基于功能化纳米材料构建的新型酶生物传感器及其应用研究
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摘要
生命科学在探究生命本质和各种生命活动内在规律的过程中,需要理清复杂样品体系的组成、性质、功能以及变异等信息。这使得其向分析科学在对复杂样品体系的分离、分析方面提出了新的要求：不仅要求分析化学提供可进行准确的定性、定量分析的检测方法,而且要能够提供最全面的整合数据信息；要求分析化学工作者在发展高灵敏度、稳定性好、重现性好的离体检测方法的同时,开展对痕量的生物活性物质进行实时、原位、活体分析的研究。其中,光电化学生物传感器可实现对多种物质的高灵敏检测,且具有检测快速以及重现性好等重要性能,为深入开展对微量、痕量物质的快速、灵敏分析检测提供了技术支持和保障。目前,光电化学生物传感器已经成为生命科学、环境科学、安全防恐、农业科学等领域探究科学原理和各种内在机制的有力工具。光电化学生物传感器是基于光电化学反应过程中产生的光电流或光电压的变化与待测物的浓度间的关系进行定量检测的。对光敏材料光电转换效率的提高以及稳定性的改善是光电生物传感器高灵敏度、高稳定性等性能的重要保障,因此对光敏材料的研究已成为光电化学生物传感器研究中的核心内容之一。
本论文的主要研究工作集中在制备了3种以二氧化钛纳米管阵列为基质的光敏半导体纳米复合材料：Ag/N-F-TNs,ZnONRs/TNs, GC-HBAP/Pty/TNs。将光敏性纳米半导体复合材料与电化学生物传感技术相结合,构建了光电化学生物传感器,并用于乙酰胆碱酯酶活性变化研究,探究了内源性神经毒素、重金属离子和抗精神类药物等物质对酶的活性损伤；进行了光电化学方法测定酶活性的研究,并对实际样品进行检测。制备了聚硫堇-金纳米粒子复合材料并构建了双电极多酶传感器检测体系,用所构建的检测体系对生物样品中葡萄糖和胆固醇进行同时检测,讨论动脉粥样硬化的高血糖机制。本论文的具体工作包含下述几个方面：
第一章绪论
本章第一节介绍电化学生物传感器的概念、工作原理、分类与应用。第二节首先介绍光电化学的基本概念与信号响应原理,然后介绍了常用的光敏活性材料及其在光电化学生物传感器中的应用研究。第三节中介绍电化学生物传感器在对与老年化疾病致病机理相关的酶活性的变化、血糖、胆固醇含量的测定中的应用。最后阐述本论文所开展工作的意义。
第二章基于可见光激发的光电化学传感器制备及其应用
第一节光电化学传感器用于内源性神经毒素对乙酰胆碱酯酶的活性损伤研究
1(R)-甲基-6,7-二羟基-1,2,3,4-四氢异喹啉[(R)-Sal]和1(R),2(N)-二甲基-6,7-二羟基-1,2,3,4-四氢异喹啉(R)-NMSal]这两种内源性神经毒素在神经退行性疾病致病机理研究中已引起广泛关注,本文中制备了一种新颖的基于可见光激发的光电化学传感器并将之应用于(R)-Sal和(?)(R)-NMSal对乙酰胆碱酯酶(Acetylcholinesterase, AChE)活性损伤的研究。光电传感器的制备分为三个步骤：首先,将金属钛片通过电化学阳极氧化法制得氮、氟元素同时掺杂的二氧化钛纳米管阵列；然后应用微波辅助加热多元醇法将银纳米粒子(AgNPs)修饰在二氧化钛纳米管阵列中；最后,以壳聚糖作为交联剂将AChE修饰在电极表面所构建的传感器标记为AChE/Ag/N-F-TNs。氮、氟元素的同时掺杂使得光电化学传感器能够在可见光辐照下产生较强的光电流,银纳米粒子可增强传感器的光电流响应。以所制备的光电化学传感器进行了(R)-Sal和(R)-NMSal对AChE活性损伤的研究。实验结果表明,(R)-Sal和(R)-NMSal对乙酰胆碱酯酶活性的损伤均表现为可逆线性混合Ⅰ型抑制,这为AChE在神经退行性疾病的致病机理、药物筛选以及酶生物传感技术等方面提供重要的实验依据。
第二节光电化学传感器测定小鼠血清中AChE活力
乙酰胆碱酯酶活性研究是开展神经退行性疾病致病机理研究的重要方面。基于光电分析构建的光电化学生物传感器具有较高的灵敏度。本文中所制备的具有可见光活性的AChE/Ag/N-F-TNs光电化学生物传感器具有宽的线性范围和较高的灵敏度,可适用于对生物样品中AChE活力的测定。实验结果表明,光电流响应与AChE活力在10U/L～2000U/L范围内呈良好的线性关系。光生电流增加值与酶活力的线性方程为：y=5.72×10-2x+15.95,相关系数为99.76%,检测限为2U/L。将该光电化学酶生物传感器用于检测小鼠血清中AChE活力,取得满意的结果。所制备的光电化学酶生物传感器灵敏度高,响应快速,为研究AChE等重要靶向分子的活性及其变化提供了新的检测方法。
第三章基于金属氧化物半导体复合纳米材料构建的光电化学传感器制备及其在Cd2+离子对乙酰胆碱酯酶活性抑制研究中的应用
镉(Cd)是一种有毒重金属,能够影响人体内多种酶活性的改变,因而即使较低剂量也会对人体健康造成慢性中毒。Cd对神经退行性疾病致病机理影响的研究已引起人们的广泛关注,临床研究中已报道了Cd急性中毒诱发帕金森综合症(PD)的病例。本文中制备了二氧化钛纳米管阵列(TNs)和氧化锌纳米棒的复合纳米材料,以此构建的新型光电化学传感器(AChE/ZnONRs/TNs)用于研究Cd2+对乙酰胆碱酯酶(AChE)活性影响的研究。实验结果表明,氧化锌纳米棒与TNs的耦合可改善复合材料对可见光的吸收,增大光电流。实验结果表明Cd2+对AChE活性的影响呈现出时间依赖和浓度依赖的特点。这不仅为Cd2+对AChE活性影响提供了重要的实验依据,而且为评估重金属离子以及微量元素对酶等生物活性物质损伤提供了一种新的研究方法。
第四章单分子聚合物纳米颗粒-二氧化钛纳米管阵列构建的光电化学传感器用于氯氮平对乙酰胆碱酯酶活性影响的研究
