纳米技术  >> Vol. 11 No. 1 (February 2021)

功能材料表面修饰用于循环肿瘤细胞的捕获
Functional Materials Surface Modified for the Capture of Circulating Tumor Cells

DOI: 10.12677/NAT.2021.111001, PDF, HTML, XML, 下载: 23  浏览: 85  科研立项经费支持

作者: 张正涛*, 陈朝会, 何荣祥:江汉大学交叉学科研究院,湖北 武汉;阮梅林:华中科技大学化学与化工学院,湖北 武汉

关键词: 微流控芯片循环肿瘤细胞抗体修饰静电纺丝捕获 Micro-Fluidic Chip Circulating Tumor Cells Antibody Modification Electr-Ostatic Spinning Capture

摘要: 本研究以循环肿瘤细胞(MCF-7)为研究对象,利用软光刻技术制备了微米尺寸的微柱,利用静电纺丝技术在含有微柱结构的基底上面纺上PLGA纳米纤维,形成一种微米和纳米复合尺寸的生物界面,在纳米纤维上修饰特异性抗体,实现肿瘤细胞(MCF-7)的捕获。这种研究方法结合了微流控技术和静电纺丝技术,制备出一种三维微柱网络支架结构,增大了细胞的捕获空间,再通过对微结构上的纳米纤维进行改性,提高了循环肿瘤细胞检测的灵敏度,这种研究方法大大提高了循环肿瘤细胞检测的效率,为临床癌症检测提供了新的研究方向。
Abstract: In this study, circulating tumor cells (McF-7) were used as the research object. Microcolumns of micron size were prepared by soft photolithography. Using electrostatic spinning technology, PLGA nanofibers were spun on the microcolumnar substrates, then a micron-nanocomposite biological interface was prepared. Specific antibodies were modified on nanofibers, circulating tumor cells can be captured at this time. This research method combines microfluidic technology and electrostatic spinning technology. The three-dimensional microcolumn network scaffold structure is prepared to increase the capture space of cells, and the sensitivity of detection of circulating tumor cells was improved by modifying the nanofibers on the microstructure. This method greatly improves the detection efficiency of circulating tumor cells, and also provides a new research direction for clinical cancer detection.

文章引用: 张正涛, 阮梅林, 陈朝会, 何荣祥. 功能材料表面修饰用于循环肿瘤细胞的捕获[J]. 纳米技术, 2021, 11(1): 1-7. https://doi.org/10.12677/NAT.2021.111001

1. 引言

循环肿瘤细胞(CTC, Circulating Tumor Cell)指的是存在于人体外周血中的各个种类的癌细胞的总称 [1],循环肿瘤细胞是从病人的原发病灶脱离,游离于外周血中的细胞,这些细胞大多数会被吞噬或者凋亡,少数可能逃逸,对人体健康构成新的重大威胁 [2]。因此,在癌症的早期检测中 [3],对患者血液中CTCs的分离与富集对于早期诊断、实时监测治疗效果以及预后评估等具有重要意义 [4] [5]。

目前常规的诊断技术主要是肿瘤活检技术 [6] [7],该方法首先从患者体内移除组织,然后通过高分辨成像技术对组织进行检测,这种技术不能满足持续监测的要求。微流控技术在微流芯片内模拟细胞在人体中的生理环境 [8] [9],可以获取大量关于人体细胞的生物学信息 [10] [11]。分析时间更快,而且对循环肿瘤细胞的侵袭性更小 [12]。仅需要5~10 mL外周血样本就可以进行检测分析。操作难度更低,方便携带。因此,这一技术在材料合成 [13]、癌症检测 [14]、基因诊断 [15]、细胞分选 [16]、器官芯片 [17]、微液滴应用等各个领域体现出了更大优势。目前,微流控技术在CTCs的富集与分选等方面已经有非常广泛的应用 [18]。

Sequist L. V.等 [19] 人利用惯性聚焦增强微流体对CTC捕获,能够以107 cell/s从全血中分选稀有CTC。Sheng W等 [20] 人开发了一种几何增强混合(GEM)芯片,用于高性能CTC捕获。通过对人字形微融合器的实验优化,实现了刺状肿瘤细胞的捕获,能够达到90%的捕获效率和高于84%的纯度。

本文将微流控芯片技术与静电纺丝技术相结合,建立了一种三维微柱纳米纤维网络支架结构,通过该结构对外周血中的循环肿瘤细胞进行捕获,得到了很好的效果。

2. 实验方法

2.1. 微流芯片制备过程

选用AZ正光刻胶(AZ9260),在单晶硅片(浙江立晶硅材料DP-8)上均匀旋涂,利用紫外光刻技术在掩膜版下曝光,然后用显影液(AZ300 MIF)去除被曝光的部分光刻胶,得到微米柱阵列的微结构。配制PDMS(A胶:B胶 = 10:1),然后浇筑于硅片上,85℃后烘,使得PDMS固化成型,此时剥离PDMS,即得到线性排列的PDMS微柱结构的芯片,图1为PDMS微芯片的制备流程,具体方法参考文献 [21]。

