【摘要】：近年來(lái),微流控芯片上的數(shù)字PCR方法得到了長(zhǎng)足的發(fā)展。數(shù)字PCR技術(shù)以其超高的靈敏度、準(zhǔn)確度和絕對(duì)定量的性質(zhì)也引起人們的強(qiáng)烈關(guān)注。數(shù)字PCR技術(shù)與微流控芯片相得益彰,互相促進(jìn),如集成流路數(shù)字PCR芯片、滑動(dòng)芯片、百萬(wàn)像素?cái)?shù)字PCR芯片和多種液滴數(shù)字PCR芯片。然而,這些現(xiàn)有的數(shù)字PCR芯片或者配有較多的管路或者配有復(fù)雜的外加設(shè)備,操作費(fèi)時(shí)費(fèi)力。并且,現(xiàn)有的幾款已經(jīng)商業(yè)化的數(shù)字PCR儀器價(jià)格昂貴,并且完全依賴進(jìn)口,限制了數(shù)字PCR技術(shù)在我國(guó)生命科學(xué)研究領(lǐng)域的普及。另外,微流控芯片的集成化是一大的發(fā)展趨勢(shì),將樣品處理、富集、分離純化、反應(yīng)等功能元件通過(guò)某種方式集成到一張芯片上,使其協(xié)同發(fā)生作用,力求實(shí)現(xiàn)真正的sample-in/answer-out。本文在總結(jié)了數(shù)字PCR技術(shù)的原理、發(fā)展和應(yīng)用的基礎(chǔ)上,結(jié)合了本實(shí)驗(yàn)室的實(shí)際情況,取得了如下的創(chuàng)新結(jié)果：1)、利用PDMS的透氣性并結(jié)合了濾器的過(guò)濾原理,我們?cè)O(shè)計(jì)并研制了一種基于PDMS透氣性的薄膜數(shù)字PCR芯片。該芯片包括如同濾器結(jié)構(gòu)的μfilter結(jié)構(gòu)和如同濾膜的薄膜芯片。其中的μfilter結(jié)構(gòu)有兩部分組成,其上有進(jìn)樣孔和抽氣孔,能實(shí)現(xiàn)一端抽氣,一端進(jìn)樣。薄膜芯片如同濾膜被夾于μfilter中間,其上有650個(gè)直徑0.2 mm,深0.23 mm的圓形小室。當(dāng)連接μfilter的注射器抽動(dòng)時(shí),薄膜芯片的一側(cè)出現(xiàn)了低于大氣壓的負(fù)壓,被樣品封閉的空氣會(huì)慢慢透過(guò)PDMS薄膜進(jìn)入到注射器中,最終將液體試劑帶入到每個(gè)小室中并充滿。充滿樣品的薄膜芯片可以在PCR儀上進(jìn)行數(shù)字PCR反應(yīng)。2)、PDMS的透氣性雖然方便了進(jìn)樣,但在PCR的反應(yīng)熱循環(huán)中也引起了水分的蒸發(fā),影響了反應(yīng)。我們沒(méi)有進(jìn)行特殊處理,而是在薄膜芯片外增加一片面積較大的補(bǔ)水層,抑制了蒸發(fā)帶來(lái)的危害,取得了很好的效果。3)、我們通過(guò)分步進(jìn)樣的方法,將核酸提取元件與數(shù)字PCR元件集成起來(lái)。首先將核酸提取的所需的試劑分段存放在特氟龍管中,當(dāng)我們?cè)诤怂崽崛≡囊欢顺閯?dòng)注射器時(shí),特氟龍管的試劑依次經(jīng)過(guò)核酸提取區(qū),樣品裂解液中的磁珠被核酸提取區(qū)的磁鐵吸附下來(lái),洗滌試劑依次通過(guò)核酸提取區(qū)完成對(duì)磁珠的清洗,PCR試劑將純化核酸從磁珠上洗脫下來(lái)。由于負(fù)壓操作,核酸提取中的樣品裂解液和洗滌液無(wú)法進(jìn)入到PCR反應(yīng)區(qū)中,防止了污染的發(fā)生。4)、我們發(fā)展了基于PDMS透氣性的進(jìn)樣方法,在微流控芯片的數(shù)字PCR小室旁放置一個(gè)面積較大的抽氣層實(shí)現(xiàn)負(fù)壓進(jìn)樣。當(dāng)連接抽氣層的注射器抽動(dòng)的時(shí)候,抽氣層內(nèi)部呈現(xiàn)負(fù)壓,芯片小室中的氣體透過(guò)PDMS進(jìn)入到抽氣層中,并將試劑帶入并充滿每個(gè)小室。同時(shí),抽氣層待進(jìn)完樣之后,又被充入水分起到了防蒸發(fā)的作用。我們同時(shí)將核酸提取元件集成到此數(shù)字PCR芯片上,實(shí)現(xiàn)了在一張芯片上進(jìn)行核酸提取和數(shù)字PCR反應(yīng)。綜上所述,基于PDMS的透氣性,我們研制了一種可以利用注射器提供的負(fù)壓就能實(shí)現(xiàn)樣品進(jìn)樣,并能完成數(shù)字PCR反應(yīng)的微流控芯片。該數(shù)字PCR系統(tǒng)比現(xiàn)有的數(shù)字PCR平臺(tái)更加簡(jiǎn)便、易操作、實(shí)用性好。我們還通過(guò)分步進(jìn)樣的方法實(shí)現(xiàn)了核酸提取元件與數(shù)字PCR反應(yīng)元件在微流控芯片上的集成,并成功的進(jìn)行了牛肉DNA的提取和數(shù)字PCR檢測(cè)。其特點(diǎn)是操作方便快速、試劑消耗少,準(zhǔn)確性好,適于普通實(shí)驗(yàn)室使用。并且該芯片還可利用到基于磁珠方法的RNA提取,細(xì)胞捕獲等方面,為生命科學(xué)領(lǐng)域的更關(guān)鍵問(wèn)題如腫瘤診斷、單細(xì)胞檢測(cè)、稀有細(xì)胞捕獲和檢測(cè)提供了一種新的方法和儀器。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R440

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