新型化学修饰电极的制备及应用
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摘要
化学修饰电极在分子层面上实现了对电极功能的设计,可以按意图赋予电极预定的功能,以便能选择性地进行检测反应。本论文中主要研究了三种化学修饰玻碳电极的制备及其在生物传感与化学传感中的应用,主要包括以下内容:
(1)通过共价键合法将转录因子Sp1修饰在玻碳电极表面,并以之作为探针使用交流阻抗法(EIS)与差示脉冲法(DPV)检测其与样品DNA的特异性结合。结果显示,我们的方法可有效地将活性Sp1修饰到电极表面,且该修饰电极不仅可用于研究Sp1与其DNA识别序列在不同条件下的结合和解离,还可用于研究点突变与抑制剂(抗癌药品光辉霉素)对结合的影响。此外,对该修饰电极的特异性、灵敏度、重复性以及再生能力都进行了研究,结果表明其能够在大量无关DNA序列中检测出低至1×10?10 mol/L的特异性结合序列(wtDNA)。
(2)在上述修饰电极的基础上,为进一步提高电极性能,以葡萄糖为原料通过水热法制备了有良好生物相容性和导电性的碳纳米球(CNP),使用CNP修饰玻碳电极后再与Sp1通过酰胺键偶联。结果显示,CNP的修饰进一步优化了该蛋白修饰电极的检测性能,具体表现在:检测信号的背景干扰降低、信噪比明显增大,对wtDNA检测限降低了5个数量级达到1 fM,且电极的再生能力得到了极大的增强,有望成为一种快速而有效的检测手段,以用于DNA结合蛋白与其识别序列间相互作用的研究,及对其新DNA识别位点的筛查和新型药物抑制剂的筛选。
(3)使用Nafion膜将半胱氨酸修饰的碳纳米球固定在玻碳电极表面,通过阳极溶出伏安法实现了对重金属离子(铅和镉)的痕量检测。结果表明,铅离子和镉离子的检测限分别为1×10~(-14) M和1×10~(-9) M,这与裸电极相比,效果得到了极大的增强,并且该修饰电极可以对溶液中的铅离子和镉离子同时进行检测。
In electrochemistry, electrodes can be functionally designed for selective detections through chemical modification of the electrode surface. In this thesis, three types of chemically modified glass carbon electrodes (GCEs) have been fabricated using different methods, and their applications in biosensing and chemical sensing have been assayed:
The first one, a biomolecule-modified GCE, was fabricated by covalently immobilizing transcription factor Sp1 on the GCE surface and the interaction of immobilized Sp1 with different DNA samples was analyzed using electrochemical impedance spectroscopy (EIS) and difference pulse voltammetry (DPV). Our results show that both the association/disassociation of Sp1 with its consensus sequence (wtDNA) under different conditions and the change of affinity caused by either point mutations or an inhibitor (anticancer drug mithramycin A) can be effectively monitored through Sp1-functionalized GCE, and as low as 1×10?10 mol/L wtDNA can be selectively detected in a DNA pool containing huge access of non-specific DNA fragments.
I further developed an enhanced electrode from the above one, taking the advantage of bio-compatible and high electrical conductive carbon nanoparticles (CNPs) derived from glucose by one step hydrothermal oxidation method. To fabricate this electrode, the bare GCE was first modified by CNPs before protein (Sp1) immobilization. The CNPs-modification greatly enhanced the performance of the electrode, and was able to lower the detection limit of Sp1-specific DNA to 1×10?15 mol/L. The CNPs-Sp1-modified GCE exihibits enhanced signal-to-noise ratio, and can be easily regenerated, suggesting a promising approach for rapid and highly sensitive DNA detection.
For the last one, L-cysteine modified CNPs were immobilized on the GCE by Nafion. This modified electrode was used to detect trace amount of heavy metal ions by anodic stripping voltammeter. Both Pb~(2+) and Cd~(2+) can be detected concurrently in a mixture, and compare to the bare GCE, the Cys-CNPs modification greatly enhanced the performance of the electrode, successfully lowering the detection limits for Pb2+ and Cd2+ to 1×10~(-14) M and 1×10~(-9) M, respectively.
