用于多巴胺电化学检测的微流控芯片研制

发布时间：2018-05-06 00:40

本文选题：微流控芯片 + 聚二甲基硅氧烷　；参考：《大连理工大学》2015年硕士论文

【摘要】：多巴胺能神经元的缺失是导致神经退行性疾病,例如帕金森病,的一种主要原因。神经干细胞经过诱导可以分化成多巴胺能神经元,因此神经干细胞移植被认为是治疗帕金森病的一种理想方法。为了更接近体内的真实环境,将微流控芯片与胶原水凝胶技术相结合,可以构建神经干细胞的体外三维培养微环境。电化学检测具有检测灵敏度高、可微型化和集成化等特点,已成为微流控芯片检测的一种主要方法。本文设计并制作了一种新的微流控芯片,在芯片上集成了用作细胞培养室的主通道、用于细胞培养基输送的两条侧通道和用于多巴胺电化学检测的3组三电极体系。该芯片有望实现神经干细胞的三维培养以及由多巴胺能神经元分泌出的多巴胺实时检测,用于研究神经干细胞向多巴胺能神经元的诱导分化情况。芯片由一片聚二甲基硅氧烷(PDMS)沟道片和一片玻璃电极片组成。沟道片采用浇注成型的方法制作完成。由于在沟道片的中间区域密集分布了两排高深宽比(3：1)的微柱阵列,这些微柱在脱模过程中极易产生断裂,为此本文研究了一种新的脱模方法。首先,将PDMS预聚物和固化剂的混合比例增大到了15：1,用于降低PDMS的弹性模量,使其在脱模时不易发生断裂。其次,整个脱模过程在无水乙醇中进行,乙醇会起到很好的润滑作用,有助于脱模。最后,利用二甲基二氯硅烷对硅模具表面进行处理,降低模具的表面能,减小PDMS与模具之间的粘附作用。为了验证二甲基二氯硅烷对硅模具表面进行处理的稳定性,对处理后的硅模具表面接触角进行了跟踪测量。表面处理后第七天的模具表面接触角与第一天相比变化量只有2.8。,而且浇注出的微柱阵列没有发生断裂,这说明了二甲基二氯硅烷的表面处理稳定性很好,可以满足模具的长期使用要求。电化学检测常用Ag/AgCl电极作为参比电极,但是Ag/AgCl在生物环境中容易出现降解现象,为此本文建立了一种Au-Au-Au三电极体系。对芯片的检测性能进行了研究。首先,以硫酸作为测试样品进行了检测,得到的伏安曲线与之前报道的Au-Ag-Au和Au-Ag-Pt三电极体系完全吻合,表明建立的三电极体系可以很好的用于电化学检测；此外,建立的三电极体系还表现出良好的片内稳定性和片问重复性。然后,对多巴胺进行了电化学检测。为了更贴近将来芯片的应用环境,本文用到的多巴胺均溶解在神经干细胞培养基中。获得的多巴胺检出限为3.92“M,线性检测范围为10μM～500μM。为了提高芯片的检测灵敏度,采用聚吡咯/聚对苯乙烯磺酸钠对Au工作电极表面进行了修饰。利用电极修饰后的芯片对多巴胺进行了检测,获得的多巴胺检出限为0.69¨M,比修饰前的芯片检出限降低了近1个数量级,显著提高了芯片的检测灵敏度。
[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.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TN492

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