振动模式扫描极化力显微镜及其在生物大分子测量上的应用
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摘要
本论文阐述了振动模式扫描极化力显微镜(vibrating mode scanning polarization force microscopy,VSPFM)的原理、构造及其在生物大分子(如DNA和抗体分子)测量上的应用。
VSPFM中是在导电的原子力显微镜(atomic force microscopy,AFM)的针尖上加一偏压，由于样品表面的极化，在针尖和样品之间诱导形成电场，这个电场力又称为极化力。与范德华力相比，极化力更强，其作用距离也更远，它叠加在范德华力上，扩大了针尖与样品的距离。与以往的扫描极化力显微镜SPFM的不同之处在于，VSPFM的针尖是在一驱动频率下振动。
本实验通过调节相关参数，如偏压、amplitude setpoint(A_(sp))的大小，实现了针尖高度在极化力介导下的非接触方式和轻敲方式之间的自由切换。在VSPFM的非接触方式中，长程的极化力叠加在范德华力上，这就克服了AFM非接触方式中范德华力力程太短而成像不稳定的缺点；在极化力介导的轻敲方式中，可以用比AFM轻敲模式中最小稳定成像力更小的力进行成像。
VSPFM中，针尖与样品的间距一般在几纳米范围内，范德华力在成像的力中仍占相当一部分；特别是在极化力介导的非接触方式和轻敲方式的转折点处，范德华力还是主要成像力，而极化力起稳定作用。这是与一般的静电力显微镜不同的。
在生物分子的AFM成像上，虽然轻敲模式比接触模式横向剪切力更小，对样品的移动和破坏也更小，但针尖压力仍然存在，使柔软的生物分子发生了很大的变形，成像高度通常小于它们的直径。这种压力实际上是样品原子和针尖原子间的排斥力，所以在AFM轻敲模式中测量柔软样品的高度存在一定问
摘要
题。本文提供了一种新的基于VSPFM的测量衬底上柔软样品高度的方法，可
以在一定程度上消除针尖压力的影响，使测量更为精确。
本文工作采用了VSPFM测量生物分子高度的两种方法:
一是在主扫描中通过改变As。来改变针尖的高度·首先在空白区域定点扫描
图内得到As。的变化值与针尖在z轴上位移的关系;然后在待测样品表面将Asp
的值不断减小，这时针尖就不断趋近样品;从样品的上表面开始到衬底表面结
束，从As。的变化值能够计算出样品的高度·
二是通过调节Lift模式下的扫描高度参数来改变针尖高度。在主扫描中维
持针尖在一个恒定的高度;而在插入扫描中，不断减小扫描高度参数;从样品
的上表面开始到衬底表面结束，扫描高度参数的差值就是样品的高度。
本文应用VSPFM测量生物分子的高度，具有以下优势:静电力的导入扩
大了针尖与样品的距离，使针尖在垂直方向上的移动更容易控制，可以在不同
的高度上稳定成像:因为原子间的排斥力只在几个A的距离内起作用，针尖的
抬升使排斥力迅速减小，也就使样品所受到的压力减小，所以发生的形变也随
之减小，测得的样品高度更接近真实值。在本论文基础上发展出新的测量方法
和测量装置与现有技术相比，装置结构比较简单，使用方便。
本实验在胶体金颗粒的高度测量上，发现VSPFM的结果与AFM轻敲模式
得到的结果类似:而对于DNA分子和抗体分子CA125，在同样的针尖、样品
和环境条件下，测量值比AFM轻敲模式所得到的结果要大得多。不同样品的
结果不同，原因在于胶体金颗粒较为坚硬，在AFM针尖的压力下变形程度小，
而DNA分子和抗体分子比较柔软，在针尖压力下变形程度大。
在高度测量的基础上，VSPFM进一步发展了对生物样品的弹性研究。AFM
摘要
对细胞、细胞器及生物分子的弹性研究己经成为一个热点，但在小分子测量上，
衬底的干扰不可忽略。VSPFM在测量小分子的弹性时，针尖对样品施加的力比
以往的力曲线测量法小得多;在最小力处，针尖压力近似为零。这使得对小分
子的测量更准确，因为此时可以避免衬底的影响。在DNA分子的弹性测量上，
本论文突破了原有的轴向弹性模量测量的局限，尝试进行径向模量的测量。
In this paper, the mechanism and construction of VSPFM are introduced as well as its application on the measurement of biomolecules such as DNAs and antibodies.
VSPFM is a deviation from SPFM. In VSPFM, a conductive AFM tip is biased to a voltage and an electric field arises between the tip and the surface as a result of the polarization of the surface. Compared with the van der Waals attractive force, the electrostatic force (also termed polarization force) is stronger and has a longer range. Tip-sample separation is then increased as a result of the combination of polarization force with van der Waals force. The difference between SPFM and VSPFM is that the biased tip of VSPFM is driven by a mechanical vibration.
