紫细菌光合蛋白纳米仿生膜及器件研究
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摘要
开发和应用各类光电功能材料，实现稳定、高效和绿色地利用太阳能是人类一直追求的梦想，也是解决各类能源问题的有效途径。另一方面，承担了地球上几乎所有生物能量来源和生命基础的光合作用因具有任何现有的人工材料或器件所无法相比的高效光电转换能力而倍受关注。近年来，针对从光合紫红细菌中分离纯化出来的，作为细菌光合过程中能够产生光致电荷分离的最小结构单元的，反应中心复合体(reaction center，简称RC)的结构和功能研究已经取得了重大进展。在对RC原初光物理和光化学过程深入理解的基础上，通过建立和运用各种先进的材料制备技术、定点的蛋白变异和基因工程、分析探索的新方法，实现高效光敏蛋白复合光电材料的研制，以及仿生膜内蛋白激发态弛豫过程的揭示，兼具了重要的理论研究价值和实际应用前景。
围绕稳定、高效的新型功能化RC纳米仿生膜的开发探索，尤其在提高基体电极上蛋白的负载，促进蛋白向基体电极的光致电子注入，控制蛋白在基体电极表面的有利方位，以及揭示蛋白复杂、快速和有效的能量／电子传递过程等方面，我们开展了一系列循序渐进的创新性工作，包括：
1、利用阳极氧化水解法制备的具有多孔结构特征的纳米晶TiO_2薄膜吸附光敏蛋白实现了RC仿生膜光电极的制备。RC在近红外区的高效捕光功能与TiO_2良好的光致电荷分离能力相结合，明显改善了仿生膜光电极的光电转化效率。
2、创新性地建立了针对RC裁剪设计的新型介孔WO_3-TiO_2膜修饰固定光敏蛋白制备仿生膜光电极的方法。首次采用具有开放孔道、均一孔径分布和理想亲水性的新型介孔WO_3-TiO_2实现了保持RC生物活性前提下对蛋白的高效捕获。利用该介孔WO_3-TiO_2能级的匹配性促进RC在光照后电子向纳米半导体电极的注入，有效降低了分离纯化后RC自身的电子传递及电荷重组对蛋白光电转换的负面影响。
3、通过对RC内色素的特定替换改变辅助因子的电荷分离态能级，进一步削弱光敏蛋白在介孔WO_3-TiO_2电极上受激后自身的电子传递及电荷重组。所获得的变异RC／WO_3-TiO_2仿生膜表现出前所未有的高效光电转换能力(相比于现有的其它RC光电极)。
4、采用自组装技术首次在基于特定设计的功能化金胶表面实现了RC的修饰。通过控制RC在金胶表面的有利取向和方位，并利用金胶粒子对电子存储和穿梭表现出的优良特性，尝试了从另一个角度研究与开发高效的光敏蛋白纳米仿生光电材料。
5、开创性地搭建了现场飞秒泵浦—探测(femtosecond pump-probe)／电化学联用技术平台，发展了探索分析光合色素—蛋白超快能量与电子传递过程的新方法。首次报道了对电化学氧化引起的紫细菌外周天线蛋白(light-harvesting complex 2，简称LH2)的快速能量陷获研究。
具体地，论文由以下六个部分构成：
一、绪论
围绕论文的主题，本章节首先明确了课题研究的重要意义。之后，概括地阐述了有关RC结构和功能的背景信息。作为本章的核心内容，RC功能性纳米仿生膜的构建及其光致电子传递的相关研究现状被作了重点介绍，对相应的文献也进行了较全面的综述。最后简要归纳了论文的主要创新性。
二、电沉积纳米晶TiO_2固定RC制备仿生膜光电极的方法与研究
本章节详细介绍了采用阳极氧化水解法合成的具有多孔结构特征的纳米晶TiO_2薄膜吸附RC制备功能性仿生膜光电极的相关研究工作。ITO／TiO_2／RC仿生膜光电极的近红外—可见吸收光谱、荧光光谱测试结果表明了修饰在纳米TiO_2薄膜上的光敏蛋白仍然保持了原有的生物活性。RC在仿生膜光电极内展现出特有的，可重现的近红外光电响应行为，其在长波长区的高效捕光功能起到了敏化纳米TiO_2的作用，提高了纳米TiO_2电极的光电转换效率。纳米半导体材料与光敏蛋白的结合既拓宽了TiO_2对太阳光的吸收利用又促进了RC的光致电荷分离，显著改善了整个光电极的光电转化效率，为研究与开发生物光电器件提供了新的借鉴。然而，同一批八通道制备的ITO／TiO_2／RC仿生膜光电极的光电流检测结果则反映出由不规则的纳米TiO_2晶粒间空隙组成的孔道在对RC的高效捕获上仍存在着较大缺陷。
三、剪裁设计的新型三维虫洞介孔WO_3-TiO_2固定RC制备仿生膜光电极的方法与研究
本部分工作的侧重点在于针对光敏蛋白的高效捕获及其光致电荷分离的有效促进，建立了采用剪裁设计的新型介孔WO_3-TiO_2膜修饰固定RC制备仿生膜光电极的方法。通过对不同孔径、结构和组成的材料在吸附RC效果上的比较表明：根据“酸碱对”概念剪裁制备的，具有开放孔道、均一孔径分布(孔径中心尺寸～7.1 mm)和理想亲水性的三维虫洞介孔WO_3-TiO_2膜对于保持RC生物活性前提下的蛋白捕获最为高效(RC负载量：0.63μmol／g)。ITO／WO_3-TiO_2／RC仿生膜光电极的荧光和光电化学测量结果证实：WO_3-TiO_2复合材料高于单一组分的光致电子空穴对分离能力以及与光敏蛋白能级的匹配性能够有效促进受激后RC向纳米半导体电极的电子注入，降低分离后RC自身的电子传递及电荷重组对仿生膜光电极光电转换的负面影响。利用新型介孔半导体材料与光敏蛋白的结合实现了RC仿生膜光电极对太阳光能尽可能高效的吸收利用，为各种功能性生物光电器件的设计迈出了坚实的一步。
四、细菌脱镁叶绿素(BPhe)被植物脱镁叶绿素(Phe)替换的RC变异体／三维虫洞介孔WO_3-TiO_2仿生膜光电极的制备与研究
