本文選題：生物大分子 + 折疊動(dòng)力學(xué)��； 參考：《華中科技大學(xué)》2013年博士論文

【摘要】：生物大分子如蛋白質(zhì)或核酸的功能與其三維結(jié)構(gòu)密切相關(guān)，折疊動(dòng)力學(xué)研究可揭示生物大分子從自由的一級(jí)結(jié)構(gòu)形成具有活性高級(jí)結(jié)構(gòu)的動(dòng)態(tài)過程，近年來倍受科學(xué)界重視。2005年，Science雜志將蛋白質(zhì)折疊列為21世紀(jì)生命科學(xué)領(lǐng)域最重要挑戰(zhàn)之一。生物大分子的折疊過程一般發(fā)生在毫秒、微秒甚至是亞微秒時(shí)間水平，而啟動(dòng)折疊反應(yīng)則需在更短的時(shí)間內(nèi)完成�？焖倩旌霞夹g(shù)能使溶液在短時(shí)間內(nèi)達(dá)到完全混合從而觸發(fā)反應(yīng)，是一種常被用于研究分子折疊反應(yīng)的有效手段。 大分子折疊動(dòng)力學(xué)研究的傳統(tǒng)工具是停流裝置，然而毫秒級(jí)的時(shí)間分辨率和大的樣品消耗量限制了其進(jìn)一步應(yīng)用，尤其是追蹤折疊早期動(dòng)力學(xué)。本文基于微流控芯片連續(xù)流概念，提出了三種用于生物大分子折疊動(dòng)力學(xué)研究的快速微混合器，為解決動(dòng)力學(xué)研究領(lǐng)域的難點(diǎn)問題提供了新技術(shù)手段。主要研究結(jié)果如下： （1）針對(duì)低粘度溶液，發(fā)展了一種混合時(shí)間短且結(jié)構(gòu)簡單的新型Z型微混合器。通過計(jì)算流體力學(xué)模擬和實(shí)驗(yàn)手段證實(shí)該混合器能在16μs內(nèi)實(shí)現(xiàn)溶液的完全混合，采用該混合器研究了化學(xué)發(fā)光反應(yīng)動(dòng)力學(xué)過程；在此基礎(chǔ)上，我們縮小該混合器微結(jié)構(gòu)的尺寸，可以使其混合時(shí)間縮短至5.5μs，，比目前最快的混沌流混合器（其混合時(shí)間是11μs）的混合速度提高了一倍；進(jìn)一步采用該Z型混合器研究了人類端粒DNA序列在金屬離子存在下形成四聚體的早期折疊動(dòng)力學(xué)過程，觀察到了該DNA分子由線性結(jié)構(gòu)坍縮成發(fā)夾結(jié)構(gòu)的實(shí)驗(yàn)證據(jù)。 （2）針對(duì)高粘度溶液，設(shè)計(jì)了一種結(jié)構(gòu)簡單，加工簡便且能快速混合的ω型混合器，通過計(jì)算流體力學(xué)模擬和實(shí)際混合實(shí)驗(yàn)，證明該ω型混合器能在579.4μs內(nèi)實(shí)現(xiàn)粘度為水的33.6倍的溶液的完全混合，其混合時(shí)間比文獻(xiàn)報(bào)道的結(jié)果縮短了約1000倍；進(jìn)一步利用該混合器研究了人類端粒DNA序列在分子擁擠環(huán)境下形成G-四聚體的早期折疊動(dòng)力學(xué)過程，發(fā)現(xiàn)G-四聚體在分子擁擠條件下存在亞毫秒尺度的折疊事件。 （3）針對(duì)樣本消耗問題，提出了微流控芯片雙水力聚焦概念，并設(shè)計(jì)了一種時(shí)間窗口寬、樣品消耗量少且結(jié)構(gòu)簡單的雙水力聚焦微混合器，通過計(jì)算流體力學(xué)模擬和實(shí)驗(yàn)評(píng)價(jià)，表明該混合器能有效實(shí)現(xiàn)兩種大分子的快速混合，且其對(duì)動(dòng)力學(xué)反應(yīng)的時(shí)間觀察范圍達(dá)4個(gè)數(shù)量級(jí)，可涵蓋從亞毫秒到數(shù)秒鐘(710s-5.36s)，而對(duì)兩種生物大分子的樣品消耗均小于0.55μL/min，比已有文獻(xiàn)減少了約1000倍；利用該混合器研究了人類端粒G-四聚體與單鏈DNA結(jié)合蛋白（SSBP）的相互作用動(dòng)力學(xué)過程，發(fā)現(xiàn)SSBP的結(jié)合能觸發(fā)G-四聚體的去折疊過程，且處于高濃度Na+溶液條件下的G-四聚體其去折疊速率相對(duì)更慢。 總之，本文針對(duì)目前生物大分子折疊動(dòng)力學(xué)研究中的一些難點(diǎn)問題提出了三種快速微混合器；針對(duì)特定的研究問題，采用計(jì)算機(jī)建模仿真和實(shí)驗(yàn)兩種方式證明它們均具有好的混合效果和時(shí)間分辨率；進(jìn)一步通過基本生物學(xué)問題的探討證明三者具備研究生物大分子折疊動(dòng)力學(xué)的能力和巨大應(yīng)用潛力。
[Abstract]:The function of biological macromolecules, such as protein or nucleic acid, is closely related to its three-dimensional structure. Folding kinetics studies can reveal that biological macromolecules form a dynamic process of active high structure from a free first order structure. In recent years, the scientific community has paid much attention to.2005 years. Science magazine listed the egg white matter as the most important field of life science in twenty-first Century. One of the challenges. The folding process of biological macromolecules usually occurs in milliseconds, microseconds or even submicroseconds, and the initiation of the folding reaction is needed in a shorter time. Fast mixing technology can make the solution complete mixing in a short time and trigger the reaction. It is an effective hand used to study the folding reaction of molecules. Paragraph.
The traditional tool for the study of macromolecular folding dynamics is a stop flow device. However, the time resolution and large sample consumption in milliseconds limit its further application, especially the tracking of early folding dynamics. Based on the concept of microfluidic chip continuum, three kinds of rapid micro mixing for the study of the dynamics of biological macromolecule folding are proposed in this paper. The combiner has provided new technical means for solving the difficult problems in the field of dynamics research. The main results are as follows:
