基于微流控芯片的浓度梯度实现新方法及其应用研究

发布时间：2018-05-01 00:32

本文选题：微流控芯片 + 浓度梯度　；参考：《华中科技大学》2013年博士论文

【摘要】：生物体能够感受周围环境中多种化学分子信号，并根据其浓度梯度改变自身的生理活动。传统的研究生物体对于外界环境反应的方法操作繁琐、耗时较长、难以定量分析、并且不具有通用性。微流控芯片技术能够将不同的操作整合在一块芯片内，从而实现分析设备的微型化、集成化、自动化，将可以解决传统方法难以解决的生物学问题。因此，利用微流控芯片模拟外界环境，建立化学物质浓度梯度，并在细胞以及个体水平上研究生物体对于外界环境的反应无疑具有重要的意义。本文建立了一种新型浓度梯度微流控芯片。不同于已报道的常见的“圣诞树”的浓度梯度形成结构，该芯片利用阶梯状微通道网络，采用了逐步分流再混流的方式逐步稀释样品溶液，从而产生液体浓度梯度。我们首先对该微流控芯片进行理论分析与优化。采用数学建模的方法对这种通过液体串行稀释而产生化学物质浓度梯度的微流控新方法进行了原理阐述。通过流体力学计算方法，对微流控芯片内微通道结构进行了数学建模，并模拟分析不同的结构对于浓度梯度的影响。结果表明采用微通道宽度为100μm、下层主通道液体分流的分叉口处于右位、入口流量为0.5μL/min时的阶梯状网络结构最有利于形成液体浓度梯度。为了验证数学模拟的结果，我们在芯片入口分别通入水和荧光素，并通过荧光显微镜进行成像分析。结果表明在芯片内混流的区域，水和荧光素实现了完全混合，并在出口的六条微通道处形成了线性浓度梯度。在出口主通道中，六路溶液呈现层流特性，形成了明显的稳定的浓度梯度，保持了较长距离。实验结果与流体动力学计算得到的模拟结果非常吻合。之后，我们利用该微流控芯片进行了细胞水平上的应用。表达有可以检测细胞凋亡的CD2探针的HeLa细胞通过正压从芯片出口处进入主通道内，并贴附其通道壁生长。通过该芯片微通道网络在主通道中形成稳定的抗癌药物—顺铂浓度梯度，从而实现不同浓度的顺铂药物对细胞的处理。利用荧光能量共振转移成像系统对顺铂诱导细胞凋亡的进行实时在体监测。实验表明：顺铂可以诱导HeLa细胞凋亡，且顺铂的浓度与细胞凋亡率呈正相关，其中蛋白酶Caspase-2介导了顺铂诱导的细胞凋亡。这些结果说明该微流控浓度梯度产生芯片可以用于细胞凋亡的研究。最后，我们利用该微流控芯片形成了传统方法难以实现的含有浓度梯度的线虫培养环境，并分析了线虫对于NaCl的趋向性以及趋向学习行为。通过正压，，我们将线虫直接注入芯片内出口主通道中，并通过芯片内阶梯状微通道网络在主通道中形成NaCl浓度梯度。为定量分析芯片内线虫的化学物质趋向性行为，我们通过成像，分析了在出口主通道中不同NaCl浓度区域内的线虫分布。结果表明野生型线虫主要趋向于浓度大于20mM的NaCl溶液，对于50-100mM之间的NaCl溶液没有偏好性，对高于300mM的NaCl溶液则表现出明显的厌恶性行为。此外，我们在该芯片内直接实现了NaCl与饥饿共处理线虫，从而将线虫的学习训练与训练后趋向性分析同在一块芯片内进行。结果表明：类似于平板上的学习行为，NaCl与饥饿共处理的线虫表现出厌恶原来喜欢的低浓度的NaCl的行为。这种芯片内学习方式可以让线虫通过学习建立低浓度NaCl与厌恶行为的联系。因此，该芯片可以为研究线虫的化学物质趋向以及学习行为提供良好的研究平台，并能够提高分析效率，降低分析时间，可以分析记忆时间很短的线虫可塑性学习行为。上述实验结果表明该微流控新技术平台具有设计新颖、结构简单以及实现效果明显的特点，能够实现可控的化学物质浓度梯度，并能够应用于不同的生物体系中。除了形成浓度梯度，该方法还可以形成温度梯度，为将来研究细胞和多细胞生物体在不同温度条件下的反应提供一种新的途径。该芯片在药物筛选、生物检测以及环境检测等方面也具有潜在的应用价值。
[Abstract]:Organisms can feel a variety of chemical molecules in the surrounding environment and change their physiological activities according to their concentration gradient. The traditional methods of studying the reaction of the environment to the external environment are tedious, time-consuming, difficult to quantify, and are not universal. Microflow chip technology can integrate different operations in one. In the chip, the microminiaturization, integration and automation of the analytical equipment can be used to solve the biological problems that are difficult to be solved by traditional methods. Therefore, it is important to use microfluidic chip to simulate the external environment and to establish the concentration gradient of chemical substances and to study the response of the organism to the external environment at the cell and individual level. Meaning.
In this paper, a novel concentration gradient microfluidic chip is developed, which is different from the reported density gradient formation of the common "Christmas tree". The chip uses a ladder like microchannel network to gradually dilute the sample solution by gradual diverting and re mixing, thus producing the concentration gradient of the liquid.
Firstly, the theoretical analysis and optimization of the microfluidic chip are carried out. The mathematical modeling method is used to explain the new method of microfluidic control, which produces the chemical substance concentration gradient through the dilution of the liquid, and the microchannel structure in the microfluidic chip is modeled by the fluid mechanics calculation method. The effects of different structures on the concentration gradient are analyzed. The results show that the microchannel width is 100 m, the bifurcation of the main channel in the lower main channel is right in the right position, and the ladder like network structure with the inlet flow rate of 0.5 Mu is the most beneficial to the formation of the liquid concentration gradient.