承接上一章的工作，本部分主要进行了将RC的色素替换变异株(Phe-RC)应用于构建仿生膜光电极的考察和研究，目的在于深入探索修饰在电极表面RC色素替换体的复杂多途径光致电子传递过程，并在此基础上尝试开发更高效的生物光电器件。近红外—可见吸收光谱和圆二色谱测试结果表明了色素替换的成功(替换效率不低于95％)。荧光发射，飞秒泵浦—探测和电化学实验的结果揭示了电荷分离态P~+Phe~-相比P~+BPhe~-更高的自由能能级，以及由此导致的1)Phe-RC自身能量／电子传递的明显受阻；2)B~*(细菌叶绿素单体激发态)，P~*(细菌叶绿素二聚体激发态)和P~+(细菌叶绿素二聚体氧化态)相比天然RC中对应物种寿命的显著增加。光电化学的测量结果进一步显示，电子传递的受阻加之介孔WO_3-TiO_2与蛋白能级的匹配性使得被WO_3-TiO_2膜捕获的Phe-RC向半导体导带的电子注入有了进一步增强，更有效地促进了仿生膜光电极电子—空穴对的分离，光电转换效率得到了极大地提高(IPCE_(800 nm)=～23％)。
五、功能化金胶控制RC在自组装仿生体系内有利方位的初步研究
本章节主要开展了在功能化的金胶表面组装RC分子并控制有利蛋白方位的相关初步研究工作。通过在合成的金胶纳米粒子上引入特定的双功能试剂及组装上带有特定官能团的分子后，可以实现光敏蛋白在金胶表面采取不同的方位进行排列(原初电子给体P／电子受体泛醌(Q_A)朝着金胶的方向)。紫外—可见吸收光谱、X射线光电子能谱、傅立叶红外光谱，以及透射电子显微镜的相关结构表征结果反映了功能化金胶的成功制备；近红外—可见吸收光谱和圆二色谱的相关检测结果则进一步证实了RC在功能化金胶表面组装的有效实现；RC／金胶自组装仿生体系的荧光和飞秒泵浦—探测实验结果揭示了修饰在不同功能化金胶表面的光敏蛋白在激发能弛豫过程和方式上的大相径庭，以及RC在受激后向金胶粒子明显的电子注入现象，暗示了控制RC在金胶表面有利方位的成功实现。利用金胶纳米颗粒优良的电子存储和穿梭特性结合有利于电荷分离和电子注入的蛋白方位的控制提供了又一设计和开发高效生物光电器件的有效途径。
六、电化学诱导的紫细菌天线色素复合物(LH2)的超快激子能弛豫研究
本章主要报道了发展现场飞秒泵浦—探测／电化学联用技术应用于电化学氧化引起的LH_2快速能量陷获的相关研究。采用近红外—可见／荧光光谱电化学方法结合超快泵浦—探测光谱电化学技术实时在线地考察了LH2内B800(细菌叶绿素单体)和B850(细菌叶绿素二聚体)不同的电化学氧化过程；色素—色素、色素—蛋白之间伴随氧化而发生的结构变化；以及电化学氧化对B800和B850参与的超快激发能弛豫的影响。结果表明：1、B800和B850环中的叶绿素分子具有几乎相同的氧化还原中点电位却表现出完全不同的电化学氧化速率(B800快于B850)。2、伴随叶绿素分子电化学氧化的进行，激发B800观察到B850自发辐射淬灭的显著增强(荧光峰下降速率明显快于吸收峰下降速率)；3、B800向B850的快速能量传递过程几乎不受电化学氧化的影响，但电化学氧化引起了B850激发能的快速陷获。现场飞秒泵浦—探测／电化学联用技术的建立不仅提供了深入分析光合色素—蛋白超快能量与电子传递的新途径，更是对现有光谱电化学技术的补充和丰富，为研究和探索复杂体系内伴随电子得失而发生的各种超快动力学变化开辟了新的方向。
Creation and exploitation of stable, high-efficient, and also environmentally friendly photoelectric materials or devices for a better utilization of solar energy is one of the long-term pursuits for human beings that might suffer from the serious problems of oil exhaustion. Photosynthesis, which has played an important role as both bio-energy source and life basis, is much more superior to the artificial apparatus on photoelectric conversion performance and has attracted great concerns. Recently, significant advancements have been achieved on both the structural and functional research of the purple bacteria photosynthetic reaction center (RC), a transmembrane pigment-protein complex that is the smallest unit in bacteria photosynthetic membrane able to perform a light-driven charge separation. It is with great value for both the theoretical study and applicable try to design effective photoelectric apparatus based on RC and fully reveal the excitation relaxation processes of the proteins encased within the functionalized films, which depends on the preparation of advanced electrode materials, introduction of site-directed protein mutation as well as genetic engineering, and establishment of new methodology for analysis and probing.
