新型铱配合物的合成、电致化学发光性质研究及在生命物质分析中的应用
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摘要
Ru(bpy)32+/TPA体系在过去的电致化学发光及其应用中发挥着重要的作用,但更多的研究者去努力开发新型的电致化学发光标记物。事实上已有很多中性铱配合物在有机溶剂中的电致化学发光效率比Ru(bpy)32+高,由于其在水中的溶解性差及缺少生物活性标记物,使得他们的实际应用受到限制。DNA作为重要的生命遗传物质信息的载体,具有贮存和传递信息的功能,研究DNA的结构和功能,可以从分子水平上了解生命现象的本质,在现代生命科学、医学领域有重要的意义。肿瘤是目前人类健康的最大威胁之一,成为现在医学领域研究的热门领域。有关肿瘤细胞和肿瘤标志物的检测方法研究已经成为肿瘤防治的热点课题之一。本文合成了一系列新型的电致化学发光铱配合物,并研究了其在电致化学发光DNA生物传感器构建中的应用及其在细胞成像检测方面的应用。具体研究内容如下:
1.合成了五种中性铱配合物,并对其结构进行了表征,对其紫外、荧光、电化学发光性质进行了研究。使用TPA作为氧化还原共反应物,分别研究了其在乙腈溶液和混合溶液中的电致化学发光性质。同时也研究了影响电致化学发光效率的几种影响因素,包括:工作电极、pH、表面活性剂等。同时表面活性剂的加入也使得其电致化学发光增强。此项研究提供了一种用环金属配合物和辅助配体设计新型的铱配合物来调节和改变电致化学发光效率的新方法。
2.合成了一种新型高效的铱配合物,并对其结构进行了表征,对其紫外、荧光、电化学及电致化学发光性质进行了研究。近来,由于聚合酶及内切酶对DNA的循环放大作用被证实,我们制作了纳米金探针,基于以上两种酶的放大作用我们对DNA进行循环放大检测。通过3σ法则计算,其检测线为8.2×10?17 M,据我们所知,这是目前最灵敏的检测方法。同时,我们也用此法对钌配合物做了对比实验,通过3σ法则计算,其检测限为9.7×10?15 M。为进一步揭示铱和钌配合物传感器灵敏度的不同,用相同的靶DNA的浓度,进行了ECL检测。实验发现,在靶DNA浓度相同的条件下,铱标记的传感器的灵敏度比钌高200倍。
3.为了研究其在实际中的应用性,我们Ramos细胞的检测液中加入了血清真样,实验结果发现当血液真样少于总体积的6%时,对细胞的ECL检测无影响。当真样加到总体积的8%,电致化学发光信号明显降低,因此6%的真样被用在后续实验中。通过3σ法则计算,细胞的检测限为6个。本实合成了一种新型的电致化学发光标记物,更重要的是,基于这种发光物质制作的生物传感器对靶DNA的检测限是传统钌配合物的200倍。在含6%真样中对Ramos细胞的检测达到6个。此种铱配合物易于合成,在空气中稳定存在,且发光效率高,为生物及临床检测提供了可靠的理论基础。
Although Ru(bpy)32+/TPA system (TPA = tripropylamine) has played a crucial role in the development of Electrochemiluminescence (ECL) and its applications, much effort has been devoted to develop new ECL labels to improve the sensitivity. In fact, although many neutral Ir(III) complexes have been demonstrated to display high ECL efficiencies in organic solutions usually with a degree of efficiency much higher than Ru(bpy)32+, their applicative field has been restricted by their very poor water-solubility and lack of active group for biological labeling. DNA is one of important genetic material to the life and is in charge of storing genetic information transferring genetic information. Studies of its structure and function contribute to realize the essence of the phenomenon of life at the molecular level , which has important significance in the field of the modern life sciences and medicine.Tumors, one of the most threatening diseases of the human being, plays an essential role in modern medicine. In recent years, many assay methods and sensors as currently popular techniques are being developed for the detection of tumor-related biomarkers. In this work, several new ECL neutral tris-chelate iridium(III) complexes were synthesized , study their application in the construction of new DNA biosensor and cell detecting. The details are given as follows:
In this work, several neutral tris-chelate iridium(III) complexes, which were prepared and their investigations of the structure, optical, and electrochemical properties are reported. Using tri-n-propylamine (TPA) as an oxidative-reductive co-reactant, their ECL properties were studied in acetonitrile (CH3CN) and mixed CH3CN/H2O (50:50, v/v) solutions, respectively. Meanwhile, the influencing factors of ECL efficiencies, including working electrode, pH, and surfactant were investigated. A remarkable ECL enhancement (up to about 13.5 times), in comparison with the commonly used Ru(bpy)32+ (2,2'-bipyridyl) ruthenium(Ⅲ), is observed from Ir(FPP)2(acac) (where FPP is 2-(4-fluorophenyl)-4-phenylpyridine, acac = acetylacetone) at Pt disk electrode. At the same time, an increase in ECL efficiency is also observed in surfactant media. This study provided a new method for further improving and tuning the ECL efficiency by designing new iridium complexes with the appropriate cyclometalated or ancillary ligands.
