区：
文献类型：
--
作者：
Ma, Hui;Wereley, Steven T.;Linnes, Jacqueline C.;Kinzer-Ursem, Tamara L.
蛋白质-蛋白质相互作用的测量和优化在其他生物分子科学和工程中的生物治疗、生物分子传感元件和基于功能蛋白质的生物材料的设计中是至关重要的。目前的金标准测定需要专门设计的核心设施、设备和专业知识来实施测量，这使得大多数实验室不方便，除非常规实施。我们开发了一种新的方法，旨在测量蛋白质结合动力学的基础上微流体和粒子扩散（PD），它只需要非常一般的实验室设备，包括荧光显微镜，注射泵，和一个简单的微通道上制作的载玻片。蛋白质结合对被固定在两种不同直径的纳米颗粒上，使用广泛可用的缀合化学。使用注射泵将两种稀释的颗粒悬浮液注射到Y型连接微通道中，在那里它们结合并形成具有增加的尺寸的颗粒复合物，从而降低颗粒的布朗运动幅度和扩散率，这可以通过PD检测。通过在沿微通道沿着的一系列特定点处拍摄图像，在引入蛋白质-蛋白质结合后的不同时间点测量颗粒扩散率。然后将这些数据用于定量蛋白结合动力学常数。这种基于颗粒的无标记方法操作简单，与当前的金标准一样准确。我们通过定量链霉亲和素-生物素结合常数（1.74 ± 0.51 × 107 M-1 s-1）证明了这种方法的可行性，与先前发表的结果进行了比较。
The measurement and optimization of protein–protein interactions are critical in the design of biotherapeutics, biomolecular sensing elements, and functional protein-based biomaterials among other biomolecular sciences and engineering. Current gold standard assays require specifically designed core facilities, equipment, and expertise to implement the measurement, making it inconvenient for most labs unless implemented routinely. We developed a new method aiming at measuring protein binding kinetics based on microfluidics and particle diffusometry (PD), which only needs very general lab equipment, including a fluorescence microscope, a syringe pump, and a simple microchannel fabricated on a glass slide. Protein binding pairs are immobilized on two kinds of nanoparticles with different diameters using widely available conjugation chemistries. The two diluted particle suspensions are injected using a syringe pump into a Y-junction microchannel, where they bind and form particle complexes with increasing size, thereby decreasing particles’ Brownian motion amplitude and diffusivity, which can be detected by PD. By taking images at a series of specific points along the microchannel, the particle diffusivity is measured at different time points after the introduction of protein–protein binding. These data are then used to quantify the protein binding kinetic constant. This label-free particle-based method is simple to operate and as accurate as the current gold standard. We demonstrate the feasibility of this accessible method by quantifying the streptavidin–biotin association constant (1.74 ± 0.51 × 107 M–1 s–1), which compares well with previously published results.
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DOI：
10.1007/978-1-4939-9654-4_11
发表时间：
2019-01-01
期刊：
BIOCONJUGATION
影响因子：
--
作者：
Ejendal, Karin F. K.;Fraseur, Julia G.;Kinzer-Ursem, Tamara L.
通讯作者：
Kinzer-Ursem, Tamara L.
影响因子：
38.3
作者：
通讯作者：
--
影响因子：
3.2
作者：
Clayton, Katherine N.;Salameh, Janelle W.;Kinzer-Ursem, Tamara L.
通讯作者：
Kinzer-Ursem, Tamara L.
影响因子：
4.6
作者：
Kim, YH;Wereley, ST;Chun, CH
通讯作者：
Chun, CH
影响因子：
2
作者：
Kumar, Aloke;Gorti, Venu M.;Wereley, Steve T.
通讯作者：
Wereley, Steve T.