Aimed at exploiting and developing the stable, high-efficient novel RC-functionalized nanofilms, a series of original work has been done especially on increasing the protein loading, promoting the photo-induced electron injection, manipulating the favorable protein orientation, and revealing the complex, ultrafast, and effective photo-induced energy/electron transfer:
1. A new kind of bio-nanocomposite photoelectrode (PE) was fabricated through direct immobilization of RC on a porous nanocrystalline TiO_2 matrix prepared by the anodic oxidative hydrolysis method. Combination of the high-efficient near-infrared (NIR) light-harvesting faculty of RC and the well performance of TiO_2 on the photo-induced charge separation led to a better photoelectric conversion efficiency of the derived bio-photoelectrode (Bio-PE) as compared with that of the separate component.
2. Novel 3D-worm-like mesoporous WO_3-TiO_2 films with tailored pore size were synthesized and applied to prepare the Bio-PEs through direct entrapping the RC molecules. These mesoporous WO_3-TiO_2 films exhibited unique characteristics, e.g. opened mesostructure, narrow-distributed pore size well-matching one 2D dimension of RC, and ideal hydrophilicity, in the specific loading of RC with high activity
retained. Thanks to the higher capability of WO_3-TiO_2 in splitting photo-induced electron hole pairs than that of the single oxide, enhanced photo-induced electron injection from photo-excited RC to the WO_3-TiO_2 matrix decreased the disadvantages of electron transfer and charge recombination within the proteins.
3. The disadvantages of electron transfer and charge recombination within RC itself were further conquered by means of the specific protein pigment exchange, which resulted in energy level change of the protein charge-separated state. The prepared Bio-PE composed of the pigment-exchanged proteins and the mesoporous WO_3-TiO_2 matrix exhibited remarkably enhanced photoelectric performances as compared with those RC PEs reported before.
4. Successful modification of RC was achieved on the surface of functionalized Au colloid through self-assembling. Favorable protein orientation manipulated and the unique property of Au colloid in storing and shuttling the electrons contributed to the new concept of research and development of high-efficient Bio-PE materials.
5. In situ femtosecond (fs) pump-probe spectroelectrochemistry methodology was developed for the first time for probing the ultrafast energy/electron transfer process inside the pigment-protein complex. The ultrafast excitation energy trapping dynamics of purple bacteria peripheral light-harvesting complexes (LH2) induced by electrochemical oxidation was investigated and reported particularly.
Detailedly, the dissertation consists of 6 chapters that tightly relate to the main topic.
1) Introduction
In this chapter, firstly, significance of the topic discussed here was briefly described. Then, the research background about the structure and function of RC was introduced and illustrated. After that, strategies for construction and fabrication of well-defined RC-functionalized nanocomposite films for probing and exploiting the photo-induced electron transfer of RC were classified and summarized mainly including five categories, and the related literatures were well-reviewed. Finally, the main novelty and innovation of the study were provided.
2) Bio-PE composed of the RC proteins adsorbed on a nanocrystalline TiO_2 film prepared by anodic electrodeposition
In this part, RC-functionalized nanocomposite PE prepared through adsorption of
the photosensitive proteins on a porous nanocrystalline TiO_2 film synthesized by the anodic oxidative hydrolysis method was studied and reported. The NIR-visible (Vis) absorption spectra and fluorescence emission spectra displayed that the native activity was well-remained for RC immobilized on the TiO_2 matrix. Obvious reproducible photoelectric responses dominated by the adsorbed RC proteins were observed when the composite PE was illuminated. The high-efficient light-harvesting faculty of RC at long wavelength region compensated the negligible NIR-light absorption of TiO_2, which enabled the fabricated composite PE to capture the light energy more effectively. Combination of RC and the nanocrystalline TiO_2 led to a better photoelectric conversion efficiency of the Bio-PE resulted from the enhanced utilization of solar energy and promoted photo-induced charge separation, which might open a new perspective to develop versatile biomimic energy convertors or photoelectric sensors. However, the dramatically different photoelectric responses of the eight ITO/TiO_2/RC PEs prepared simultaneously strongly implied that the widely distributed nanopores derived from the inter-crystalline voids of TiO_2 may dampen the high-efficient immobilization of proteins with a certain specific dimensional size.
