| Rapid methods for bacterial detection are essential in food, industrial and environmental monitoring, clinical diagnostics, and biodefence to allow faster decisions to be made with respect to food poisoning, water contamination, the presence of disease and, therefore, treatment options. Escherichia coli (E. coli) are one of the most common types of bacteria and are a normal inhabitant of the large intestine of warm-blooded animals. Some of the strains of E. coli are particularly virulent and cause a wide spectrum of human diseases. Most conventional methods for bacterial detection are time-consuming, often requiring1-2days to obtain results. A rapid, quantitative, sensitive, and specific method for one-step detection of E. coli is therefore highly sought after. Traditionally, cancers are diagnosed mostly based on the morphology of tumor tissues or the contents increase of cancer biomarkers. However, these methods are difficult to be used to carry out early cancer diagnosis or to evaluate the complex molecular alterations that lead to cancer progression. Mounting evidence suggests that alterations to glycan structures can contribute to the development and progression of cancer and other diseases. Lectins, a group of proteins extracted from plants or animals, can strongly bind to specific carbohydrate moieties on the surface of bacteria or cancer cells and thus are particularly interesting candidates as molecular recognition elements to efficiently profile the variation in glycosylation of cancer cells. Therefore, the biosensors using lectins as recognition elements for the dectection of cancer cells have recently attracted much attention.ECL biosensors combine the advantages of both electrochemical and chemiluminescent biosensors, such as high sensitivity, ease of control and using of simple equipment and specific selectivity offered by the biological recognition elements, which has widely been used in pharmaceutical analysis, bioanalysis, environmental analysis and clinical analysis.The aim of this thesis is to design and fabricate ECL biosensors for determination of bacteria and cancer cells with high sensitivity. Lectins are particularly interesting candidates for use as molecular recognition elements because of their ease of production and intrinsic stability. In this thesis, taking advantage of the unique properties of lectins and the specificity of biological molecular recognition elements, such as Con A/lipidpolysacchrides (LPS), WGA/GlcNAc on PC-3cells, we have designed one type of "signal off" ECL biosensors for determination of E. coli. Two types of "signal on" ECL biosensors for determination of cancer cells were fabricated.The thesis includes three parts. First part, chapter1, is general introduction while second part consisting of three chapters, is a research report. The last one, chapter5, is the conclusion of the thesis.In Chapter1, general introduction to glycobiology and lectin, ECL biosensor and research development of bacterial and cancer cells sensing methods using lectin as molecular recognition elements, and the purpose of this research work were presented.In Chapter2, a novel electrogenerated chemiluminescence (ECL) biosensor for highly sensitive detection of Escherichia coli (E. coli) was first developed by employing Concanavalin A (Con A) as a biological recognition element and bis(2,2’-bipyridine)-4’-methyl-4-carboxybipy-ridine ruthenium (II)(Rul) complex as the detector. The ECL biosensor was fabricated by adsorbing carboxyl-functionalised single-wall carbon nanotubes (SWNTs) onto a paraffin-impregnated graphite electrode and further covalently coupling the Rul-Con A probe onto the surface of the SWNT-modified electrode. Upon the binding of E. coli O157:H7(as a model target), the biosensor showed a decreased ECL intensity in the presence of tri-n-propylamine (TPrA), which was in logarithmically direct proportion to the concentration of E. coli over a range from5.0×102to5.0×105cells/mL. The detection limit of this sensor was127cells/mL. Additionally, the ECL biosensor also showed satisfactory selectivity in discriminating gram-negative E. coli from gram-positive bacteria. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and rapid detection of other desired bacteria.In Chapter3, a multi-label ECL biosensor for highly sensitive detection of Ramos cells was developed on bases of Ramos cells-specific oligonucleotide (TD05) served as molecular recognition element and Rul as an ECL signal complex. The ECL probe of (G5-Rul-TD05) was prepared by covalently coupling Rul and TD05with the generation fifth polyamidoamine (G5-PAMAM). The ECL biosensor was fabricated by covalently coupling the reorganization molecular of TD05onto the glass carbon electrode (GCE) modified with single-wall carbon nanotubes through a spacer5’-amino-(CH2)6-TD05. Upon the binding of Ramos cells by TD05binding with the mIgM proteins on Ramos cells surfaces, then the biosensor incubates with the ECL probe G5-Rul-TD05, results a high ECL intensity. The newly developed biosensor showed a low detection limit of55cells/mL, which is2.5-fold lower than that of the biosensor using Rul-TD05as ECL probe. The effect of length of the spacer of capture probe and signal probe were investigated. This work demonstrates that using G5-PAMAM as a carrier of ruthenium complex and molecular recognition element for the fabrication of ECL biosensor for detection of cancer cells with sensitivity is a promising approaches.In Chapter4, a novel sandwich ECL biosensor for sensitive detection of prostate PC-3cancer, was developed by employing antibody PSA as capture probe and ruthenium complex labeled wheat germ agglutinin (WGA) as the ECL probe. The ECL biosensor was fabricated by adsorbing graphene oxide (GO) onto a GCE and further covalently coupling the capture probe antibody (anti-PSA) onto the surface of the GO-modified electrode. The GO was employed to attach the primary antibody due to it large surface area and promising biocompatible. The biomarker of prostate cancer specific antigen (PSA) used as a model to optimize the fabrication and the detection conditions of the ECL biosensor. After incubated with PSA, the biosensor binding with the ECL probe based on the recognition between WGA and N-glycan at PSA, the ECL biosensor showed strong ECL intensity in the presence of TPrA, which was in logarithmically direct proportion to the concentration of PSA over a range from0.5to20ng/mL. The detection limit of PSA was0.1ng/mL (signal-to-noise ratio of3). The ECL biosensor was also detected PC-3prostate cancer cells concentration with the limit of156cells/mL and dynamic range from700to10000cells/mL. Moreover, the proposed ECL lecitn-based immunosorbent exhibited excellent specificity and high sensitivity. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related glycoproteins.In Chapter5, the conclusion was presented. This work establishes one "signal off" and two "signal on" ECL biosensor for the detection of bacteria and cancer cells with high sensitivity. This work demonstrates that suing G5-PAMAM as a carrier of ruthenium complex and recognition molecular element and covalently coupling method for the fabrication of the ECL biosensor with high sensitivity and good stability are promising approach. |
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