(1)通过共价键合法将转录因子Sp1修饰在玻碳电极表面,并以之作为探针使用交流阻抗法(EIS)与差示脉冲法(DPV)检测其与样品DNA的特异性结合。结果显示,我们的方法可有效地将活性Sp1修饰到电极表面,且该修饰电极不仅可用于研究Sp1与其DNA识别序列在不同条件下的结合和解离,还可用于研究点突变与抑制剂(抗癌药品光辉霉素)对结合的影响。此外,对该修饰电极的特异性、灵敏度、重复性以及再生能力都进行了研究,结果表明其能够在大量无关DNA序列中检测出低至1×10?10 mol/L的特异性结合序列(wtDNA)。
(2)在上述修饰电极的基础上,为进一步提高电极性能,以葡萄糖为原料通过水热法制备了有良好生物相容性和导电性的碳纳米球(CNP),使用CNP修饰玻碳电极后再与Sp1通过酰胺键偶联。结果显示,CNP的修饰进一步优化了该蛋白修饰电极的检测性能,具体表现在:检测信号的背景干扰降低、信噪比明显增大,对wtDNA检测限降低了5个数量级达到1 fM,且电极的再生能力得到了极大的增强,有望成为一种快速而有效的检测手段,以用于DNA结合蛋白与其识别序列间相互作用的研究,及对其新DNA识别位点的筛查和新型药物抑制剂的筛选。
(3)使用Nafion膜将半胱氨酸修饰的碳纳米球固定在玻碳电极表面,通过阳极溶出伏安法实现了对重金属离子(铅和镉)的痕量检测。结果表明,铅离子和镉离子的检测限分别为1×10~(-14) M和1×10~(-9) M,这与裸电极相比,效果得到了极大的增强,并且该修饰电极可以对溶液中的铅离子和镉离子同时进行检测。
In electrochemistry, electrodes can be functionally designed for selective detections through chemical modification of the electrode surface. In this thesis, three types of chemically modified glass carbon electrodes (GCEs) have been fabricated using different methods, and their applications in biosensing and chemical sensing have been assayed:
The first one, a biomolecule-modified GCE, was fabricated by covalently immobilizing transcription factor Sp1 on the GCE surface and the interaction of immobilized Sp1 with different DNA samples was analyzed using electrochemical impedance spectroscopy (EIS) and difference pulse voltammetry (DPV). Our results show that both the association/disassociation of Sp1 with its consensus sequence (wtDNA) under different conditions and the change of affinity caused by either point mutations or an inhibitor (anticancer drug mithramycin A) can be effectively monitored through Sp1-functionalized GCE, and as low as 1×10?10 mol/L wtDNA can be selectively detected in a DNA pool containing huge access of non-specific DNA fragments.
I further developed an enhanced electrode from the above one, taking the advantage of bio-compatible and high electrical conductive carbon nanoparticles (CNPs) derived from glucose by one step hydrothermal oxidation method. To fabricate this electrode, the bare GCE was first modified by CNPs before protein (Sp1) immobilization. The CNPs-modification greatly enhanced the performance of the electrode, and was able to lower the detection limit of Sp1-specific DNA to 1×10?15 mol/L. The CNPs-Sp1-modified GCE exihibits enhanced signal-to-noise ratio, and can be easily regenerated, suggesting a promising approach for rapid and highly sensitive DNA detection.
For the last one, L-cysteine modified CNPs were immobilized on the GCE by Nafion. This modified electrode was used to detect trace amount of heavy metal ions by anodic stripping voltammeter. Both Pb~(2+) and Cd~(2+) can be detected concurrently in a mixture, and compare to the bare GCE, the Cys-CNPs modification greatly enhanced the performance of the electrode, successfully lowering the detection limits for Pb2+ and Cd2+ to 1×10~(-14) M and 1×10~(-9) M, respectively.
引文
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