3) Bio-PE composed of the RC proteins entrapped on a tailor-made mesoporous WO3-TiO_2 matrix
In this chapter, novel 3D-worm-like mesoporous WO_3-TiO_2 films with tailored pore size were applied to entrap the RC proteins for obtaining increased protein loading as well as promoted charge separation. By analyzing the relationship between the matrix properties (pore size, structural topology, and composing) and the immobilized protein amount, the optimized RC entrapment was obtained for the tailor-made 3D-worm-like mesoporous WO_3-TiO_2 with opened pore structure, narrow-distributed and matched pore size (- 7.1 nm), and ideal hydrophilicity. The fluorescence and photoelectric measurements of the ITO/WO_3-TiO_2/RC PE confirmed the improved photo-induced electron injection from the photo-excited proteins to the matrix as compared with that for the ITO/TiO_2/RC PE mentioned above, thanks to the higher capability of WO_3-TiO_2 in splitting photo-induced electron hole pairs than that of single TiO_2 or WO_3 as well as the matched energy level between the mesoporous semiconductor and RC. Such strategy for fabricating PE based on well-designed mesoporous metal oxides and RC contributed to a better utilization of solar energy, which might open a new perspective to develop versatile bio-photoelectric devices.
4) Bio-PE composed of the pigment-exchanged RC mutant (containing Phe instead of BPhe) entrapped on the mesoporous WO_3-TiO_2 films
With the aim at well probing the photo-induced multiple-pathway electron transfer and further promoting the photoelectric performances of the photosensitive proteins entrapped on the above-mentioned mesoporous WO_3-TiO_2 films, a RC mutant (containing plant pheophytin (Phe) instead of bacteriopheophytin (BPhe), termed as Phe-RC) was introduced here for replacement of native-RC adsorbed. The NIR-Vis absorption and circular dichroism (CD) spectra displayed the successful replacement of BPhe by Phe with a high yield of more than 95%. The fluorescence emission, fs pump-probe and electrochemical experiments indicated that the free energy level of P~+Phe" is higher than that of P~+BPhe", which resulted in 1) the deferred evolution of ultrafast excited-state dynamics of Phe-RC; 2) increased life time of B* (excited-state of bacteriochlorophyll (BChl) monomer), P* (excited-state of BChl dimer), and P~+ (oxidated-state of BChl dimer) in the RC mutant as compared with that in its native counterpart. The photoelectric measurements further demonstrated that the delayed electron transfer as well as the matched energy level between RC and the WO_3-TiO_2 matrix contributed to the further enhanced electron injection intensity, and thus the promoted electron-hole pair separation. Accordingly, remarkably improved photoelectric performance of the ITO/WO_3-TiO_2/Phe-RC PE was clearly observed
(IPCE_(800nm)=-23%)
5) Manipulating the favorable RC orientation on functionalized Au colloids
In this chapter, RC-capped functionalized Au colloids were prepared for controlling the favorable protein orientation. Dramatically different protein orientations were achieved on Au colloids derivated with different bifunctional reagents (P or the neighborhood/primary quinone (Q_A) facing to the Au colloids). The ultraviolet (UV)-Vis absorption, X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) measurements reflected the structural properties of the functionalized Au colloids, which suggested the successful derivation of Au colloids with the selected bifunctional reagents. The NIR absorption and CD spectra showed that both the native structure and function of RC assembled on the derivative Au colloids remained unaltered. The fluorescence emission and fs pump-probe spectra results revealed the significantly different excitation relaxation pathway and dynamics of different RC/Au
colloid systems, and the existence of photo-induced electron injection from the photo-excited proteins to the Au colloids, which suggested the successful manipulation of favorable RC orientation. Combination of the favorable protein orientation and the unique properties of Au colloids in storing and shuttling the electrons provided a potential strategy for research and development of high-efficient Bio-PE materials.
6) Ultrafast excitation energy relaxation of electrochemical-oxidized LH2
Herein, in situ fs pump-probe spectroelectrochemistry methodology was established for probing the ultrafast energy entrapment dynamics resulted from electrochemical oxidation of LH2 from purple bacteria. Fs pump-probe spectroelectrochemical techniques coupled with NIR absorption spectroelectrochemical and fluorescence emission spectroelectrochemical methods were employed to investigate the different electrochemical oxidative behaviors of B800 (BChl monomer) and B850 (BChl dimer), address the change of pigment-pigment, pigment-protein arrangements during the oxidation, and reveal the effect of oxidation on the ultrafast energy transfer. The results showed that 1) Although the degradation of both B800 and B850 Qy bands took place at practically the same potential, yet the B800 band bleached faster as compared with the B850 band. 2) Dramatically quenching of the fluorescence emission from the B850 ring was observed during the electrochemical oxidation and the peak decrease rate was much faster as compared with the bleaching rate of absorption. 3) The BChl-B850 radical cation might act as an additional channel to compete with the unoxidized BChl-B850 molecules for rapidly releasing the excitation energy, however the B800-B850 energy transfer rate remained almost unchanged during the oxidation process. The development of in situ fs pump-probe spectroelectrochemical method might not only supply a new approach for monitoring and analysis of the ultrafast energy/electron transfer inside the photosynthetic proteins but also inspire more concerns on study and probing of various ultrafast excited-state dynamics with electrochemical oxidation concomitantly occurring.