A new Ir complex was synthesized, its UV-vis absorption, fluorescence, electrochemistry and ECL characteristics were explored. Recently, circular amplifications based on DNA polymerase and endonuclease were confirmed that they were effective means for improving sensitivity. Encouraged by the above experiment results and to demonstrate the generality of Ir complex as a luminescent reagent, the above developed nanoprobe was designed to construct a protocol based on strand-displacement DNA polymerization and nicking endonuclease assisted cycle。The detection limit of 8.2×10?17 M could be estimated by using 3σruler, which is one of the most sensitive methods for detection and analysis of DNA to the best of our knowledge. Also, a simultaneous control experiment was carried out by labeling with Ru complex. The detection limit of 9.7×10?15 M was calculated according to 3σ. It is obvious that the sensitivity of the proposed protocol is improved 2 orders of magnitude by replacing conventional Ru complex. To further reveal the difference in sensitivity of biosensor between employing Ir complex and Ru complex label, the ECL responses of the biosensor with the same concentration of target DNA were tested. It could be found that, in the presence of the same concentration of target DNA, the ECL signal labeled with Ir complex is about 200 times higher than that based on Ru complex. It was found there was no matrix effect on the responses of detection when added blood is less than 6% in volume。However, a significant decrease of the signal was observed after adding more than 8% of blood. Herefore, 6% of human blood in volume was used in the following experiments. The study has presented a new luminescence labeling reagent for ECL, and it has been successfully applied to construct ultrasensitive biosensor. More importantly, base on the reagent, the sensitivity of biosensor for target DNA detection is improved more than 200 times compared to that employing the well-known ECL reagent Ru complex. As evidenced by our research, this assay allows us to determine cancer cell down to 6 cells and exhibits a significant specificity for Ramos even in 6% real human blood sample. It is expected that the easily synthetic, air-stable and very efficient phosphorescent emitter can be used in more fields for biological assays and clinical diagnoses.
1.合成了五种中性铱配合物,并对其结构进行了表征,对其紫外、荧光、电化学发光性质进行了研究。使用TPA作为氧化还原共反应物,分别研究了其在乙腈溶液和混合溶液中的电致化学发光性质。同时也研究了影响电致化学发光效率的几种影响因素,包括:工作电极、pH、表面活性剂等。同时表面活性剂的加入也使得其电致化学发光增强。此项研究提供了一种用环金属配合物和辅助配体设计新型的铱配合物来调节和改变电致化学发光效率的新方法。
2.合成了一种新型高效的铱配合物,并对其结构进行了表征,对其紫外、荧光、电化学及电致化学发光性质进行了研究。近来,由于聚合酶及内切酶对DNA的循环放大作用被证实,我们制作了纳米金探针,基于以上两种酶的放大作用我们对DNA进行循环放大检测。通过3σ法则计算,其检测线为8.2×10?17 M,据我们所知,这是目前最灵敏的检测方法。同时,我们也用此法对钌配合物做了对比实验,通过3σ法则计算,其检测限为9.7×10?15 M。为进一步揭示铱和钌配合物传感器灵敏度的不同,用相同的靶DNA的浓度,进行了ECL检测。实验发现,在靶DNA浓度相同的条件下,铱标记的传感器的灵敏度比钌高200倍。
3.为了研究其在实际中的应用性,我们Ramos细胞的检测液中加入了血清真样,实验结果发现当血液真样少于总体积的6%时,对细胞的ECL检测无影响。当真样加到总体积的8%,电致化学发光信号明显降低,因此6%的真样被用在后续实验中。通过3σ法则计算,细胞的检测限为6个。本实合成了一种新型的电致化学发光标记物,更重要的是,基于这种发光物质制作的生物传感器对靶DNA的检测限是传统钌配合物的200倍。在含6%真样中对Ramos细胞的检测达到6个。此种铱配合物易于合成,在空气中稳定存在,且发光效率高,为生物及临床检测提供了可靠的理论基础。
Although Ru(bpy)32+/TPA system (TPA = tripropylamine) has played a crucial role in the development of Electrochemiluminescence (ECL) and its applications, much effort has been devoted to develop new ECL labels to improve the sensitivity. In fact, although many neutral Ir(III) complexes have been demonstrated to display high ECL efficiencies in organic solutions usually with a degree of efficiency much higher than Ru(bpy)32+, their applicative field has been restricted by their very poor water-solubility and lack of active group for biological labeling. DNA is one of important genetic material to the life and is in charge of storing genetic information transferring genetic information. Studies of its structure and function contribute to realize the essence of the phenomenon of life at the molecular level , which has important significance in the field of the modern life sciences and medicine.Tumors, one of the most threatening diseases of the human being, plays an essential role in modern medicine. In recent years, many assay methods and sensors as currently popular techniques are being developed for the detection of tumor-related biomarkers. In this work, several new ECL neutral tris-chelate iridium(III) complexes were synthesized , study their application in the construction of new DNA biosensor and cell detecting. The details are given as follows:
In this work, several neutral tris-chelate iridium(III) complexes, which were prepared and their investigations of the structure, optical, and electrochemical properties are reported. Using tri-n-propylamine (TPA) as an oxidative-reductive co-reactant, their ECL properties were studied in acetonitrile (CH3CN) and mixed CH3CN/H2O (50:50, v/v) solutions, respectively. Meanwhile, the influencing factors of ECL efficiencies, including working electrode, pH, and surfactant were investigated. A remarkable ECL enhancement (up to about 13.5 times), in comparison with the commonly used Ru(bpy)32+ (2,2'-bipyridyl) ruthenium(Ⅲ), is observed from Ir(FPP)2(acac) (where FPP is 2-(4-fluorophenyl)-4-phenylpyridine, acac = acetylacetone) at Pt disk electrode. At the same time, an increase in ECL efficiency is also observed in surfactant media. This study provided a new method for further improving and tuning the ECL efficiency by designing new iridium complexes with the appropriate cyclometalated or ancillary ligands.
A new Ir complex was synthesized, its UV-vis absorption, fluorescence, electrochemistry and ECL characteristics were explored. Recently, circular amplifications based on DNA polymerase and endonuclease were confirmed that they were effective means for improving sensitivity. Encouraged by the above experiment results and to demonstrate the generality of Ir complex as a luminescent reagent, the above developed nanoprobe was designed to construct a protocol based on strand-displacement DNA polymerization and nicking endonuclease assisted cycle。The detection limit of 8.2×10?17 M could be estimated by using 3σruler, which is one of the most sensitive methods for detection and analysis of DNA to the best of our knowledge. Also, a simultaneous control experiment was carried out by labeling with Ru complex. The detection limit of 9.7×10?15 M was calculated according to 3σ. It is obvious that the sensitivity of the proposed protocol is improved 2 orders of magnitude by replacing conventional Ru complex. To further reveal the difference in sensitivity of biosensor between employing Ir complex and Ru complex label, the ECL responses of the biosensor with the same concentration of target DNA were tested. It could be found that, in the presence of the same concentration of target DNA, the ECL signal labeled with Ir complex is about 200 times higher than that based on Ru complex. It was found there was no matrix effect on the responses of detection when added blood is less than 6% in volume。However, a significant decrease of the signal was observed after adding more than 8% of blood. Herefore, 6% of human blood in volume was used in the following experiments. The study has presented a new luminescence labeling reagent for ECL, and it has been successfully applied to construct ultrasensitive biosensor. More importantly, base on the reagent, the sensitivity of biosensor for target DNA detection is improved more than 200 times compared to that employing the well-known ECL reagent Ru complex. As evidenced by our research, this assay allows us to determine cancer cell down to 6 cells and exhibits a significant specificity for Ramos even in 6% real human blood sample. It is expected that the easily synthetic, air-stable and very efficient phosphorescent emitter can be used in more fields for biological assays and clinical diagnoses.
引文
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