基于原子力显微镜的高效率DNA纳米操纵系统设计与应用
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摘要
本论文分析了目前在基于原子力显微镜的纳米操纵,尤其是DNA单分子操纵中,影响效率和精度的主要因素。通过设计有针对性的解决方案,实现了高效率高精度的单分子纳米操纵,并提出了一种智能化的操纵策略。在此基础上,本论文对DNA纳米操纵的机制进行了初步探索,通过建立DNA操纵过程模型,为解释实验中遇到的各种现象提供了依据。
纳米操纵技术是上世纪80年代末期随着扫描探针显微技术的发展而发展起来的新兴科学技术,它使得真正意义上的单原子/分子研究成为可能,在半导体、材料、化学、医学等领域都展现出了巨大的前景和价值。其中,原子力显微镜由于其对样品和环境条件几乎没有限制,并且能够对样品施加多种相互作用,成为单分子纳米操纵的主要工具。在生物医学领域,传统的生化分析方法通常是基于大量分子的统计信息,失去了“空间分辨率”。原子力显微镜能够实现单分子分析,在纳米尺度上直接观测生物分子及其相互作用,对于揭示各种重要生命过程的本质以及促进新一代个性化医疗方法具有重要的意义。例如对DNA分子的纳米操纵有助于单分子序列信息的研究,通过机械力拉伸操纵能够研究DNA分子折叠结构的力学信息,对DNA的机械力操纵还有助于了解机械力对于特定序列突变的诱导等等。
然而,目前原子力显微镜纳米操纵的效率还比较低,其原因主要是:
(1)原子力显微镜针尖既是成像工具又是操纵工具。因此在操纵过程中需要不断的在操纵模式和成像模式之间切换,耗费大量的操作和时间;此外原子力显微镜栅格扫描模式本身也存在成像速度较慢的问题;
(2)设备本身存在热漂移。热漂移是设备部件由于温度波动导致的微小形变(膨胀/收缩),是一种普遍存在的现象。在宏观环境中,热漂移的影响并不明显,但在纳米操纵过程中,热漂移对操纵准确性的影响不可忽略,必须发展快速有效的热漂移的补偿方法,提高操纵的精度。
(3)纳米操纵过程中,针尖与分子的相互作用非常复杂,包括范德华力、毛吸力、静电力、斥力、甚至化学键作用,使得通过模型进行定量分析相当困难,无法将已有的自动化技术直接应用于纳米操纵过程;例如,针尖拾取纳米颗粒是依靠非特异性的吸附力,主要是范德华力和毛细力。这两种力均与环境的湿度条件密切相关,但是环境湿度如何影响吸附力还没有一个明确的理论可供参照。
针对上述问题,本论文的研究内容主要可以分为以下几个方面:
(1)根据不同的操纵目的,如DNA操纵、纳米颗粒操纵和纳米刻蚀,分别设计了高效率的针尖移动方式,结合原子力显微镜设备提供的开放接口,实现了大范围成像与小范围(局域化)操纵兼容的问题,有效消除了操纵过程中扫描范围的切换,从而简化连续操纵的步骤,提高操纵的效率。
(2)发展了一套基于“图像配准”的原子力显微镜热漂移补偿技术,并通过引入最小二乘估计和卡尔曼滤波器等方法实现了软件方式的亚像素精度热漂移补偿,提高了操纵的精度。此外,该方法还能够用于校正由热漂移引起的原子力显微镜图像失真,提高图像测量的准确性。
(3)发展了一套针对低信噪比原子力显微镜图像的图像识别改进算法并成功应用于DNA和纳米颗粒的操纵,进一步提高了操纵的效率,为自动化及智能化的纳米操纵提供了一种可行的策略。
(4)结合一些较为公认的纳米尺度相互作用模型对原子力显微镜纳米操纵,尤其是DNA分子操纵的物理过程和机制进行了初步的探讨,并分析了适用于纳米操纵的APTES衬底的性质,为解释实验中的各种现象提供了依据。
基于原子力显微镜的高效率纳米操纵的研究目前尚处于起步阶段,对于生物单分子高效率操纵的研究更不多见。本论文在大量实验总结的经验基础上,对提高基于原子力显微镜的DNA纳米操纵效率和精度做出了有益的尝试。文中涉及的研究内容和发展的方法策略有助于原子力显微镜在生物大分子操纵领域的广泛应用,对单分子水平上DNA、RNA、病毒颗粒的研究具有重要意义。
The thesis focuses on improving the efficiency and precision of nano-manipulation, especially single DNA manipulation, with Atomic Force Microscope (AFM). A high efficiency nano-manipulation system has been realized, and an intelligent manipulation strategy has been developed. For the explanation of experimental results, models of DNA manipulation processes have also been developed, and the manipulation mechanics therein have been discussed.