围绕稳定、高效的新型功能化RC纳米仿生膜的开发探索，尤其在提高基体电极上蛋白的负载，促进蛋白向基体电极的光致电子注入，控制蛋白在基体电极表面的有利方位，以及揭示蛋白复杂、快速和有效的能量／电子传递过程等方面，我们开展了一系列循序渐进的创新性工作，包括：
1、利用阳极氧化水解法制备的具有多孔结构特征的纳米晶TiO_2薄膜吸附光敏蛋白实现了RC仿生膜光电极的制备。RC在近红外区的高效捕光功能与TiO_2良好的光致电荷分离能力相结合，明显改善了仿生膜光电极的光电转化效率。
2、创新性地建立了针对RC裁剪设计的新型介孔WO_3-TiO_2膜修饰固定光敏蛋白制备仿生膜光电极的方法。首次采用具有开放孔道、均一孔径分布和理想亲水性的新型介孔WO_3-TiO_2实现了保持RC生物活性前提下对蛋白的高效捕获。利用该介孔WO_3-TiO_2能级的匹配性促进RC在光照后电子向纳米半导体电极的注入，有效降低了分离纯化后RC自身的电子传递及电荷重组对蛋白光电转换的负面影响。
3、通过对RC内色素的特定替换改变辅助因子的电荷分离态能级，进一步削弱光敏蛋白在介孔WO_3-TiO_2电极上受激后自身的电子传递及电荷重组。所获得的变异RC／WO_3-TiO_2仿生膜表现出前所未有的高效光电转换能力(相比于现有的其它RC光电极)。
4、采用自组装技术首次在基于特定设计的功能化金胶表面实现了RC的修饰。通过控制RC在金胶表面的有利取向和方位，并利用金胶粒子对电子存储和穿梭表现出的优良特性，尝试了从另一个角度研究与开发高效的光敏蛋白纳米仿生光电材料。
5、开创性地搭建了现场飞秒泵浦—探测(femtosecond pump-probe)／电化学联用技术平台，发展了探索分析光合色素—蛋白超快能量与电子传递过程的新方法。首次报道了对电化学氧化引起的紫细菌外周天线蛋白(light-harvesting complex 2，简称LH2)的快速能量陷获研究。
具体地，论文由以下六个部分构成：
一、绪论
围绕论文的主题，本章节首先明确了课题研究的重要意义。之后，概括地阐述了有关RC结构和功能的背景信息。作为本章的核心内容，RC功能性纳米仿生膜的构建及其光致电子传递的相关研究现状被作了重点介绍，对相应的文献也进行了较全面的综述。最后简要归纳了论文的主要创新性。
二、电沉积纳米晶TiO_2固定RC制备仿生膜光电极的方法与研究
本章节详细介绍了采用阳极氧化水解法合成的具有多孔结构特征的纳米晶TiO_2薄膜吸附RC制备功能性仿生膜光电极的相关研究工作。ITO／TiO_2／RC仿生膜光电极的近红外—可见吸收光谱、荧光光谱测试结果表明了修饰在纳米TiO_2薄膜上的光敏蛋白仍然保持了原有的生物活性。RC在仿生膜光电极内展现出特有的，可重现的近红外光电响应行为，其在长波长区的高效捕光功能起到了敏化纳米TiO_2的作用，提高了纳米TiO_2电极的光电转换效率。纳米半导体材料与光敏蛋白的结合既拓宽了TiO_2对太阳光的吸收利用又促进了RC的光致电荷分离，显著改善了整个光电极的光电转化效率，为研究与开发生物光电器件提供了新的借鉴。然而，同一批八通道制备的ITO／TiO_2／RC仿生膜光电极的光电流检测结果则反映出由不规则的纳米TiO_2晶粒间空隙组成的孔道在对RC的高效捕获上仍存在着较大缺陷。
三、剪裁设计的新型三维虫洞介孔WO_3-TiO_2固定RC制备仿生膜光电极的方法与研究
本部分工作的侧重点在于针对光敏蛋白的高效捕获及其光致电荷分离的有效促进，建立了采用剪裁设计的新型介孔WO_3-TiO_2膜修饰固定RC制备仿生膜光电极的方法。通过对不同孔径、结构和组成的材料在吸附RC效果上的比较表明：根据“酸碱对”概念剪裁制备的，具有开放孔道、均一孔径分布(孔径中心尺寸～7.1 mm)和理想亲水性的三维虫洞介孔WO_3-TiO_2膜对于保持RC生物活性前提下的蛋白捕获最为高效(RC负载量：0.63μmol／g)。ITO／WO_3-TiO_2／RC仿生膜光电极的荧光和光电化学测量结果证实：WO_3-TiO_2复合材料高于单一组分的光致电子空穴对分离能力以及与光敏蛋白能级的匹配性能够有效促进受激后RC向纳米半导体电极的电子注入，降低分离后RC自身的电子传递及电荷重组对仿生膜光电极光电转换的负面影响。利用新型介孔半导体材料与光敏蛋白的结合实现了RC仿生膜光电极对太阳光能尽可能高效的吸收利用，为各种功能性生物光电器件的设计迈出了坚实的一步。
四、细菌脱镁叶绿素(BPhe)被植物脱镁叶绿素(Phe)替换的RC变异体／三维虫洞介孔WO_3-TiO_2仿生膜光电极的制备与研究
承接上一章的工作，本部分主要进行了将RC的色素替换变异株(Phe-RC)应用于构建仿生膜光电极的考察和研究，目的在于深入探索修饰在电极表面RC色素替换体的复杂多途径光致电子传递过程，并在此基础上尝试开发更高效的生物光电器件。近红外—可见吸收光谱和圆二色谱测试结果表明了色素替换的成功(替换效率不低于95％)。荧光发射，飞秒泵浦—探测和电化学实验的结果揭示了电荷分离态P~+Phe~-相比P~+BPhe~-更高的自由能能级，以及由此导致的1)Phe-RC自身能量／电子传递的明显受阻；2)B~*(细菌叶绿素单体激发态)，P~*(细菌叶绿素二聚体激发态)和P~+(细菌叶绿素二聚体氧化态)相比天然RC中对应物种寿命的显著增加。光电化学的测量结果进一步显示，电子传递的受阻加之介孔WO_3-TiO_2与蛋白能级的匹配性使得被WO_3-TiO_2膜捕获的Phe-RC向半导体导带的电子注入有了进一步增强，更有效地促进了仿生膜光电极电子—空穴对的分离，光电转换效率得到了极大地提高(IPCE_(800 nm)=～23％)。
