Studies On The Ecl And Fluorescence Biosensors Based On The Bipyridine-Ruthenium Cyclodextrins Supramolecular Compounds And The Host-guest Recognition

Study on the molecular biology has long been the most valuable thing for people and has been growing faster than before since the turn of the century. The detection of biomolecules such as nucleic acid, proteins and ATP is of great importance for clinical detection and disease diagnosis. Electrochemiluminescence and fluorescence biosensing using Ru(bpy)32+as signal label are popular and powerful analytical techniques because of their high sensitivity, low background, simple instrumentation, and rapid sample analysis, which have been widely used in biomolecule detection. Herein, to improve the analytical sensitivity and extend its application, new luminophores with higher ECL efficiencies and techniques for the labeling of biomolecules while maintaining their specific biological activity are desired.Metallocyclodextrin, presenting excellent electronic and photoactive properties owing to its assured luminescent structure and molecular-recognition function of cyclodextrin cavity as the host model, is a highly useful signal supermolecule for the construction of supramolecular devices and biosensors, which has increasingly attracted the interest of researchers from many fields in recent years.Based on the host-guest recognition of the bipyridine-ruthenium cyclodextrins supramolecular compounds, several ECL and fluorescence biosensors have been developed for the detection of nucleic acid, proteins and ATP. By taking full advantages of the bipyridine-ruthenium cyclodextrins compounds, the proposed ECL and fluorescence biosensors not only exhibited excellent sensitivity but also performed high efficiency and simplicity without any other signal labeling or amplification procedures. Therefore, this approach offered great potentials for the ultrasensitive, simple and efficient detections of diverse target biomolecules.Chapter1:IntroductionIn chapter1, the excellent properties of supramolecular compounds especially for the metallocyclodextrins have been introduced generally. The host-guest recognition properties of the supramolecular compounds and their applications in biosensing have been studied systematically. We have illustrated the principle and characteristics of Electrochemiluminescence (ECL) and the development of new type luminophor. The current applications of biosensors based on ECL and fluorescence were highlighted. Finally, we summarized the significance, main content and the innovation of this thesis.Chapter2:The studies of the electrochemiluminescence properties of multi(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin supramolecular compoundsMetallocyclodextrins, multi(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin supramolecular compounds (multi-Ru-(3-CD) in particular, have special luminescent properties due to their multiple photoactive units. Thus we studied the electrochemiluminescence (ECL) properties of multi-Ru-β-CD systematically. It is observed that tris(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin compound [tris(bpyRu)-β-CD] could be applied in the construction of ECL sensors owing to its high photovoltaic efficiency stemming from multiple chromophores of a single molecule and unique host-guest recognition properties based on the supramolecular structure. And we studied the effects of several guests including methylene bule,4-dimethylaminoazobenzene-4’-carboxylicacid, ursodesoxycholic acid and nucleic acid on the ECL behavior of tris(bpyRu)-β-CD. Chapter3:The studies of the electrochemiluminescence properties of tris(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin solid-state film based on host-guest recognition and application in DNA sensingIn chapter3, the tris(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin compound [tris(bpyRu)-P-CD] was electropolymerized on an electrode surface to fabricate a stable solid-state film for considerable improvement of luminescence. Exploiting the luminescent properties and supramolecular structure of the tris(bpyRu)-β-CD film, we developed a sensitive ECL sensor for DNA using a Dab-labeled hairpin DNA. After hybridization with target DNA, the Dab group of the double-stranded (ds)-DNA was captured by the cyclodextrin cavity of the host ECL film, quenching the ECL response of the sensor. This process was closely related to the concentration of target DNA. The proposed ECL-DNA sensor exhibited high sensitivity and selectivity, demonstrating its promising