微流控核酸浓度检测系统的研究

论文总字数：28029字

摘 要

在利用数字PCR技术实现DNA分子的绝对定量之前，需要对核酸样本进行较高精度的浓度定量检测以预估样本浓度。传统的紫外可见分光光度计在测量核酸的吸光度时需要较多的样本量，且不能进行实时检测。本课题基于紫外分光光度法，研究了用于测量少量核酸样本核酸浓度的微流控芯片。

第一版微流控芯片使用直通道作为流体通道，并在光纤接收端设计了狭缝，被测通道宽度分别为200µm，400µm,500µm,750µm时所测得工作曲线的灵敏度在合理的范围内（将使用分光光度计测量时单链DNA的工作曲线灵敏度作为高值，双链DNA的工作曲线灵敏度作为低值）。且验证了狭缝的存在对于灵敏度的增加有一定的贡献。但是被测通道长度为1000µm时，工作曲线的灵敏度偏离了合理的范围。

第二版芯片使用Z字形微流体通道，并将中间的直通道作为被测通道，使用仅有微流体通道和光纤通道的原始结构作了工作曲线，该工作曲线适合低浓度DNA溶液的测量。研究了在该微流体通道周围分布有吸光剂通道对线性范围了的影响，得出了分布于被测通道周围的吸光剂通道不益于减少杂散光影响的结论。将这一结构应用于直通道并加入透镜结构后，吸光度-浓度曲线在浓度范围0-400ng/µL的拟合曲线相比于第一版芯片灵敏度有所提升，吸光度-浓度曲线在浓度范围0-350ng/µL的拟合曲线可以作为工作曲线使用。这意味着透镜结构有助于减少杂散光的影响。

该微流控芯片由微通道和玻璃片键合而成。模具由基于SLA的3D打印技术代替软光刻技术制备，使用倒模工艺得到微通道，倒模使用的材料为PDMS。第一版微流控芯片由一层具有微通道的PDMS和玻璃片键合，第二版微流控芯片首先键合两层具有不同微通道的PDMS，之后再与玻璃片键合。

关键词：3D打印，核酸浓度检测，吸光度，杂散光

Abstract

Before using the digital PCR technology to achieve absolute quantification of DNA molecµLes, it is necessary to perform high-accuracy quantitative detection of nucleic acid samples to estimate the sample concentration. Conventional UV-Vis spectrophotometers require more sample size when measuring the absorbance of nucleic acids and cannot be detected in real time. Based on the µLtraviolet spectrophotometry, this study manufactured a kind of microfluidic chips for measuring the nucleic acid concentration of a small amount of nucleic acid volµme.

The first version of the microfluidic chip used a straight channel as the fluid channel, and a slit is designed at the end of the fiber aisle. The measured channel width were 200µm, 400µm, 500µm, 750µm, and the sensitivity of the measured curve is within a reasonable range. (The sensitivity of the working curve of single-stranded DNA when using a spectrophotometer is taken as a high value, and the sensitivity of the working curve of double-stranded DNA is taken as a low value). It was also verified that the presence of the slit contributed to the increase in sensitivity. However, when the measured channel length is 1000µm, the sensitivity of the working curve deviates from a reasonable range.

The second version of the chip uses a zigzag microfluidic channel, and the middle straight channel is used as the channel to be measured. The working curve is made using the original structure with only the microfluidic channel and the fiber channel. This working curve is suitable for the measurement of low concentration DNA solutions. The light-absorption agent channel is not conducive to reducing the influence of stray light. After applying this structure to the straight channel and adding the lens structure, The absorbance-concentration curve has a higher sensitivity than the first version of the chip in the concentration range of 0-400 ng/µL. The fit curve of the absorbance-concentration curve in the concentration range of 0-350 ng/μL can be used as the working curve. This means that the microlen helps to reduce the effects of stray light.

The microfluidic chip is formed by bonding microchannels and glass sheets. The mold was prepared by SLA-based 3D printing technology instead of soft lithography, and the microchannel was obtained by the mold-cutting process. The material used for the mold was PDMS. The first version of the microfluidic chip is bonded by a layer of PDMS and glass sheets with microchannels. The second version of the microfluidic chip is first bonded to two layers of PDMS with different microchannels, and then bonded to the glass sheets.

KEY WORDS: 3D printing, nucleic acid concentration detection, absorbance, stray light

目录

摘要 I

Abstract II

目录 III

第一章 绪论 1

1.1引言 1

1.2 基于吸光度法检测物质浓度的微流控技术研究进展 1

1.3 微流控芯片实验室 4

1.4 核酸简介 4

1.5 光纤光谱仪简介 5

1.6 抗紫外辐照光纤简介 5

1.7 立体光刻成型3D打印技术在微流控芯片中的应用 6

1.8 本文的研究内容及目的 6

第二章 原理简介 8

2.1 紫外分光光度法定量检测核酸浓度原理 8

2.1.1 分光光度法 8

2.1.2 分光光度法在测量核酸浓度中的应用 8

2.2 朗伯比尔定律与工作曲线 9

2.3 杂散光 10

第三章 用于检测DNA浓度的光纤直插式微流控芯片的设计与制造 12

3.1模具制造 12

3.1.1 solidworks与模具设计 12

3.1.2 3D打印平台与打印流程 13

3.2 模具的硅烷化与倒模 14

3.3 芯片键合 14

3.4 吸光剂加载 14

第四章 实验方法与流程 16

4.1 鲑鱼精DNA溶液的稀释 16

4.2 测量系统的搭建 16

4.3 测量流程 17

4.4 数据处理 17

第五章 实验结果与分析 18

5.1 第一版芯片 18

5.2 第二版芯片 21

第六章 总结与展望 28

致谢 29

参考文献 30

附录 32

1. 绪论

1.1 引言

数字PCR是新发展起来的一种可以进行绝对定量分析的技术。将每个DNA分子移入单独的反应池，在PCR扩增之后，在统计和分析荧光信号之后，实现DNA的绝对定量检测，数字PCR检测的绝对精度不仅受限于有效反应单元的数量，也依赖于每个单元中的分子数。每个单元中的分子数又由样本核酸原始浓度及其预处理稀释方式决定。因此，为利用数字PCR技术实现DNA分子的绝对定量，需在PCR检测之前对核酸样本浓度进行预估计和定量检测，其后进行样本稀释，以达到最佳浓度。本课题的研究任务在于研制一种用于DNA浓度测量的微流控芯片，该微流控芯片需要在测量中低浓度的溶液时有较高的灵敏度和精度，以及在测量高浓度的溶液时尽量避免杂散光的影响，尽量提高芯片所能测试的浓度上限。芯片的制造需要经过3D打印，倒模，键合，注入吸光剂等多个工艺流程，制造良好的芯片再与光源，光纤光谱仪等一起搭建测量系统，之后使用一定浓度分布的DNA溶液来制作工作曲线，依此曲线为标准测量未知浓度DNA溶液的浓度，用于制作工作曲线的DNA溶液样本数量越多，浓度分布越密，所测曲线就越准确。

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