本论文研究中基于单分子聚合物纳米颗粒和二氧化钛纳米管阵列研制了一种新型的光电化学酶传感器,并用于研究氯氮平对乙酰胆碱酯酶活性的影响。首先制备了金纳米粒子-超分子聚合物的单分子纳米颗粒(GC-HBAP),将其修饰在功能化的TNs表面,通过交联剂固定乙酰胆碱酯酶制备了具有可见光活性的光电化学酶传感器,并应用于氯氮平对乙酰胆碱酯酶活性影响的研究。研究表明,氯氮平对乙酰胆碱酯酶活性的影响体现出底物依赖的特点,即低浓度底物时表现为抑制,随着底物浓度的增加,体现为激活作用。研究结果为临床研究中应用抗精神类药物辅助治疗晚期帕金森综合症提供了有价值的体外实验依据,同时所构建的检测体系也适用于评价抗精神类药物对酶活性的影响。
第五章双电极多酶传感器检测体系同时检测大鼠血清与腹腔巨噬细胞内葡萄糖与胆固醇的研究
本文中,制备了一种新颖的双电极多酶传感器检测体系,通过该体系实现了对大鼠血清和腹腔巨噬细胞内葡萄糖与胆固醇的同时检测,并应用于“对高血糖诱发动脉粥样硬化”模型的解释。传感器通过三步法进行制备：首先以循环伏安法在玻碳电极表面电聚合聚硫堇(PTH)膜层；然后通过吸附作用将金纳米粒子(GNPs)修饰在聚硫堇膜层上；最后以壳聚糖作为交联剂将葡萄糖氧化酶(GOx)修饰在电极1上制得葡萄糖电化学传感器,将胆固醇酯酶(ChE)和胆固醇氧化酶(ChOx)修饰在电极2上制得胆固醇电化学传感器。葡萄糖传感器上信号响应与葡萄糖浓度在0.008～6.0mM范围内呈良好线性关系,检测限仅为2.0μM。胆固醇传感器上信号响应与胆固醇浓度在0.002～1.0mM范围内呈良好线性关系,检测限仅为0.6μM。检测体系对大鼠样品检测结果表明：正常大鼠和糖尿病模型大鼠血清中葡萄糖含量与胆固醇含量的变化无相关性,且胆固醇含量随血糖含量的升高增加不明显。但糖尿病模型大鼠巨噬细胞内葡萄糖和胆固醇含量随血清中血糖的增大而增加。本论文研究结果为解释“高血糖诱发巨噬细胞泡沫化促进动脉粥样硬化风险”的病理学假说模型提供了重要的实验依据和补充说明。
The rapid development of life science, which studies the nature of life and all kinds of the internal regular patterns about life activities, needs the information about the complicated biosamples such as composition, property, function and variation. This requires analytical chemistry to provide not only analytical methods and technologies but also comprehensive and abundant information for in-situ, on line and in vivo analysis. Photoelectrochemical biosensors is a burgeoning analytical technique presenting extremely high sensitivity and it has been widely applied in many areas such as life science, environmental monitoring, security inspection and agriculture science. Photoelectrochemical biosensing employs current or potential as a detection signal while uses light as an excitation source. Therefore, how to choose appropriate photosensitive materials and improve the photoelectric conversion efficiency and stability is an important subject in the research of potoelectrochemical biosensors.
In this dissertation, we laid our emphasis on the development of the photoelectrochemical biosensors based on TiO2nanotube assays, and their applications in the analysis of enzyme activity damage in vitro. The fabricated photoelectrochemical biosensors are used to study the inhibition of acetylcholinesterase (AChE) activity induced by two endogenous neurotoxins, Cadmium ions (Cd2+) and Clozapine. Furthermore, we studied an innovative detection method of enzyme activity by photoelectorchemical analysis with the prepared photoelectrochemical biosensor. In addition, a novel dual enzymatic-biosensor was described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. This dissertation includes five chapters:
Chapter1. Overview
In this chapter, the information of electrochemical biosensors including the concept, working principle, classification and application was first introduced. Then, the contents of photoelectrochemistry which focused on photoelectrochemical materials and their applications in biosensors were presented. Followed was the brief introduction of Parkinson's disease, diabetes mellitus and atherosclerosis which are three diseases related to the research work. Finally, we emphatically indicated the purpose and significance of the dissertation, innovation spot and content as well.
Charpt2. Preparation and amplification of the Visible-light-actived photoelectrochemical biosensor
Section One. Visible-light-actived photoelectrochemical biosensor for the study of acetylcholinesterase inhibition induced by endogenous neurotoxins
In this section, a novel visible-light-actived photoelectrochemical biosensor was fabricated to study the inhibition of acetylcholinesterase (AChE) activity induced by two endogenous neurotoxins,1(R)-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-Sal] and1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetra-hydroisoquinoline [(R)-NMSal] which have drawn much attention in the study of the pathogenesis of neurodegenerati ve diseases such as Parkinson's disease. The photoelectrode was prepared by three steps as follows. At first, nitrogen and fluorine co-doped TiO2nanotubes (TNs) were obtained by anodic oxidation of Ti sheet. Secondly, silver nanoparticles (AgNPs) were deposited onto the TNs through microwave-assisted heating polyol (MAHP) process. At last, AChE was immobilized on the obtained photoelectrode and the biosensor was marked as AChE/Ag/N-F-TNs. Due to the nitrogen and fluorine co-doping, the photoelectrochemical biosensors can produce high photocurrent under the visible light irradiation. Moreover, the presence of AgNPs greatly increased the photocurrent response of the biosensor. AChE/Ag/N-F-TNs hybrid system was used to study AChE inhibition induced by (R)-Sal and (R)-NMSal and the result proved that both (R)-Sal and (R)-NMSal all exhibited mixed and reversible inhibition against AChE. This strategy is of great significance for the development of novel photoelectrochemical biosensors in the future.