By changing variables such as the bias and Asp, the tip can be located from non-contact to tapping region with the assistance of polarization force. In VSPFM non-contact mode, the difficulty of short range of imaging force in AFM is overcome because of the combination of polarization force with van der Waals force. In VSPFM tapping mode, the tip-sample interaction is even smaller than the minimum force in AFM tapping mode stable imaging with the assistance of polarization force.
When tip-sample separation is below several nanometers in VSPFM, van der Waals force constitutes the dominant part of tip-sample interactions especially in the transitional region between non-contact and tapping mode, and the polarization force only assists in stabilizing the imaging. However, in normal Electrostatic Force Microscopy (EFM), electrostatic force is the main force in imaging.
In the application of AFM on bimolecules, the tip pressure still exists in tapping mode and results in great deformation of these soft bimolecules, though the lateral force in tapping mode is much less than that in contact mode. Tip pressure is essentially a result of the repulsive force between the tip and the substrate. The
apparent heights of these molecules in AFM tapping mode are questionable since they are always smaller than their actual diameters. In this paper, we provide a new method for height measurement of soft samples on substrates by VSPFM, in which tip pressure can be decreased to a certain extent and the measurement will be more precise.
There are two ways in height measurement by VSPFM:
One is to change tip-sample separation by Asp. Firstly, the relationship between AAsp and DZ0 (vertical displacement of the tip in zero-scale scan) can be calculated and calibrated in zero-scale scan (scan size is 0 nm). Then the tip is positioned on a molecule and approaches to the surface with the decrease of Asp. From the top of the molecule to the substrate, the vertical displacement of the tip can be calculated from AAsp and is close to the true height of this molecule.
The other is to change tip-sample separation by altering the parameter of lift scan height in lift mode. The height of the tip is maintained in main scan, and in interleave scan, the tip is lowered from the top of the molecule to the substrate with the decrease of lift scan height. The difference in the parameter of lift scan height approximates the true height of this molecule.
The height measurement carried out by VSPFM has some advantages. The vertical movement of the tip is more controllable with the assistance of long-range polarization force, and stable images can be obtained at different tip-sample seperation. With the lift of the tip, repulsive force (tip pressure) decreases rapidly because its effective range is only several angstroms and the deformation is reduced consistently. The new method and apparatus of the measurement derived from this paper is simple and accessible compared with the existing techniques.
The measured heights of colloidal gold particles by VSPFM are almost the same as
those in TM-AFM, but the results of dsDNA and antibodies behave differently even under the same conditions such as tips and samples. A possible reason is that the tip pressure in AFM tapping mode results in large deformation on soft DNA and antibodies b
VSPFM中是在导电的原子力显微镜(atomic force microscopy,AFM)的针尖上加一偏压，由于样品表面的极化，在针尖和样品之间诱导形成电场，这个电场力又称为极化力。与范德华力相比，极化力更强，其作用距离也更远，它叠加在范德华力上，扩大了针尖与样品的距离。与以往的扫描极化力显微镜SPFM的不同之处在于，VSPFM的针尖是在一驱动频率下振动。
本实验通过调节相关参数，如偏压、amplitude setpoint(A_(sp))的大小，实现了针尖高度在极化力介导下的非接触方式和轻敲方式之间的自由切换。在VSPFM的非接触方式中，长程的极化力叠加在范德华力上，这就克服了AFM非接触方式中范德华力力程太短而成像不稳定的缺点；在极化力介导的轻敲方式中，可以用比AFM轻敲模式中最小稳定成像力更小的力进行成像。
VSPFM中，针尖与样品的间距一般在几纳米范围内，范德华力在成像的力中仍占相当一部分；特别是在极化力介导的非接触方式和轻敲方式的转折点处，范德华力还是主要成像力，而极化力起稳定作用。这是与一般的静电力显微镜不同的。
在生物分子的AFM成像上，虽然轻敲模式比接触模式横向剪切力更小，对样品的移动和破坏也更小，但针尖压力仍然存在，使柔软的生物分子发生了很大的变形，成像高度通常小于它们的直径。这种压力实际上是样品原子和针尖原子间的排斥力，所以在AFM轻敲模式中测量柔软样品的高度存在一定问
摘要
题。本文提供了一种新的基于VSPFM的测量衬底上柔软样品高度的方法，可
以在一定程度上消除针尖压力的影响，使测量更为精确。
本文工作采用了VSPFM测量生物分子高度的两种方法:
一是在主扫描中通过改变As。来改变针尖的高度·首先在空白区域定点扫描
图内得到As。的变化值与针尖在z轴上位移的关系;然后在待测样品表面将Asp
的值不断减小，这时针尖就不断趋近样品;从样品的上表面开始到衬底表面结
束，从As。的变化值能够计算出样品的高度·
二是通过调节Lift模式下的扫描高度参数来改变针尖高度。在主扫描中维
持针尖在一个恒定的高度;而在插入扫描中，不断减小扫描高度参数;从样品
的上表面开始到衬底表面结束，扫描高度参数的差值就是样品的高度。
本文应用VSPFM测量生物分子的高度，具有以下优势:静电力的导入扩
大了针尖与样品的距离，使针尖在垂直方向上的移动更容易控制，可以在不同
的高度上稳定成像:因为原子间的排斥力只在几个A的距离内起作用，针尖的
抬升使排斥力迅速减小，也就使样品所受到的压力减小，所以发生的形变也随
之减小，测得的样品高度更接近真实值。在本论文基础上发展出新的测量方法
和测量装置与现有技术相比，装置结构比较简单，使用方便。
本实验在胶体金颗粒的高度测量上，发现VSPFM的结果与AFM轻敲模式