(1) a new type of Z type micro mixer with short mixing time and simple structure was developed for low viscosity solution. Through computational fluid dynamics simulation and experimental means, it was proved that the mixer could complete the complete mixing of the solution in 16 mu, and the chemiluminescence reaction kinetics was studied by the mixer. On this basis, we reduced the mixture. The size of the microstructure can shorten the mixing time to 5.5 s, which is more than twice as fast as the fastest chaotic mixer (its mixing time is 11 s). Further, the Z mixer was used to study the early folding kinetics of the human telomere DNA sequence in the presence of metal ions to form four polymer. Experimental evidence for the collapse of the DNA molecule from a linear structure to a hairpin structure is presented.
(2) a kind of Omega mixer with simple structure, simple processing and fast mixing is designed for high viscosity solution. Through the calculation of fluid mechanics simulation and actual mixing experiment, it is proved that the Omega mixer can realize the complete mixing of the solution with the viscosity of 33.6 times the water in 579.4 s. The mixing time is about 10 shorter than the reported result. 00 times, the early folding kinetics of the human telomere DNA sequence formed G- four polymer in a crowded environment was further studied. It was found that there was a submillisecond folding event of the G- four polymer under the molecular crowding condition.
(3) aiming at the problem of sample consumption, a dual hydraulic focusing concept of microfluidic chip is proposed, and a dual hydraulic focusing micro mixer with wide time window, less sample consumption and simple structure is designed. Through the calculation of fluid mechanics simulation and experimental evaluation, it is shown that the mixer can effectively realize the fast mixing of two kinds of macromolecules, and the power of the mixer is effective. The time observation range of the reaction is 4 orders of magnitude, covering from sub milliseconds to several seconds (710s-5.36s), and the consumption of two biological macromolecules is less than 0.55 mu L/min, which is about 1000 times less than that of the existing literature. The interaction kinetics of human telomere G- four polymer and single strand DNA binding protein (SSBP) is studied by this mixer. It was found that the binding of SSBP could trigger the unfolding process of G- four dimers, and the G- four dimers in high concentration Na+ solution had relatively slower rate of unfolding.
In conclusion, three fast micromixers are proposed in this paper for some difficult problems in the study of biological macromolecule folding dynamics. For specific research problems, two ways of computer modeling, simulation and experiment are used to prove that they have good mixing effect and time resolution rate, and further through the basic biological problems. It is proved that the three have the ability to study the folding kinetics of biological macromolecules and great potential for application.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318

【共引文獻(xiàn)】

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本文編號(hào)：1905351