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基于原子力显微镜技术的生物大分子与纳米颗粒的力学性质研究
周星飞
Subtype博士
Thesis Advisor沈文庆
2005
Degree Grantor中国科学院上海应用物理研究所
Place of Conferral中国科学院上海应用物理研究所
Degree Discipline粒子物理与原子核物理
Keyword原子力显微镜 Dna与igg蛋白质分子 Ppv纳米颗粒 压弹性
Abstract近年来随着纳米测量技术(如原子力显微镜)的发展使人们能够直接探测甚到桑纵单个生物大分子(像DNA和蛋白质分子),从而能够在单分子水平上来探索生物分子的特异性。目前单个生物大分子的力学性质(弹性或柔性)引起了人们很大的兴趣,这是因为生物大分子一般都具有一定的柔性,即在微小的外力作用下会发生构像的改变,从而可能导致其功能的变化,单分子柔性(弹性)的研究将有助于揭示生物分子结构与功能之间的动力学规律。最近利用纳米探测技术对单个生物分子弹性的研究主要集中在拉伸特性的测量方面,但是拉弹性不能很好地全面反映DNA分子结构和动力学性质的关系。从某种意义上讲,拉弹性(如链状分子DNA的拉伸特性)反映的也是一条链上许多分子的集体行为;在拉伸蛋白分子时,蛋白分子的结构己经远离了其正常构型,从而增加了复杂性,然而单分子的压弹性则能反映单个分子更加局域的力学性质,更好的体现单个分子的特异性。从原理上讲,利用AFM可以测量单个生物大分子的压弹性,而且国际上也有过尝试,但据我们所知,单个DNA分子直径方向的压弹性数据还未见报道,而且利用原子力显微镜的力曲线得到的单个蛋白分子压弹性的数据往往偏大同时又有较大的争议。这主要是受到目前实验仪器的限制,在精度上不能满足测量要求。本论文首先从实验与理论两方面详细介绍了我们最近发展的振动模式扫描极化力显微镜(Vibratingmodescanningpolarizationforcemicroscopy,VSPFM)技术,它能够在几十皮牛到几纳牛范围(精度一0.1nN)内精确测量物体微小的形变(精度一o·In"),非常清合直接测量单个生物分子在小力区(<0·5nN)的压弹性,另外通过改变环境湿度还可以测量生物分子的含水量对其弹性的影响,因此利用vs户FM技术可以为在单个生物大分子局域压弹性的研究中开辟一个新的领域。在介绍VSPFM技术的基础上,我们详细讨论了利用该技术测量单个DNA分子直径方向(与双螺旋方向垂直)的压弹性。研究结果表明同一根DNA分子上各个区域的压弹性差异很大,这可能是由于DNA分子本身结构的差异(碱基对序列不同)也可能是局域的环境不同(如温度、湿度不同)引起的。实验中还发现两根交叉DNA分子的交叉点与比较均匀的单个DNA分子的弹性模量几乎没有差异,说明衬底对测量的影响不大。我们还比较了不经过化学修饰的B一DNA分子与溟化一锭(EB)修饰的DNA分子的压弹性差异,在我们的实验精度范围内没有发现两者的区别。另外冲了直观地描述"DNA分子有多软?",我们试图给DNA分子径向压弹性模量的大小,在论文中我们测量了大约3时良DNA分子的压弹性,估算了其小力区(<0.5nN)的弹性模量。DNA分子压弹性的测量受到很多因素的影响,随着测量精度的提高,以后可以对这些因素进一步深入细致地研究。除了DNA分子压弹性l狗测量以外,论文还讨论了纳米颗粒状物体(即V纳米颗粒与蛋白质分子)的弹性,同时与胶体金颗粒进行了比较,利用赫兹模型计算得到即V纳米颗粒的压弹性模量大约为16士6MI,a,略小于文献报道的即V薄膜的弹,险模量(-50MPa),这可能是由于即v成膜时分子间的侧向支撑使其变得相对较硬。另外,实验结果还表明工gG蛋白质分子要比胶体金颗粒软得多,我们还尝试对一个蛋白分子上不同亚基(亚单元)的压弹性进行分别钡l量,试图通过比较亚基压弹性的差异来实现分子识别。另外,我是宁波大学的在职博士,在论文的最后一章简要介绍了我在博士学习期间从事宁波市纳米实验室建设的情况:我主要负责了原子力显微镜的采购,安装、调试工作,维护其正常的工作。同时也在宁波大学建立了一套VSpl:M系统,今后可以深入的开展生物分子力学性质、水与生物分子相互作用以及水的纳米性质等单分子水平上的各项研究。
Other AbstractThe advance in nanotechnology has made it possible to investigate the dynamic behaviors of single biomolecules (DNA and protein) and directly manipulate it to explore the "molecular individualism". During the past fifteen years, the elastic properties of single biological molecules have attracted tremendous attentions. The understanding of mechanical responses of individual macromolecules under a small external force is of profound importance partly because the induced conformationai transitions can result in the change of biological functions, and partly because, in a language of physics, investigation of single molecules would inspire new concepts in polymer physics, which go far beyond the classical elastic models.Recently, measurement of force and extension on single biomolecules (DNA and protein) has been achieved and revealed the unforeseen structural transitions. However, the stretching properties of DNA are unable to reveal the local dynamic behaviors, for it also describes the collective behaviors of many molecules on one DNA strand. Moreover, when single protein molecule is extracted, its structure would be far from the normal conformation and has little biological means. It is the compression elasticity that can be used to character the local properties of biomolecules and thus the "molecular individualism" can be well rendered.In principle, the compression elasticity can be measured