基于微流控芯片的膀胱癌循环肿瘤细胞的特异性捕获

发布时间：2018-12-18 01:01

【摘要】：[目的]借助全新膀胱癌特异性单克隆抗体BCMabl、生物素-亲和素系统及微流控芯片技术,设计并制作膀胱癌循环肿瘤细胞特异性筛选芯片,实现对膀胱癌循环肿瘤细胞的特异性捕获,并探讨影响芯片捕获靶细胞的因素。[方法]1.筛选芯片的设计:借助计算机辅助设计软件设计筛选芯片。整个筛选芯片由上下两部分组成,且每部分均有微结构,上部分为鱼骨结构,用于改变流体性状,下部分为微通道结构,用于捕获靶细胞。2.筛选芯片的制作:制作硅片模板、用聚二甲基硅氧烷(PDMS)制作基片、修剪、打孔、清洗处理,等离子处理键合封接上下两部分,制成筛选芯片。3.筛选芯片的表面修饰:首先,将链霉亲和素包被到微通道内;其次,对膀胱癌单克隆抗体BCMabl进行生物素化修饰;最后,将生物素化的抗体灌入微通道内,链霉亲和素与生物素化抗体结合,最终形成链霉亲和素-生物素-抗体系统。4.样本液的制备:取膀胱癌细胞T24作为靶细胞,实验前用荧光染料5(6)-羧基二乙酸荧光素琥珀酰亚胺酯(CFDA-SE)按说明对靶细胞进行荧光标记。取得实验参与者知情同意,取健康人外周血,用生理盐水稀释10倍。将标记的膀胱癌细胞T24加入到稀释10倍的人外周血液中,并根据实验需要制备不同细胞浓度的样本液。5.芯片的设计分组:在实验中,为了研究抗体对靶细胞是否具有捕获作用,根据芯片表面抗体包被情况,将芯片制作成两种:有鱼骨结构及BCMabl抗体的芯片;有鱼骨结构无BCMabl抗体的芯片。6.芯片捕获靶细胞:将60 μ 1、靶细胞浓度为5 000个/ml的样本液以流速为10μl/mmin分别泵入到有鱼骨结构及BCMabl抗体的芯片和有鱼骨结构无BCMabl抗体的两组芯片中,冲洗后,于显微镜下观察,计数,统计捕获率。7.探讨影响芯片捕获靶细胞的因素:①为了探讨流速对芯片捕获率的影响,将60 μl、浓度为5 000个/ml的样本液分别以流速为10、15、20、25和30μl/min泵入到含有抗体及鱼骨结构的芯片中;②为了探讨细胞浓度对芯片捕获率的影响,将细胞浓度为500、5 000和50 000个/ml的样本液,以10μl/min的速度泵入到含有抗体及鱼骨结构的芯片通道内进行筛选;③为研究鱼骨结构对芯片捕获靶细胞的影响,我们设计了两种不同的芯片,即含有鱼骨结构和未含有鱼骨结构的芯片,二者均包被BCMabl抗体,将60 μ l细胞浓度为5 000个/ml的样本液,借助注射泵,以流速为10 μ l/min分别泵入到有鱼骨结构及BCMabl抗体的芯片和无鱼骨结构有BCMabl抗体的芯片,冲洗后,于显微镜下观察,计数。[结果]1.制作成特异性筛选膀胱癌循环肿瘤细胞的微流控芯片,大小为40mm×5mm×3mm。芯片上下两层,上层为鱼骨样结构,宽50 μm,高45 μm,相邻两个鱼骨结构之间的间距为100～300 μ m;下层为微通道,微通道高为50 μ m。2.高倍显微镜下观察到,含有抗体及鱼骨结构的芯片对膀胱癌细胞T4具有明显的捕获作用。包被有BCMabl抗体的芯片对靶细胞捕获平均(20.8 ±6.3)个,未含抗体的芯片对靶细胞捕获为平均(1.2± 1.1)个,二者差异有统计学意义(P＜0.05)。3.在不同细胞浓度下,芯片对靶细胞的捕获率维持在89%左右。随着流速的增加芯片对靶细胞的捕获率下降;当流速在10 ul/min时,无论是含有鱼骨结构的筛选芯片还是不含鱼骨结构的筛选芯片,对靶细胞的捕获率最大。在同一流速下,含有抗体及鱼骨结构的芯片捕获率明显高于含有抗体而未含有鱼骨结构的芯片捕获率为(P0.05)。[结论]设计的捕获膀胱癌循环肿瘤细胞微流控芯片成本低,操作简单,具有高特异性捕获膀胱癌循环肿瘤细胞。
[Abstract]:[Objective] To design and manufacture a specific screening chip for bladder cancer circulating tumor cells by means of the novel human bladder cancer specific monoclonal antibody BABl, the biotin-affinity system and the micro-flow control chip technology, and to realize the specific capture of the bladder cancer circulating tumor cells, and to explore the factors that affect the capture of target cells by the wafer.[Method] 1. The design of the filter chip is to filter the chip with the aid of the computer-aided design software. the whole screening chip consists of an upper part and a lower part, and each part has a microstructure, the upper part is a fishbone structure, is used for changing the fluid property, and the lower part is a micro-channel structure and is used for capturing a target cell. The preparation method of the screening chip comprises the following steps of: manufacturing a silicon wafer template, preparing a substrate by using a polydimethylsiloxane (PDMS), trimming, punching, cleaning and processing, and sealing the plasma processing key to form a screening chip. The method for screening the surface of the chip comprises the following steps of: firstly, coating the streptavidin in the micro-channel; secondly, carrying out the biotinylation modification on the bladder cancer monoclonal antibody 1abl; and finally, injecting the biotinylated antibody into the micro-channel, and the streptavidin is combined with the biotinylated antibody, Finally, a streptavidin-biotin-antibody system is formed. The preparation of the sample solution: the bladder cancer cell T24 was taken as the target cell, and the fluorescent dye 5 (6)-Kentucky diacetate (CFDA-SE) was used to mark the target cell according to the instructions before the experiment. To obtain the informed consent of the experimental participants, the peripheral blood of the healthy person was taken, and the peripheral blood of the healthy person was diluted 10 times with normal saline. The labeled bladder cancer cell T24 was added to the diluted 10-fold human peripheral blood