Section Two. Detection of AChE activity in rat serum with photoelectrochemical biosensor
The study of Acetylcholinesterase (AChE) activity is one of the most important aspects in the research on pathogenesis mechanism of neurodegenerative disease. Thus, a sensitive and rapid determination technology for AChE activity is urgently needed. In this section, the AChE activity in rat serum was determined by photoelectrochemical analysis (PECA) method with the visible-light-actived photoelectrochemical biosensor prepared in section one. This method showed an excellent analytical performance, which exhibited a wide dynamic response range of AChE activity from10U/L to2000U/L and with a detection limit of2U/L. The results demonstrate that the developed PECA method offered a new approach for AChE activity study, which is related to the pathogenesis mechanism. Overall, this propsed established PECA method was very promising and could be easily extended to measure the other enzyme activity.
Charpt3. A photoelectrochemical biosensor for the study of acetylcholinesterase inhibition induced by cadmium ions based on metal-oxide semiconductor nanocomposites
Cadmium (Cd) is a heavy metal with a high toxicity. It is toxic at very low dose and it has acute and chronic effects on human health since it can influence the activity of numerous enzymes in body. A representative case about Parkinson's disease (PD) after acute Cd poisoning in clinic treatment has been reported. In this report, a novel photoelectrochemical biosensor was fabricated to study the effect on AChE activity induced by Cd2+ions which have drawn much attention in the study of the pathogenesis of neurodegenerative diseases such as PD, Alzheimer's disease (AD) and Myasthenia Gravis (MG). The photoelectrode was prepared by depositing zinc oxide nanorodes (ZnONRs) onto the TNs through cathodic deposition method and then AChE was immobilized on the obtained photoelectrode and the biosensor was marked as AChE/ZnONRs/TNs. The photoelectrochemical biosensor showed good performance in the monitoring of acetylthiocholine (ATCh) with a rapid response. Experimental results show that Cd ion exhibited dose-dependent and time-dependent effects on AChE. In addition, we proposed a newly effective method to estimate the damage effect of enzyme activity caused by heavy metal ions.
Charpt4. A photoelectrochemical biosensor based on TiO2NTs cooperated with hyperbranched polymer hybrid for studying the effect of clozapine on acetylcholinesterase activity
In this report, a novel photoelectrochemical biosensor was fabricated to study the inhibition of AChE activity induced by clozapine, which have drawn much attention in the study of the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Herein, thiol-functionalized hyperbranched azo-polymers (HBAP) decorated with gold colloids (GC-HBAP) via covalent bond were utilized as the biosensing platform, which have been demonstrated to not only anchor larger amounts of capture antibodies, but also act as hole scavengers to facilitate the improvement of the photoelectrochemical performance of semiconductive nanomaterials. Tyrosine was initially electropolymerized on the TNs electrode surface to perform abundant carboxyl groups to bonding the prepared GC-HBAP nanoparticals. And then, AChE immobilized on the surface of the photoelectrode and marked as AChE/GC-HBAP/Pty/TNs. The effects of clozapine on AChE activity in vitro were studied by using AChE/GC-HBAP/Pty/TNs hybrid system. Experimental results show that clozapine exhibited dose-dependent effects on AChE. This established method successfully provies a robust way for evaluating the effects of enzyme on other antipsychotics. In addition, this strategy could be easily used to find new therapies and screen new drugs.
Charpt5. A dual electrode detection system with multienzyme biosensor for simultaneous determination of Glucose and Cholesterol in serum and peritoneal macrophages of diabetic mice:evaluation of the diabetes-accelerated atherosclerosis risk
In this paper, a novel dual electrode detection system with multienzyme biosensor is described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. The biosensor was constructed by a three-step method. First, a poly-thionine (PTH) film was assembled on the surface of glassy carbon electrode by cyclic voltammetric electropolymerization of thionine, which serves as an electron transfer mediator (ETM). Second, gold nanoparticles (GNPs) were covered on the surface of PTH facilitating the electron transfer between glucose oxidase (GOx), cholesterol oxidase (ChOx) and electrode. Finally, the enzymes, GOx, cholesterol esterase (ChE), ChOx, were covalent attached to the PTH layer through a chitosan (CH) linker. The PTH coupled with GNPs provides good selectivity, high sensitivity and little crosstalk for the dual enzymatic-biosensor. The constructed detection system had good electrocatalytic activity toward the oxidations of glucose and cholesterol, exhibiting a linear range from0.008mM to6.0mM for glucose with a detection limit of2.0μM, and a linear range from0.002mM to1.0mM for cholesterol with a detection limit of0.6μM. The results of the diabetic mice demonstrated that the cholesterol level did not change obviously with the increase of glucose level in serum, while the cholesterol level was induced with the increase of the glucose level in PMs. Previous studies have shown that the large accumulation of cholesterol in macrophage could lead to macrophage foam cell formation, which is the hallmark of early atherosclerosis. This study provides useful further evidences for the development of diabetes-accelerated atherosclerosis.
