本文選題：微流控芯片 + 聚二甲基硅氧烷　； 參考：《大連理工大學(xué)》2015年碩士論文

【摘要】：多巴胺能神經(jīng)元的缺失是導(dǎo)致神經(jīng)退行性疾病,例如帕金森病,的一種主要原因。神經(jīng)干細(xì)胞經(jīng)過(guò)誘導(dǎo)可以分化成多巴胺能神經(jīng)元,因此神經(jīng)干細(xì)胞移植被認(rèn)為是治療帕金森病的一種理想方法。為了更接近體內(nèi)的真實(shí)環(huán)境,將微流控芯片與膠原水凝膠技術(shù)相結(jié)合,可以構(gòu)建神經(jīng)干細(xì)胞的體外三維培養(yǎng)微環(huán)境。電化學(xué)檢測(cè)具有檢測(cè)靈敏度高、可微型化和集成化等特點(diǎn),已成為微流控芯片檢測(cè)的一種主要方法。本文設(shè)計(jì)并制作了一種新的微流控芯片,在芯片上集成了用作細(xì)胞培養(yǎng)室的主通道、用于細(xì)胞培養(yǎng)基輸送的兩條側(cè)通道和用于多巴胺電化學(xué)檢測(cè)的3組三電極體系。該芯片有望實(shí)現(xiàn)神經(jīng)干細(xì)胞的三維培養(yǎng)以及由多巴胺能神經(jīng)元分泌出的多巴胺實(shí)時(shí)檢測(cè),用于研究神經(jīng)干細(xì)胞向多巴胺能神經(jīng)元的誘導(dǎo)分化情況。芯片由一片聚二甲基硅氧烷(PDMS)溝道片和一片玻璃電極片組成。溝道片采用澆注成型的方法制作完成。由于在溝道片的中間區(qū)域密集分布了兩排高深寬比(3：1)的微柱陣列,這些微柱在脫模過(guò)程中極易產(chǎn)生斷裂,為此本文研究了一種新的脫模方法。首先,將PDMS預(yù)聚物和固化劑的混合比例增大到了15：1,用于降低PDMS的彈性模量,使其在脫模時(shí)不易發(fā)生斷裂。其次,整個(gè)脫模過(guò)程在無(wú)水乙醇中進(jìn)行,乙醇會(huì)起到很好的潤(rùn)滑作用,有助于脫模。最后,利用二甲基二氯硅烷對(duì)硅模具表面進(jìn)行處理,降低模具的表面能,減小PDMS與模具之間的粘附作用。為了驗(yàn)證二甲基二氯硅烷對(duì)硅模具表面進(jìn)行處理的穩(wěn)定性,對(duì)處理后的硅模具表面接觸角進(jìn)行了跟蹤測(cè)量。表面處理后第七天的模具表面接觸角與第一天相比變化量只有2.8。,而且澆注出的微柱陣列沒(méi)有發(fā)生斷裂,這說(shuō)明了二甲基二氯硅烷的表面處理穩(wěn)定性很好,可以滿足模具的長(zhǎng)期使用要求。電化學(xué)檢測(cè)常用Ag/AgCl電極作為參比電極,但是Ag/AgCl在生物環(huán)境中容易出現(xiàn)降解現(xiàn)象,為此本文建立了一種Au-Au-Au三電極體系。對(duì)芯片的檢測(cè)性能進(jìn)行了研究。首先,以硫酸作為測(cè)試樣品進(jìn)行了檢測(cè),得到的伏安曲線與之前報(bào)道的Au-Ag-Au和Au-Ag-Pt三電極體系完全吻合,表明建立的三電極體系可以很好的用于電化學(xué)檢測(cè)；此外,建立的三電極體系還表現(xiàn)出良好的片內(nèi)穩(wěn)定性和片問(wèn)重復(fù)性。然后,對(duì)多巴胺進(jìn)行了電化學(xué)檢測(cè)。為了更貼近將來(lái)芯片的應(yīng)用環(huán)境,本文用到的多巴胺均溶解在神經(jīng)干細(xì)胞培養(yǎng)基中。獲得的多巴胺檢出限為3.92“M,線性檢測(cè)范圍為10μM～500μM。為了提高芯片的檢測(cè)靈敏度,采用聚吡咯/聚對(duì)苯乙烯磺酸鈉對(duì)Au工作電極表面進(jìn)行了修飾。利用電極修飾后的芯片對(duì)多巴胺進(jìn)行了檢測(cè),獲得的多巴胺檢出限為0.69¨M,比修飾前的芯片檢出限降低了近1個(gè)數(shù)量級(jí),顯著提高了芯片的檢測(cè)靈敏度。
[Abstract]:The loss of dopaminergic neurons is a major cause of neurodegenerative diseases, such as Parkinson's disease. Neural stem cells can be induced to differentiate into dopaminergic neurons, so neural stem cell transplantation is considered to be an ideal method for the treatment of Parkinson's disease. In order to be closer to the real environment in vivo, the microfluidic chip and collagen hydrogel technology can be combined to construct the three-dimensional culture microenvironment of neural stem cells in vitro. Electrochemical detection, with its high sensitivity, miniaturization and integration, has become a main method for microfluidic chip detection. In this paper, a new microfluidic chip is designed and fabricated. The microfluidic chip is integrated into three groups of three electrode systems: the main channel used as the cell culture chamber, the two side channels for the transport of cell culture medium and the three groups of three electrodes for the electrochemical detection of dopamine. The chip is expected to realize the three-dimensional culture of neural stem cells and the