10,000-fold concentration increase in proteins in a cascade microchip using anionic ITP by a 3-D numerical simulation with experimental results.
10,000-fold concentration increase in proteins in a cascade microchip using anionic ITP by a 3-D numerical simulation with experimental results.
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DOI：
10.1002/elps.201000510
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发表时间：
2011-02
期刊：
影响因子：
2.9
通讯作者：
Ivory CF
中科院分区：
文献类型：
--
作者：
Bottenus D;Jubery TZ;Dutta P;Ivory CF
本文描述了利用阳离子等速电泳法(ITP)在3.9 cm长的聚甲基丙烯酸甲酯(PMMA)微流控芯片中预富集生物标志物心肌肌钙蛋白I(CTnI)和荧光蛋白(R-藻红蛋白)。该微流控芯片包括深度减少5倍、宽度减少10倍的通道。因此，从入口(阳极)到出口(阴极)的总横截面积减小了50倍。浓度与横截面积成反比，因此当蛋白质通过还原迁移时，浓度成比例增加。此外，通过ITP，蛋白质获得了额外的浓度。我们观察到，在还原微流控芯片的横截面上进行ITP，我们可以获得大于10000的浓缩因子。微流控芯片中cTnI的起始浓度为2.3μg m L-1，最终浓度为2 5.5 2±1.2 5 mg m L-1。据笔者了解，这是微流控芯片首次尝试浓缩心脏生物标志物cTn I。这种实验方法可以与基于免疫分析的技术相结合，并有可能降低检测下限，提高灵敏度，并定量检测不同的孤立cTnI磷酸化状态。
This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10 000. The starting concentration of cTnI was 2.3 μg mL–1 and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL–1. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.
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