毛细管电泳-双光子荧光偏振单分子检测装置

Currently, single molecule detection is mainly restricted to the biomolecules located in the solid/liquid interface. However, the complex and the adsorption of the solid/liquid interface definitely impact the behavior of the biomolecules and their interactions. On the basis of our study on the apparatus construction and capillary electrophoresis-laser induced fluorescence polarization analysis, we for the first time propose to develop capillary electrophoresis-two photon fluorescence polarization apparatus. The new apparatus will take advantage of two-photon excitation, allowing us to detect single molecules in aqueous solution rather than in the solid/liquid interface. On the other hand, by the use of fluorescence polarization, the three-dimension resolution for single molecule detection will be enhanced. As well-known facts, the coupled capillary electrophoresis separation will reduce the interferences. Interestingly, following two photon excitation, the wavelengths of the fluorescence is much shorted than that of the excited light, eliminating the light interference resulting from Raman scattering and Rayleigh scattering. Moreover, the process of the two-photon absorbance is more sensitive to the rotation state of the biomolecules, and thus, the two-photon fluorescence polarization may exquisitely reflect the structural and/or functional changes of biomolecules. The apparatus will consist of two photon excitation system equipped with cooling accessary, highly efficient separation system, fluorescence polarization system and signal collection and data acquisition system. The two-photon excitation system will be established using femtosecond, pulsed, model-locked IR Ti:Sapphire Laser. The attenuated energy and broaden pulse of the laser beam for two photon excitation will be overcome using a well-designed feto-second delay reimbursement optics. The built apparatus will be applied to the study of protein-DNA interactions.

目前，由于技术限制，单分子检测主要应用于固液界面处的生物分子分析，而固液界面本身的复杂性和吸附均会影响待测分子的物理化学特性以及它们的相互作用行为，并且难以应用于复杂样品的分析。在此，我们提出研制具有原创性的毛细管电泳-双光子荧光偏振分析装置。采用双光子激发，可实现本体溶液中的单分子检测；采用荧光偏振，可提高单分子检测的分辨率；采用毛细管电泳，可减少复杂基质的干扰。因此，有望解决目前单分子检测的某些局限性。同时，双光子激发所产生荧光的波长更短，可有效地避免各种散射的干扰。更重要的是，双光子吸收对分子的转动状态更为敏感，因而其产生的荧光偏振更为精细地反映生物分子构象和运动状态。我们拟在过去研制毛细管电泳-激光诱导荧光偏振装置的基础上，设计、加工和研制新装置。针对双光子激发系统存在飞秒脉冲光变宽、光强降低的难题，设计和研制先进的飞秒延迟补偿光路。利用研制装置，探讨蛋白质与DNA的相互作用。

按照计划书要求，研制出具有自主知识产权的毛细管电泳-双光子荧光偏振分析装置1套。该装置将毛细管电泳高效分离技术、高灵敏的双光子激光诱导荧光检测技术和分子转动关联的荧光偏振检测技术结合为一体。该装置不仅具有很高的分离效率和检测灵敏度（可达单分子水平），同时由于采用双光子激发模式可以实现飞升级小体积选择性激发，并且能够拓展荧光偏振的测量范围，突破单光子荧光偏振测量极限(Pmax = 0.5)。我们的研究表明，所研制的装置对生物大分子（蛋白质和核酸）的结构、动态构象变化以及相互作用的形成更为敏感。为解决生物化学分析和生物分子相互作用中的前沿问题提供一种先进而有效手段。该装置已应用于蛋白质-DNA相互作用分析研究，探讨了同源重组修复过程中重组酶RecA在单链DNA上如何组装的机理，发现不饱和RecA-ssDNA丝状复合物是介导原核生物同源重组的结构基础，为同源重组研究提出新的思路。另外，这项研究成果也显示了该装置在许多蛋白质-DNA相互作用研究中的重要而广泛的应用前景。通过研制项目，除了获得专用装置及其产权、特色方法和评价数据等科研成果外，已培养出一支具有设备研发能力的专业人才队伍。已发表署名基金号的高水平论文12篇，包括1篇Cell，1篇Cell Discovery, 6篇Anal Chem, 1篇ACS Chem Biol。已完成设定的任务目标，达到了验收的要求，并取得预期成果。培养博士5名、硕士1名，并建立了一支专业队伍。