五、功能化金胶控制RC在自组装仿生体系内有利方位的初步研究
本章节主要开展了在功能化的金胶表面组装RC分子并控制有利蛋白方位的相关初步研究工作。通过在合成的金胶纳米粒子上引入特定的双功能试剂及组装上带有特定官能团的分子后，可以实现光敏蛋白在金胶表面采取不同的方位进行排列(原初电子给体P／电子受体泛醌(Q_A)朝着金胶的方向)。紫外—可见吸收光谱、X射线光电子能谱、傅立叶红外光谱，以及透射电子显微镜的相关结构表征结果反映了功能化金胶的成功制备；近红外—可见吸收光谱和圆二色谱的相关检测结果则进一步证实了RC在功能化金胶表面组装的有效实现；RC／金胶自组装仿生体系的荧光和飞秒泵浦—探测实验结果揭示了修饰在不同功能化金胶表面的光敏蛋白在激发能弛豫过程和方式上的大相径庭，以及RC在受激后向金胶粒子明显的电子注入现象，暗示了控制RC在金胶表面有利方位的成功实现。利用金胶纳米颗粒优良的电子存储和穿梭特性结合有利于电荷分离和电子注入的蛋白方位的控制提供了又一设计和开发高效生物光电器件的有效途径。
六、电化学诱导的紫细菌天线色素复合物(LH2)的超快激子能弛豫研究
本章主要报道了发展现场飞秒泵浦—探测／电化学联用技术应用于电化学氧化引起的LH_2快速能量陷获的相关研究。采用近红外—可见／荧光光谱电化学方法结合超快泵浦—探测光谱电化学技术实时在线地考察了LH2内B800(细菌叶绿素单体)和B850(细菌叶绿素二聚体)不同的电化学氧化过程；色素—色素、色素—蛋白之间伴随氧化而发生的结构变化；以及电化学氧化对B800和B850参与的超快激发能弛豫的影响。结果表明：1、B800和B850环中的叶绿素分子具有几乎相同的氧化还原中点电位却表现出完全不同的电化学氧化速率(B800快于B850)。2、伴随叶绿素分子电化学氧化的进行，激发B800观察到B850自发辐射淬灭的显著增强(荧光峰下降速率明显快于吸收峰下降速率)；3、B800向B850的快速能量传递过程几乎不受电化学氧化的影响，但电化学氧化引起了B850激发能的快速陷获。现场飞秒泵浦—探测／电化学联用技术的建立不仅提供了深入分析光合色素—蛋白超快能量与电子传递的新途径，更是对现有光谱电化学技术的补充和丰富，为研究和探索复杂体系内伴随电子得失而发生的各种超快动力学变化开辟了新的方向。
Creation and exploitation of stable, high-efficient, and also environmentally friendly photoelectric materials or devices for a better utilization of solar energy is one of the long-term pursuits for human beings that might suffer from the serious problems of oil exhaustion. Photosynthesis, which has played an important role as both bio-energy source and life basis, is much more superior to the artificial apparatus on photoelectric conversion performance and has attracted great concerns. Recently, significant advancements have been achieved on both the structural and functional research of the purple bacteria photosynthetic reaction center (RC), a transmembrane pigment-protein complex that is the smallest unit in bacteria photosynthetic membrane able to perform a light-driven charge separation. It is with great value for both the theoretical study and applicable try to design effective photoelectric apparatus based on RC and fully reveal the excitation relaxation processes of the proteins encased within the functionalized films, which depends on the preparation of advanced electrode materials, introduction of site-directed protein mutation as well as genetic engineering, and establishment of new methodology for analysis and probing.
