CeO2纳米晶修饰的细菌视紫红质薄膜M态寿命的研究

紫膜中的蛋白质细菌视紫红质(bR)具有独特的光循环和光致变色特性 ,在分子电子学和生物电子技术领域具有广泛的潜在应用价值. 本文采用稀土纳米晶CeO₂对细菌视紫红质 聚乙烯醇(bR-PVA)薄膜进行化学修饰 .研究了纳米晶对bR光循环中的重要中间态M态的寿命的影响. 发现上述稀土纳米晶可延长M态寿命, 且晶粒尺寸越小对M态寿命的影响越大. 纳米晶周围的羟基有助于阻碍席夫碱获得质子, 从而使M态寿命延长.     

利用化学修饰的细菌视紫红质薄膜进行光学信息存储的研究

细菌视紫红质(bacteriorhodopsin,bR)具有独特的光、化学和热稳定性.bR光循环中间态M态的最大吸收峰相对于始态明显地发生蓝移,所以基于bR(→)M的光致变色模型可以为光学应用尤其是信息存储提供一个机制.但是野生型bR 中M态的寿命很短,不适合用来进行信息存储.本文采用化学添加剂的方法,将bR/聚乙烯醇(PVA)薄膜的中间态M态的寿命显著延长.用此化学修饰的bR薄膜作为记录介质进行缩微图像存储,所得到的图像具有较高的对比度和较长的保存时间.此实验首次实现了在化学修饰的bR薄膜上基于bR始态和中间态M态的双稳态模型在光学信息存储上的应用.     

细菌视紫红质／聚乙烯醇复合膜的制备及相关功能研究

细菌视紫红质（bR)是一种独特的光敏蛋白，具有光致变色和光驱质子泵功能 。将bR蛋白包埋于聚乙烯醇（PVA）基质中，制备了bR/PVA复合膜。利用紫外－可 见分光光度计和自制的毫秒级动力学光谱仪，检测了样品的吸收光谱和光循环M中 间体在脉冲光激发下随时间的变化；同时，利用凝胶扫描成像仪及相关分析软件考 察了样品成膜后的均匀程度。实验表明：bR/PVA复合膜具有良好的均匀性、透明性 和力学性能，而且bR蛋白保持了原有的生物活性和光学性质，bR与M中间体之间能 达到一种光可控制的双稳态，M中间体的寿命也得到了显著的延长，证实了bR可以 提供一个用于信息存储的模型材料。     

细菌视紫红质多重突变体结构变化及其中间态的寿命

采用基因定点突变的方法, 构建了细菌视紫红质(Bacteriorhodopsin, BR)的3种突变体蛋白, 即单突变体BRE194Q、三突变体BRI119T/T121S/A126T和四突变体BRI119T/T121S/A126T/E194Q. 测定了突变体和野生型BR在水溶液和聚乙烯醇(PVA)膜中的紫外-可见吸收光谱和拉曼光谱, 采用显微视频录像技术记录了PVA膜中野生型和3个突变体样品的M态寿命. 与野生型BR相比较, 在水溶液中, 单突变体的可见吸收光谱的最大吸收峰发生了轻微红移, 三突变体和四突变体的最大吸收峰则分别发生了11.0和12.0 nm的明显蓝移. 在PVA膜中, 3个突变体BR的可见吸收光谱的最大吸收峰均发生蓝移, 四突变体BR的最大吸收峰为557 nm, 蓝移达15.0 nm. 四突变体BR在水溶液中的共振拉曼光谱不仅表现有与M态特征相关的1567和1573 cm-1谱带, 还有L态特征带1334 cm-1及N态特征带1200, 1328, 1530和1549 cm-1. 在PVA膜中的样品与在水溶液中的比较, 四突变体共振拉曼光谱的1334和1549 cm-1带消失, 同时1187 cm-1带的强度下降. 显微视频录像技术记录的PVA膜中样品的M态寿命表明, 野生型BR的M态寿命最短, 单突变体的M态寿命小于1.0 s, 三突变体的寿命为3.0 s, 四突变体的寿命为2.0 s.     

