CN1558222A - 生物光敏蛋白-纳米半导体复合光电极的制备方法 - Google Patents
生物光敏蛋白-纳米半导体复合光电极的制备方法 Download PDFInfo
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Abstract
本发明提供了一种构建高性能生物光电极的有效途径。通过将提取的紫细菌光合反应中心蛋白(RC)的各类变异体修饰于特定的纳米半导体基底上，可以得到在非常宽的波长范围内，特别是在近红外区有着高效光电转换功能的复合光电极。一方面，这些人工改性的RC在该体系中具有比天然RC更高效的光电转换性能；另一方面，采用纳米半导体材料，特别是介孔半导体材料可以实现在RC高效固定的前提下对蛋白光电转换的积极促进。改性并优化后的RC对纳米半导体的敏化作用大大提高了整个复合光电极对太阳能的吸收和利用，为开发高效的太阳能电池提供了借鉴。
Description
技术领域
本发明涉及了生物技术和纳米技术领域中制备改性和优化的生物光敏蛋白——纳米半导体复合光电极的制备方法。
背景技术
对太阳光能的有效开发和高效利用，一直以来作为关系到人类生存与发展的重大课题而引起了各国科研工作者的关注。近年来，有关纳米结构和染料敏化的半导体光电极或光电池(M.Gratzel，Nature 2001，414，343.，)以及人工合成或组装的包括捕光系统、给体、桥、受体结构的分子内仿生光合反应中心体系(D.Gust，T. A.Moore，A.L.Moore，Acc.Chem.Res，2001，34，40.，)被大量报道，成为光电转换研究的一大热点。另一方面，设计和制作包含紫细菌光合反应中心蛋白(RC)的各类生物光电极或光电转换器件提供了利用光敏蛋白天然高效的光电转换功能的有效途径。光合反应中心蛋白极高的光致电荷分离的量子产率，特别是对于近红外长波长光的高效吸收是其作为光电转换材料具有的巨大优势。
在先前的工作中曾报道了采用电沉积TiO2固定修饰上RC，制作生物光电极的方法(03141612.8)。该复合光电极有效拓宽了半导体光电材料对太阳光的吸收，光电转换效率得到了大幅提高。然而，RC被激发后自发的电子传递过程及随时可能发生的反向电荷复合而回到基态的竞争反应，极大地削弱了光激发后RC对溶液中介体的氧化，进而影响到整个光电极的光电转换效率。虽然采用能极匹配性良好的纳米材料可以促进光激发后RC电子——空穴对的有效分离，在一定程度上降低RC受激后电荷的重组对蛋白光电流产生的负面影响，但是该方法并不能从更本上解决RC受激后发生的竞争反应对整个光电极光电转换的干扰。
发明内容
本发明的目的是获得光电转换效率高的系列人工改性和优化的生物光敏蛋白——纳米半导体复合光电极。
本发明制备了系列的RC变异体，即通过控制替换色素与天然RC的摩尔比、反应温度及反应时间等条件，利用植物脱镁叶绿素(Phe)及其它相似的人工色素如卟啉类色素替代RC内的细胞脱镁叶绿素(BPhe)，从而减缓或部分地阻断RC受激后自发的电子传递过程，推动光激发后RC电子——空穴对的有效分离，延长电荷分离态的寿命。将上述替代即变异后的RC修饰固定于特定的介孔半导体氧化物基体电极上可以得到性能优良的复合生物光电极。
本发明提出的人工改性并优化的生物光敏蛋白——纳米半导体复合光电极的制备包括以下几个步骤：
第一步：色素替换的RC变异体的获得。取一定体积的RC溶液，加入一定量含植物脱镁叶绿素或其它卟啉类色素的丙酮溶液。加入的丙酮溶液与RC溶液的体积比在5％～20％间；混合溶液中，植物脱镁叶绿素或其它卟啉类色素与RC的摩尔比大于20-30％。上述溶液在搅拌后置于40℃～45℃下共保温0.5-2小时，随后在DEAE-纤维素层析柱(DE-52)上用NaCl的TL缓冲稀溶液洗去游离的叶绿素。选用浓度稍大的NaCl的TL缓冲溶液将修饰的RC洗脱下来，再将待纯化的RC溶液铺在10％～40％的蔗糖梯度上，4℃条件下离心14～16h，从上至下取第二层淡黄色上清液，即得到纯化后的色素替换RC。采用pH 8.0的Tris-HCl缓冲溶液稀释上述色素替换的RC，得到浓度为0.5～2mM的变异RC的稀溶液，置于4℃冰箱内保存待用。
