| With the success of dolly and the comletion of the human Genome project, we have entered in Post Genome Era after experienced molecule biology and Structural genomics era in last century. In this era, the major research focus on functional genomics, including genome and proteomic. The basic element for genome and proteomic are protein and DNA. The data of gene sequence is growing faster than ever before. However, How to figure out the function of all the gene during the life process has became the common topic for the scientific researcher all over the world. Therefore, it is extremely improtant to develop a new method for hybridization and sequence detection which can be used to assay and analyse a large number of genetic Information effectively and rapidly.Electrochemiluminescence (ECL), which triggers optical signal by electrochemical reactions, offers the advantages of versatile employment, simplified optical setup, low background disturbance, and good temporal and spatial control. Therefore, it has been widely used in the field of immunoassay, food and water analysis and for the detection of biowarfare agents. it also has been used as a detector for HPLC, FIA and CE. Among the electrochemiluminescence compounds, the mostly used one is tris(2,2'-bipyridyl)ruthenium(Ⅱ) (Ru(bpy)32+), which excites more sensitive ECL signal. Recently, solid-state Ru(bpy)32+-ECL system has been developed. Compared with the solution phase-based ones, it has enhanced the ECL intensity as well as consumed less expensive Ru(bpy)32+, simplified experimental design, and created regenerable sensing component by recycling Ru(bpy)32+modified electrode.In this thesis, we had developed a novel ECL array biosensor and solid ECL array sensor with high sensitivity, specificity and selectivity which had been used to detect targets such as DNA and TPrA. It provided a simple, rapid and effecitve method for the fabrication of solid ECL array and ECL DNA chips. It would broaden the research field of ECLChapter 1:PrefaceIn this chapter, we elaborated review from the principles, characteristics of the electrochemiluminescence, the five most primary types of ECL reaction and their application in analytical chemistry field. We highlight the character and application of Ru(bpy)32+. Furthermore, we introduce the sort, character and research progress of ECL biosensor. Finally, expounded the aim and the significance, pointed out the research content and the innovation in this paper.Chapter 2:Electrogenerated chemiluminescence Immunoassay for Human IgG with Electrochemical polymerization-based immobilization methodRabbit-anti-human IgG was immobilized on gold electrode by electrochemical polymerization with pyrrole. An efficient immunoassay strategy for human IgG determine in human serum was developed based on the specific immunological reaction between the rabbit-anti-human IgG, human IgG and Ru(bpy)32+-labled goat-anti-human IgG, which forming sandwich-typed immunocomplex. Then the target IgG in the sandwich-typed structure was determined by electrogenerated chemiluminescence. The parameters of polymerization time, incubation time and the pH of detection solution were optimized. The protocol has been characterized with impendence, cyclic voltammogram and ECL. A good linear relationship between ECL intensity and human IgG in the concentration range between 50μg/L and 2 mg/L was obtained, and the detection limit was 20μg/L.Chapter 3:An electrochemiluminescence immunosensor based on diazonium reaction modified carbon nanotubes electrodeAn electrochemiluminescence (ECL) immunoassay biosensing strategy involving diazonium reaction modified carbon nanotubes electrode was developed. The multi-walled carbon nanotubes (MWCNTs) modified glassy carbon (GC) electrode was firstly functionalized with carboxyl groups by the electrochemical reduction of diazonium cation onto it, and then covalently linked antibody. The target antigen was then detected via a sandwich detection approach, in which the rabbit anti-human IgG modified MWCNTs/GC electrode was as the capture component, and Ru(bpy)32+-labeled goat anti-human IgG was as the signal producing element. Such immunoassay strategy was applied for the determination of human IgG. Due to the excellent electric conductivity of MWCNTs and strong covalent bond between antibody and electrode, a good linear relationship between ECL intensity and human IgG concentration in the ranging from 2 ng mL-1 to 120 ng mL-1 was obtained, and the detection limit was 0.5 ng mL-1.Chapter 4:Solid state electrochemiluminescence sensor through the electrodeposition of Ru(bpy)32+/AuNPs/chitosan composite film onto electrodeTris (2,2'-bipyridyl) ruthenium(Ⅱ) (Ru(bpy)32+) has been successfully immobilized onto electrode through the electrodeposition of Ru(bpy)32+/AuNPs/chitosan composite film. In the experiments, chitosan solution was first mixed with Au nanoparticles (AuNPs) and Ru(bpy)32+. Then, during chronopotentiometry experiments in this mixed solution, a porous 3-D network structured film containing Ru(bpy)32+, AuNPs and chitosan has been electrodeposited onto cathode due to the deposition of chitosan when pH value is over its pKa (6.3). The applied current density is crutial to the film thickness and the amount of the entrapped Ru(bpy)32+. Additionally, these doping Ru(bpy)32+ in the composite film maintained their intrinsic electrochemical and electrochemiluminescence activities. Consequently, this Ru(bpy)32+/AuNPs/chitosan modified electrode has been used in ECL to detect tripropylamine, and the detection limit was 5×10-10 M.Chapter 5:Selective immobilization of tris(2,2'-bipyridyl)ruthenium (Ⅱ) onto array electrode for solid-state electrochemiluminescene sensor fabricationAn effective method for selective immobilization of tris(2,2'-bipyridyl)ruthenium(Ⅱ) (Ru(bpy)3+) onto one target electrode surface based on the electrodeposition of RuDS NPs (Ru(bpy)32+ doped silica nanoparticles)/chitosan composite film is presented in this paper. Ru(bpy)3+ was selectively deposited onto four individual electrodes in an Au electrode array. The results demonstrate the possibility of selective immobilization of Ru(bpy)32+ onto array electrodes and selective immobilization of different ECL composite. Therefore have potential applications in bioanalysis, capillary electrophresis and drug screening. RuDS NPs and the resulting composite film were characterized by UV-vis spectroscopy, transmission electron microscopy (TEM) and field-emitted scanning electro microscopy (FE-SEM). The electrochemiluminescence (ECL) sensor based on the composite film modified electrode exhibited excellent reproducibility, stability and sensitivity for the detection of tri-n-propylamine (TPrA). The linear range was from 1×10-10 to 1×10-6 M(R2=0.9954) with the detection limit of 5×10-11 M.Chapter 6:A Sensitive Electrogenerated Chemiluminescence DNA Biosensing Array for the Simultaneous Multianalyte DetectionA novel electrogenerated chemiluminescence (ECL) array for simultaneous multianalyte assay is developed. Three different DNA target sequences, which were respectively related to the disease of HCV, SARS and HIV, were selectively immobilized onto one pointed electrode in one Au array electrode with the aid of electrodeposited chitosan film. Ru(bpy)32+-labeled DNA was used as the signal producing element to signal the target DNA sequences using sandwich detection format. Due to the special addressable immobilization method, the electrochemical and cross-reactivity between the neighboring electrode sites was negligible for the ECL array sensor, which showing good sensitivity and selectivity for multi-target sequence detection and thus having potential usage for clinical, environmental and biodefense applications. |
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