自吸分液式数字PCR集成流路芯片的研制及其在单分子核酸和蛋白质分子检测中的应用

发布时间：2018-06-03 11:15

  本文选题：单分子核酸扩增 + 数字PCR　； 参考：《浙江大学》2013年博士论文

【摘要】：数字PCR,以其能通过单分子扩增实现核酸拷贝数绝对定量被称为第三代PCR技术。特别是以微流控芯片为基础的数字PCR方法,因其超高的灵敏度、准确度及低试剂消耗等优点而得到迅速发展。然而现有的芯片型和液滴型数字PCR平台操作复杂费时,需要许多外接管路和配套控制系统,并未完全发挥微流控芯片的优势,由此商品化的仪器价格昂贵且全部依赖进口,制约了数字PCR技术在我国生命科学技术领域的应用和普及。本论文在总结数字PCR原理、方法及学习研制基于气动微阀大规模集成流路数字PCR芯片技术的基础上,优化了纳升以下体积的单分子PCR反应的反应体系与反应条件,在此基础上取得了如下创新性结果：1)首次成功研制具有完全自主知识产权的自吸分液式集成流路(Integrated Fluidic Circuits, IFC)数字PCR芯片。该芯片利用PDMS材料的储气特性和缓慢的气体分子扩散过程,将负压预先储存在PDMS芯片内为芯片进样提供动力源,芯片由与由主通道相连接的微反应室阵列组成,利用油水两相不相容的性质以及两相之间的界面张力,连续的将PCR反应液和油相吸入芯片,实现样品的随机分配和隔离。该芯片无动力源,以油相代替微阀,实现了芯片的整合集成化。2)芯片内部采用一种新的氟硅烷纳米涂层,厚度仅10 nm就能有效的防治反应样品的蒸发,与已有的parylene C防水涂层相比,操作简单,无需活化,成本低廉,具有无可比拟的优越性。油相可以在加热时固化,防止样品溶液在高温时对流,避免了反应的交叉污染。3)设计了密度为1156反应/cm2的数字PCR芯片,尺寸与与盖玻片(50 mm × 24 mm× 4 mm)大小相当,分为四个检测区域,每个区域包含1280个小室,小室尺寸150 pm × 150 pm × 250μm,体积为6.5 nL,可同时检测四个样品或者同一样品的四个浓度。应用该芯片分别进行了数字环介导等温扩增反应(digital LAMP)和数字PCR反应(digital PCR),对芯片的灵敏度、准确度、测量误差进行了验证,建立了该芯片的数据统计方法,成功对人类持家基因和肿瘤相关基因进行了精确定量。4)设计了密度为1562500反应/cm2,总反应小室个数达到6,400,000的数字LAMP芯片,每个区域包含1,600,000个小室,小室尺寸为4 μmx4μm×10μm,体积160飞升,反应总体积仅仅1此,理论上检测能力可达到7个数量级,准确度可达99%,该芯片的检测能力大大超过了现有的数字PCR芯片,具有超高通量、低消耗、微型化、集成化和超大规模平行处理等方面的优势,可用于现场即时检测。5)基于蛋白质定量PLA技术,利用该芯片首次成功建立了蛋白质单分子精确定量的数字亲和连接反应(digital PLA),对EGFR蛋白质分子进行单分子计数精确定量。与数字ELISA相比,该检测方法更加简单,特异性、准确度、灵敏性等更高,为临床蛋白质标志物的检测提供了新工具。综上,该自吸分液式数字PCR芯片自动进样,无需动力驱动及微阀门控制等系统,消除了数字PCR芯片对外界控制系统的依赖,使数字核酸扩增芯片从操控芯片各个功能单元的仪器中独立出来,解决了芯片与外界系统的连接问题,比现有的数字PCR平台更加简单、实用。具有易于操作、敏感度高(单分子)、准确性好(绝对定量)、节省样品和试剂(数纳升臌反应室)、交叉污染小等特点,适合于任何实验室使用。为解决生命科学领域中基因拷贝数精确定量难的关键问题包括肿瘤早期诊断、非侵入性产前诊断、细菌和病毒检测、单细胞基因组、单分子蛋白质检测等研究提供一种新仪器。
[Abstract]:Digital PCR is known as the third generation PCR technology with its single molecule amplification by single molecule amplification. In particular, the digital PCR method based on microfluidic chips has developed rapidly because of its high sensitivity, accuracy and low reagent consumption. However, some chip and drop digital PCR platforms operate complex. Many external pipelines and matching control systems are needed in the miscellaneous fee, and the advantages of microfluidic chips are not fully played. The commercialized instruments are expensive and all depend on import, which restricts the application and popularization of digital PCR technology in the field of life science and technology in our country. This paper summarizes the principle of digital PCR, methods and study and development based on gas. On the basis of large scale integrated flow digital PCR chip technology of dynamic micro valve, the reaction system and reaction conditions of the single molecule PCR reaction of the volume below the NL are optimized. On this basis, the following innovative results are obtained: 1) the self-priming liquid integrated flow path (Integrated Fluidic Circuits) with fully autonomous intellectual property rights is successfully developed for the first time. IFC) digital PCR chip. The chip uses the gas storage characteristics of the PDMS material and the slow gas molecular diffusion process, and stores the negative pressure in the PDMS chip in advance to provide the power source for the chip sample. The chip is composed of the microreaction chamber array connected with the main channel, using the incompatible properties of the two phase of the oil and water and the interfacial tension between the two phases. Continuous PCR reacting liquid and oil phase are inhaled to realize the random distribution and isolation of the samples. The chip has no power source, the oil phase is replaced by