real-time detection of dopamine secreted by dopaminergic neurons, which can be used to study the induction and differentiation of neural stem cells into dopaminergic neurons. The chip consists of a polydimethylsiloxane (PDMS) channel and a glass electrode. The channel sheet is made by pouring molding method. Due to the dense distribution of two arrays of microcolumns with high aspect ratio of 3: 1 in the middle region of the channel, these microcolumns are prone to fracture during the demoulding process. Therefore, a new demoulding method is studied in this paper. Firstly, the mixing ratio of PDMS prepolymer and curing agent is increased to 15: 1, which is used to reduce the elastic modulus of PDMS and make it difficult to break in the demoulding process. Secondly, the whole demoulding process is carried out in anhydrous ethanol, which plays a good lubricating role and is helpful to demoulding. Finally, dimethyldichlorosilane was used to treat the surface of silicon mould, which reduced the surface energy of die and the adhesion between PDMS and mould. In order to verify the stability of dimethyldichlorosilane on the surface of silicon mould, the contact angle of the treated silicon mold surface was measured. The surface contact angle of the mold on the seventh day after surface treatment was only 2.8.The microcolumn array was not broken, which indicated that the surface treatment stability of dimethyl dichlorosilane was very good. Can meet the mold long-term use requirements. Ag/AgCl electrode is commonly used as reference electrode for electrochemical detection, but Ag/AgCl is easy to be degraded in biological environment. Therefore, a three-electrode system of Au-Au-Au is established in this paper. The detection performance of the chip is studied. Firstly, the voltammetric curves obtained by using sulfuric acid as the test sample are in good agreement with the reported three-electrode system of Au-Ag-Au and Au-Ag-Pt, which indicates that the three-electrode system can be used for electrochemical detection. The three-electrode system also showed good stability and reproducibility. Then, the dopamine was detected by electrochemical method. In order to be closer to the future microarray application environment, the dopamine used in this paper is dissolved in neural stem cell medium. The detection limit of dopamine was 3.92 "M. the linear detection range was 10 渭 M ~ 500 渭 M. In order to improve the detection sensitivity, polypyrrole / sodium polystyrene sulfonate was used to modify the surface of au working electrode. The detection limit of dopamine was 0.69? M, which was about 1 order of magnitude lower than that of the modified chip, and the sensitivity of the chip was improved significantly.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：TN492

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