by AFM. However, to our best knowledge, the reasonable data on radial compression elasticity of single DNA molecules has not been reported hitherto probable due to the limitations of current experimental techniques. And the data on compression elasticity of single protein molecules are also disputable.In this thesis, our newly developed vibrating mode scanning polarization force microscopy (VSPFM) is detailedly described from both theoretical and experimental aspects. The biggest advantage of VSPFM is its stable performance. both. in non-contact and tapping modes thus the tip/sample interaction can be well controlled to be from tens pN to several nN, which renders it potentially useful not only for imaging soft specimens but also for measuring the compression elasticity of soft materials.With our VSPFM-based approach, we investigate the compressionelasticity of single DNA molecules in radial direction (perpendicular to the DNAstrand). It shows that the compression properties of single DNA moleculesstrongly depend on the locations along the strand, which was likely due to thelocal structure of DNA (e.g. different base-pair sequence) and localenvironmental conditions (e.g. condensation of irons and humidity). We alsonotice that the compression elasticity of a crossing point on two crossed DNAmolecules is nearly the same as that of single uniform DNA, indicating that thesubstrate had little effect on the measurement. In addition, in order to givepeople direct impression on "How soft a single DNA molecules is", theelasticity modulus of ~ 30 DNA fragments is calculated. The statistical result onradial compression elastic modulus of these DNA molecules is about 30-95MPa, comparable to the Young's modulus of human cartilage (~ 24 MPa). Themeasurement of DNA elasticity is subject to many factors; with theimprovement of experimental precision we can further reveal how thesefactors modify the molecules' mechanical properties.In addition to DNA, the compression elasticity of nanoparticle-like objects (PPV nanoparticle and IgG protein) is also discussed. With the well-known Hertz model the compression elasticity of PPV particle is estimated to be about 16±6 MPa, reasonably lower than that of PPV film (~50 MPa) probably due to the lack of lateral constraint. In this thesis we also try to tell apart the difference in compression elasticity of sub-units of single IgG protein so as to achieve the molecular recognition.Last but not the least, as a staff of Ningbo University I actively take part in the constructions of the nanomaterilas laboratory during the periods of my pursuing Ph.D. In the end of this thesis, the nanomaterilas laboratory of Ningbo University is also briefly introduced. It is worthwhile to stress that AFM in Ningbo University has also been rebuilt into VSPFM system, with which the mechanical properties of biomolecuels, water/biomolecules interaction as well as the nature of water at nanometer scale could be explored.
Language中文
Document Type学位论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/7413
Collection中科院上海应用物理研究所2004-2010年
Recommended Citation
GB/T 7714
周星飞. 基于原子力显微镜技术的生物大分子与纳米颗粒的力学性质研究[D]. 中国科学院上海应用物理研究所. 中国科学院上海应用物理研究所,2005.
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