the micro valve and the integrated integrated.2 of the chip is implemented. A new fluorossilane nano coating is used inside the chip. The thickness of only 10 nm can effectively prevent the evaporation of the reaction samples from the existing parylene C. Compared with the water coating, the operation is simple, no activation, low cost and incomparable superiority. The oil phase can be cured at heating, prevent the sample solution from convection in high temperature and avoid the cross contamination.3 of the reaction. The digital PCR chip with a density of 1156 reaction /cm2 is designed, and the size is equal to the size of the cover glass (50 mm * 24 mm * 4 mm). For four detection regions, each area contains 1280 chambers, the size of the chamber is 150 PM * 150 PM x 250 mu m, and the volume is 6.5 nL, and four samples or four concentrations of the same sample can be detected simultaneously. The chip is used for the digital ring mediated isothermal amplification reaction (digital LAMP) and the digital PCR reaction (digital PCR), and the sensitivity of the chip to the chip. The degree, accuracy, measurement error was verified, and the data statistics method of the chip was established. The accurate quantitative.4 of the human family and tumor related genes was successfully designed. The digital LAMP chip with a density of 1562500 reaction /cm2 and the total number of small cells with a total number of 6400000 had 1600000 small chambers in each area and the size of the chamber was 4. MX4 mu m x 10 mu m, the volume 160 fly up, the total volume of the reaction is only 1. In theory, the detection ability can reach 7 orders of magnitude and the accuracy is up to 99%. The detection ability of the chip is much more than the existing digital PCR chip. It has the advantages of super high throughput, low consumption, miniaturization, integration and large scale parallel processing. Time detection.5) based on the protein quantitative PLA technology, the digital affinity connection reaction (digital PLA) of the protein single molecule was established for the first time, and the single molecule count of EGFR protein molecules was quantified. Compared with the digital ELISA, the detection method was more simple, more specific, more accurate, and more sensitive, and so on. The detection of clinical protein markers provides a new tool. To sum up, the automatic sampling of the self absorption digital PCR chip, without power drive and micro valve control system, eliminates the dependence of the digital PCR chip on the external control system, and makes the digital nucleic acid amplification chip independent from the instrument of each functional unit of the control core and solves the problem. The connection between the chip and the external system is simpler and more practical than the existing digital PCR platform. It has the characteristics of easy operation, high sensitivity (single molecule), good accuracy (absolute quantification), saving of samples and reagents (number of liters and heave reaction rooms), small cross contamination and so on. It is suitable for the use of any laboratory in the life science field. The key problems of precision and quantification of the number of shells include early diagnosis of tumor, non-invasive prenatal diagnosis, bacterial and viral detection, single cell genome, single molecule protein detection, and other research instruments.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R440
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本文编号：1972585