凝血酶电化学适体传感器的研究
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摘要
凝血酶是一种由凝血酶前体形成的丝氨酸蛋白质水解酶,具有催化纤维蛋白元变成纤维蛋白,促进血液凝固和调控凝血等作用,在揭示肿瘤的发生机制及作为早期诊断、疗效及愈后判断依据等方面有着非常重大的意义。由于血液中凝血酶的浓度达nmol·L-1,建立简单、快速、高灵敏度检测凝血酶的方法具有非常重要的意义。将SELEX技术筛选出的适体作为识别元件与电化学生物传感器相结合构建电化学适体传感器,集成了适体和电化学传感器两方面的优势,既具有电化学传感器的高灵敏度、快响应、简单操作、低成本,又具有适体的高选择性和特异性,在凝血酶检测方面具有广阔的应用前景。本文的研究目的是构建简单、可靠、快速、高灵敏度的电化学适体传感器用于凝血酶的检测。围绕本研究目的主要从电化学适体传感器敏感界面的构建,多功能化复合纳米材料的制备以及新型电化学信号放大策略等进行了探索和研究。本文分以下几个部分组成：
第一章绪论简要对适体的体外筛选方法、适体的特点、适体与凝血酶的相互作用进行了概述。详细介绍了电化学适体传感器的基本原理和其分类,适体的固定化方法以及相关的电化学分析技术,重点评述了纳米粒子、酶催化、杂交链式反应和目标循环信号放大技术在电化学适体传感器中的应用。最后引出本论文的选题背景、研究意义、研究目的和研究内容。
第二章由于适体本身为非电活性,或者适体中A、G和C碱基的电氧化还原信号低,且电化学调制不利于适体亲合性质的保持,大部分电化学适体传感器都需要功能性标记物以得到可定量检测的电化学信息。然而标记物的标记过程复杂、耗时、成本较高,且标记过程可能会影响其生物活性。基于层层自组装技术在金电极表面组装了{nano-Au/Thi}n多层膜,并以此为氧化还原探针和凝血酶适体固载基质构建了一种signal-off型免标记电化学适体传感器用于凝血酶的检测。该方法无需对适体进行电活性物质标记,一定程度上大大增加了电活性物质在电极表面的固载量和减少了电活性物质与电极表面的距离,具有操作简单、响应快、特异性强等特点。同时,该适体传感器的构建方法适用于其他各种适体的固载,在蛋白质检测和疾病诊断等领域都有很广的应用前景。
第三章G-四链体-卟啉铁(hemin/G-quadruplex)作为酶催化标记能有效放大电化学响应信号。与普通的蛋白酶相比较它更易于标记、成本低,稳定性好和抗水解性强。利用凝血酶适体富含G碱基能与卟啉铁hemin结合形成具有类似辣根过氧化物酶电催化增强性质的催化结构hemin/G-quadruplex,构建了电催化放大信号的signal-off型电化学适体传感器用于凝血酶的检测。并首次提出使用HRP封闭电化学适体传感器表面的非特异性吸附位点,且同时催化放大响应电流信号,进而提高电化学适体传感器的灵敏度的新方法。该传感器实现了对凝血酶高特异性和高灵敏度的识别,免去了电活性物质和酶的标记。经实验研究表明,该传感器构建方法简单,切实可行,大大提高了电化学适体传感器的灵敏度。
第四章金属纳米催化信号放大与酶相比,其热稳定性有很大的改善。以电惰性物质十八酸OCA和具有双功能的Au-PtNPs电催化双重放大响应信号制备了目标物引起的signal-on型电化学适体传感器用于凝血酶的检测。在测试底液中存在H2O2情况下,Au-PtNPs对亚甲蓝MB的氧化还原反应起到明显的增强作用。进行定量分析时,传感器表面阻碍电子传输的十八酸OCA修饰的凝血酶适体捕获目标分子后离开电极表面,电极表面空间位阻减少,同时Au-PtNPs的催化活性增加,使所得的响应信号进一步增加,从而有效地提高了电极灵敏度。该适体传感器构建方法简单、响应快、灵敏度高、稳定性好,将其用于临床样品检测,得到满意结果。
第五章通过π-π堆垛制备了具有一对很好氧化还原峰和类似辣根过氧化物酶催化作用的苝四甲酸/卟啉铁复合纳米材料(PTCA/hemin)。并结合葡萄糖氧化酶(GOx)作为封闭剂构建了一种基于PTCA/hemin纳米复合材料为氧化还原探针及催化剂的仿双酶放大信号signal-on型电化学适体传感器用于凝血酶的检测。PTCA/hemin复合纳米材料良好的成膜性避免了传统固载电活性物质和酶的复杂过程,同时也减少了固载电活性物质过程中电惰性物质的加入,操作简单、灵敏度高。GOx作为封闭剂封闭传感器表面的非特异性吸附位点,且同时构建了仿双酶催化放大响应电流信号,进而进一步提高了电化学适体传感器的灵敏度。
第六章双酶催化放大信号优于单酶催化体系,通过将hemin/G-quadruplex同时作为辣根过氧化物模拟酶和NADH氧化酶构建了仿双酶夹心式电化学适体传感器用于凝血酶的检测。与传统的酶标适体传感器相比,该传感器更易于标记,成本低,稳定性好、抗水解性强。更为重要的是hemin/G-quadruplex同时作为辣根过氧化物模拟酶和NADH氧化酶避免了繁琐的酶标记过程、解决了酶分布比例,多少及活性等问题,为构建简单、高灵敏的传感器提供了新的构建方法。
Thrombin, a kind of serine protease that involved in thrombosis and hemostasis, plays an important role in revealing tumorigenesis mechanism and judgment of early diagnosis, curative effect and prognosis. Since the concentration of thrombin in blood reaches to nmol·L-1, create a simple, rapid and high-sensitivity method for detection of thrombin is extremely important. Electrochemical aptasensor, the combination of SELEX-screened aptamers and electrochemical sensors, integrates the advantages of high sensitivity, fast response, simple operation and low cost of electrochemical sensors as well as high selectivity and specificity of aptamer, which thus provids very broad application prospects in the field of thrombin detection. The aim of present work is to develop simple, reliable, fast, high-sensitivity electrochemical aptasensor for the detection of thrombin. Therefore, this work focuses on the construction of the sensitive interface, the preparation of multi-functionalized nanoparticles and the development of novel electrochemical signal amplificatory strategy. The detail contents are as follows:
