Development And Application Of Novel Biosensing Technology Based On Graphene Oxide And Telomerase

In recent years, nanomaterials have been regarded as the research focus, and applied in biosensors more and more widespreadly, due to their excellent optical and electrochemical properties. The combination of nanomaterials and biosensors is concerned with the fields of biotechnology, information technology, nanoscience and so on. Therefore, the study of nanomaterials in biosensors provides a lot of innovative ideas for basic research, and plays a most important role in clinical testing, medical diagnosis, and environmental monitoring. Nanomaterials are also facing an unprecedented opportunity for development. In this paper, a seris of novel nano-biotechology as a simple and low-cost platform for sensitive detection of DNA and proteins, screening antitumor drugs. Because reported methods for detecting telomerase activity were time-consuming, low-sensitive and false negative, a seris of novel biosensing strategys were developed for sensitive detection of telomerase activity. The detailed contents are described as follows:(1) In chapter2, we developed a novel biosensing strategy for high-sensitive detection of DNA based on Exo Ⅲ-assisted target recycling amplification. In this assay, we designed a fluorescein amidite (FAM)-labeled hairpin signal probe which with extensions7bases at its3’terminus. In the absence of target DNA, signal probe first adsorbed onto the surface of graphene oxide through π-stacking interaction between the ring structrue in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye was quenched. While the target DNA was introduced, signal probe hybridezed with target DNA to form double-stranded DNA structure, which led to the releasing of signal probe from the surface of graphene oxide and the fluorescence intensity recovered. The duplex DNA was digested by Exo Ⅲ, a sequence-independent nuclease that catalyzes the removal of mononucleaotides from blunt or recessed3’-hydroxyl termini of duplex DNA and is not active on single-stranded DNA and3’-protruding termini with extensions4bases or longer. This digestion not only make the dye far from the graphene oxide, but also released the target DNA, which then hybridize with fresh signal probes and restart the digestion process. The results revealed that this strategy offered a sensitive and selective method for the detection of target DNA over the concentration range of1pM to50nM with the detection limit of1pM. The sensitivity of the assay is3orders magnitude better than previously reported methods.(2) In chapter3, we constructed a novel biosensing platform for screening antitumor drugs based on graphene oxide sheets. In this method, we chose human telomeric DNA as the template to design a fluorescein amidite (FAM)-labeled signal probe which adsorbed onto the surface of graphene oxide sheets, and the fluorescence of the FAM was quenched. When the quadruplex-binding ligands were introduced, the signal probe folded to form intramolecular antiparallel G-quadruplex structure through the π-π conjugated interactions and hydrogen bindings between ligands and bases of signal probe. It led to the releasing of FAM-labeled signal probe from the surface of graphene oxide and the fluorescence intensity recovered. Three series of Chinese medicine monomers were investigated by the proposed method, and the flavonoids were demonstrated to be the potential quadruplex-binding ligands. Furthermore, the strategy could find wide applications in the discovery of new antitumor drugs.(3) In order to further extend the application range of graphene oxide, we exploited a universal biosensing platform for protease detection and intracellular imaging in live cells based on graphene oxide-peptide conjugate in chapter4. In this strategy, peptide probe was conjugated to the surface of graphene oxide by covalently crosslinking method to reduce the background signal. In the presence of target protease, peptide probe was cleaved by target protease specificly, which led to the releasing of FAM-labeled peptide probe from the surface of graphene oxide and the fluorescence intensity enhanced substantially. On one hand, graphene oxide was a fluorescence quencher for fluorophores adjacent to its surface. On the other hand, graphene oxide is intrinsically a nanocarrier for delivering peptide cargos inside live cells. After being transported into cells followed by cleavage of the peptide by intracellular proteases, the GO-peptide conjugate provided greatly enhanced fluorescence imaging. The results demonstrated that this strategy could afford a simple, sensitive and selective biosensor for the detection of caspase-3in the dynamic range of7.25-362ng/mL. In addition, it could be extended to multiplex in vitro assays or live-cell imaging of multiple proteases by use of the conjugate of GO to different peptide substrates with multicolor fluorophore tags.(4) In chapter5, we presented a novel biosensing technology for the detection of telomerase activity in cancer cells by using gold nanoparticles as a fluorescence quencher. Firstly, thiolated capture probe was mixed with signal probe and slowly cooled to room temperature. Then the DNA duplexes were added to gold nanoparticles (GNPs) to form the DNA-GNPs composite probes by self-assembly technology. The TS primer contained at the thiolated capture probe elonged in the presence of target telomerase. While the telomerase extension products could folded into intramolecular hairpin structure with the complementary sequences at the capture probe and released signal probe to enhance the fluorescence intensity. The results indicated that the method could be used for sensitive determination of telomerase in a concentration range from100to30000HeLa cells with the linear range of0to1600HeLa cells. Moreover, it could be extended to detection of Pb2+sensitively and selectively with the detection limit of2nM.(5) In chapter6, we developed a novel eletrochemical DNA sensor for sensitive and selective detection of telomerase based on target-induced structure-switching DNA. In this assay, capture probe with the sequences of TS primer at its3’terminus was firstly immobilized on the gold electrode via self-assembly of the terminal thiol moiety and then hybridized with a ferrocene-tagged signal probe, leading to a high redox current. In the presence of telomerase, the TS primer elonged and the telomerase extension products could fold into intramolecular hairpin structure with the complementary sequences at the capture probe, resulting in the release of the ferrocence-tagged signal probe far from the electrode with a substantially decreased redox current. The results show that the eletrochemical DNA sensor displayed a quantitative analysis of telomerase with the linear range of100to60000HeLa cells besides desirable specificity and sensitivity.(6) In chapter7, we exploited a high-sensitive fluorescence strategy for telomerase detection in cancer cells base on T7Exonuclease-assisted target recycling amplification. In this assay, we designed a Taqman probe modified with6-carboxy-fluorescein (FAM) as a fluorophore at its5’terminus and tetramethyl-6-carboxyrhodamine (TAMRA) as a quencher at the neighboring three-nucleotide position. The T7Exonuclease, a sequence-independent nuclease that catalyzes the removal of5’mononucleotides from5’termini of double-stranded DNA, couldnot digest the Taqman probe, hence this probe gives very weak fluorescence signal from FAM due to fluorescence resonant energy transfer from FAM to TAMRA. The telomerase can bind to the substrate sequence (TS) and enzymatically elongate it with TTAGGG repeats in the presence of dNTPs. Once the telomerase extension products hybridize with the Taqman probes, the T7Exonuclease will digest product-bound Taqman probes. This digestion not only makes the quencher-fluorophore pair of Taqman probe separate from each other, leading to significantly enhanced fluorescence, but also releases the intact product strands, which then hybridize with fresh Taqman probes and re-start the digestion process. Therefore, the fluorescence signal is amplified repeatedly through recycle of the telomerase extension products. The results revealed that it offered a sensitive and selective biosensing strategy for the detection of telomerase over the concentration range of5to1000HeLa cells with the detection limit of5HeLa cells. This strategy holds great promise as a simple, sensitive method for telomerase detection in proteomics and clinical diagnostics.