液相沉积法制备ZnO/CdS复合纳米棒阵列薄膜及其光电性质

采用两步化学溶液沉积法在氧化铟锡(ITO)导电玻璃衬底上制备了ZnO/CdS复合纳米棒阵列薄膜.利用X射线衍射(XRD)仪、扫描电子显微镜(SEM)、紫外-可见(UV-Vis)吸收分光光度计、荧光(PL)光谱仪及表面光电压谱(SPS)研究了不同CdS沉积时间对复合薄膜的晶体结构、形貌、光电性质的影响.研究结果表明:ZnO纳米棒阵列表面包覆CdS纳米颗粒后,其吸收光谱可拓展到可见光区;与吸收光谱相对应在可见光区出现新的光电压谱响应区,这一现象证实,通过与CdS复合可显著提高ZnO纳米棒阵列在可见光区的光电转换性能;随着CdS纳米颗粒沉积时间的延长,复合纳米棒阵列薄膜在大于383nm波长区域的光电压强度逐渐减弱,而在小于383nm波长区域的光电压强度逐渐增强.用两种不同的电荷产生和分离机制对这一截然相反的光响应过程进行了详细的讨论和解释.     

Bi2MoO6纳米薄膜的制备及其光电性能

采用非晶态配合物法在ITO导电玻璃上制备了Bi2MoO6薄膜. 采用扫描电子显微镜(SEM)、X射线衍射(XRD)、激光拉曼光谱(LRS)、紫外-可见漫反射谱(DRS)、光电流响应谱、光电转换量子效率(IPCE)等技术研究了Bi2MoO6薄膜的制备工艺、形貌、结构与薄膜光电性能的关系. 结果表明, 500 ℃、1 h焙烧后的Bi2MoO6薄膜为γ-Bi2MoO6晶相, 沿(131)晶面方向生长, 薄膜厚度约为69 nm. 随着焙烧温度的升高和焙烧时间的延长, Bi2MoO6薄膜的平均颗粒度增大, 并且在525 ℃焙烧出现β-Bi2MoO6和γ’-Bi2MoO6晶相. Bi2MoO6薄膜具有可见光响应活性, 在可见光照射下可以产生光电流, 优化条件下的Bi2MoO6薄膜在400 nm的光电转换量子效率可以达到2.14%. 薄膜的光电响应和光电转换量子效率受薄膜形貌及结晶状态影响, 可以通过控制薄膜的制备条件来提高薄膜的光电转换量子效率.     

电沉积表面修饰对染料敏化太阳电池微观性能影响机理研究

为了改善染料敏化太阳电池内电子的传输复合过程, 研究者尝试不同方法制备或改性TiO₂薄膜. 对TiO₂薄膜进行后处理, 在其表面引入一层小颗粒层, 是一种有效的方法并被广泛研究. 通过对TiO₂薄膜不同时间的电沉积表面修饰, 细致研究了表面修饰后染料敏化太阳电池微观性能的变化机制. 采用阳极氧化法在TiCl₃水溶液中对TiO₂薄膜进行电沉积后处理, 将溶液pH值调至2.2, 装置的反应速率由恒电位仪控制. 不同沉积时间电池带边移动以及电子传输复合的动力学过程, 借助强度调制光电流谱(IMPS)/强度调制光电压谱(IMVS)和电化学阻抗谱(EIS)等探测技术表征. 研究表明, 电沉积在TiO₂薄膜表面引入了大量浅能级陷阱态, 以致电势较高时电容随沉积时间延长增加明显. 不同时间的电沉积表面修饰在TiO₂薄膜表面形成了新的小颗粒层并改善了TiO₂颗粒间接触, 在改善电子注入及收集过程的同时, 也有效抑制了内部电子复合. IMPS/IMVS结果表明, 电沉积对动力学过程改善的效果受光强影响明显, 弱光下作用更为突出. 此外, 电池开路电压主要受带边移动及内部复合变化影响, 随沉积时间延长, 表面电荷的增多使TiO₂薄膜带边逐渐正移, 有效改善了光电流却限制了开路电压的提升. 在适合的电沉积时间下, 电沉积表面修饰可以同时改善光电流和光电压.     

基于光波导分光光谱技术研究蛋白质与亚甲基蓝的竞争吸附行为

通过利用时间分辨光波导分光光谱技术原位测量从蛋白质-亚甲基蓝(MB)混合水溶液吸附到亲水玻璃光波导表面的MB可见光吸收谱,观测到在溶液pH值低于蛋白质等电点时MB与牛血清蛋白(BSA)以及MB与血红蛋白(Hb)存在竞争吸附行为,进一步测得这种竞争吸附行为对蛋白质浓度十分敏感,可以用于简单测定溶液中的蛋白质含量.基于Langmuir等温吸附理论推导出了两种分子竞争吸附的动力学方程,并利用该动力学方程对实验测得的吸光度随时间变化曲线进行了最佳拟合,揭示了玻璃表面吸附的MB分子个数在达到最大值后随时间呈指数衰减,同时得出拟合参数与蛋白质浓度呈准线性关系.     