本论文的主要研究工作集中在制备了3种以二氧化钛纳米管阵列为基质的光敏半导体纳米复合材料：Ag/N-F-TNs,ZnONRs/TNs, GC-HBAP/Pty/TNs。将光敏性纳米半导体复合材料与电化学生物传感技术相结合,构建了光电化学生物传感器,并用于乙酰胆碱酯酶活性变化研究,探究了内源性神经毒素、重金属离子和抗精神类药物等物质对酶的活性损伤；进行了光电化学方法测定酶活性的研究,并对实际样品进行检测。制备了聚硫堇-金纳米粒子复合材料并构建了双电极多酶传感器检测体系,用所构建的检测体系对生物样品中葡萄糖和胆固醇进行同时检测,讨论动脉粥样硬化的高血糖机制。本论文的具体工作包含下述几个方面：
第一章绪论
本章第一节介绍电化学生物传感器的概念、工作原理、分类与应用。第二节首先介绍光电化学的基本概念与信号响应原理,然后介绍了常用的光敏活性材料及其在光电化学生物传感器中的应用研究。第三节中介绍电化学生物传感器在对与老年化疾病致病机理相关的酶活性的变化、血糖、胆固醇含量的测定中的应用。最后阐述本论文所开展工作的意义。
第二章基于可见光激发的光电化学传感器制备及其应用
第一节光电化学传感器用于内源性神经毒素对乙酰胆碱酯酶的活性损伤研究
1(R)-甲基-6,7-二羟基-1,2,3,4-四氢异喹啉[(R)-Sal]和1(R),2(N)-二甲基-6,7-二羟基-1,2,3,4-四氢异喹啉(R)-NMSal]这两种内源性神经毒素在神经退行性疾病致病机理研究中已引起广泛关注,本文中制备了一种新颖的基于可见光激发的光电化学传感器并将之应用于(R)-Sal和(?)(R)-NMSal对乙酰胆碱酯酶(Acetylcholinesterase, AChE)活性损伤的研究。光电传感器的制备分为三个步骤：首先,将金属钛片通过电化学阳极氧化法制得氮、氟元素同时掺杂的二氧化钛纳米管阵列；然后应用微波辅助加热多元醇法将银纳米粒子(AgNPs)修饰在二氧化钛纳米管阵列中；最后,以壳聚糖作为交联剂将AChE修饰在电极表面所构建的传感器标记为AChE/Ag/N-F-TNs。氮、氟元素的同时掺杂使得光电化学传感器能够在可见光辐照下产生较强的光电流,银纳米粒子可增强传感器的光电流响应。以所制备的光电化学传感器进行了(R)-Sal和(R)-NMSal对AChE活性损伤的研究。实验结果表明,(R)-Sal和(R)-NMSal对乙酰胆碱酯酶活性的损伤均表现为可逆线性混合Ⅰ型抑制,这为AChE在神经退行性疾病的致病机理、药物筛选以及酶生物传感技术等方面提供重要的实验依据。
第二节光电化学传感器测定小鼠血清中AChE活力
乙酰胆碱酯酶活性研究是开展神经退行性疾病致病机理研究的重要方面。基于光电分析构建的光电化学生物传感器具有较高的灵敏度。本文中所制备的具有可见光活性的AChE/Ag/N-F-TNs光电化学生物传感器具有宽的线性范围和较高的灵敏度,可适用于对生物样品中AChE活力的测定。实验结果表明,光电流响应与AChE活力在10U/L～2000U/L范围内呈良好的线性关系。光生电流增加值与酶活力的线性方程为：y=5.72×10-2x+15.95,相关系数为99.76%,检测限为2U/L。将该光电化学酶生物传感器用于检测小鼠血清中AChE活力,取得满意的结果。所制备的光电化学酶生物传感器灵敏度高,响应快速,为研究AChE等重要靶向分子的活性及其变化提供了新的检测方法。
第三章基于金属氧化物半导体复合纳米材料构建的光电化学传感器制备及其在Cd2+离子对乙酰胆碱酯酶活性抑制研究中的应用
镉(Cd)是一种有毒重金属,能够影响人体内多种酶活性的改变,因而即使较低剂量也会对人体健康造成慢性中毒。Cd对神经退行性疾病致病机理影响的研究已引起人们的广泛关注,临床研究中已报道了Cd急性中毒诱发帕金森综合症(PD)的病例。本文中制备了二氧化钛纳米管阵列(TNs)和氧化锌纳米棒的复合纳米材料,以此构建的新型光电化学传感器(AChE/ZnONRs/TNs)用于研究Cd2+对乙酰胆碱酯酶(AChE)活性影响的研究。实验结果表明,氧化锌纳米棒与TNs的耦合可改善复合材料对可见光的吸收,增大光电流。实验结果表明Cd2+对AChE活性的影响呈现出时间依赖和浓度依赖的特点。这不仅为Cd2+对AChE活性影响提供了重要的实验依据,而且为评估重金属离子以及微量元素对酶等生物活性物质损伤提供了一种新的研究方法。
第四章单分子聚合物纳米颗粒-二氧化钛纳米管阵列构建的光电化学传感器用于氯氮平对乙酰胆碱酯酶活性影响的研究
