Coupling isotachophoresis with affinity chromatography for rapid and selective purification with high column utilization, part 1: theory.
Coupling isotachophoresis with affinity chromatography for rapid and selective purification with high column utilization, part 1: theory.
复制标题
将等速电泳与亲和层析结合，实现高柱利用率的快速选择性纯化，第 1 部分：理论。
DOI：
10.1021/ac5011052
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发表时间：
2014
影响因子：
7.4
通讯作者：
Santiago,JuanG
中科院分区：
文献类型：
--
作者：
Shkolnikov,Viktor;Santiago,JuanG
我们提出了一种将等速电泳法(ITP)与亲和层析(AC)相结合的新技术，以实现快速、选择性的纯化，并提高了柱的利用率。ITP根据样品组分的流动性差异，同时对分析物进行预浓缩和提纯，排除了可能污染亲和底物或与目标竞争的物种。ITP预浓缩加速了亲和反应，缩短了分析时间，提高了柱利用率，并允许捕获解离常数较高的目标。此外，ITP-AC将目标和污染物分离到非扩散区，从而在短距离和短时间内实现高分辨率。提出了ITP-AC时空动力学的解析模型。我们确定并探索了关键工艺参数，包括靶标分布的宽度和高度、ITP区速度、正向和反向反应常数以及探针浓度对所需亲和区长度、分析时间和捕获效率的影响。我们的分析方法显示这些变量崩溃为三个无量纲参数。该分析得出了相关ITP-AC体系中捕获长度和捕获时间的简单解析关系，并展示了ITP如何极大地缩短分析时间和提高柱利用率。在这个由两部分组成的系列的第二部分中，我们将对我们的模型进行实验验证，并演示ITP-AC将目标从10,000倍更丰富的污染物中分离出来。
We present a novel technique that couples isotachophoresis (ITP) with affinity chromatography (AC) to achieve rapid, selective purification with high column utilization. ITP simultaneously preconcentrates an analyte and purifies it, based on differences in mobility of sample components, excluding species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization, and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into nondiffusing zones, thus achieving high resolution in a short distance and time. We present an analytical model for spatiotemporal dynamics of ITP-AC. We identify and explore the effect of key process parameters, including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our analytical approach shows collapse of these variables to three nondimensional parameters. The analysis yields simple analytical relations for capture length and capture time in relevant ITP-AC regimes, and it demonstrates how ITP greatly reduces assay time and improves column utilization. In the second part of this two-part series, we will present experimental validation of our model and demonstrate ITP-AC separation of the target from 10,000-fold more-abundant contaminants.