电化学生物传感技术用于重金属和蛋白质的检测
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摘要
环境中的重金属离子难以被微生物降解,通过生物链的累积进入人体,严重威胁着人们的身体健康及生命安全。蛋白质是生物体最基本的物质之一,在生物体内含量大,种类繁多,参与生命体每一步反应和活动,与生命的起源和进化、物质运转、遗传等息息相关。而各种蛋白质在人体内的浓度变化直接与人体的新陈代谢情况密切相关,已成为疾病判别与诊断的依据。因此,建立简单、快捷、灵敏、实用的蛋白质检测方法对药物研究、临床诊治等医学研究具有重要的意义。电化学生物传感器具有实时、稳定、在线、快捷、选择、直观的超灵敏分析检测的优点,检测对象涉及医药、环境、食品和金属等多领域,使得以电化学方法为基础的生物分子技术具有极其广阔的发展空间和应用前景。因此,在本研究论文中,我们建构了一下几种电化学传感器。
(1)本文建构了一种基于目标物诱导DNA链折叠的新型电化学传感器实现对汞离子的超灵敏检测。实验设计了一条巯基标记的包含了33个碱基的DNA链,该链由7对胸腺嘧啶-胸腺嘧啶错配碱基对分隔5对互补杂交碱基对而成,其通过巯基自组装固定在金电极表面,然后与一条二茂铁标记的包含10个碱基的DNA链杂交,产生很强的电化学氧化还原信号。在汞离子的作用下,由于T-Hg-T结构的形成,汞离子诱导33个碱基的DNA链折叠成发卡结构,二茂铁标记的DNA链从电极表面释放下来,电化学信号降低。本传感器的响应信号通过循环伏安,差示脉冲伏安以及电化学阻抗谱图进行了完整的表征。结果表明,该传感器实现了对汞离子的超灵敏检测,浓度范围从0.1 nM到5μM,检测限为0.06 nM。此外,该方法还具有很好的选择性,能实现对实际环境样品中Hg2+的检测。
(2)本章建立了一种基于免疫脂质体的电化学传感器用于甲胎蛋白AFP的超灵敏检测。通过在脂质体中包埋大量的电活性物质标记的DNA链结合电化学方法的灵敏性进行放大检测,使灵敏度大大提高,显示了很好的线性范围、稳定性和高选择性,检测范围从1 fg/mL到10 ng/mL,使其检测限大大降低,最低检测下限为0.7 fg/mL。
(3)建立了一种基于包酶脂质体和生物催化金属沉积进行信号二次放大的新型电化学免疫传感器检测前列腺特异性抗原PSA。包裹的碱性磷酸酶和脂质体表面功能化的检测探针作为检测试剂,通过夹心免疫反应,目标分析物PSA和功能化的脂质体被依次固定到电极表面,在表面活性剂的作用下,脂质体被破解释放出内部包裹的碱性磷酸酶ALP, ALP催化抗坏血酸磷酸酯产生的抗坏血酸还原银离子形成金属银纳米颗粒沉积在电极表面。本传感器的响应信号用线性扫描伏安法进行检测。结果显示,阳极溶出峰电流与PSA的浓度在0.01 ng/mL到100 ng/mL具有很好的线性关系,最低检测下限为0.007 ng/mL。由于医学检测人血清中PSA的最低浓度为4.0 ng/mL,该电化学免疫传感器在医疗诊断领域具有可行性和广泛地应用空间。
Heavy metals in the environment are difficult to degrade, which can be accumulated into the human body through the food chain, leading a serious threat to people's health and life. Proteins as the basic material of living organisms with large content and wide variety take part in every step of the reaction and activities, and being closely related to the origin, evolution of life, physical functioning, genetic and so on. Moreover, the concentration of proteins is closely related to the body's metabolism, becoming the basis for identification and diagnosis. Therefore, it is of great significance to establish a simple, fast, sensitive protein detection method used in drug research, clinical treatment of medical research. Electrochemical biosensor with real-time, stable, online, quick, selective, visual inspection involved in pharmaceutical, environment, food and metal areas, indicating its broad space to develop and apply. In this research paper, we construct several electrochemical sensors, the details are described as follows:
(1) A novel electrochemical sensor has been developed for sensitive and selective detection of mercury(II) based on target-induced structure-switching DNA. A 33-mer oligonucleotide 1 with five self-complementary base pairs separated by seven thymine-thymine mismatches was first immobilized on the electrode via self-assembly of the terminal thiol moiety and then hybridized with a ferrocene-tagged oligonucleotide 2, leading to a high redox current. In the presence of Hg2+, mercury-mediated base pairs (T-Hg-T) induced the folding of the oligonucleotidel into a hairpin structure, resulting in the release of the ferrocene-tagged oligonucleotide 2 from the electrode surface with a substantially decreased redox current. The response characteristics of the sensor were thoroughly investigated using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The effect of the reaction temperature on the response of the sensor was also studied in detail. The results revealed that the sensor showed sensitive response to Hg2+ in a concentration range from 0.1 nM to 5μM with a detection limit of 0.06 nM. In addition, this strategy afforded exquisite selectivity for Hg2+ against other environmentally related metal ions, which was superior to that of previous anodic stripping voltammetry (ASV)-based techniques. The excellent sensitivity and selectivity signified the potential of the sensor for Hg2+ detection in real environmental samples.