本论文研究中基于单分子聚合物纳米颗粒和二氧化钛纳米管阵列研制了一种新型的光电化学酶传感器,并用于研究氯氮平对乙酰胆碱酯酶活性的影响。首先制备了金纳米粒子-超分子聚合物的单分子纳米颗粒(GC-HBAP),将其修饰在功能化的TNs表面,通过交联剂固定乙酰胆碱酯酶制备了具有可见光活性的光电化学酶传感器,并应用于氯氮平对乙酰胆碱酯酶活性影响的研究。研究表明,氯氮平对乙酰胆碱酯酶活性的影响体现出底物依赖的特点,即低浓度底物时表现为抑制,随着底物浓度的增加,体现为激活作用。研究结果为临床研究中应用抗精神类药物辅助治疗晚期帕金森综合症提供了有价值的体外实验依据,同时所构建的检测体系也适用于评价抗精神类药物对酶活性的影响。
第五章双电极多酶传感器检测体系同时检测大鼠血清与腹腔巨噬细胞内葡萄糖与胆固醇的研究
本文中,制备了一种新颖的双电极多酶传感器检测体系,通过该体系实现了对大鼠血清和腹腔巨噬细胞内葡萄糖与胆固醇的同时检测,并应用于“对高血糖诱发动脉粥样硬化”模型的解释。传感器通过三步法进行制备：首先以循环伏安法在玻碳电极表面电聚合聚硫堇(PTH)膜层；然后通过吸附作用将金纳米粒子(GNPs)修饰在聚硫堇膜层上；最后以壳聚糖作为交联剂将葡萄糖氧化酶(GOx)修饰在电极1上制得葡萄糖电化学传感器,将胆固醇酯酶(ChE)和胆固醇氧化酶(ChOx)修饰在电极2上制得胆固醇电化学传感器。葡萄糖传感器上信号响应与葡萄糖浓度在0.008～6.0mM范围内呈良好线性关系,检测限仅为2.0μM。胆固醇传感器上信号响应与胆固醇浓度在0.002～1.0mM范围内呈良好线性关系,检测限仅为0.6μM。检测体系对大鼠样品检测结果表明：正常大鼠和糖尿病模型大鼠血清中葡萄糖含量与胆固醇含量的变化无相关性,且胆固醇含量随血糖含量的升高增加不明显。但糖尿病模型大鼠巨噬细胞内葡萄糖和胆固醇含量随血清中血糖的增大而增加。本论文研究结果为解释“高血糖诱发巨噬细胞泡沫化促进动脉粥样硬化风险”的病理学假说模型提供了重要的实验依据和补充说明。
The rapid development of life science, which studies the nature of life and all kinds of the internal regular patterns about life activities, needs the information about the complicated biosamples such as composition, property, function and variation. This requires analytical chemistry to provide not only analytical methods and technologies but also comprehensive and abundant information for in-situ, on line and in vivo analysis. Photoelectrochemical biosensors is a burgeoning analytical technique presenting extremely high sensitivity and it has been widely applied in many areas such as life science, environmental monitoring, security inspection and agriculture science. Photoelectrochemical biosensing employs current or potential as a detection signal while uses light as an excitation source. Therefore, how to choose appropriate photosensitive materials and improve the photoelectric conversion efficiency and stability is an important subject in the research of potoelectrochemical biosensors.