溶胶-凝胶多孔性硅胶薄膜的制备及吸附特性研究

研究了纳米硅溶胶和多孔性硅胶薄膜的制备与性能.一定条件下Na2SiO3溶液经过阳离子交挟树脂除去Na+离子后形成纳米硅溶胶,再经过调节pH值和添加化学添加剂如丙三醇、聚乙烯醇(PVA)形成硅溶胶前体.用甩膜法制备的凝胶二氧化硅薄膜在约550℃下热处理30min后,薄膜孔径分布在55～310nm之间.对硅溶胶的粒度分布、粘度和多孔性硅胶薄膜的形貌、孔率分布和吸附性能及相关参数进行了表征,从中得知,通过化学添加剂、调节硅溶胶pH值可控制硅溶胶粒度分布,从而达到控制多孔性硅胶薄膜的徼结构;硅胶薄膜热处理温度在很大程度上影响着硅胶薄膜孔径、孔率分布和吸附量.考虑到硅胶薄膜微结构和吸附特性以及高温下多孔硅胶薄膜会出现熔结现象等因素,二氧化硅溶胶pH为7.5时,凝胶薄膜热处理温度550℃最佳.     

一种具有光致变色特性的膜蛋白/聚合物复合膜

通过基因工程技术将古紫质4(archaerhodopsin-4,AR4)膜蛋白的基因转入本身不表达该蛋白质、也不表达玉红素的嗜盐菌株L33中,去除了天然菌株xz515所获得的AR4膜蛋白脂环境中的玉红素,优化了AR4的光致变色性能.将重组古紫质4包埋于聚乙烯醇中制成复合膜,并通过提高体系的pH值,延长了M态的寿命,进一步实现了简单图案记录,验证了该活性蛋白质与聚合物的复合材料具有光信息存储功能.     

BIOSYNTHETIC INCORPORATION OF M-FLUOROTYROSINE INTO BACTERIORHODOPSIN

Halobacterium halobium, grown in a defined medium where tyrosine had been largely replaced with m-fluorotyrosine, biosynthetically produced purple membrane. Analysis of this membrane by high pressure liquid chromatography of phenylthiocarbamyl derivatized amino acids of membrane acid hydrolysates revealed that up to 50% of the tyrosine was present as the m-fluorotyrosine form. Yields of the purple membrane decreased as the level of incorporation increased. The experimental purple membrane showed a single 19F NMR resonance at -61.983 ppm (relative to trifluoroacetic acid). The bacteriorhodopsin (bR) in the purple membrane was normal as assayed by gel electrophoresis, isoelectric focusing, circular dichroic spectra, and UV-visible spectra. However, the fluorinated tyrosine bacteriorhodopsins at near neutral pH exhibited slightly slower rates of proton uptake and a slower M-state decay with biphasic kinetics reminiscent of alkaline solutions of bR (pH > 9). These results imply that the tyrosines in bacteriorhodopsin may play a role in the photoactivated proton translocation process of this pigment.     

EVIDENCE FOR BRANCHING IN THE PHOTOCYCLE OF BACTERIORHODOPSIN AND CONCENTRATION CHANGES OF LATE INTERMEDIATE FORMS

Abstract— Flash photolysis transients of bacteriorhodopsin were recorded with a spectrograph -multielement photodiode array combination and the recordings were analyzed to determine the concentrations of bacteriorhodopsin intermediates "M" and "O" relative to the amount of "bR" cycling (pH 7.1,10–40°C). Estimated concentration time courses were simulated with solutions to two kinetic decay models which could account for photocycle temperature dependence. A unidirectional unbranched decay model overpredicts our estimated levels of [O(r)], whereas a model branched at the "M" intermediate describes each of the later intermediate levels well (with no evidence for an independent "N" form). Our results are consistent with "M" decay regulating the level and rates of change of [bR (t)] and (bR(f)]- and also suggest that two temperature-dependent pathways form "bR" from "M", one directly, and the other indirectly through "O".     

