Department of Mechanical Engineering, McMaster University, Hamilton, ON, Canada.
School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada.
Mikrochim Acta. 2024 Feb 13;191(3):131. doi: 10.1007/s00604-024-06223-5.
Histones are basic proteins with an isoelectric point around 11. It has been shown that the level of plasma circulating histones increases significantly during sepsis, and circulating free histones are associated with sepsis severity and mortality. It was found that the median plasma total free histone concentration of sepsis ICU non-survivors is higher compared to survivors. Therefore, histone concentration can serve as a prognostic indicator and there is a need for a simple, low-cost, and rapid method for measuring histone levels. In this work, we have developed a microfluidic device containing an isoelectric membrane made of dehydrated agarose gel of a specific pH embedded in a porous membrane for isoelectric trapping of histones rapidly. Although isoelectric gates have been used for trapping proteins before, they have to be introduced at the time of the experiment. Here, we show that isoelectric gates formed by gels loaded in a scaffold can be integrated directly into the fabrication process flow, dehydrated for storage, and rehydrated during the experiment and still function effectively to achieve isoelectric trapping. A low-cost and rapid microfabrication technique, xurography, was used for agarose integration and device fabrication. The integrated device was tested with samples containing buffered histone, histone in the presence of high-concentration bovine serum albumin (BSA), and histone spiked in blood plasma. The results show that the device can be used to distinguish between survivors and non-survivors of sepsis in less than 10 min, making it suitable as a point-of-care device for sepsis prognosis.
组蛋白是等电点约为 11 的碱性蛋白。已有研究表明，脓毒症患者血浆中循环组蛋白水平显著升高，且游离组蛋白与脓毒症的严重程度和死亡率相关。研究发现，脓毒症 ICU 死亡患者的血浆总游离组蛋白浓度中位数高于存活患者。因此，组蛋白浓度可作为预后标志物，需要开发一种简单、低成本且快速的组蛋白水平测量方法。在这项工作中，我们开发了一种包含等电膜的微流控装置，该等电膜由特定 pH 值的脱水琼脂糖凝胶制成，嵌入多孔膜中，可快速进行组蛋白等电捕获。尽管之前已经使用等电门来捕获蛋白质，但它们必须在实验时引入。在这里，我们证明了通过支架加载的凝胶形成的等电门可以直接集成到制造工艺流中，脱水储存，并在实验过程中重新水合，仍然可以有效地实现等电捕获。我们使用低成本且快速的微制造技术 xurography 进行琼脂糖集成和器件制造。该集成器件经过缓冲组蛋白样品、高浓度牛血清白蛋白（BSA）存在下的组蛋白样品以及组蛋白掺入血浆样品的测试。结果表明，该设备可在 10 分钟内区分脓毒症的存活患者和死亡患者，适合用作脓毒症预后的即时护理设备。