In this dissertation, we laid our emphasis on the development of the photoelectrochemical biosensors based on TiO2nanotube assays, and their applications in the analysis of enzyme activity damage in vitro. The fabricated photoelectrochemical biosensors are used to study the inhibition of acetylcholinesterase (AChE) activity induced by two endogenous neurotoxins, Cadmium ions (Cd2+) and Clozapine. Furthermore, we studied an innovative detection method of enzyme activity by photoelectorchemical analysis with the prepared photoelectrochemical biosensor. In addition, a novel dual enzymatic-biosensor was described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. This dissertation includes five chapters:
Chapter1. Overview
In this chapter, the information of electrochemical biosensors including the concept, working principle, classification and application was first introduced. Then, the contents of photoelectrochemistry which focused on photoelectrochemical materials and their applications in biosensors were presented. Followed was the brief introduction of Parkinson's disease, diabetes mellitus and atherosclerosis which are three diseases related to the research work. Finally, we emphatically indicated the purpose and significance of the dissertation, innovation spot and content as well.
Charpt2. Preparation and amplification of the Visible-light-actived photoelectrochemical biosensor
Section One. Visible-light-actived photoelectrochemical biosensor for the study of acetylcholinesterase inhibition induced by endogenous neurotoxins
In this section, a novel visible-light-actived photoelectrochemical biosensor was fabricated to study the inhibition of acetylcholinesterase (AChE) activity induced by two endogenous neurotoxins,1(R)-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-Sal] and1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetra-hydroisoquinoline [(R)-NMSal] which have drawn much attention in the study of the pathogenesis of neurodegenerati ve diseases such as Parkinson's disease. The photoelectrode was prepared by three steps as follows. At first, nitrogen and fluorine co-doped TiO2nanotubes (TNs) were obtained by anodic oxidation of Ti sheet. Secondly, silver nanoparticles (AgNPs) were deposited onto the TNs through microwave-assisted heating polyol (MAHP) process. At last, AChE was immobilized on the obtained photoelectrode and the biosensor was marked as AChE/Ag/N-F-TNs. Due to the nitrogen and fluorine co-doping, the photoelectrochemical biosensors can produce high photocurrent under the visible light irradiation. Moreover, the presence of AgNPs greatly increased the photocurrent response of the biosensor. AChE/Ag/N-F-TNs hybrid system was used to study AChE inhibition induced by (R)-Sal and (R)-NMSal and the result proved that both (R)-Sal and (R)-NMSal all exhibited mixed and reversible inhibition against AChE. This strategy is of great significance for the development of novel photoelectrochemical biosensors in the future.
Section Two. Detection of AChE activity in rat serum with photoelectrochemical biosensor
The study of Acetylcholinesterase (AChE) activity is one of the most important aspects in the research on pathogenesis mechanism of neurodegenerative disease. Thus, a sensitive and rapid determination technology for AChE activity is urgently needed. In this section, the AChE activity in rat serum was determined by photoelectrochemical analysis (PECA) method with the visible-light-actived photoelectrochemical biosensor prepared in section one. This method showed an excellent analytical performance, which exhibited a wide dynamic response range of AChE activity from10U/L to2000U/L and with a detection limit of2U/L. The results demonstrate that the developed PECA method offered a new approach for AChE activity study, which is related to the pathogenesis mechanism. Overall, this propsed established PECA method was very promising and could be easily extended to measure the other enzyme activity.