Nano-manipulation technique was introduced with the development of Scanning Probe Microscopy (SPM) in 1980s. It enables‘true’single atoms/molecules research for the first time, and shows great potential in the fields of semi-conductors, materials, chemistry, medical etc. Among the SPM family, the AFM has become the most popular tool for single molecule manipulation applications, as there are almost no limitation for samples and environment conditions. For example, in bio-medical researches, traditional biochemical analysis is based on statistic information of large amount of molecules, losing the“spacial resolution”. The atomic force microscope could observe and manipulate directly single molecules and their interactions, thus essential for revealing important life secrets and accelerate next-generation“personalized medical care”. Such as the mechanical properties of DNA folding structures could be studied by nano stretching, and mechanical interaction induced mutation could be systematically analyzed.
However, current efficiency of AFM manipulation is not satisfactory, mainly because of the following reasons:
(1) The AFM tip is not only for imaging but also for manipulation. Therefore, during manipulation, one has to frequently switch between imaging mode and manipulation mode, costing a lot of time and operations. Besides, the AFM scanning is inherently slow due to the raster scanning pattern.
(2) There are thermal drifts. Thermal drift is the small deformation of instruments due to thermal fluctuations. It is not obvious in the macro world, however, it matters in the nano scale, and must be compensated during manipulation for high precision.
(3) The tip-sample interactions during manipulation are complicated. There are van der Waals force, capillary force, electrostatic forces, chemistry force etc., making the understanding of manipulation process very difficult.
To address above issues, the main topics of the thesis includes the following aspects:
(1) To improve manipulation efficiency. Designed high efficiency tip movements according to different manipulation purposes. Simplified the manipulation processes by eliminating mode switching through localized manipulation.
(2) To improve manipulation precision. Developed a thermal drift compensation method based on image registration. Realized sub-pixel compensation precision by introducing Least Square Estimation and Kalman Filter Estimaiton. Besides, this compensation method could also be used to calibrate AFM image distortion caused by thermal drift during scanning.
(3) To further improve the efficiency. Developed an image recognition algorithm for low signal-to-noise ratio AFM images, e.g. DNA image, and successfully applied in nano-manipulation, providing an automatic and intelligent manipulation stragetgy.
(4) Developed models of DNA manipulation processes. Discussed important factors in nano-manipulation.
Though the nano-manipulation with AFM has demonstrated great potential, there are few studies on improving the manipulation efficiency, especially for biomolecule manipulations. The developed methods and models in the thesis could shed some lights for high efficiency AFM manipulation, thus facilitating the application of this single molecule manipulation strategy. Keywords: Atomic Force Microscopy, DNA, Nano-manipulation, Automatization
纳米操纵技术是上世纪80年代末期随着扫描探针显微技术的发展而发展起来的新兴科学技术,它使得真正意义上的单原子/分子研究成为可能,在半导体、材料、化学、医学等领域都展现出了巨大的前景和价值。其中,原子力显微镜由于其对样品和环境条件几乎没有限制,并且能够对样品施加多种相互作用,成为单分子纳米操纵的主要工具。在生物医学领域,传统的生化分析方法通常是基于大量分子的统计信息,失去了“空间分辨率”。原子力显微镜能够实现单分子分析,在纳米尺度上直接观测生物分子及其相互作用,对于揭示各种重要生命过程的本质以及促进新一代个性化医疗方法具有重要的意义。例如对DNA分子的纳米操纵有助于单分子序列信息的研究,通过机械力拉伸操纵能够研究DNA分子折叠结构的力学信息,对DNA的机械力操纵还有助于了解机械力对于特定序列突变的诱导等等。
然而,目前原子力显微镜纳米操纵的效率还比较低,其原因主要是:
(1)原子力显微镜针尖既是成像工具又是操纵工具。因此在操纵过程中需要不断的在操纵模式和成像模式之间切换,耗费大量的操作和时间;此外原子力显微镜栅格扫描模式本身也存在成像速度较慢的问题;
(2)设备本身存在热漂移。热漂移是设备部件由于温度波动导致的微小形变(膨胀/收缩),是一种普遍存在的现象。在宏观环境中,热漂移的影响并不明显,但在纳米操纵过程中,热漂移对操纵准确性的影响不可忽略,必须发展快速有效的热漂移的补偿方法,提高操纵的精度。
(3)纳米操纵过程中,针尖与分子的相互作用非常复杂,包括范德华力、毛吸力、静电力、斥力、甚至化学键作用,使得通过模型进行定量分析相当困难,无法将已有的自动化技术直接应用于纳米操纵过程;例如,针尖拾取纳米颗粒是依靠非特异性的吸附力,主要是范德华力和毛细力。这两种力均与环境的湿度条件密切相关,但是环境湿度如何影响吸附力还没有一个明确的理论可供参照。
针对上述问题,本论文的研究内容主要可以分为以下几个方面:
(1)根据不同的操纵目的,如DNA操纵、纳米颗粒操纵和纳米刻蚀,分别设计了高效率的针尖移动方式,结合原子力显微镜设备提供的开放接口,实现了大范围成像与小范围(局域化)操纵兼容的问题,有效消除了操纵过程中扫描范围的切换,从而简化连续操纵的步骤,提高操纵的效率。