Aimed at exploiting and developing the stable, high-efficient novel RC-functionalized nanofilms, a series of original work has been done especially on increasing the protein loading, promoting the photo-induced electron injection, manipulating the favorable protein orientation, and revealing the complex, ultrafast, and effective photo-induced energy/electron transfer:
1. A new kind of bio-nanocomposite photoelectrode (PE) was fabricated through direct immobilization of RC on a porous nanocrystalline TiO_2 matrix prepared by the anodic oxidative hydrolysis method. Combination of the high-efficient near-infrared (NIR) light-harvesting faculty of RC and the well performance of TiO_2 on the photo-induced charge separation led to a better photoelectric conversion efficiency of the derived bio-photoelectrode (Bio-PE) as compared with that of the separate component.
2. Novel 3D-worm-like mesoporous WO_3-TiO_2 films with tailored pore size were synthesized and applied to prepare the Bio-PEs through direct entrapping the RC molecules. These mesoporous WO_3-TiO_2 films exhibited unique characteristics, e.g. opened mesostructure, narrow-distributed pore size well-matching one 2D dimension of RC, and ideal hydrophilicity, in the specific loading of RC with high activity
retained. Thanks to the higher capability of WO_3-TiO_2 in splitting photo-induced electron hole pairs than that of the single oxide, enhanced photo-induced electron injection from photo-excited RC to the WO_3-TiO_2 matrix decreased the disadvantages of electron transfer and charge recombination within the proteins.
3. The disadvantages of electron transfer and charge recombination within RC itself were further conquered by means of the specific protein pigment exchange, which resulted in energy level change of the protein charge-separated state. The prepared Bio-PE composed of the pigment-exchanged proteins and the mesoporous WO_3-TiO_2 matrix exhibited remarkably enhanced photoelectric performances as compared with those RC PEs reported before.
4. Successful modification of RC was achieved on the surface of functionalized Au colloid through self-assembling. Favorable protein orientation manipulated and the unique property of Au colloid in storing and shuttling the electrons contributed to the new concept of research and development of high-efficient Bio-PE materials.
5. In situ femtosecond (fs) pump-probe spectroelectrochemistry methodology was developed for the first time for probing the ultrafast energy/electron transfer process inside the pigment-protein complex. The ultrafast excitation energy trapping dynamics of purple bacteria peripheral light-harvesting complexes (LH2) induced by electrochemical oxidation was investigated and reported particularly.
Detailedly, the dissertation consists of 6 chapters that tightly relate to the main topic.
1) Introduction
In this chapter, firstly, significance of the topic discussed here was briefly described. Then, the research background about the structure and function of RC was introduced and illustrated. After that, strategies for construction and fabrication of well-defined RC-functionalized nanocomposite films for probing and exploiting the photo-induced electron transfer of RC were classified and summarized mainly including five categories, and the related literatures were well-reviewed. Finally, the main novelty and innovation of the study were provided.
2) Bio-PE composed of the RC proteins adsorbed on a nanocrystalline TiO_2 film prepared by anodic electrodeposition
In this part, RC-functionalized nanocomposite PE prepared through adsorption of
the photosensitive proteins on a porous nanocrystalline TiO_2 film synthesized by the anodic oxidative hydrolysis method was studied and reported. The NIR-visible (Vis) absorption spectra and fluorescence emission spectra displayed that the native activity was well-remained for RC immobilized on the TiO_2 matrix. Obvious reproducible photoelectric responses dominated by the adsorbed RC proteins were observed when the composite PE was illuminated. The high-efficient light-harvesting faculty of RC at long wavelength region compensated the negligible NIR-light absorption of TiO_2, which enabled the fabricated composite PE to capture the light energy more effectively. Combination of RC and the nanocrystalline TiO_2 led to a better photoelectric conversion efficiency of the Bio-PE resulted from the enhanced utilization of solar energy and promoted photo-induced charge separation, which might open a new perspective to develop versatile biomimic energy convertors or photoelectric sensors. However, the dramatically different photoelectric responses of the eight ITO/TiO_2/RC PEs prepared simultaneously strongly implied that the widely distributed nanopores derived from the inter-crystalline voids of TiO_2 may dampen the high-efficient immobilization of proteins with a certain specific dimensional size.
