Study On Novel Electrochemical Sensors Based On Functionalized Nanomaterials And Their Applications In Biomacromolecular Analysis

Biosensors are analytical devices comprising a biological or biologically derived sensing element either integrated within or intimately associated with a physicochemical transducer. Owing to their intrinsic advantages of high sensitivity, inherent miniaturization, in vivo monitoring and real-time determination, biosensors have attracted immense interest and thus played a significant analytical role in clinical diagnosis, environmental monitoring, drug development, food safety inspection and other fields. The emergence of nanoscience and nanotechnology is opening new horizons for biosensors. Recent years have witnessed the development of a variety of nanomaterial-based biosensors exhibiting novel functions. Nanoparticles, nanowires and nanotubes have already made a major impact on the field of biosensors, due to their unique structural, electrical, mechanical properties, and the capacity that improved stability, minimization of sensor's surface fouling, increased sensitivity, multiplexing capacity along with an improved cost-efficiency. The use of nanomaterials in such biosensors has taken off rapidly and will surely continue to expand the realm.In this dissertation, we laid our emphasis on the fabrication of novel nanomaterials, such as composite materials, multi-functional polymers, and bioconjugates. We combined electrochemical biosensors with nanomaterials and photoelectrochemistry, and developed novel biosensors and photoelectrochemical system for determination of a-synuclein, SirTl and enzyme inhibition. This dissertation includes six chapters:Chapter1. OverviewIn this chapter, a detailed outlines and reviews related with the development of biosensor was given, which introduced the concepts, fundamental principles and applications of the biosensors. According to the difference of bioreceptors, biosensors can be classified into several types and the characteristics of electrochemical enzyme biosensor and electrochemical immunosensor were exploited. Then, the development and application of nanotechnology in biosensors was highlighted. In addition, photoelectrochemistry was outlined with regard to concepts, mechanism, and application in electrochemical biosensors. We also briefly reviewed the current development of sensitive detection methods of two types of significant proteins, and introduced neurodegenerative disorders. Finally, we emphatically indicated the purpose and significance of the dissertation, innovation spot and content as well.Chapter2. Photoelectrochemical immunosensor based on Au-doped TiO2nanotube arrays for detection of a-synucleina-Synuclein (α-SYN) is a very important neuronal protein which is associated with Parkinson's disease. In this paper, we utilized Au-doped TiO2nanotube arrays to design a novel photoelectrochemical immunosensor for detection of a-SYN. The highly ordered TiO2nanotubes were fabricated by electrochemical anodization technique on a pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au-doped TiO2nanotubes constructed effective antibody immobilization arrays and made primary antibodies (Ab1) immobilized with high stability and bioactivity to bind target a-SYN. The enhanced sensitivity was obtained by using{Ab2-Au-GOx} bioconjugates, which was featured with secondary antibody (Ab2) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in detection solution to produce H2O2, which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO2nanotubes upon irradiation of the other side of Ti foil and led to a prompt photocurrent. The photocurrents were proportional to a-SYN concentrations, and the linear range of the developed immunosensor was from50pg/mL to100ng/mL with a detection limit of34pg/mL. The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.Chapter3. Sensitive electrochemical immunosensor for a-synuclein based on dual signal amplification using PAMAM dendrimer-encapsulated Au and enhanced gold nanoparticle labelsA novel electrochemical immunosensor for sensitive detection of α-synuclein (a-SYN), a very important neuronal protein, has been developed based on dual signal amplification strategy. Herein, G4-polyamidoamine dendrimer-encapsulated Au nanoparticles (PAMAM-Au nanocomposites) were covalently bound on the poly-o-aminobenzoic acid (poly-o-ABA), which was initially electropolymerized on the electrode surface to perform abundant carboxyl groups. The formed immunosensor platform, PAMAM-Au, was proved to provide numerous amino