Two orientations and photochromism of polyelectrolyte/bacteriorhodopsin alternative deposition multilayers

A series of organized multilayers have been formed by the alternative adsorption of positively charged poly(dimethyldiallylammonium chloride) (PDAC) and purple membrane (PM) fragments in suspensions at pH = 4—11. Both UV-vis spectrophotometry and quartz crystal micro-balance (QCM) technique were used to monitor the deposition process of PDAC/bacteriorhodopsin (bR) multilayers, suggesting that PM fragments and PDAC are deposited alternatively on the substrate uniformly. Upon illumination, all these multilayers generate photovoltages with defined signs. The negative sign of photovoltage accompanying the formation of M-state at pH <7 indicates that the extracellular side of PM fragments is directed toward the substrate; and the positive sign at pH≥7 indicates that the cytoplasmic side of PM fragments is directed toward the substrate. In addition, the long-lived multiple M-state has been observed in all multilayer films. Moreover, M-state at high pH, which shows the longer lifetime than that at low pH, de     

Studies on pyrylretinal analogues of bacteriorhodopsin

The retinal analogues 3-methyl-5-(1-pyryl)-2E,4E-pentadienal (1) and 3,7-dimethyl-9-(1-pyryl)-2E,4E,6E,8E-nonatetr aenal (2), which contain the tetra aromatic pyryl system, have been synthesized and characterized in order to examine the effect of the extended ring system on the binding capabilities and the function of bacteriorhodopsin (bR). The two bR mutants, E194Q and E204Q, known to have distinct proton-pumping patterns, were also examined so that the effect of the bulky ring system on the proton-pumping mechanism could be studied. Both retinals formed pigments with all three bacterioopsins, and these pigments were found to have absorption maxima in the range 498-516 nm. All the analogue pigments showed activity as proton pumps. The pigment formed from wild-type apoprotein bR with 1 (with the shortened polyene side chain) showed an M intermediate at 400 nm and exhibited fast proton release followed by proton uptake. Extending the polyene side chain to the length identical with retinal, analogue 2 with wild-type apoprotein gave a pigment that shows M and O intermediates at 435 nm and 650 nm, respectively. This pigment shows both fast and slow proton release at pH 7, suggesting that the pKa of the proton release group (in the M-state) is higher in this pigment compared to native bR. Hydrogen azide ions were found to accelerate the rise and decay of the O intermediate at neutral pH in pyryl 2 pigment. The pigments formed between 2 and E194Q and E204Q showed proton-pumping behavior similar to pigments formed with the native retinal, suggesting that the size of the chromophore ring does not alter the protein conformation at these sites.     

Adsorption behavior and photoelectric response characteristics of bacteriorhodopsin thin films fabricated by self-assembly technique

The characteristics of bacteriorhodopsin (bR)-based thin films fabricated by self-assembly (SA) technique were investigated. Self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (11-MUA) were spontaneously formed onto a pretreated gold substrate by soaking it into the ethanolic solution of 11-MUA, and used as a template for the adsorption of bR. Using poly- -lysine as a bridging molecule for bR adsorption onto SAMs of 11-MUA, bR-embedded purple membrane fragments were adsorbed by electrostatic attractive force. By ellipsometry and atomic force microscopy, the properties of the prepared bR-based thin films were investigated with the various fabrication conditions, such as bR suspension concentration and pH. An artificial photoreceptor was then fabricated with a sandwich-type structure of ITO/electrolyte gel/bR-based thin films/gold substrate. According to the monochromatic light illumination (560 nm) using Xenon lamp system, photoelectric responses of the fabricated photoreceptor were detected and analyzed. The stability of photoreceptors composed of the bR films fabricated by different technique was also examined over the period of 60 days. It is concluded that the SA technique could be usefully applied to the protein-based thin films preparation for the development of bioelectronic devices.     

Structural changes of purple membrane and bacteriorhodopsin during its denaturation induced by high pH

Bacteriorhodopsin (bR) trimers naturally form two-dimensional hexagonal crystals in purple membrane (PM), which make it very stable. However, the dnaturation of bR was found to occur during a very narrow pH range when the pH was increased above 12.0, as indicated by inactivation of the photochemical cycle observed by flash photolysis kinetic spectra. Here, atomic force microscopy was used to study the surface structural changes of PM during the denaturation process induced by high pH. Together with the absorption and fluorescence spectra, it was found that the structural changes could be divided into three steps. First, some hydrophobic amino acids of bR become exposed to the aqueous environment and PM loses its 2D crystalline structure, transforming into the so-called "nonisland" structure. Second, bR molecules are extracted out of membrane and form protrusions on the surface like islands in the sea; therefore, the "nonisland" structure transforms into the "island" structure. Finally, most bRs break off from the membrane and form large depositions.     