(2)发展了一套基于“图像配准”的原子力显微镜热漂移补偿技术,并通过引入最小二乘估计和卡尔曼滤波器等方法实现了软件方式的亚像素精度热漂移补偿,提高了操纵的精度。此外,该方法还能够用于校正由热漂移引起的原子力显微镜图像失真,提高图像测量的准确性。
(3)发展了一套针对低信噪比原子力显微镜图像的图像识别改进算法并成功应用于DNA和纳米颗粒的操纵,进一步提高了操纵的效率,为自动化及智能化的纳米操纵提供了一种可行的策略。
(4)结合一些较为公认的纳米尺度相互作用模型对原子力显微镜纳米操纵,尤其是DNA分子操纵的物理过程和机制进行了初步的探讨,并分析了适用于纳米操纵的APTES衬底的性质,为解释实验中的各种现象提供了依据。
基于原子力显微镜的高效率纳米操纵的研究目前尚处于起步阶段,对于生物单分子高效率操纵的研究更不多见。本论文在大量实验总结的经验基础上,对提高基于原子力显微镜的DNA纳米操纵效率和精度做出了有益的尝试。文中涉及的研究内容和发展的方法策略有助于原子力显微镜在生物大分子操纵领域的广泛应用,对单分子水平上DNA、RNA、病毒颗粒的研究具有重要意义。
The thesis focuses on improving the efficiency and precision of nano-manipulation, especially single DNA manipulation, with Atomic Force Microscope (AFM). A high efficiency nano-manipulation system has been realized, and an intelligent manipulation strategy has been developed. For the explanation of experimental results, models of DNA manipulation processes have also been developed, and the manipulation mechanics therein have been discussed.
Nano-manipulation technique was introduced with the development of Scanning Probe Microscopy (SPM) in 1980s. It enables‘true’single atoms/molecules research for the first time, and shows great potential in the fields of semi-conductors, materials, chemistry, medical etc. Among the SPM family, the AFM has become the most popular tool for single molecule manipulation applications, as there are almost no limitation for samples and environment conditions. For example, in bio-medical researches, traditional biochemical analysis is based on statistic information of large amount of molecules, losing the“spacial resolution”. The atomic force microscope could observe and manipulate directly single molecules and their interactions, thus essential for revealing important life secrets and accelerate next-generation“personalized medical care”. Such as the mechanical properties of DNA folding structures could be studied by nano stretching, and mechanical interaction induced mutation could be systematically analyzed.
However, current efficiency of AFM manipulation is not satisfactory, mainly because of the following reasons:
(1) The AFM tip is not only for imaging but also for manipulation. Therefore, during manipulation, one has to frequently switch between imaging mode and manipulation mode, costing a lot of time and operations. Besides, the AFM scanning is inherently slow due to the raster scanning pattern.
(2) There are thermal drifts. Thermal drift is the small deformation of instruments due to thermal fluctuations. It is not obvious in the macro world, however, it matters in the nano scale, and must be compensated during manipulation for high precision.
(3) The tip-sample interactions during manipulation are complicated. There are van der Waals force, capillary force, electrostatic forces, chemistry force etc., making the understanding of manipulation process very difficult.
To address above issues, the main topics of the thesis includes the following aspects:
(1) To improve manipulation efficiency. Designed high efficiency tip movements according to different manipulation purposes. Simplified the manipulation processes by eliminating mode switching through localized manipulation.
(2) To improve manipulation precision. Developed a thermal drift compensation method based on image registration. Realized sub-pixel compensation precision by introducing Least Square Estimation and Kalman Filter Estimaiton. Besides, this compensation method could also be used to calibrate AFM image distortion caused by thermal drift during scanning.
(3) To further improve the efficiency. Developed an image recognition algorithm for low signal-to-noise ratio AFM images, e.g. DNA image, and successfully applied in nano-manipulation, providing an automatic and intelligent manipulation stragetgy.
(4) Developed models of DNA manipulation processes. Discussed important factors in nano-manipulation.
Though the nano-manipulation with AFM has demonstrated great potential, there are few studies on improving the manipulation efficiency, especially for biomolecule manipulations. The developed methods and models in the thesis could shed some lights for high efficiency AFM manipulation, thus facilitating the application of this single molecule manipulation strategy. Keywords: Atomic Force Microscopy, DNA, Nano-manipulation, Automatization
引文
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