In chapter one, after general introduction of aptamer including its in vitro selection, characteristic and interaction with thrombin, the principles, classification, immobilization method and associated electrochemical analysis techniques of the electrochemical aptasensor were described in detail. Moreover, the application of signal amplificatory strategy for electrochemical aptasenor was highlighted. Lastly, the aim and the significance of this thesis were briefly introduced.
In chapter two, since the aptamers are intrinsically unable to act as redox partners in an electrochemical reaction, most electrochemical aptasensors are relied on the label of either aptamer or its complementary strand. The labeling process, however, is complicated, time-consuming, and may affect the biological activity of aptamer. In this experiment, we described a signal-off and label-free electrochemical aptasensor for the detection of thrombin based on{nano-Au/Thi}n multilayer films acted as redox probe and thrombin aptamer (TBA) immobilized matrix. Herein, the {nano-Au/Thi}n multilayer films are formed on the electrode surface via the layer-by-layer self-assembly technique, which avoids the fuzzy labeling process of aptamer, thus leads to a great increase of redox probe amount on electrode surface and a decrease of distance between the redox probe and electrode surface to a certain extent. This approach possesses some dramatic advantages, such as simple operation, fast response, high specificity and so on. At the same time, the constructing method of this aptasensor is applicable to many other aptamers, providing very broad application prospects in the field of protein detection and disease diagnosis.
In chapter three, hemin/G-quadruplex used for enzyme catalysis can effectively amplify the electrochemical signal. Compared with protein enzymes, hemin/G-quadruplex is relatively easy to label, less expensive to produce, and more stable against hydrolysis and heat treatment. Take advantage of the G base-riched thrombin aptamer can combine with hemin to form a horseradish peroxidase-like hemin/G-quadruplex catalytic structure, we described a signal-off electrochemical aptasensor with electrocatalytic amplification for the detection of thrombin. Moreover, the new strategy of employing HRP to block the possible remaining active sites and thus then amplify the response of the TBA-thrombin recognition was proposed. The proposed electrochemical aptasensor realized the recognition to thrombin with high specificity and high sensitivity, and avoided the fuzzy labeling process of electroactive substance and the enzyme. Experimental studies have shown that this approach is simple, practical and greatly improved the sensitivity of electrochemical aptasensor.