Figure 1. Preparation process of PDMS microchip

图1. PDMS微芯片的制备流程

2.2. 微芯片结构表征

利用磁控溅射镀膜机(常熟虞华CS-3000)对PDMS微柱芯片表面进行喷金处理,然后在SEM (HITACHI S3400N)下进行拍摄。如图2所示,制备的微芯片表面有线性排布的微柱结构。在SEM下测量得到微柱的直径为:50 µm,高度为500 µm,间距为250 µm。

Figure 2. (a) SEM image of 100 times microcolumn structure; (b) SEM image of 300 times microcolumn structure

图2.(a) SEM放大倍数100倍微柱结构SEM图;(b) SEM放大倍数300倍微柱结构SEM图

2.3. 静电纺丝

利用静电纺丝机(深圳通力微纳TL-03)在制备的PDMS微芯片微柱表面均匀的纺上PLGA纳米纤维,过程如下:配置15% (w/v)PLGA溶液,取0.9 g PLGA,溶于4.5 mL DMF和1.5 mLTHF混合溶液,均匀搅拌20 min,混合均匀后,将混匀的溶液连接PE管,放置在静电纺丝机中,将静电纺丝机加热器升温至38℃,设置好流速,扫描距离、速度等各项参数后开始纺丝,最后得到纳米纤维,将得到的具有PLGA纳米纤维的PDMS微芯片在扫描电子显微镜(SEM)下成像观察。下图3为不同放大倍数下纺丝后微柱结构的SEM图。可以看到纳米纤维均匀的纺在微柱结构表面并呈线性排列,纳米纤维直径约为350 nm。

Figure 3. (a) SEM image of microcolumn spinning magnified by 200 times; (b) SEM image of microcolumn spinning magnified by 500 times

图3. (a) SEM放大倍数200倍微柱纺丝SEM图;(b) SEM放大倍数500倍微柱纺丝SEM图

Figure 4. (a) Unmodified anti-EpCAM microchip; (b) Modified anti-EpCAM microchip; (c) Unprocessed microchip

图4. (a) 未修饰anti-EpCAM的微芯片;(b) 修饰anti-EpCAM的微芯片;(c) 未经过任何处理的微芯片

2.4. anti-EpCAM的修饰验证

为验证PLGA纳米纤维经过化学修饰连接上了anti-EpCAM,在相同纺丝密度的基底上,修饰抗体后再加入二抗IFKine Green AffiniPure Donkey Anti-Goat IgG,然后在荧光下观察基底。如图4所示。(a)为未修饰anti-EpCAM的微芯片;(b)为修饰anti-EpCAM的微芯片;(c)为未经过任何处理的微芯片。可以观察到,在经过anti-EpCAM修饰的微芯片上出现了明显的绿色荧光,没有修饰anti-EpCAM的微芯片与无任何修饰的微芯片均无绿色荧光。可以证明PLGA纳米纤维经过改性修饰上了anti-EpCAM抗体。

3. 结果和讨论

在微流芯片的微柱结构上面纺丝并进行抗体修饰后,在芯片中通入MCF-7细胞悬液,观察细胞捕获的结果,如下图5,a、b分别为明场和荧光下拍摄的细胞捕获结果图。通过实验结果我们可以观察到,通过微柱结构尺寸以及抗原–抗体之间的特异性免疫反应,可以很好的捕获癌细胞。

Figure 5. (a) Cell capture results in open field; (b) Cell capture results in the fluorescence

图5. (a) 明场下细胞捕获结果图;(b) 荧光下细胞捕获结果图

4. 结论

本文将微流控技术、静电纺丝技术相结合,建立了一种三维微柱纳米纤维网络支架结构并总结了该结构对循环肿瘤细胞的捕获效果,得到如下结论:

不同的基底捕获循环肿瘤细胞的结果表明:既含微柱又含纳米纺丝的PDMS基底比仅含微柱或者纺丝基底的细胞捕获效果好,说明我们构建的三维网络微柱纺丝结构具有较好的细胞。

捕获能力。单独的微柱结构对细胞有较强的非特异性吸附,加入BSA可以降低微柱的非特异性吸附。在构建的三维网络微柱支架结构上,由于微柱及纳米纤维的存在,会增加细胞吸附效果,并且在网络状的纳米纤维上经过抗体修饰,也增加了细胞检测的灵敏度。

综上所述,我们利用微流控芯片技术、静电纺丝技术相结合的方法构建的三维微柱网络支架能够对细胞进行特异性捕获,由于微柱支撑纳米纤维能够增加捕获空间,纳米纤维经过改性修饰上抗体,也提高了循环肿瘤细胞检测的灵敏度,这种结构为后续实验提供了一定的实验基础,也希望在肿瘤细胞检测方面发挥潜在的临床应用价值。

基金项目

感谢武汉市科技局应用基础前沿项目(2019020701011440)的支持。

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