Charpt3. A photoelectrochemical biosensor for the study of acetylcholinesterase inhibition induced by cadmium ions based on metal-oxide semiconductor nanocomposites
Cadmium (Cd) is a heavy metal with a high toxicity. It is toxic at very low dose and it has acute and chronic effects on human health since it can influence the activity of numerous enzymes in body. A representative case about Parkinson's disease (PD) after acute Cd poisoning in clinic treatment has been reported. In this report, a novel photoelectrochemical biosensor was fabricated to study the effect on AChE activity induced by Cd2+ions which have drawn much attention in the study of the pathogenesis of neurodegenerative diseases such as PD, Alzheimer's disease (AD) and Myasthenia Gravis (MG). The photoelectrode was prepared by depositing zinc oxide nanorodes (ZnONRs) onto the TNs through cathodic deposition method and then AChE was immobilized on the obtained photoelectrode and the biosensor was marked as AChE/ZnONRs/TNs. The photoelectrochemical biosensor showed good performance in the monitoring of acetylthiocholine (ATCh) with a rapid response. Experimental results show that Cd ion exhibited dose-dependent and time-dependent effects on AChE. In addition, we proposed a newly effective method to estimate the damage effect of enzyme activity caused by heavy metal ions.
Charpt4. A photoelectrochemical biosensor based on TiO2NTs cooperated with hyperbranched polymer hybrid for studying the effect of clozapine on acetylcholinesterase activity
In this report, a novel photoelectrochemical biosensor was fabricated to study the inhibition of AChE activity induced by clozapine, which have drawn much attention in the study of the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Herein, thiol-functionalized hyperbranched azo-polymers (HBAP) decorated with gold colloids (GC-HBAP) via covalent bond were utilized as the biosensing platform, which have been demonstrated to not only anchor larger amounts of capture antibodies, but also act as hole scavengers to facilitate the improvement of the photoelectrochemical performance of semiconductive nanomaterials. Tyrosine was initially electropolymerized on the TNs electrode surface to perform abundant carboxyl groups to bonding the prepared GC-HBAP nanoparticals. And then, AChE immobilized on the surface of the photoelectrode and marked as AChE/GC-HBAP/Pty/TNs. The effects of clozapine on AChE activity in vitro were studied by using AChE/GC-HBAP/Pty/TNs hybrid system. Experimental results show that clozapine exhibited dose-dependent effects on AChE. This established method successfully provies a robust way for evaluating the effects of enzyme on other antipsychotics. In addition, this strategy could be easily used to find new therapies and screen new drugs.
Charpt5. A dual electrode detection system with multienzyme biosensor for simultaneous determination of Glucose and Cholesterol in serum and peritoneal macrophages of diabetic mice:evaluation of the diabetes-accelerated atherosclerosis risk
In this paper, a novel dual electrode detection system with multienzyme biosensor is described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. The biosensor was constructed by a three-step method. First, a poly-thionine (PTH) film was assembled on the surface of glassy carbon electrode by cyclic voltammetric electropolymerization of thionine, which serves as an electron transfer mediator (ETM). Second, gold nanoparticles (GNPs) were covered on the surface of PTH facilitating the electron transfer between glucose oxidase (GOx), cholesterol oxidase (ChOx) and electrode. Finally, the enzymes, GOx, cholesterol esterase (ChE), ChOx, were covalent attached to the PTH layer through a chitosan (CH) linker. The PTH coupled with GNPs provides good selectivity, high sensitivity and little crosstalk for the dual enzymatic-biosensor. The constructed detection system had good electrocatalytic activity toward the oxidations of glucose and cholesterol, exhibiting a linear range from0.008mM to6.0mM for glucose with a detection limit of2.0μM, and a linear range from0.002mM to1.0mM for cholesterol with a detection limit of0.6μM. The results of the diabetic mice demonstrated that the cholesterol level did not change obviously with the increase of glucose level in serum, while the cholesterol level was induced with the increase of the glucose level in PMs. Previous studies have shown that the large accumulation of cholesterol in macrophage could lead to macrophage foam cell formation, which is the hallmark of early atherosclerosis. This study provides useful further evidences for the development of diabetes-accelerated atherosclerosis.
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