本文選題：納米生物界面　切入點：靜態(tài)調(diào)控　出處：《中國科學(xué)院研究生院(上海應(yīng)用物理研究所)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：納米生物界面精確調(diào)控可以很好地實現(xiàn)器件的構(gòu)建并發(fā)揮其良好的生物傳感性能,DNA納米技術(shù)基于堿基互補配對的特性可以很好地實現(xiàn)對距離、結(jié)構(gòu)、大小和構(gòu)象的精確調(diào)控。本研究主要基于DNA納米技術(shù)構(gòu)建了一系列可以精確調(diào)控的納米生物界面并利用其實現(xiàn)了對目前缺乏有效檢測手段的前列腺癌靶標小分子的精確檢測。最后在構(gòu)建納米生物界面的基礎(chǔ)上,實現(xiàn)了對細胞表面的精確組裝并成功地實現(xiàn)了可視化定量檢測乳腺癌細胞。具體內(nèi)容如下:一、對納米生物界面進行了靜態(tài)結(jié)構(gòu)的構(gòu)建和調(diào)控,利用DNA納米技術(shù)的可編輯性同時引入DNA折紙技術(shù)構(gòu)建了二維(2D)的納米生物界面,并運用該界面實現(xiàn)了對兩種生物大分子之間距離的精確調(diào)控,可以從10nm到70nm不等。在2D納米生物界面構(gòu)建基礎(chǔ)上,我們又利用DNA四面體技術(shù)構(gòu)建了三維(3D)的納米生物界面,實現(xiàn)了對生物大分子從準納米到納米距離的精確調(diào)控。在成功實現(xiàn)對組裝分子從2D到3D的距離調(diào)控基礎(chǔ)上,又設(shè)計了五種不同構(gòu)象的四面體,構(gòu)建了五種不同構(gòu)象的納米生物界面并發(fā)現(xiàn)不同構(gòu)象的納米生物界面確實會影響其生物傳感性能。二、在成功實現(xiàn)對納米生物界面的靜態(tài)調(diào)控后,我們又對納米生物界面實現(xiàn)了動態(tài)的調(diào)節(jié)。借鑒生物酶研究中的別構(gòu)效應(yīng),基于靜態(tài)構(gòu)象調(diào)控的基礎(chǔ),引入帶有莖環(huán)結(jié)構(gòu)的四面體并通過加入刺激因子,包括響應(yīng)鏈(effector)、抑制鏈、p H、雜交時間等調(diào)控四面體構(gòu)象改變從而實現(xiàn)對納米生物界面的動態(tài)調(diào)控。同時發(fā)現(xiàn)納米生物界面的動態(tài)調(diào)控可以影響相同探針和靶序列的結(jié)合能力從而影響生物傳感性能,我們利用構(gòu)象改變導(dǎo)致的探針和靶序列結(jié)合能力的不同實現(xiàn)了對靶序列捕獲和釋放的精確調(diào)控。三、在成功實現(xiàn)精確調(diào)控納米生物界面從2D到3D、從靜態(tài)到動態(tài)、從距離到構(gòu)象改變的基礎(chǔ)上,利用設(shè)計的納米生物界面來構(gòu)建生物傳感器。肌氨酸作為前列腺癌檢測的潛在靶標小分子由于分子量小、體內(nèi)含量低等特點導(dǎo)致目前仍然缺乏高靈敏度、高特異性的手段來檢測。我們利用肌氨酸氧化酶、辣根過氧化物酶在肌氨酸存在時可以發(fā)生級聯(lián)反應(yīng)從而提高檢測的特異性,并且運用構(gòu)建的納米生物界面可以精確調(diào)控酶之間的距離,同時結(jié)合電化學(xué)技術(shù)實現(xiàn)了對肌氨酸的高靈敏檢測,檢測限可以達到50n M,最后運用該方法進行了大量前列腺癌、前列腺腫大、正常男性血清樣本的檢測。四、在納米生物界面構(gòu)建技術(shù)基礎(chǔ)上,我們利用DNA適配體和乳腺癌細胞MCF-7表面高表達的上皮細胞粘附因子(Ep CAM)結(jié)合,實現(xiàn)了在細胞界面組裝DNA的技術(shù)。然后利用DNA雜交技術(shù)將引發(fā)鏈雜交到細胞表面,這樣帶有引發(fā)鏈的細胞就可以引發(fā)3D的雜交鏈式反應(yīng)從而形成DNA水凝膠,在這過程中加入DNA保護的納米金就可以實現(xiàn)對癌細胞定量、可視化的檢測。
[Abstract]:The precise regulation of nanoscale biological interface can realize the construction of devices and give full play to their good biosensor performance. DNA nanotechnology based on the characteristics of base complementary pairing can well realize the distance and structure of the devices. In this study, a series of accurately regulated nanoscale biological interfaces were constructed based on DNA nanotechnology and used to realize the detection of prostate cancer target small molecules that lack effective detection methods. Finally, based on the construction of nanoscale biological interface, The precise assembly of the cell surface and the visual quantitative detection of breast cancer cells have been successfully realized. The main contents are as follows: firstly, the static structure of the nanoscale biological interface is constructed and regulated. Using the editable property of DNA nanotechnology and the introduction of DNA origami technology, the two-dimensional biologic interface was constructed, and the precise control of the distance between the two biomolecules was realized by the interface. It can range from 10 nm to 70 nm. On the basis