3) Bio-PE composed of the RC proteins entrapped on a tailor-made mesoporous WO3-TiO_2 matrix
In this chapter, novel 3D-worm-like mesoporous WO_3-TiO_2 films with tailored pore size were applied to entrap the RC proteins for obtaining increased protein loading as well as promoted charge separation. By analyzing the relationship between the matrix properties (pore size, structural topology, and composing) and the immobilized protein amount, the optimized RC entrapment was obtained for the tailor-made 3D-worm-like mesoporous WO_3-TiO_2 with opened pore structure, narrow-distributed and matched pore size (- 7.1 nm), and ideal hydrophilicity. The fluorescence and photoelectric measurements of the ITO/WO_3-TiO_2/RC PE confirmed the improved photo-induced electron injection from the photo-excited proteins to the matrix as compared with that for the ITO/TiO_2/RC PE mentioned above, thanks to the higher capability of WO_3-TiO_2 in splitting photo-induced electron hole pairs than that of single TiO_2 or WO_3 as well as the matched energy level between the mesoporous semiconductor and RC. Such strategy for fabricating PE based on well-designed mesoporous metal oxides and RC contributed to a better utilization of solar energy, which might open a new perspective to develop versatile bio-photoelectric devices.
4) Bio-PE composed of the pigment-exchanged RC mutant (containing Phe instead of BPhe) entrapped on the mesoporous WO_3-TiO_2 films
With the aim at well probing the photo-induced multiple-pathway electron transfer and further promoting the photoelectric performances of the photosensitive proteins entrapped on the above-mentioned mesoporous WO_3-TiO_2 films, a RC mutant (containing plant pheophytin (Phe) instead of bacteriopheophytin (BPhe), termed as Phe-RC) was introduced here for replacement of native-RC adsorbed. The NIR-Vis absorption and circular dichroism (CD) spectra displayed the successful replacement of BPhe by Phe with a high yield of more than 95%. The fluorescence emission, fs pump-probe and electrochemical experiments indicated that the free energy level of P~+Phe" is higher than that of P~+BPhe", which resulted in 1) the deferred evolution of ultrafast excited-state dynamics of Phe-RC; 2) increased life time of B* (excited-state of bacteriochlorophyll (BChl) monomer), P* (excited-state of BChl dimer), and P~+ (oxidated-state of BChl dimer) in the RC mutant as compared with that in its native counterpart. The photoelectric measurements further demonstrated that the delayed electron transfer as well as the matched energy level between RC and the WO_3-TiO_2 matrix contributed to the further enhanced electron injection intensity, and thus the promoted electron-hole pair separation. Accordingly, remarkably improved photoelectric performance of the ITO/WO_3-TiO_2/Phe-RC PE was clearly observed
(IPCE_(800nm)=-23%)
5) Manipulating the favorable RC orientation on functionalized Au colloids
In this chapter, RC-capped functionalized Au colloids were prepared for controlling the favorable protein orientation. Dramatically different protein orientations were achieved on Au colloids derivated with different bifunctional reagents (P or the neighborhood/primary quinone (Q_A) facing to the Au colloids). The ultraviolet (UV)-Vis absorption, X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) measurements reflected the structural properties of the functionalized Au colloids, which suggested the successful derivation of Au colloids with the selected bifunctional reagents. The NIR absorption and CD spectra showed that both the native structure and function of RC assembled on the derivative Au colloids remained unaltered. The fluorescence emission and fs pump-probe spectra results revealed the significantly different excitation relaxation pathway and dynamics of different RC/Au
colloid systems, and the existence of photo-induced electron injection from the photo-excited proteins to the Au colloids, which suggested the successful manipulation of favorable RC orientation. Combination of the favorable protein orientation and the unique properties of Au colloids in storing and shuttling the electrons provided a potential strategy for research and development of high-efficient Bio-PE materials.
6) Ultrafast excitation energy relaxation of electrochemical-oxidized LH2
Herein, in situ fs pump-probe spectroelectrochemistry methodology was established for probing the ultrafast energy entrapment dynamics resulted from electrochemical oxidation of LH2 from purple bacteria. Fs pump-probe spectroelectrochemical techniques coupled with NIR absorption spectroelectrochemical and fluorescence emission spectroelectrochemical methods were employed to investigate the different electrochemical oxidative behaviors of B800 (BChl monomer) and B850 (BChl dimer), address the change of pigment-pigment, pigment-protein arrangements during the oxidation, and reveal the effect of oxidation on the ultrafast energy transfer. The results showed that 1) Although the degradation of both B800 and B850 Qy bands took place at practically the same potential, yet the B800 band bleached faster as compared with the B850 band. 2) Dramatically quenching of the fluorescence emission from the B850 ring was observed during the electrochemical oxidation and the peak decrease rate was much faster as compared with the bleaching rate of absorption. 3) The BChl-B850 radical cation might act as an additional channel to compete with the unoxidized BChl-B850 molecules for rapidly releasing the excitation energy, however the B800-B850 energy transfer rate remained almost unchanged during the oxidation process. The development of in situ fs pump-probe spectroelectrochemical method might not only supply a new approach for monitoring and analysis of the ultrafast energy/electron transfer inside the photosynthetic proteins but also inspire more concerns on study and probing of various ultrafast excited-state dynamics with electrochemical oxidation concomitantly occurring.
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