Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells
Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells
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DOI：
10.1016/j.chroma.2007.05.064
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发表时间：
2007-08-31
影响因子：
4.1
通讯作者：
Tai, Yu-Chong
中科院分区：
文献类型：
--
作者：
Zheng, Siyang;Lin, Henry;Tai, Yu-Chong
本文介绍了一种用于单级捕获和电解人类血液中循环肿瘤细胞 (CTC) 的聚对二甲苯膜微过滤器装置的开发，以及该装置进行基因组分析的潜力。最近已证明血液中 CTC 的存在和数量可以为转移性乳腺癌患者提供重要的预后信息。虽然在约 7.5 mL 血液（即 10(10) 个血细胞）中发现少至 5 个 CTC 具有临床意义，但目前检测 CTC 既困难又耗时。 CTC 富集是通过基于 CTC 浮力密度的梯度离心或上皮 CTC 的磁分离来进行的，这两种方法都是费力的过程，效率各异，CTC 鉴定通常由训练有素的病理学家通过目视观察染色的细胞角蛋白阳性上皮 CTC 来完成。这些过程可能需要数小时甚至数天的时间。这里介绍的工作提供了一种基于微机电系统 (MEMS) 的选项，使这个过程更简单、更快、更好、更便宜。我们利用 CTC 和人类血细胞之间的大小差异，在过滤器上实现 CTC 捕获，并在 10 分钟内回收率接近 90%，这优于当前的方法。捕获后，我们通过使用嵌入电极到达 16,000 个过滤孔中的每一个进行膜上电解，促进基于聚合酶链式反应 (PCR) 的基因组分析。这种膜上原位细胞裂解的最大优点是效率高，因为细胞被固定，允许它们与电极直接接触。作为原理验证，我们展示了 β 肌动蛋白基因 PCR，相同的技术可以轻松扩展到 CTC 特异性转录本的实时 PCR，从而实现 CTC 的分子鉴定及其进一步表征。 (c) 2007 Elsevier B.V. 保留所有权利。
This paper presents development of a parylene membrane microfilter device for single stage capture and electrolysis of circulating tumor cells (CTCs) in human blood, and the potential of this device to allow genomic analysis. The presence and number of CTCs in blood has recently been demonstrated to provide significant prognostic information for patients with metastatic breast cancer. While finding as few as five CTCs in about 7.5 mL of blood (i.e., 10(10) blood cells in) is clinically significant, detection of CTCs is currently difficult and time consuming. CTC enrichment is performed by either gradient centrifugation of CTC based on their buoyant density or magnetic separation of epithelial CTC, both of which are laborious procedures with variable efficiency, and CTC identification is typically done by trained pathologists through visual observation of stained cytokeratin-positive epithelial CTC. These processes may take hours, if not days. Work presented here provides a micro-electro-mechanical system (MEMS)-based option to make this process simpler, faster, better and cheaper. We exploited the size difference between CTCs and human blood cells to achieve the CTC capture on filter with similar to 90% recovery within 10 min, which is superior to current approaches. Following capture, we facilitated polymerase chain reaction (PCR)-based genomic analysis by performing on-membrane electrolysis with embedded electrodes reaching each of the individual 16,000 filtering pores. The biggest advantage for this on-membrane in situ cell lysis is the high efficiency since cells are immobilized, allowing their direct contact with electrodes. As a proof-of-principle, we show beta actin gene PCR, the same technology can be easily extended to real time PCR for CTC-specific transcript to allow molecular identification of CTC and their further characterization. (c) 2007 Elsevier B.V. All rights reserved.