In chapter four, compared with protein enzyme, the nano-metals for electrocatalytic amplification possess better thermal stability. In this experiment, we described a target-induced signal-on electrochemical aptasensor for the detection of thrombin based on the dual-amplification of inert material stearic acid OCA and dual-functional Au-PtNPs. With the addition of H2O2to the electrolytic cell, the Au-PtNPs on the aptasensor surface effectively electrocatalyzed the oxidation-reduction reaction of methylene blue MB, which provided an extremely amplified electrochemical signal. When the quantitative analysis was carried out, the OCA label TBA on electrode surface captured thrombin and followed with the leave of the electrode surface, which led to a decrease in steric hindrance of electrode surface and an increase in catalytic efficiency of Au-PtNPs, significantly amplified the electrochemical signal, and thereby effectively improved the sensitivity of electrochemical aptasensor. The proposed aptasensor has been demonstrated to offer a simple, rapid and sensitive method for the detection of thrombin, providing a satisfactory result in clinical detection.
In chapter five, a novel3,4,9,10-perylenetetracarboxylic acid/hemin nanocomposites (PTCA/hemin) with a pair of well-defined redox peaks and intrinsic horseradish peroxidase-like activity was prepared through π-π interactions. And a pseudobienzyme-channeling amplified signal-on electrochemical aptasensor based on glucose oxidase (GOx) as blocking reagent and PTCA/hemin nanocomposites simultaneously as redox probes and electrocatalysts was prepared for the detection of thrombin. The superior membrane-forming property of PTCA/hemin nanocomposites not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer, providing a simple and sensitive method of detection of thrombin. Moreover, the new strategy of employing GOx to block the possible remaining active sites and thus then construct pseudobienzyme-channeling amplified system for amplifying the response of the TBA-thrombin recognition was proposed, which further improved the sensitivity of electrochemical aptasensor.
In chapter six, since bienzyme catalytic signal amplification is obviously larger that of single enzyme catalytic system, we described a pseudobienzyme amplifying sandwich-type electrochemical aptasensor for the detection of thrombin with hemin/G-quadruplex simultaneously serve as NADH oxidase and an HRP-mimicking DNAzyme. Compared with protein enzymes labeled aptasensor, the proposed aptasensor are relatively easy to label, less expensive to produce, and more stable against hydrolysis and heat treatment. More importantly, this strategy realized the simultaneous action of the hemin/G-quadruplex as an NADH oxidase and HRP-mimicking DNAzyme in bioelectrocatalytic amplification, which avoided the fussy labeling process, resolved the spatial distribution of each sequentially acting enzyme and provided a new strategy for constructing simple and sensitive aptasensor for the detection of thrombin.
第一章绪论简要对适体的体外筛选方法、适体的特点、适体与凝血酶的相互作用进行了概述。详细介绍了电化学适体传感器的基本原理和其分类,适体的固定化方法以及相关的电化学分析技术,重点评述了纳米粒子、酶催化、杂交链式反应和目标循环信号放大技术在电化学适体传感器中的应用。最后引出本论文的选题背景、研究意义、研究目的和研究内容。