application in DNA sensing.Chapter4:Electrochemiluminescence aptasensor for adenosine triphosphate detection using host-guest recognition between tris(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin and aptamerIn this chapter, based on the host-guest recognition between tris(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin compound [tris(bpyRu)-β-CD] and an ATP-binding aptamer which has been performed in Chapter2, a sensitive and label-free electrochemiluminescence (ECL) aptasensor for the detection of adenosine triphosphate (ATP) was successfully designed. In the protocol, the NH2-terminated aptamer was immobilized on a glassy carbon electrode (GCE) by a coupling interaction. After host-guest recognition with tris(bpyRu)-β-CD, the tris(bpyRu)-β-CD/aptamer/GCE produced a strong ECL signal as a result of the photoactive properties of tris(bpyRu)-β-CD. However, in the presence of ATP, the ATP/aptamer complex was formed preferentially, which restricted host-guest recognition, and therefore less tris(bpyRu)-β-CD was attached to the GCE surface, resulting in an obvious decrease in the ECL intensity. Under optimal determination conditions, an excellent logarithmic linear relationship between the ECL decrease and ATP concentration, with a detection limit of1.0×10-11mol/L at the S/N ratio of3. The proposed ECL-based ATP aptasensor exhibited high sensitivity and selectivity, without time-consuming signal-labeling procedures, and is considered to be a promising model for detection of aptamer-specific targets.Chapter5:Ultrasensitive and signal-on electrochemiluminescence detector of proteins using the multi(bipyridine)ruthenium(Ⅱ)-∞-cyclodextrin supramolecular compoundsAn ultrasensitive and signal-on electrochemiluminescence (ECL) aptasensor to detect target protein (thrombin or lysozyme) was developed using the host-guest recognition between multi(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin supramolecular compounds (multi-Ru-P-CD) and single-stranded DNA (ss-DNA). The aptasensor uses both the photoactive properties of the multi-Ru-P-CD and their specific recognition with ss-DNA, which amplified the ECL signal without luminophore labeling. After investigating the ECL performance of different multi-Ru-β-CD, tris(bipyridine)ruthenium(Ⅱ)-β-cyclodextrin (tris(bpyRu)-β-CD) was selected as a suitable host to construct an aptasensor. First, double-stranded DNA (ds-DNA) formed by hybridization of the aptamer and its target DNA was attached to a glassy carbon electrode via coupling interaction, which showed low ECL intensity with2-(dibutylamino) ethanol (DBAE) as coreactant because of the weak recognition between ds-DNA and tris(bpyRu)-P-CD. Upon addition of the corresponding protein, the ECL intensity increased when target ss-DNA was released because of the higher stability of the aptamer-protein complex than the aptamer-DNA one. A linear relationship was observed in the range of1.0×10-10～1.0×10-14mol/L between ECL intensity and thrombin concentrations with a directly limited detection of1.0×10-14mol/L. Meanwhile, the measured concentration of lysozyme was from5.0×10-10to5.0×10-14mol/L and the directly measured detection limit was5.0×10-14mol/L. The investigations of proteins in human real serum samples were also performed to demonstrate the validity of detection in real clinical samples. The simplicity, high sensitivity and selectivity of this aptasensor show great promise for practical applications in protein monitoring and disease diagnosis.Chapter6:Sensitive fluorescence detection of lysozyme using (bipyridine)ruthenium(Ⅱ)-multi-β-cyclodextrin supramolecular compounds and aptamerPhotophysical studies of a series of metallocyclodextrins named (bipyridine)ruthenium(Ⅱ)-multi-β-cyclodextrin supramolecular compounds (Ru-multi-P-CD), comprising a ruthenium core and multiple cyclodextrins indicated their higher emission efficiency than the parent compound tris(bipyridine)ruthenium(Ⅱ) thanks to shielding of the metal cores from fluorescence quenching. Host-guest recognition between the Ru-multi-β-CD and DNA aptamer further shielded against quenching. Based on this a sensitive fluorescence detector for protein was proposed. First, the fluorescence intensity of the Ru-multi-β-CD was enhanced upon binding aptamer. While, addition of lysozyme causes aptamer/lysozyme complexes to form, reducing aptamer binding to the cyclodextrin and leading to reduced fluorescence intensity. This reduction can be used to determine the lysozyme concentration with a low detection limit of1.0×10-10mol/L.