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Joule Heating-Induced Dispersion in Open Microfluidic Electrophoretic Cytometry
Analytical Chemistry ( IF 7.4 ) Pub Date : 2017-11-15 00:00:00 , DOI: 10.1021/acs.analchem.7b03096
Julea Vlassakis 1 , Amy E. Herr 1
Affiliation  

While protein electrophoresis conducted in capillaries and microchannels offers high-resolution separations, such formats can be cumbersome to parallelize for single-cell analysis. One approach for realizing large numbers of concurrent separations is open microfluidics (i.e., no microchannels). In an open microfluidic device adapted for single-cell electrophoresis, we perform 100s to 1000s of simultaneous separations of endogenous proteins. The microscope slide-sized device contains cells isolated in microwells located in a ∼40 μm polyacrylamide gel. The gel supports protein electrophoresis after concurrent in situ chemical lysis of each isolated cell. During electrophoresis, Joule (or resistive) heating degrades separation performance. Joule heating effects are expected to be acute in open microfluidic devices, where a single, high-conductivity buffer expedites the transition from cell lysis to protein electrophoresis. Here, we test three key assertions. First, Joule heating substantially impacts analytical sensitivity due to diffusive losses of protein out of the open microfluidic electrophoretic (EP) cytometry device. Second, increased analyte diffusivity due to autothermal runaway Joule heating is a dominant mechanism that reduces separation resolution in EP cytometry. Finally, buffer exchange reduces diffusive losses and band broadening, even when handling single-cell lysate protein concentrations in an open device. We develop numerical simulations of Joule heating-enhanced diffusion during electrophoresis and observe ∼50% protein loss out of the gel, which is reduced using the buffer exchange. Informed by analytical model predictions of separation resolution (with Joule heating), we empirically demonstrate nearly fully resolved separations of proteins with molecular mass differences of just 4 kDa or 12% (GAPDH, 36 kDa; PS6, 32 kDa) in each of 129 single cells. The attained separation performance with buffer exchange is relevant to detection of currently unmeasurable protein isoforms responsible for cancer progression.

中文翻译:

开式微流控电泳细胞术中焦耳热诱导的分散

尽管在毛细管和微通道中进行蛋白质电泳可提供高分辨率的分离,但这种格式对于单细胞分析而言很难并行化。一种实现大量并发分离的方法是开放式微流体技术(即无微通道)。在适用于单细胞电泳的开放式微流控设备中,我们执行100s至1000s的内源蛋白质同时分离。显微镜载玻片大小的装置包含分离在约40μm聚丙烯酰胺凝胶中的微孔中的细胞。每个分离的细胞同时原位化学裂解后,凝胶支持蛋白质电泳。电泳期间,焦耳(或电阻)加热会降低分离性能。在开放式微流控设备中,预计焦耳热效应会非常严重,其中单个,高电导率缓冲液可加快从细胞裂解到蛋白质电泳的过渡。在这里,我们测试三个关键断言。首先,焦耳热实质上会影响分析灵敏度,这是由于蛋白质从开放式微流控电泳(EP)细胞仪设备中扩散出来所致。其次,由于自热失控的焦耳热导致分析物扩散性增加,这是降低EP细胞计数分离分辨率的主要机制。最后,即使在开放设备中处理单细胞裂解物蛋白浓度时,缓冲液交换也可以减少扩散损失和谱带展宽。我们开发了在电泳过程中焦耳加热增强扩散的数值模拟,并观察到凝胶中约50%的蛋白质损失,使用缓冲液交换可以减少蛋白质损失。通过分析模型预测的分离分辨率(焦耳加热),我们可以凭经验证明几乎完全分辨的蛋白质分离,其分子质量差异仅为4 kDa或12%(GAPDH,36 kDa; PS6,32 kDa)细胞。通过缓冲液交换获得的分离性能与检测导致癌症进展的当前无法测量的蛋白质同工型有关。
更新日期:2017-11-16
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