第二章由于适体本身为非电活性,或者适体中A、G和C碱基的电氧化还原信号低,且电化学调制不利于适体亲合性质的保持,大部分电化学适体传感器都需要功能性标记物以得到可定量检测的电化学信息。然而标记物的标记过程复杂、耗时、成本较高,且标记过程可能会影响其生物活性。基于层层自组装技术在金电极表面组装了{nano-Au/Thi}n多层膜,并以此为氧化还原探针和凝血酶适体固载基质构建了一种signal-off型免标记电化学适体传感器用于凝血酶的检测。该方法无需对适体进行电活性物质标记,一定程度上大大增加了电活性物质在电极表面的固载量和减少了电活性物质与电极表面的距离,具有操作简单、响应快、特异性强等特点。同时,该适体传感器的构建方法适用于其他各种适体的固载,在蛋白质检测和疾病诊断等领域都有很广的应用前景。
第三章G-四链体-卟啉铁(hemin/G-quadruplex)作为酶催化标记能有效放大电化学响应信号。与普通的蛋白酶相比较它更易于标记、成本低,稳定性好和抗水解性强。利用凝血酶适体富含G碱基能与卟啉铁hemin结合形成具有类似辣根过氧化物酶电催化增强性质的催化结构hemin/G-quadruplex,构建了电催化放大信号的signal-off型电化学适体传感器用于凝血酶的检测。并首次提出使用HRP封闭电化学适体传感器表面的非特异性吸附位点,且同时催化放大响应电流信号,进而提高电化学适体传感器的灵敏度的新方法。该传感器实现了对凝血酶高特异性和高灵敏度的识别,免去了电活性物质和酶的标记。经实验研究表明,该传感器构建方法简单,切实可行,大大提高了电化学适体传感器的灵敏度。
第四章金属纳米催化信号放大与酶相比,其热稳定性有很大的改善。以电惰性物质十八酸OCA和具有双功能的Au-PtNPs电催化双重放大响应信号制备了目标物引起的signal-on型电化学适体传感器用于凝血酶的检测。在测试底液中存在H2O2情况下,Au-PtNPs对亚甲蓝MB的氧化还原反应起到明显的增强作用。进行定量分析时,传感器表面阻碍电子传输的十八酸OCA修饰的凝血酶适体捕获目标分子后离开电极表面,电极表面空间位阻减少,同时Au-PtNPs的催化活性增加,使所得的响应信号进一步增加,从而有效地提高了电极灵敏度。该适体传感器构建方法简单、响应快、灵敏度高、稳定性好,将其用于临床样品检测,得到满意结果。
第五章通过π-π堆垛制备了具有一对很好氧化还原峰和类似辣根过氧化物酶催化作用的苝四甲酸/卟啉铁复合纳米材料(PTCA/hemin)。并结合葡萄糖氧化酶(GOx)作为封闭剂构建了一种基于PTCA/hemin纳米复合材料为氧化还原探针及催化剂的仿双酶放大信号signal-on型电化学适体传感器用于凝血酶的检测。PTCA/hemin复合纳米材料良好的成膜性避免了传统固载电活性物质和酶的复杂过程,同时也减少了固载电活性物质过程中电惰性物质的加入,操作简单、灵敏度高。GOx作为封闭剂封闭传感器表面的非特异性吸附位点,且同时构建了仿双酶催化放大响应电流信号,进而进一步提高了电化学适体传感器的灵敏度。
第六章双酶催化放大信号优于单酶催化体系,通过将hemin/G-quadruplex同时作为辣根过氧化物模拟酶和NADH氧化酶构建了仿双酶夹心式电化学适体传感器用于凝血酶的检测。与传统的酶标适体传感器相比,该传感器更易于标记,成本低,稳定性好、抗水解性强。更为重要的是hemin/G-quadruplex同时作为辣根过氧化物模拟酶和NADH氧化酶避免了繁琐的酶标记过程、解决了酶分布比例,多少及活性等问题,为构建简单、高灵敏的传感器提供了新的构建方法。
Thrombin, a kind of serine protease that involved in thrombosis and hemostasis, plays an important role in revealing tumorigenesis mechanism and judgment of early diagnosis, curative effect and prognosis. Since the concentration of thrombin in blood reaches to nmol·L-1, create a simple, rapid and high-sensitivity method for detection of thrombin is extremely important. Electrochemical aptasensor, the combination of SELEX-screened aptamers and electrochemical sensors, integrates the advantages of high sensitivity, fast response, simple operation and low cost of electrochemical sensors as well as high selectivity and specificity of aptamer, which thus provids very broad application prospects in the field of thrombin detection. The aim of present work is to develop simple, reliable, fast, high-sensitivity electrochemical aptasensor for the detection of thrombin. Therefore, this work focuses on the construction of the sensitive interface, the preparation of multi-functionalized nanoparticles and the development of novel electrochemical signal amplificatory strategy. The detail contents are as follows:
In chapter one, after general introduction of aptamer including its in vitro selection, characteristic and interaction with thrombin, the principles, classification, immobilization method and associated electrochemical analysis techniques of the electrochemical aptasensor were described in detail. Moreover, the application of signal amplificatory strategy for electrochemical aptasenor was highlighted. Lastly, the aim and the significance of this thesis were briefly introduced.
