核酸纯化扩增及检测一体化数字PCR集成流路芯片的研制与应用

发布时间：2019-03-13 13:00

【摘要】：近年来,微流控芯片上的数字PCR方法得到了长足的发展。数字PCR技术以其超高的灵敏度、准确度和绝对定量的性质也引起人们的强烈关注。数字PCR技术与微流控芯片相得益彰,互相促进,如集成流路数字PCR芯片、滑动芯片、百万像素数字PCR芯片和多种液滴数字PCR芯片。然而,这些现有的数字PCR芯片或者配有较多的管路或者配有复杂的外加设备,操作费时费力。并且,现有的几款已经商业化的数字PCR仪器价格昂贵,并且完全依赖进口,限制了数字PCR技术在我国生命科学研究领域的普及。另外,微流控芯片的集成化是一大的发展趋势,将样品处理、富集、分离纯化、反应等功能元件通过某种方式集成到一张芯片上,使其协同发生作用,力求实现真正的sample-in/answer-out。本文在总结了数字PCR技术的原理、发展和应用的基础上,结合了本实验室的实际情况,取得了如下的创新结果：1)、利用PDMS的透气性并结合了滤器的过滤原理,我们设计并研制了一种基于PDMS透气性的薄膜数字PCR芯片。该芯片包括如同滤器结构的μfilter结构和如同滤膜的薄膜芯片。其中的μfilter结构有两部分组成,其上有进样孔和抽气孔,能实现一端抽气,一端进样。薄膜芯片如同滤膜被夹于μfilter中间,其上有650个直径0.2 mm,深0.23 mm的圆形小室。当连接μfilter的注射器抽动时,薄膜芯片的一侧出现了低于大气压的负压,被样品封闭的空气会慢慢透过PDMS薄膜进入到注射器中,最终将液体试剂带入到每个小室中并充满。充满样品的薄膜芯片可以在PCR仪上进行数字PCR反应。2)、PDMS的透气性虽然方便了进样,但在PCR的反应热循环中也引起了水分的蒸发,影响了反应。我们没有进行特殊处理,而是在薄膜芯片外增加一片面积较大的补水层,抑制了蒸发带来的危害,取得了很好的效果。3)、我们通过分步进样的方法,将核酸提取元件与数字PCR元件集成起来。首先将核酸提取的所需的试剂分段存放在特氟龙管中,当我们在核酸提取元件的一端抽动注射器时,特氟龙管的试剂依次经过核酸提取区,样品裂解液中的磁珠被核酸提取区的磁铁吸附下来,洗涤试剂依次通过核酸提取区完成对磁珠的清洗,PCR试剂将纯化核酸从磁珠上洗脱下来。由于负压操作,核酸提取中的样品裂解液和洗涤液无法进入到PCR反应区中,防止了污染的发生。4)、我们发展了基于PDMS透气性的进样方法,在微流控芯片的数字PCR小室旁放置一个面积较大的抽气层实现负压进样。当连接抽气层的注射器抽动的时候,抽气层内部呈现负压,芯片小室中的气体透过PDMS进入到抽气层中,并将试剂带入并充满每个小室。同时,抽气层待进完样之后,又被充入水分起到了防蒸发的作用。我们同时将核酸提取元件集成到此数字PCR芯片上,实现了在一张芯片上进行核酸提取和数字PCR反应。综上所述,基于PDMS的透气性,我们研制了一种可以利用注射器提供的负压就能实现样品进样,并能完成数字PCR反应的微流控芯片。该数字PCR系统比现有的数字PCR平台更加简便、易操作、实用性好。我们还通过分步进样的方法实现了核酸提取元件与数字PCR反应元件在微流控芯片上的集成,并成功的进行了牛肉DNA的提取和数字PCR检测。其特点是操作方便快速、试剂消耗少,准确性好,适于普通实验室使用。并且该芯片还可利用到基于磁珠方法的RNA提取,细胞捕获等方面,为生命科学领域的更关键问题如肿瘤诊断、单细胞检测、稀有细胞捕获和检测提供了一种新的方法和仪器。
[Abstract]:In recent years, the digital PCR method on the micro-fluidic chip has been developed. The high sensitivity, accuracy and absolute quantitative properties of the digital PCR technology also give rise to the strong interest of people. The digital PCR technology is complementary to the micro-fluidic chip, and is mutually promoted, such as an integrated flow path digital PCR chip, a sliding chip, a million pixel digital PCR chip and a plurality of liquid drop digital PCR chips. However, these existing digital PCR chips, or with more lines or with complex external equipment, are laborious and time-consuming. Moreover, the existing several commercial digital PCR instruments are expensive and rely on the import completely, limiting the popularization of the digital PCR technology in the field of life science research in China. In addition, the integration of the micro-fluidic chip is a major development trend, and the functional elements such as sample processing, enrichment, separation and purification, reaction and the like are integrated into a single chip in a certain way, so that the micro-fluidic chip can cooperate with each other, and the real sample-in/ answer-out can be realized. Based on the summary of the principle, development and application of the digital PCR technology, this paper, combined with the actual situation of the laboratory, has obtained the following innovative results:1), using the air permeability of PDMS and combining the filter principle of the filter, We designed and developed a film digital PCR chip based on PDMS air permeability. The chip includes a mufilter structure like a filter structure and a thin film chip like a filter membrane. Wherein, the filter structure is composed of two parts, a sample injection hole and an air extraction hole are arranged on the filter structure, one end of the filter structure can be pumped, and one end of the sample is injected. The membrane chip is clamped in the middle of the. filter with a filter membrane with 650 circular cells with a diameter of 0.2 mm and a depth of 0.23 mm. When the syringe connected to