中国化学会第32届学术年会
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基于硅纳米线场效应晶体管的单分子马达蛋白F1水解动力学研究
刘文哲 郭雪峰*

分会

第二十五分会:有机固体

摘要

在高度进化的生命体中存在着专门负责能量转换的的蛋白机器(ATPase),这些蛋白机器又被称为分子马达,可高效率地将贮藏在三磷酸腺苷(ATP)分子中的化学能直接转换为机械能,产生协调的定向运动并做功,从而驱动着各种生命活动有序进行。了解生命体高度保守的马达蛋白这一独特而高效的能量转换过程的细致机理,对于理解最本征的生命活动——能量转换是至关重要的,并且成为分子生物学、物理学、生物化学等诸多学科共同面临的一个极具挑战性的科学研究领域。 本论文提出用单分子灵敏度的纳米线生物传感器件,来实时监测免标记的单分子F1-ATPase (F1)动态水解过程。该实验体系主要利用了纳米电子器件超高灵敏度和电学检测超高的时间分辨率优势,并具有免标记长时间检测的特点,可以更真实的在单分子层面研究本征的F1水解ATP的实时动态过程,是成熟的光学检测方法的有益补充。实验过程中我们获取了F1的动态水解过程中诱导器件产生快慢交错的双态电学信号,这种信号对应了单分子F1的实时动态水解过程,进一步我们可以分析数据的分布特征来解析出了F1的γ旋转轴分步旋转步骤。对该体系在不同浓度温度条件下进行长时间的检测,我们获得了本征 F1 的水解动力学速率参数,比常规的荧光检测方法高出约一个数量级。新方法新思路的引入可以使研究者从新的角度去认识、研究分子马达 F1 的能量转换分子机制。这种纳米线微电子传感器件平台也能够拓展应用于其他分子大生物作用体系的实时监测应用和研究。 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.

关键词

硅纳米线;场效应晶体管;单分子检测;F1-ATP合酶

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