用于数字PCR的微滴生成及检测芯片研究

发布时间：2018-01-12 22:12

本文关键词：用于数字PCR的微滴生成及检测芯片研究　出处：《中国科学院研究生院(长春光学精密机械与物理研究所)》2015年硕士论文　论文类型：学位论文

【摘要】：微流控芯片微滴技术是一种基于微流控芯片的操控微小体积液体的新技术,它以分散的微滴单元作为微反应器,大大强化了微流控芯片的高灵敏度、低消耗、高通量和自动化等优点。微滴技术生成的微滴具有分散性好、体积小、样品间无交叉污染、生成速率及反应条件稳定等优点。基于液滴技术的数字PCR检测技术具有小体积及高通量的特点,微滴技术能够显著地优化传统数字PCR的检测流程,提高检测效率,对生物化学反应的高通量分析意义重大。本文的研究目标是分别设计出微滴生成芯片和检测芯片,实现微滴快速、均匀、稳定的生成,微滴直径约为0.1毫米,生成速率为1500个/秒。在对微滴进行收集后,对微滴内的样品进行PCR扩增,随后采用检测芯片将微滴排成等距序列,微滴间距约为1毫米,等距微滴依次通过流式细胞仪检测系统,便可实现对扩增样品的快速精确检测,微滴检测速率约为200个/秒。论文的主要研究内容和成果如下:1.利用COMSOL仿真软件对微滴生成及检测模块进行仿真,讨论通道结构、管壁性质、负压大小等因素对微滴的直径大小、生成速率及间距所造成的影响,根据仿真结果确定微滴验收指标,设计微滴生成芯片及检测芯片的几何结构,使芯片产生的微滴能够满足数字PCR系统的反应和检测需求。2.对SU-8光刻胶工艺进行研究,制作出理想的结构阳模,选取PDMS材料,采用模塑技术及PDMS键和技术得到具有微结构通道的微流控芯片,芯片键合强度、通道表面平整程度及疏水特性满足数字PCR系统的反应和检测需求。3.本文采用负压系统驱动微滴进行运动,研究了微流体在微通道结构内受到的摩擦阻力与其流程的关系,并应用沿程损失理论得到理想的负压驱动力。最后对在该负压驱动下生成的微滴进行了大小均一性分析。结论:1.利用扫描电子显微镜对制作完成的芯片结构进行观察测量,微通道深度约为100微米,深度一致性良好,内壁表面平整,陡直性良好,深宽、宽度误差可控制在10微米以内,测量结果表明利用MEMS加工工艺制作的芯片效果完全满足芯片的设计要求;2.在高速摄像机(7500帧/秒)的辅助下,对制作出的微滴生成及检测芯片进行微滴效果实验。对微滴进行计数分析,可得到微滴的生成速率及检测速率,采用图形对比方法,可得到微滴的直径及相邻微滴的间距。实验表明,本文设计制作的微滴及检测芯片满足数字PCR系统的使用需求。
[Abstract]:Droplet microfluidic chip technology is a new technology of micro volume liquid control based on microfluidic chip, it dispersed droplet unit as a micro reactor, which greatly enhanced the sensitivity of microfluidic chip, low consumption, high throughput and automation. The advantages of micro droplet technology into droplets having dispersed good, small size, no cross contamination between samples, the advantages of formation rate and stable reaction condition. The characteristics of digital PCR droplet detection technology has the advantages of small size and high throughput detection process based on micro droplet technology can significantly optimize the traditional digital PCR, improve the detection efficiency, high throughput analysis of bio chemical reaction of great significance. The goal of this paper is to separately design droplet formation and detection chip chip, micro drop fast, uniform, stable production, the droplet diameter is about 0.1 mm, the formation rate is 1500 in the micro / sec. Drops were collected, the droplets within the samples were amplified by PCR, then detected by chip micro droplet arranged in equidistance sequence, the droplet spacing is about 1 mm, isometric droplet detection system followed by flow cytometry, can achieve rapid and accurate detection of the amplified samples, the droplet detection rate is about 200 / sec. The main research contents and results are as follows: 1. using COMSOL software to simulate the droplet generation and detection module, discuss the channel structure, wall properties, negative pressure and other factors on the droplet diameter, affect the formation rate and spacing caused by the droplets is determined according to the acceptance index of simulation the structural design of micro droplet generation and detection chip chip, the chip to produce droplets can meet the reaction of digital PCR system and test requirement of.2. to study the SU-8 photoresist process, make structure mold ideal, select P DMS materials, molding technology and using the PDMS key and technology of microfluidic chip with micro channel structure, bonding strength, surface roughness and channel hydrophobic properties meet the digital PCR system and the test requirement of.3. reaction system of negative pressure actuated micro droplet motion, studied the relationship between the micro fluid friction resistance in micro channel structure in the process of its application, and the loss along the way to get the ideal theory of negative pressure driving force. At the end of the negative pressure generated in the driven droplet are analyzed with uniform size. Conclusion: the chip structure using 1. scanning electron microscope on the made were observed. The depth of the micro channel is about 100 microns depth, good consistency, wall surface roughness, steep well depth and width, width error can be controlled within 10 microns, the measurement results show that the chip made by end use MEMS processing technology Meet the design requirements of the chip; 2. in high-speed camera (7500 frames / sec) under the assistance of droplet generation and detection of chip production of micro droplet experiments. Counting analysis of droplet formation rate and detection rate, can be obtained by droplets, graphic contrast method, spacing can be obtained in diameter and the adjacent droplet droplet. Experimental results show that the droplet and detecting chip are designed and made to meet the demand for the use of digital PCR system.

【学位授予单位】：中国科学院研究生院(长春光学精密机械与物理研究所)
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TN492

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