In chapter two, since the aptamers are intrinsically unable to act as redox partners in an electrochemical reaction, most electrochemical aptasensors are relied on the label of either aptamer or its complementary strand. The labeling process, however, is complicated, time-consuming, and may affect the biological activity of aptamer. In this experiment, we described a signal-off and label-free electrochemical aptasensor for the detection of thrombin based on{nano-Au/Thi}n multilayer films acted as redox probe and thrombin aptamer (TBA) immobilized matrix. Herein, the {nano-Au/Thi}n multilayer films are formed on the electrode surface via the layer-by-layer self-assembly technique, which avoids the fuzzy labeling process of aptamer, thus leads to a great increase of redox probe amount on electrode surface and a decrease of distance between the redox probe and electrode surface to a certain extent. This approach possesses some dramatic advantages, such as simple operation, fast response, high specificity and so on. At the same time, the constructing method of this aptasensor is applicable to many other aptamers, providing very broad application prospects in the field of protein detection and disease diagnosis.
In chapter three, hemin/G-quadruplex used for enzyme catalysis can effectively amplify the electrochemical signal. Compared with protein enzymes, hemin/G-quadruplex is relatively easy to label, less expensive to produce, and more stable against hydrolysis and heat treatment. Take advantage of the G base-riched thrombin aptamer can combine with hemin to form a horseradish peroxidase-like hemin/G-quadruplex catalytic structure, we described a signal-off electrochemical aptasensor with electrocatalytic amplification for the detection of thrombin. Moreover, the new strategy of employing HRP to block the possible remaining active sites and thus then amplify the response of the TBA-thrombin recognition was proposed. The proposed electrochemical aptasensor realized the recognition to thrombin with high specificity and high sensitivity, and avoided the fuzzy labeling process of electroactive substance and the enzyme. Experimental studies have shown that this approach is simple, practical and greatly improved the sensitivity of electrochemical aptasensor.
In chapter four, compared with protein enzyme, the nano-metals for electrocatalytic amplification possess better thermal stability. In this experiment, we described a target-induced signal-on electrochemical aptasensor for the detection of thrombin based on the dual-amplification of inert material stearic acid OCA and dual-functional Au-PtNPs. With the addition of H2O2to the electrolytic cell, the Au-PtNPs on the aptasensor surface effectively electrocatalyzed the oxidation-reduction reaction of methylene blue MB, which provided an extremely amplified electrochemical signal. When the quantitative analysis was carried out, the OCA label TBA on electrode surface captured thrombin and followed with the leave of the electrode surface, which led to a decrease in steric hindrance of electrode surface and an increase in catalytic efficiency of Au-PtNPs, significantly amplified the electrochemical signal, and thereby effectively improved the sensitivity of electrochemical aptasensor. The proposed aptasensor has been demonstrated to offer a simple, rapid and sensitive method for the detection of thrombin, providing a satisfactory result in clinical detection.
In chapter five, a novel3,4,9,10-perylenetetracarboxylic acid/hemin nanocomposites (PTCA/hemin) with a pair of well-defined redox peaks and intrinsic horseradish peroxidase-like activity was prepared through π-π interactions. And a pseudobienzyme-channeling amplified signal-on electrochemical aptasensor based on glucose oxidase (GOx) as blocking reagent and PTCA/hemin nanocomposites simultaneously as redox probes and electrocatalysts was prepared for the detection of thrombin. The superior membrane-forming property of PTCA/hemin nanocomposites not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer, providing a simple and sensitive method of detection of thrombin. Moreover, the new strategy of employing GOx to block the possible remaining active sites and thus then construct pseudobienzyme-channeling amplified system for amplifying the response of the TBA-thrombin recognition was proposed, which further improved the sensitivity of electrochemical aptasensor.
In chapter six, since bienzyme catalytic signal amplification is obviously larger that of single enzyme catalytic system, we described a pseudobienzyme amplifying sandwich-type electrochemical aptasensor for the detection of thrombin with hemin/G-quadruplex simultaneously serve as NADH oxidase and an HRP-mimicking DNAzyme. Compared with protein enzymes labeled aptasensor, the proposed aptasensor are relatively easy to label, less expensive to produce, and more stable against hydrolysis and heat treatment. More importantly, this strategy realized the simultaneous action of the hemin/G-quadruplex as an NADH oxidase and HRP-mimicking DNAzyme in bioelectrocatalytic amplification, which avoided the fussy labeling process, resolved the spatial distribution of each sequentially acting enzyme and provided a new strategy for constructing simple and sensitive aptasensor for the detection of thrombin.
引文
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