and a sample solution of different cell concentrations was prepared according to the experiment. The design group of the chip: in the experiment, in order to study whether the antibody has a capture effect on the target cell, according to the antibody coating condition of the surface of the chip, the chip is made into two chips with the fishbone structure and the ababl antibody; and the chip with the fishbone structure and the ababl antibody is provided. the chip captures the target cell: the sample liquid with the concentration of 60 & mu; l and the target cell concentration of 5,000/ ml is pumped into two groups of chips with the fishbone structure and the ababl antibody respectively at a flow rate of 10 & mu; l/ mmin, Statistical capture rate. 7. In order to study the effect of the flow rate on the capture rate of the chip, 60. m u.l of sample liquid with a concentration of 5,000/ ml was pumped into the chip containing the antibody and the fishbone structure at a flow rate of 10, 15, 20, 25 and 30 & mu; l/ min, respectively. In order to study the effect of cell concentration on the capture rate of the chip, a sample solution with a cell concentration of 500, 5,000 and 50,000/ ml was pumped into a chip channel containing an antibody and a fishbone structure at a rate of 10. m u.l/ min, and the effect of the fishbone structure on the capture target cells of the chip was studied. we designed two different chips, i. e., a chip containing a fishbone structure and a non-fish-bone structure, both of which were coated with a babl antibody, a sample solution of 60. m u.l of cell concentration of 5,000/ ml, the chip with the fishbone structure and the ababl antibody and the chip with the ababl antibody are respectively pumped into the chip with the fishbone structure and the ababl antibody at a flow rate of 10 & mu; l/ min, and then the chip is observed under the microscope and counted under the microscope.[Results] 1. The micro-flow control chip is prepared to specifically screen the circulating tumor cells of the bladder cancer, and the size of the micro-flow control chip is 40mm-5mm and 3mm. the upper layer and the lower layer of the chip are fishbone-like structures, the width of the upper layer is 50 & mu; m, the height is 45 & mu; m, the spacing between the two adjacent fishbone structures is 100-300 & mu; m, the lower layer is a micro-channel, and the micro-channel is high by 50 & mu; m. Under the high-power microscope, the chip containing the antibody and the fishbone structure has obvious capture effect on the bladder cancer cell T4. The average of the target cells (20.8 to 6.3) and the average (1.2-1.1) of the non-antibody-containing chips on target cells was statistically significant (P <0.05). The capture rate of the target cells was maintained at about 89% at different cell concentrations. As the flow rate increases, the capture rate of the target cells is decreased; when the flow rate is in the range of 10 ul/ min, the capture rate of the target cells is the most, whether the screening chip containing the fishbone structure or the filter chip without the fishbone structure. At the same flow rate, the capture rate of the chip containing the antibody and the fishbone structure was significantly higher than that of the chip containing the antibody without the fishbone structure (P0.05).[Conclusion] The design of the micro-flow control chip for the bladder cancer circulating tumor cell is low in cost, simple in operation, and has high specificity for capturing the circulating tumor cells of the bladder cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

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