第二步：介孔半导体基体的合成。介孔半导体氧化物(如TiO2，WO3等)是根据“酸碱对”概念制备的。取一定量的表面活性剂(如P123)溶于乙醇中(质量比0.05～0.2)，将两种介孔半导体基体的无机前驱体(如TiCl4和Ti(Opr)4)混合后加入溶液中，并在常温下搅拌1～4h。得到的母液在20℃～40℃，湿度20％～30％的条件下充分蒸发0.5-2小时。将该母液旋涂或蘸涂于表面经过清洁处理的ITO(掺铟的氧化锡)玻璃上，在40℃～60℃条件下放置一天后，得到的半导体膜置于350℃马福炉内恒温数小时，最终得到表面均匀的介孔结构半导体薄膜电极。
第三步：RC(色素替换)——介孔半导体复合光电极的制备。将上述新制备的介孔半导体薄膜电极置于浓度为0.5～2mM的变异RC的pH 8.0 Tris-HCl缓冲液中，在4℃冰箱内吸附1～5天，最终得到人工改性并优化的生物光敏蛋白——纳米半导体复合膜电极。该复合膜电极可以在干态或同种蛋白的稀溶液中于4℃冰箱内保存。
上述第二步中表面活性剂的用量是无机前驱体的5-35％。
制备电极时，母液在20-40℃、湿度20-30％条件下蒸发0.5-2小时为好。
本发明电极适用于对太阳能的吸收利用，效果显著。
本发明制备的人工改性和优化的生物光敏蛋白——纳米半导体复合光电极表现出极佳的光电转换性能。一方面，电荷分离态寿命的延长使得经过色素替换后的RC在该复合光电极中的光电转换效率要远好于相同条件下天然RC的转换效率；另一方面，利用特定的介孔半导体材料可以实现RC高效固定的前提下对光激发后RC电荷分离的有效促进。改性并优化后的RC对纳米半导体的敏化作用大大提高了整个复合光电极对太阳能的吸收和利用。本发明制备方法不复杂，操作步骤为该领域技术人员现有技术，具有很好推广应用前景。
具体实施方式
实施例一：以菠菜作为原料，获得植物叶绿素粗提掖，在4℃条件下，进行DEAE-Sephaerose CL-6B柱层析。先用约100ml石油醚清洗，然后用含0.5％正丙醇的石油醚将植物色素依次洗脱下来，弃去先洗脱的类胡萝卜素，收集接着洗脱下来的叶绿素a，在N2中干燥后，溶于1g/L的吡啶溶液中于4℃下暗态保存。取10ml稀释的植物叶绿素吡啶溶液(0.1mmol/L)，于N2中干燥，加0.5ml冰醋酸。得到的植物脱镁叶绿素在N2中干燥后，溶于10ml丙酮，4℃下暗态保存。取一定体积的RC溶液，加入10％体积上述植物脱镁叶绿素的丙酮溶液，使植物脱镁叶绿素与RC的摩尔比大于20％。搅拌后，在43.5±0.5℃条件下共保温一段时间，随后在DEAE-纤维素层析柱(DE-52)上用0.09mol/LNaCl的TL缓冲稀溶液约600ml洗去游离的叶绿素，用0.15mol/L的NaCl的TL缓冲溶液将修饰的RC洗脱下来，再将待纯化的RC溶液铺在10％～40％的蔗糖梯度上，4℃条件下离心16h，从上至下取第二层淡黄色上清液，即得到纯化后的色素替换RC。采用pH 8.0的Tris-HCl缓冲溶液稀释上述色素替换的RC，得到浓度为2mM的变异RC的稀溶液，置于4℃冰箱内保存。取1g P123溶于10g乙醇中。然后将0.8g WCl6和2.4g Ti(OBu)4混合后加入上述溶液中，并在常温下搅拌2h。得到的母液在30℃，湿度20％-30％的条件下充分蒸发。将该WO3-TiO2母液旋涂于表面经过清洁处理的ITO(掺铟的氧化锡)玻璃上，在45℃的条件下放置一天后，将该WO3-TiO2/ITO膜置于350℃马福炉内恒温2h，最终得到孔径分布均一的虫洞结构介孔WO3-TiO2薄膜电极。将新制备的介孔WO3-TiO2薄膜电极置于上述浓度为2mM的变异RC的pH 8.0 Tris-HCl缓冲液中，在4℃冰箱内吸附3天，得到的人工改性的生物光敏蛋白——纳米半导体复合膜电极置于干态或同种蛋白的稀溶液中于4℃冰箱内保存。该复合膜光电极表现出优良的光电转化性能。在8mM的连二亚硫酸钠的Tris-HCl溶液中以60W的白织灯为光源，测得的色素替换RC——介孔WO3-TiO2复合光电极的短路光电流约为3.2微安，是相同条件下制备和测试的不含RC的介孔WO3-TiO2电极短路光电流(0.7微安)的4.6倍。上述制备的复合膜电极具有很好的重现性。该复合膜电极置于4℃冰箱内保存，在一定时间内表现出良好的稳定性与重复性。
