Study On Novel Enzyme Biosensors Based On Functionalized Nanomaterials And Their Applications

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. OverviewIn 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 biosensorSection One. Visible-light-actived photoelectrochemical biosensor for the study of acetylcholinesterase inhibition induced by endogenous neurotoxinsIn 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 biosensorThe 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 nanocompositesCadmium (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 activityIn 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 riskIn 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.