of 2D nanoscale biological interface construction, we have used DNA tetrahedron technology to construct three dimensional (3D) nanoscale biological interface. On the basis of the successful distance control of the assembled molecules from 2D to 3D, five different conformation tetrahedrons have been designed. Five nanoscale biological interfaces with different conformations were constructed, and it was found that different conformational nanoscale biological interfaces did affect their biosensor performance. Secondly, after the static regulation of nanoscale biological interfaces was successfully realized, Based on the static conformational regulation, the tetrahedron with stem ring structure was introduced and the stimulant factor was added. It includes response chain effector, inhibition of chain Hand, hybridization time and so on to control tetrahedron conformation to realize dynamic regulation of nano-biological interface. At the same time, it is found that the dynamic regulation of nano-biological interface can affect the same probe and target sequence. Binding ability to affect biosensor performance, We make use of the different binding ability of probe and target sequence caused by conformation change to realize the accurate regulation of target sequence capture and release. Third, in the successful implementation of precise regulation of nano-biological interface from 2D to 3D, from static to dynamic, Based on the distance to conformation change, the biosensor was constructed by using the designed nanoscale biological interface. Creatine is used as a potential target for prostate cancer detection because of its small molecular weight. Because of the low content in the body, there is still a lack of high sensitivity and high specificity for detection. We use sarcosine oxidase and horseradish peroxidase to produce cascade reaction in the presence of sarcosine to improve the specificity of the detection. At the same time, the high sensitivity detection of sarcosine can be realized with electrochemical technology, and the detection limit can reach 50nM. finally, a large number of prostate cancer was carried out by this method. Detection of benign prostatic enlargement and normal male serum samples. Fourthly, based on the technique of constructing nanoscale biological interface, we combine DNA aptamer with epithelial cell adhesion factor (Ep CAM), which is highly expressed on MCF-7 surface of breast cancer cells. The technique of assembling DNA at the cell interface is realized, and then using DNA hybridization, the initiation chain is hybridized onto the cell surface, so that the cells with the initiation chain can initiate a 3D hybridization chain reaction to form a DNA hydrogel. DNA-protected nanocrystalline gold can be used to detect cancer cells quantitatively and visually.
【學(xué)位授予單位】：中國科學(xué)院研究生院(上海應(yīng)用物理研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：TB383.1;TP212

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