得到的结果类似:而对于DNA分子和抗体分子CA125，在同样的针尖、样品
和环境条件下，测量值比AFM轻敲模式所得到的结果要大得多。不同样品的
结果不同，原因在于胶体金颗粒较为坚硬，在AFM针尖的压力下变形程度小，
而DNA分子和抗体分子比较柔软，在针尖压力下变形程度大。
在高度测量的基础上，VSPFM进一步发展了对生物样品的弹性研究。AFM
摘要
对细胞、细胞器及生物分子的弹性研究己经成为一个热点，但在小分子测量上，
衬底的干扰不可忽略。VSPFM在测量小分子的弹性时，针尖对样品施加的力比
以往的力曲线测量法小得多;在最小力处，针尖压力近似为零。这使得对小分
子的测量更准确，因为此时可以避免衬底的影响。在DNA分子的弹性测量上，
本论文突破了原有的轴向弹性模量测量的局限，尝试进行径向模量的测量。
In this paper, the mechanism and construction of VSPFM are introduced as well as its application on the measurement of biomolecules such as DNAs and antibodies.
VSPFM is a deviation from SPFM. In VSPFM, a conductive AFM tip is biased to a voltage and an electric field arises between the tip and the surface as a result of the polarization of the surface. Compared with the van der Waals attractive force, the electrostatic force (also termed polarization force) is stronger and has a longer range. Tip-sample separation is then increased as a result of the combination of polarization force with van der Waals force. The difference between SPFM and VSPFM is that the biased tip of VSPFM is driven by a mechanical vibration.
By changing variables such as the bias and Asp, the tip can be located from non-contact to tapping region with the assistance of polarization force. In VSPFM non-contact mode, the difficulty of short range of imaging force in AFM is overcome because of the combination of polarization force with van der Waals force. In VSPFM tapping mode, the tip-sample interaction is even smaller than the minimum force in AFM tapping mode stable imaging with the assistance of polarization force.
When tip-sample separation is below several nanometers in VSPFM, van der Waals force constitutes the dominant part of tip-sample interactions especially in the transitional region between non-contact and tapping mode, and the polarization force only assists in stabilizing the imaging. However, in normal Electrostatic Force Microscopy (EFM), electrostatic force is the main force in imaging.
In the application of AFM on bimolecules, the tip pressure still exists in tapping mode and results in great deformation of these soft bimolecules, though the lateral force in tapping mode is much less than that in contact mode. Tip pressure is essentially a result of the repulsive force between the tip and the substrate. The
apparent heights of these molecules in AFM tapping mode are questionable since they are always smaller than their actual diameters. In this paper, we provide a new method for height measurement of soft samples on substrates by VSPFM, in which tip pressure can be decreased to a certain extent and the measurement will be more precise.
There are two ways in height measurement by VSPFM:
One is to change tip-sample separation by Asp. Firstly, the relationship between AAsp and DZ0 (vertical displacement of the tip in zero-scale scan) can be calculated and calibrated in zero-scale scan (scan size is 0 nm). Then the tip is positioned on a molecule and approaches to the surface with the decrease of Asp. From the top of the molecule to the substrate, the vertical displacement of the tip can be calculated from AAsp and is close to the true height of this molecule.
The other is to change tip-sample separation by altering the parameter of lift scan height in lift mode. The height of the tip is maintained in main scan, and in interleave scan, the tip is lowered from the top of the molecule to the substrate with the decrease of lift scan height. The difference in the parameter of lift scan height approximates the true height of this molecule.
The height measurement carried out by VSPFM has some advantages. The vertical movement of the tip is more controllable with the assistance of long-range polarization force, and stable images can be obtained at different tip-sample seperation. With the lift of the tip, repulsive force (tip pressure) decreases rapidly because its effective range is only several angstroms and the deformation is reduced consistently. The new method and apparatus of the measurement derived from this paper is simple and accessible compared with the existing techniques.
The measured heights of colloidal gold particles by VSPFM are almost the same as
those in TM-AFM, but the results of dsDNA and antibodies behave differently even under the same conditions such as tips and samples. A possible reason is that the tip pressure in AFM tapping mode results in large deformation on soft DNA and antibodies b
引文
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