groups to allow highly dense immobilization of antigen, and facilitate the improvement of electrochemical responses as well. Subsequently, the enhanced gold nanoparticle labels ({HRP-Ab2-GNPs}) were fabricated by immobilizing horseradish peroxidase-secondary antibody (HRP-Ab2) on the surface of gold nanoparticles (GNPs). After an immunoassay process, the{HRP-Ab2-GNPs} labels were introduced onto the electrode surface, and produced an electrocatalytic response by reduction of hydrogen peroxide (H2O2) in the presence of enzymatically oxidized thionine. On the basis of the dual signal amplification of PAMAM-Au and{HRP-Ab2-GNPs} labels, the designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This developed strategy was successfully proved as a simple, cost-effective method, and could be easily extended to other protein analysis schemes.Chapter4. SirTl regulation study with a novel immunosensor based on TiO2photoelectrochemical label cooperated with hyperbranched polymer hybridWe report an investigation of a novel photoelectrochemical immunosensor using TiO2-Au nanocomposite labels cooperated with hyperbranched polymer hybrids for SirTl assay. 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. The typical sandwich-type immunoassay procedure comprised of the immunoreaction of capture antibody (Ab1#1) with target SirT1, followed by the attachment of detection antibody (Ab1#2), and{TiO2-Au/Ab2}, which featured secondary antibody (Ab2) linked to TiO2-Au nanocomposites as "photoelectrochemical label". When this immunosensor was upon visible-light irradiation at400nm, the methanol acted as a electron donor to scavenge the holes generated in the valence band of TiO2, meanwhile, GC-HBAP polymer induce the electron transfer to the ITO electrode owing to larger amounts of azo groups in the backbones of the polymer, thus leading to an enhanced photocurrent signal. The SirTl concentration, which was proportional to that of the Ab2linked to TiO2-Au nanocomposites, could be readily examined through measurement of the photocurrent derived from the system of TiO2-Au nanocomposites. The proposed photoelectrochemical immunosensor showed good performance in the monitoring of SirTl with a rapid response, wide concentration range, and high sensitivity. The photoelectrochemical immunosensor was also successfully used to quantify SirTl in H1299cells and Hela cells. Overall, the established method provides a robust approach for the photoelectrochemical detection for other significant proteins in the future.Chapter5. Functionalized Au@SiO2shell-core structure for signal amplification in electrochemical immunoassay of SirTlA novel strategy using functionalized Au@SiO2nanoparticles for the amplified electrochemical immunosensor was developed in the present work. The horseradish peroxidase-secondary antibody (HRP-Ab2) was immobilized onto the surface of Au@SiO2nanoparticles, denoted as{Au@SiO2/HRP-Ab2} labels. Subsequently, the MWNTP-Au nanocomposites were fabricated by immobilizing PAMAM-Au on the surface of multiwall carbon nanotubes (MWNTs). Greatly amplified signal was achieved in the sandwich-type immunosensor employing{Au@SiO2/HRP-Ab2} labels and MWNTP-Au nanocomposites. For SirTl assay, the designed immunoassay showed an excellent analytical performance, and exhibited a wide dynamic response range of SirTl concentration from0.02to500ng/mL with a detection limit of12.5pg/mL. This proposed signal amplification strategy was very promising and could be easily extended to measure other biorecognition events.Chapter6. Study on acetylcholinesterase inhibition induced by endogenous neurotoxin with enzyme-semiconductor photoelectrochemical system The inhibition of acetylcholinesterase (AChE) induced by N-Methyl-(R)-salsolinol, a dopamine-derived endogenous neurotoxin, has been considered to involve in the pathogenesis of Parkinson's disease. Thus, a sensitive and simple method for the evaluation of AChE inhibition is imminently needed. In this chapter, the well-ordered TiO2nanotubes are fabricated by electrochemical anodic oxidation and modified with Au nanoparticles by a novel photoelectrochemical deposition. Then, the integration of semiconductor TiO2nanotubes with enzyme AChE yields a novel TiO2-Au-AChE hybrid system, which provides a novel, valid and rapid photoelectrochemical approach to the detection of AChE inhibition induced by endogenous neurotoxin. The results showed that the semiconductor TiO2nanotubes can be also coupled with other enzymes to design diverse photoelectrochemical sensors for the inhibition of enzymes.