the. mufilter is pumped, a negative pressure below atmospheric pressure appears on one side of the film wafer, and the air trapped by the sample is slowly introduced into the syringe through the PDMS film, and finally the liquid reagent is brought into each chamber and is filled. The membrane chip filled with the sample can carry out the digital PCR reaction on the PCR instrument.2) The air permeability of the PDMS is convenient for injection, but also the evaporation of the water is caused in the reaction thermal cycle of the PCR, and the reaction is influenced. We do not carry out special treatment, but add a large area of water-supplementing layer outside the film chip, which can inhibit the damage caused by evaporation.3) We integrate the nucleic acid extracting element and the digital PCR element through the method of separating the step-like sample. firstly, the required reagent segments of the nucleic acid extraction are stored in a Teflon tube; when we pump the syringe at one end of the nucleic acid extraction element, the reagent of the Teflon tube passes through the nucleic acid extraction region in sequence, the magnetic beads in the sample cracking liquid are absorbed by the magnet of the nucleic acid extraction region, The washing reagent sequentially performs the cleaning on the magnetic beads through the nucleic acid extraction region, and the PCR reagent is used for eluting the purified nucleic acid from the magnetic beads. As a result of the negative pressure operation, the sample lysing liquid and the washing liquid in the nucleic acid extraction cannot enter the PCR reaction zone to prevent the occurrence of the pollution. And a large area of the air extraction layer is arranged beside the digital PCR chamber of the micro-fluidic chip to realize the negative pressure injection. When the syringe connected to the suction layer is withdrawn, a negative pressure is present inside the suction layer, and the gas in the wafer chamber is introduced into the suction layer through the PDMS, and the reagent is brought into and filled with each cell. At the same time, after the air extraction layer is finished, the air is filled with water to act as an anti-evaporation function. At the same time, the nucleic acid extracting element is integrated on the digital PCR chip, and the nucleic acid extraction and the digital PCR reaction on a single chip are realized. To sum up, based on the air permeability of PDMS, we have developed a micro-fluidic chip which can realize the sample injection by using the negative pressure provided by the syringe, and can complete the digital PCR reaction. Compared with the prior digital PCR platform, the digital PCR system is more convenient, easy to operate and good in practicability. The integration of the nucleic acid extraction element and the digital PCR reaction element on the microfluidic chip is also realized by a step-by-step method, and the extraction of the beef DNA and the detection of the digital PCR are successfully carried out. The method is characterized in that the operation is convenient and rapid, the reagent consumption is small, the accuracy is good, and the method is suitable for common laboratory use. And the chip can also be used in the aspects of RNA extraction and cell capture based on the magnetic bead method, and provides a new method and an instrument for the more important problems in the field of life science such as tumor diagnosis, single cell detection, rare cell capture and detection.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R440

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