Khosravi Farhad, Trainor Patrick, Rai Shesh N, Kloecker Goetz, Wickstrom Eric, Panchapakesan Balaji
Department of Mechanical Engineering, Small Systems Laboratory, Worcester Polytechnic Institute, Worcester, MA 01532, USA.
Nanotechnology. 2016 Apr 1;27(13):13LT02. doi: 10.1088/0957-4484/27/13/13LT02. Epub 2016 Feb 22.
We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.
我们展示了使用纳米管-抗体微阵列对掺入血沉棕黄层中的乳腺癌细胞进行快速且无标记的捕获。单壁碳纳米管阵列是使用光刻、金属沉积和蚀刻技术制造的。使用1-芘丁酸琥珀酰亚胺酯功能化方法将抗上皮细胞粘附分子（EpCAM）抗体功能化到纳米管装置表面。功能化后，将普通血沉棕黄层和掺入MCF7细胞的血沉棕黄层吸附到纳米管装置上，并记录电信号以检测样品间相互作用的差异。开发了一种用于“液体活检”的统计分类器，以基于动态时间规整创建预测模型，对与普通（对照）或掺入血沉棕黄层（病例）相对应的装置电信号进行分类。在训练测试中，来自血沉棕黄层样品与掺入血沉棕黄层样品的装置电信号分类的灵敏度约为100%，特异性约为91%，准确率约为96%。在盲测中，信号分类的灵敏度约为91%，特异性约为82%，准确率约为86%。生成了热图以直观地捕捉电信号与样品状态之间的关系。基于电信号被分类为掺入血沉棕黄层的装置的共聚焦显微镜分析证实了癌细胞的存在、它们在装置上的附着以及EpCAM受体的过表达。计数每5微升每个装置中的细胞数约为1-17个细胞，表明掺入血沉棕黄层中的单细胞灵敏度可通过微阵列扩展到更高体积。