Photolytic cleavage of 1-(2-nitrophenyl)ethyl ethers involves two parallel pathways and product release is rate-limited by decomposition of a common hemiacetal intermediate

Time-resolved FTIR spectroscopic studies of the flash photolysis of several 1-(2-nitrophenyl)ethyl ethers derived from aliphatic alcohols showed that a long-lived hemiacetal intermediate was formed during the reaction. Breakdown of this intermediate was rate-limiting for product release. One of these compounds (methyl 2-[1-(2-nitrophenyl)ethoxy]ethyl phosphate, 9) was studied in detail by a combination of time-resolved FTIR and UV-vis spectroscopy. In addition, product studies confirmed clean photolytic decomposition to the expected alcohol, 2-hydroxyethyl methyl phosphate, and the 2-nitrosoacetophenone byproduct. At pH 7.0, 1 degrees C, the rate constant for product release was 0.11 s(-1), very much slower than the 5020 s(-1) rate constant for decay of the photochemically generated aci-nitro intermediate (pH 7.0, 2 degrees C). Time-resolved UV-vis measurements showed that the hemiacetal intermediate is formed by two competing pathways, with fast (approximately 80% of the reaction flux) and slow (approximately 20% of the flux) components. Only the minor, slower path is responsible for the observed aci-nitro decay process. These competing reactions are interpreted with the aid of semiempirical PM3 calculations of reaction barriers. Furthermore, AMSOL calculations indicate that the pK(a) of the nitronic acid isomer formed by photolysis is likely to determine partition into the alternate paths. These unusual results appear to be general for 1-(2-nitrophenyl)ethyl ethers and contrast with a related 2-nitrobenzyl ether that photolyzed without involvement of a long-lived hemiacetal.     

Partial dehydration of the retinal binding pocket and proof for photochemical deprotonation of the retinal Schiff base in bicelle bacteriorhodopsin crystals

In bicelle bacteriorhodopsin (bcbR) crystals, the protein has a different structure from both native bacteriorhodopsin (bR) and in-cubo bR (cbR) crystals. Recently, we studied the ability of bcbR crystals to undergo the photocycle upon laser excitation, characterized by the appearance of the M intermediate by single crystal resonance Raman spectroscopy. Calculation of the M lifetime by flash photolysis experiments demonstrated that in our bcbR crystals, the M rise time is much faster than in the native or cbR crystals, with a decay time that is much slower than these other two forms. Although it is now known that the bcbR crystals are capable of photochemical deprotonation, it is not known whether photochemical deprotonation is the only way to create the deprotonated Schiff base in the bcbR crystals. We measured both the visible and Raman spectra of crystals dried under ambient lighting and dried in the dark in order to determine whether the retinal Schiff base is able to thermally deprotonate in the dark. In addition, changes in the visible spectrum of single bcbR crystals under varying degrees of hydration and light exposure were examined to better understand the retinal binding environment.     

Time-resolved FTIR spectroscopy of the photointermediates involved in fast transient H+ release by proteorhodopsin

Proteorhodopsin (pR) is a homologue of bacteriorhodopsin (bR) that has been recently discovered in oceanic bacterioplankton. Like bR, pR functions as a light-driven proton pump. As previously characterized by laser flash induced absorption spectroscopy (Krebs, R. A.; Alexiev, U.; Partha, R.; DeVita, A. M.; Braiman, M. S. BMC Physiol. 2002, 2, 5), the pR photocycle shows evidence of light-induced H(+) release on the 10-50 micros time scale, and of substantial accumulation of the M intermediate, only at pH values above 9 and after reconstitution into phospholipid followed by extensive washing to remove detergent. We have therefore measured the time-resolved FTIR difference spectra of pR intermediates reconstituted into DMPC vesicles at pH 9.5. A mixture of K- and L-like intermediates, characterized by a 1516 cm(-1) positive band and a 1742 cm(-1) negative band respectively, appears within 20 micros after photolysis. This mixture decays to an M-like state, with a clear band at 1756 cm(-1) due to protonation of Asp-97. The 50-70 micros rise of M at pH 9.5 is similar to (but a little slower than) the rise times for M formation and H(+) release that were reported earlier based on flash photolysis measurements of pR reconstituted into phospholipids with shorter acyl chains. We conclude that, at pH 9.5, H(+) release occurs while Asp-97 is still protonated; i.e., this aspartic acid cannot be the H(+) release group observed by flash photolysis under similar conditions.