本文選題：微流控芯片 + 濃度梯度��； 參考：《華中科技大學(xué)》2013年博士論文

【摘要】：生物體能夠感受周圍環(huán)境中多種化學(xué)分子信號(hào)，并根據(jù)其濃度梯度改變自身的生理活動(dòng)。傳統(tǒng)的研究生物體對(duì)于外界環(huán)境反應(yīng)的方法操作繁瑣、耗時(shí)較長(zhǎng)、難以定量分析、并且不具有通用性。微流控芯片技術(shù)能夠?qū)⒉煌牟僮髡显谝粔K芯片內(nèi)，從而實(shí)現(xiàn)分析設(shè)備的微型化、集成化、自動(dòng)化，將可以解決傳統(tǒng)方法難以解決的生物學(xué)問題。因此，利用微流控芯片模擬外界環(huán)境，建立化學(xué)物質(zhì)濃度梯度，并在細(xì)胞以及個(gè)體水平上研究生物體對(duì)于外界環(huán)境的反應(yīng)無疑具有重要的意義。 本文建立了一種新型濃度梯度微流控芯片。不同于已報(bào)道的常見的“圣誕樹”的濃度梯度形成結(jié)構(gòu)，該芯片利用階梯狀微通道網(wǎng)絡(luò)，采用了逐步分流再混流的方式逐步稀釋樣品溶液，從而產(chǎn)生液體濃度梯度。 我們首先對(duì)該微流控芯片進(jìn)行理論分析與優(yōu)化。采用數(shù)學(xué)建模的方法對(duì)這種通過液體串行稀釋而產(chǎn)生化學(xué)物質(zhì)濃度梯度的微流控新方法進(jìn)行了原理闡述。通過流體力學(xué)計(jì)算方法，對(duì)微流控芯片內(nèi)微通道結(jié)構(gòu)進(jìn)行了數(shù)學(xué)建模，并模擬分析不同的結(jié)構(gòu)對(duì)于濃度梯度的影響。結(jié)果表明采用微通道寬度為100μm、下層主通道液體分流的分叉口處于右位、入口流量為0.5μL/min時(shí)的階梯狀網(wǎng)絡(luò)結(jié)構(gòu)最有利于形成液體濃度梯度。 為了驗(yàn)證數(shù)學(xué)模擬的結(jié)果，我們?cè)谛酒肟诜謩e通入水和熒光素，并通過熒光顯微鏡進(jìn)行成像分析。結(jié)果表明在芯片內(nèi)混流的區(qū)域，水和熒光素實(shí)現(xiàn)了完全混合，并在出口的六條微通道處形成了線性濃度梯度。在出口主通道中，六路溶液呈現(xiàn)層流特性，形成了明顯的穩(wěn)定的濃度梯度，保持了較長(zhǎng)距離。實(shí)驗(yàn)結(jié)果與流體動(dòng)力學(xué)計(jì)算得到的模擬結(jié)果非常吻合。 之后，我們利用該微流控芯片進(jìn)行了細(xì)胞水平上的應(yīng)用。表達(dá)有可以檢測(cè)細(xì)胞凋亡的CD2探針的HeLa細(xì)胞通過正壓從芯片出口處進(jìn)入主通道內(nèi)，并貼附其通道壁生長(zhǎng)。通過該芯片微通道網(wǎng)絡(luò)在主通道中形成穩(wěn)定的抗癌藥物—順鉑濃度梯度，從而實(shí)現(xiàn)不同濃度的順鉑藥物對(duì)細(xì)胞的處理。利用熒光能量共振轉(zhuǎn)移成像系統(tǒng)對(duì)順鉑誘導(dǎo)細(xì)胞凋亡的進(jìn)行實(shí)時(shí)在體監(jiān)測(cè)。實(shí)驗(yàn)表明：順鉑可以誘導(dǎo)HeLa細(xì)胞凋亡，且順鉑的濃度與細(xì)胞凋亡率呈正相關(guān)，其中蛋白酶Caspase-2介導(dǎo)了順鉑誘導(dǎo)的細(xì)胞凋亡。這些結(jié)果說明該微流控濃度梯度產(chǎn)生芯片可以用于細(xì)胞凋亡的研究。 最后，我們利用該微流控芯片形成了傳統(tǒng)方法難以實(shí)現(xiàn)的含有濃度梯度的線蟲培養(yǎng)環(huán)境，并分析了線蟲對(duì)于NaCl的趨向性以及趨向?qū)W習(xí)行為。通過正壓，，我們將線蟲直接注入芯片內(nèi)出口主通道中，并通過芯片內(nèi)階梯狀微通道網(wǎng)絡(luò)在主通道中形成NaCl濃度梯度。為定量分析芯片內(nèi)線蟲的化學(xué)物質(zhì)趨向性行為，我們通過成像，分析了在出口主通道中不同NaCl濃度區(qū)域內(nèi)的線蟲分布。結(jié)果表明野生型線蟲主要趨向于濃度大于20mM的NaCl溶液，對(duì)于50-100mM之間的NaCl溶液沒有偏好性，對(duì)高于300mM的NaCl溶液則表現(xiàn)出明顯的厭惡性行為。此外，我們?cè)谠撔酒瑑?nèi)直接實(shí)現(xiàn)了NaCl與饑餓共處理線蟲，從而將線蟲的學(xué)習(xí)訓(xùn)練與訓(xùn)練后趨向性分析同在一塊芯片內(nèi)進(jìn)行。結(jié)果表明：類似于平板上的學(xué)習(xí)行為，NaCl與饑餓共處理的線蟲表現(xiàn)出厭惡原來喜歡的低濃度的NaCl的行為。這種芯片內(nèi)學(xué)習(xí)方式可以讓線蟲通過學(xué)習(xí)建立低濃度NaCl與厭惡行為的聯(lián)系。因此，該芯片可以為研究線蟲的化學(xué)物質(zhì)趨向以及學(xué)習(xí)行為提供良好的研究平臺(tái)，并能夠提高分析效率，降低分析時(shí)間，可以分析記憶時(shí)間很短的線蟲可塑性學(xué)習(xí)行為。 上述實(shí)驗(yàn)結(jié)果表明該微流控新技術(shù)平臺(tái)具有設(shè)計(jì)新穎、結(jié)構(gòu)簡(jiǎn)單以及實(shí)現(xiàn)效果明顯的特點(diǎn)，能夠?qū)崿F(xiàn)可控的化學(xué)物質(zhì)濃度梯度，并能夠應(yīng)用于不同的生物體系中。除了形成濃度梯度，該方法還可以形成溫度梯度，為將來研究細(xì)胞和多細(xì)胞生物體在不同溫度條件下的反應(yīng)提供一種新的途徑。該芯片在藥物篩選、生物檢測(cè)以及環(huán)境檢測(cè)等方面也具有潛在的應(yīng)用價(jià)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Adverse effects of metal exposure on chemotaxis towards water-soluble attractants regulated mainly by ASE sensory neuron in nematode Caenorhabditis elegans[J];Journal of Environmental Sciences;2009年12期

本文編號(hào)：1826951