In order to verify the results of the mathematical simulation, we put water and fluorescein at the entrance of the chip and analyzed the imaging by fluorescence microscopy. The results showed that the water and fluorescein were completely mixed in the region of the mixed flow in the chip, and the linear concentration gradient was formed at the six microchannels at the exit. In the main outlet of the export, the six road solution was in the main outlet. The laminar flow characteristics are shown to form a clear stable concentration gradient and maintain a long distance. The experimental results are in good agreement with the simulation results obtained by the fluid dynamics calculation.
Then, we use the microfluidic chip to carry out the application of the cell level. It expresses that HeLa cells with CD2 probe that can detect cell apoptosis enter the main channel from the chip outlet and attach its channel wall growth. The microchannel network of the chip forms a stable anticancer drug cisplatin concentration gradient in the main channel through the microchannel network. The experiments show that cisplatin can induce apoptosis in HeLa cells, and the concentration of cisplatin and the rate of apoptosis of cisplatin and the rate of apoptosis are Cheng Zhengxiang, in which protease Caspase-2 mediates cisplatin induced by cisplatin. These results indicate that the microfluidic gradient generation chip can be used for apoptosis research.
Finally, we use the microfluidic chip to form a traditional method which is difficult to achieve with the concentration gradient of the nematode culture environment, and analyzes the tendency of the nematode to NaCl and the tendency to learn. Through positive pressure, we directly inject the nematode into the main channel of the chip and make the main passageway through the microchannel network in the chip. NaCl concentration gradient was formed in the channel. In order to quantitatively analyze the chemical substance tendency of nematodes, we analyzed the distribution of nematodes in different NaCl concentration regions in the main outlet of the outlet. The results showed that the wild type nematodes were mainly concentrated in the NaCl solution with a concentration greater than 20mM, and there was no preference for NaCl solution between 50-100mM. In addition, we directly realized the NaCl and starvation co processing nematodes in this chip, and then carried out the learning training of the nematode and the trend analysis after training in one chip. The results showed that the learning behavior on the plate was similar to the learning behavior on the flat plate, and the NaCl and the starvation were co treated with the 300mM. The nematodes show the behavior of aversion to the low concentration of NaCl that they originally liked. This chip learning way allows nematodes to establish a link between low concentration of NaCl and aversion by learning. Therefore, the chip can provide a good research platform for studying the chemical tendency and learning behavior of the nematodes, and can improve the efficiency of analysis and decrease the efficiency of analysis. Analyzing time can analyze the learning behavior of nematode with short memory time.
The experimental results show that the new technology has the characteristics of novel design, simple structure and obvious effect. It can be used in different biological systems, and can be applied to different biological systems. In addition to forming a concentration gradient, the method can also form a temperature gradient for the future study of cells and how fine. Cellular organisms provide a new way of reaction under different temperature conditions. This chip is also of potential application value in drug screening, biological detection and environmental detection.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318

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