实施例二：采用卟啉正丙醚醇作为替换色素，在一定体积的RC溶液中加入10％体积替换色素的丙酮溶液，使卟啉正丙醚醇与RC的摩尔比大于25％。搅拌后，在43.5±0.2℃条件下共保温一段时间，随后在DEAE-纤维素层析柱(DE-52)上用0.09mol/L NaCl的TL缓冲稀溶液约600ml洗去游离的叶绿素，用0.15mol/L的NaCl-的TL缓冲溶液将修饰的RC洗脱下来，再将待纯化的RC溶液铺在10％～40％的蔗糖梯度上，4℃条件下离心16h，从上至下取第二层淡黄色上清液，即得到纯化后的色素替换RC。采用pH 8.0的Tris-HCl缓冲溶液稀释上述色素替换的RC，得到浓度为2mM的变异RC的稀溶液，置于4℃冰箱内保存。介孔WO3-TiO2薄膜电极的制备同实施例一。将新制备的介孔WO3-TiO2薄膜电极置于上述浓度为2mM的变异RC的pH8.0 Tris-HCl缓冲液中，在4℃冰箱内吸附4天，得到的人工改性的生物光敏蛋白——纳米半导体复合膜电极置于干态或同种蛋白的稀溶液中于4℃冰箱内保存。在8mM的连二亚硫酸钠的Tris-HCl溶液中以60W的白织灯为光源，测得的色素替换RC——介孔WO3-TiO2复合光电极的短路光电流约为2.6微安，是相同条件下制备和测试的不含RC的介孔WO3-TiO2电极短路光电流(0.7微安)的3.7倍。上述制备的复合膜电极具有很好的重现性。该复合膜电极置于4℃冰箱内保存，在一定时间内表现出良好的稳定性与重复性。
实施例三：采用卟啉双甲醚双醇作为替换色素。卟啉双甲醚双醇替换RC的Tris-HCl缓冲溶液的制备同实施例二。介孔WO3-TiO2薄膜电极的制备同实施例一。色素替换RC——介孔WO3-TiO2复合光电极的制备及光电性能的测定过程同实施例二。该色素替换RC——介孔WO3-TiO2复合光电极在上述条件下的短路光电流约为2.6微安，是相同条件下制备和测试的不含RC的介孔WO3-TiO2电极短路光电流(0.7微安)的3.7倍。上述制备的复合膜电极具有很好的重现性。该复合膜电极置于4℃冰箱内保存，在一定时间内表现出良好的稳定性与重复性。
实施例四：采用次卟啉二甲酯作为替换色素。其它实验条件同实施例三。该色素替换RC——介孔WO3-TiO2复合光电极在上述条件下的短路光电流约为2.2微安，是相同条件下制备和测试的不含RC的介孔WO3-TiO2电极短路光电流(0.7微安)的3.1倍。上述制备的复合膜电极具有很好的重现性。该复合膜电极置于4℃冰箱内保存，在一定时间内表现出良好的稳定性与重复性。
Claims (4)
1、一种生物光敏蛋白与纳米半导体复合光电极的制备方法，其特征是：
(1)将紫细菌光合反应中心蛋白RC与植物脱镁叶绿素或卟啉类色素的丙酮溶液搅拌混合，在40℃～45℃温度下共保温0.5-2小时，然后在层析柱上洗去游离的叶绿素，再将RC洗脱下来后离心纯化，用缓冲溶液稀释至浓度为0.5～2mM的色素替换RC溶液待用；上述混合时丙酮溶液与RC溶液的体积比是5-20％，植物脱镁叶绿素或其它卟啉类色素与RC的摩尔比大于20-30％；
(2)将两种制备介孔体半导体基体的无机前驱体混合后溶于表面活性剂溶液，常温下搅拌1～4h得到母液，母液蒸发后旋涂或蘸涂于掺铟氧化锡玻璃上，干燥，恒温，得到介孔半导体薄膜电极；
(3)将上述介孔半导体薄膜电极置于浓度为0.5～2mM的色素替换RC缓冲溶液4℃下吸附1～5天即可。
2、根据权利要求1所述的生物光敏蛋白与纳米半导体复合光电极的制备方法，其特征是制备介孔半导体薄膜电极时表面活性剂用量是无机前驱体的5-35％。
3、根据权利要求1所述的生物光敏蛋白与纳米半导体复合光电极的制备方法，其特征是制备介孔半导体薄膜电极时，母液在温度20-40℃，湿度20-30％条件下蒸发0.5-2小时。
4、根据权利要求1所述的生物光敏蛋白与纳米半导体复合光电极在太阳能的吸收方面的运用。
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