分會

第二十五分會：有機固體

摘要

在高度進化的生命體中存在著專門負責能量轉(zhuǎn)換的的蛋白機器(ATPase)，這些蛋白機器又被稱為分子馬達，可高效率地將貯藏在三磷酸腺苷(ATP)分子中的化學能直接轉(zhuǎn)換為機械能，產(chǎn)生協(xié)調(diào)的定向運動并做功，從而驅(qū)動著各種生命活動有序進行。了解生命體高度保守的馬達蛋白這一獨特而高效的能量轉(zhuǎn)換過程的細致機理，對于理解最本征的生命活動——能量轉(zhuǎn)換是至關(guān)重要的，并且成為分子生物學、物理學、生物化學等諸多學科共同面臨的一個極具挑戰(zhàn)性的科學研究領(lǐng)域。 本論文提出用單分子靈敏度的納米線生物傳感器件，來實時監(jiān)測免標記的單分子F1-ATPase (F1)動態(tài)水解過程。該實驗體系主要利用了納米電子器件超高靈敏度和電學檢測超高的時間分辨率優(yōu)勢，并具有免標記長時間檢測的特點，可以更真實的在單分子層面研究本征的F1水解ATP的實時動態(tài)過程，是成熟的光學檢測方法的有益補充。實驗過程中我們獲取了F1的動態(tài)水解過程中誘導器件產(chǎn)生快慢交錯的雙態(tài)電學信號，這種信號對應了單分子F1的實時動態(tài)水解過程，進一步我們可以分析數(shù)據(jù)的分布特征來解析出了F1的γ旋轉(zhuǎn)軸分步旋轉(zhuǎn)步驟。對該體系在不同濃度溫度條件下進行長時間的檢測，我們獲得了本征 F1 的水解動力學速率參數(shù)，比常規(guī)的熒光檢測方法高出約一個數(shù)量級。新方法新思路的引入可以使研究者從新的角度去認識、研究分子馬達 F1 的能量轉(zhuǎn)換分子機制。這種納米線微電子傳感器件平臺也能夠拓展應用于其他分子大生物作用體系的實時監(jiān)測應用和研究。 Highly evolved organisms have protein machines (ATPase) dedicated to energy conversion. These protein machines, also known as molecular motors, can efficiently convert chemical energy stored in adenosine triphosphate (ATP) molecules into mechanical energy, thus driving a variety of life activities in an orderly manner. Understanding the detailed mechanism of the unique and efficient energy conversion process of the highly conserved motor protein in living organisms, is very important for understanding the most intrinsic life activities and it has become a challenging scientific research field in many disciplines, such as molecular biology, physics, biochemistry and so on. In this paper, a nanowire biosensor with single-molecule-sensitivity is proposed to study the dynamic hydrolysis process of label-free single molecule F1-ATPase (F1). The nano-electronic devices possess advantages of ultra-high sensitivity and ultra-high time resolution, and label-free long-time detection. We built the dynamic single molecule monitoring system based on silicon nanowires field-effect-transistors (SiNW FET). We tested the system under different ATP concentration and temperature, and revealed the intrinsic kinetics of label-free F1 hydrolysis rate parameters. The rate is one order of magnitude higher than conventional optical observation obtained, of which needs a large label on F1 to overcome the photophysical limitation as F1 is too small. Large load label hampers better understanding of the intrinsic kinetic behavior of ATP hydrolysis. We reproducibly observed the regular current signal fluctuations with two distinct levels, which induced by the binding dwell and the catalytic dwell, respectively, in both concentration- and temperature- dependent experiments. The results of our experiments have demonstrated the ability of nanowire nanocircuits to direct probe the intrinsic dynamic processes of the biological activities with single-molecule/single-event sensitivity. This approach is complementary to traditional optical methods, offering endless opportunities to unravel molecular mechanisms of a variety of dynamic biosystems under realistic physiological conditions.

關(guān)鍵詞

硅納米線;場效應晶體管;單分子檢測;F1-ATP合酶

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