Chemiluminescence Functionalized Nanprobe And Their Applications In Biosensors For Tuberculosis

In this dissertation, the state of arts in the field of chemiluminescence (CL), Nanomaterial-amplified CL systems, nucleic acid probe based CL biosensors, CL functionalized nanomaterials and their applications in bioassays were reviewed. Chemiluminescence has become a powerful analytical tool and been widely used in bioassays for its advantages such as high sensitivity, wide linear range, low background and simple instrumentation. In CL bioassays, quantitative assays commonly rely on the indirect approaches based on labeling strategies. In the commercially available CL bioassays based on labeling strategies, only one signal reporter was attached to a signal probe. Challenge remains in improving the sensitivity of bioassays to meet the increasing demand for detection of biomarkers at ultra-trace amount level. Accordingly, some multi-labeling strategies have been explored, in which one signal probe can carry a number of signal reporters. Among them, the development of CL functionalized nanoparticles(CF-NPs) becomes a new trend with innovative concepts nanoscience, which makes hundreds of CL signal-generating molecules be coated on or encaspsulated in a nanoparticle host. Our previous work has demonstrated the direct synthesis of CL functionalized gold nanoparticles using luminol as reducent and stabilizing reagent. This new type of CF-NPs would be used as labels for nucleic acid probe based CL biosensors with excellent signal amplification, improving the sensitivity of the biosensors. The aim of this dissertation is to explore the synthesis of new CF-NPs and their applications in nucleic acid probe based biosensors for tuberculosis diagnosis. The assembly of the luminol functionalized gold nanoparticles with DNA and aptamer molecules to build nucleic acid nanoprobes was studied. On the basis, a sandwich-type electrochemiluminescence (ECL) DNA sensor and a homogeneous CL aptamer sensor were developed for tuberculosis diagnosis. Moreover, a kind of new CF-NPs, i.e carbon nanodots with strong photoluminescence were synthesized. Carbon nanodots involved new CL system was designed and its CL behavior and mechanism were explored. The main results are as follows:1. A novel ECL DNA sensor was developed for a fast test for Mycobacterium tuberculosis (MTB), which was based on luminol functionalized gold nanoprobes with excellent signal amplification functionality. This nanoprobe was formed by the conjugation of luminol functionalized gold nanoparticles (lum-AuNPs) with signal DNA probes. An81bp segment derived from the Mycobacterium tuberculosis specific insertion sequence IS6110was chosen as the target strand and corresponding probes were designed to specifically hybridize with it. Biotinylated capture probes can be effectively immobilized directly on a streptavidin coated AuNP modified indium tin oxide electrode. After catching the TB target strand, signal probes tagged with lum-AuNPs were attached to the assembled electrode surface to.form a sandwich-type TB sensor. Extremely high sensitivity for detecting the synthetic TB target strand was achieved with a detection limit of6.7×10-15mol/L, which was superior to other genetic methodologies for TB tests based on gold nanoprobes. Genomic DNA from other pathogenic bacterias (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) had a negligible effect on its detection, which guaranteed the good selectivity of the TB sensor. The efficacy of the TB sensor was also evaluated for genomic DNA extracted from cultured M. tuberculosis. The TB sensor is sensitive, highly selective, convenient and cost-effective. The simplicity of the assay and the lack of a requirement for sophisticated equipment render the TB sensor a promising candidate as a rapid molecular test for the detection of M. tuberculosis.2. A homogeneous hemin/G-quadruplex DNAzyme (HGDNAzyme) based turn-on chemiluminescence aptasensor for interferon-y detection is developed, via dynamic in-situ assembly of luminol functionalized gold nanoparticles, DNA, IFN-y and hemin. The G-quadruplex oligomer of the HGDNAzyme was split into two halves, which was connected with the complementary sequence of P1(IFN-γ-binding aptamer) to form P2. P2hybridized with IFN-y-binding aptamer and meanwhile assembled onto lum-AuNPs through biotin-SA specific interaction. When IFN-γ was recognized by aptemer, P2was released into the solution. The two lateral portions of P2combined with hemin to yield the catalytic hemin/G-quadruplex DNAzyme, which amplified the luminol oxidation for a turn-on chemiluminescence signaling. Based on this strategy, the homogeneous aptasensor enables the facile detection of IFN-y with high sensitivity (0.4nM) and satisfactory specificity, pointing to great potential applications in clinical analysis.3. A general strategy for the production of carbon nanodots (CDs) has been developed involving microwave irradiation of amino acids in the presence of acids or alkali. Transmission electron microscopy, UV-visible spectroscopy, X-ray photoelectron spectroscopy and powder X-ray diffraction analysis were used to characterize the morphology and surface component of the obtained CDs. The result demonstrated that the CDs were spherical in morphology and well dispersed with diameter of1-4nm. The obtained CDs exhibited highly photoluminescent activities, excellent upconversion photoluminescent properties and could effectively enhance the ultra-weak chemiluminescence from the reaction of NaIO4with H2O2. This investigation could be valuable to obtain new insight into the optical characteristics of the CDs and broaden the application of the novel materials in analytical fields. Because of their high photoluminescence and effective enhancement of chemiluminescence, CDs would offer great potential for a broad range of applications, including optoelectronic devices, bioassays, biomedical and bioimaging applications.