(2)We establish a new electrochemical sensor based on immunoliposome for sensitive and selective detection of alpha-fetoprotein. Sensitive electrochemical methods and encapsulated labled DNA probe in liposome, lead to inceasing of sensitivity. At the same time, The results revealed that the sensor showed sensitive response to AFP in a concentration range from 1 fg/mL to 10 ng/mL, with a detection limit of 0.7 fg/mL.
(3)A novel electrochemical immunosensor based on double signal amplification of enzyme-encapsulated liposomes and biocatalytic metal deposition was developed for the detection of human prostate specific antigen (PSA). Alkaline phosphatase (ALP)-encapsulated and detection antibody-functionalized liposomes were first prepared and used as the detection reagent. In the sandwich immunoassay, the model analyte PSA was first captured by anti-PSA capture antibody immobilized on the electrode and then sandwiched with the functionalized liposomes. The bound liposomes were then lysed with surfactant to release the encapsulated ALP, which served as secondary signal amplification means. ALP on the electrode surface initiated the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid. The latter, in turn, reduced silver ions on the electrode surface, leading to deposition of the metal silver on the electrode surface. Linear sweep voltammetry (LSV) was chosen to detect the amount of the deposited silver. The results showed that the anodic stripping peak current was linearly dependent on the PSA concentration in the range of 0.01 ng/mL to100.0 ng/mL, and a detection limit as low as 0.007 ng/mL can be obtained. Since the cut-off value of human PSA is 4.0 ng/mL, the proposed electrochemical immunosensor would be expected to gain widespread applications for the detection of PSA in clinical diagnosis.
(1)本文建构了一种基于目标物诱导DNA链折叠的新型电化学传感器实现对汞离子的超灵敏检测。实验设计了一条巯基标记的包含了33个碱基的DNA链,该链由7对胸腺嘧啶-胸腺嘧啶错配碱基对分隔5对互补杂交碱基对而成,其通过巯基自组装固定在金电极表面,然后与一条二茂铁标记的包含10个碱基的DNA链杂交,产生很强的电化学氧化还原信号。在汞离子的作用下,由于T-Hg-T结构的形成,汞离子诱导33个碱基的DNA链折叠成发卡结构,二茂铁标记的DNA链从电极表面释放下来,电化学信号降低。本传感器的响应信号通过循环伏安,差示脉冲伏安以及电化学阻抗谱图进行了完整的表征。结果表明,该传感器实现了对汞离子的超灵敏检测,浓度范围从0.1 nM到5μM,检测限为0.06 nM。此外,该方法还具有很好的选择性,能实现对实际环境样品中Hg2+的检测。
(2)本章建立了一种基于免疫脂质体的电化学传感器用于甲胎蛋白AFP的超灵敏检测。通过在脂质体中包埋大量的电活性物质标记的DNA链结合电化学方法的灵敏性进行放大检测,使灵敏度大大提高,显示了很好的线性范围、稳定性和高选择性,检测范围从1 fg/mL到10 ng/mL,使其检测限大大降低,最低检测下限为0.7 fg/mL。
(3)建立了一种基于包酶脂质体和生物催化金属沉积进行信号二次放大的新型电化学免疫传感器检测前列腺特异性抗原PSA。包裹的碱性磷酸酶和脂质体表面功能化的检测探针作为检测试剂,通过夹心免疫反应,目标分析物PSA和功能化的脂质体被依次固定到电极表面,在表面活性剂的作用下,脂质体被破解释放出内部包裹的碱性磷酸酶ALP, ALP催化抗坏血酸磷酸酯产生的抗坏血酸还原银离子形成金属银纳米颗粒沉积在电极表面。本传感器的响应信号用线性扫描伏安法进行检测。结果显示,阳极溶出峰电流与PSA的浓度在0.01 ng/mL到100 ng/mL具有很好的线性关系,最低检测下限为0.007 ng/mL。由于医学检测人血清中PSA的最低浓度为4.0 ng/mL,该电化学免疫传感器在医疗诊断领域具有可行性和广泛地应用空间。
Heavy metals in the environment are difficult to degrade, which can be accumulated into the human body through the food chain, leading a serious threat to people's health and life. Proteins as the basic material of living organisms with large content and wide variety take part in every step of the reaction and activities, and being closely related to the origin, evolution of life, physical functioning, genetic and so on. Moreover, the concentration of proteins is closely related to the body's metabolism, becoming the basis for identification and diagnosis. Therefore, it is of great significance to establish a simple, fast, sensitive protein detection method used in drug research, clinical treatment of medical research. Electrochemical biosensor with real-time, stable, online, quick, selective, visual inspection involved in pharmaceutical, environment, food and metal areas, indicating its broad space to develop and apply. In this research paper, we construct several electrochemical sensors, the details are described as follows:
(1) A novel electrochemical sensor has been developed for sensitive and selective detection of mercury(II) based on target-induced structure-switching DNA. A 33-mer oligonucleotide 1 with five self-complementary base pairs separated by seven thymine-thymine mismatches was first immobilized on the electrode via self-assembly of the terminal thiol moiety and then hybridized with a ferrocene-tagged oligonucleotide 2, leading to a high redox current. In the presence of Hg2+, mercury-mediated base pairs (T-Hg-T) induced the folding of the oligonucleotidel into a hairpin structure, resulting in the release of the ferrocene-tagged oligonucleotide 2 from the electrode surface with a substantially decreased redox current. The response characteristics of the sensor were thoroughly investigated using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The effect of the reaction temperature on the response of the sensor was also studied in detail. The results revealed that the sensor showed sensitive response to Hg2+ in a concentration range from 0.1 nM to 5μM with a detection limit of 0.06 nM. In addition, this strategy afforded exquisite selectivity for Hg2+ against other environmentally related metal ions, which was superior to that of previous anodic stripping voltammetry (ASV)-based techniques. The excellent sensitivity and selectivity signified the potential of the sensor for Hg2+ detection in real environmental samples.
(2)We establish a new electrochemical sensor based on immunoliposome for sensitive and selective detection of alpha-fetoprotein. Sensitive electrochemical methods and encapsulated labled DNA probe in liposome, lead to inceasing of sensitivity. At the same time, The results revealed that the sensor showed sensitive response to AFP in a concentration range from 1 fg/mL to 10 ng/mL, with a detection limit of 0.7 fg/mL.
(3)A novel electrochemical immunosensor based on double signal amplification of enzyme-encapsulated liposomes and biocatalytic metal deposition was developed for the detection of human prostate specific antigen (PSA). Alkaline phosphatase (ALP)-encapsulated and detection antibody-functionalized liposomes were first prepared and used as the detection reagent. In the sandwich immunoassay, the model analyte PSA was first captured by anti-PSA capture antibody immobilized on the electrode and then sandwiched with the functionalized liposomes. The bound liposomes were then lysed with surfactant to release the encapsulated ALP, which served as secondary signal amplification means. ALP on the electrode surface initiated the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid. The latter, in turn, reduced silver ions on the electrode surface, leading to deposition of the metal silver on the electrode surface. Linear sweep voltammetry (LSV) was chosen to detect the amount of the deposited silver. The results showed that the anodic stripping peak current was linearly dependent on the PSA concentration in the range of 0.01 ng/mL to100.0 ng/mL, and a detection limit as low as 0.007 ng/mL can be obtained. Since the cut-off value of human PSA is 4.0 ng/mL, the proposed electrochemical immunosensor would be expected to gain widespread applications for the detection of PSA in clinical diagnosis.
引文
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