Application Of Graphene And Signal Amplification Technology In Optical Analysis Method To Detect The Bioactive Substances

Graphene is a nanophase material with the lattice structure of monolayer two-dimensional honeycomb. It has been paid wide attention for its unique physical and chemical properties. Optical analysis is convenient and practical in operation, with high sensitivity and selectivity. In the thesis, through the graphene as well as the combination of optical analysis with signal amplification technology, we’ve achieved the highly-sensitive detection of DNA, Thrombin, Glutathione, DNA methylase. Our work was conducted mainly according to the following three aspects.1. The Research about the detection DNA and thrombin based on the Luminol-H2O2-HRP-fluorescein-graphene oxide chemi luminescence resonance energy transfer systemA new Chemiluminescence biosensor to detect the sensitivity of DNA and Thrombin was invented based on the quenching effect of Graphene oxide on Luminol-H202-HRP-fluorescein chemiluminescence resonance energy transfer system. The fluorescence will be quenched when the fluorescein-labeled ssDNA is absorbed on the surface of GO through π-π stacking. When the GO surface meet the target DNA, the ss-DNA will form a double helix structure with it, thus dissociating from the surface. With the Luminol-H202-HRP-fluorescein chemiluminescence resonance energy transfer system, the detection limit of H1V1DNA can reach0.1pM. Then in accorDNAce with the specific bonding of aptamer with thrombin, making the fluorescein-labeled thrombin aptamer adsorbed on the surface of GO,thus achieving the highly sensitive and selective detection, with its limit reaching1.OpM. It expand the application field of chemiluminescence in biochemistry.2. The preparation of Graphene oxide-hemin nanoprobe and its application in the detection of GlutathioneThe synthesis of the Graphene oxide-hemin nanoprobe to achieve the detection of Glutathione:firstly, combining mercapto bead with2-pyridyldithio ethyl amine to form the bead containing a dis-μL fide bond and terminal amino; Then,modifying the dis-μL fide bond on the surface of GO through the reaction of carboxyl of GO with amino of the bead.Meanwhile, synthetising graphene oxide-hemin nanoprobe throμgh the adsorption mechanism of GO adsorbed on Hemin. For the synthetic nanoprobe, characterizing it with UV,IR,Raman, AFM,SEM respectively. For the GSH can break the dis-μL fide bonds, the GO-hemin probe can dissociate in solution.Detecting the UV-visible absorption values of the supernatant by magnetic separation to achieve the highly sensitive detection of GSH. We can apply this method to the analysis of complex samples and tumor cell which shows a good selectivity.3. The Research about the detection of DNA methylation through chemiluminescence method based on a new hybrid chain reaction-rolling circle replication zoom technologyA novel hybridization chain reaction-rolling circle replication amplification technology (HCR-BRCA) has been designed to detect the activity of DNA methylase through the method of chemiluminescence. The experiment is divided into three stages:1) methylation and the cyclization process of DNA:first, after the two complementary ssDNA hybridize,adding the DNA methyltransferase as well as the Dpn I restriction endonuclease that has the same recognition sites. Then cutting the DNA chain between the recognition sites and obtaining two ssDNA with different lengths.second, adding DNA primer DNA Pi DNA P2(with the terminal the amino-containing magnetic beads) that are complementary to the long chain DNA and end-phosphorylating. Adding T4ligase to make the long-chain DNA into a ring.2) HCR-BRCA process:Under the action of the Klenow polymerase/dNTPs, rolling circle replication is triggered. Introducing primer DNA H1, DNA H2(with the end containing biotin) which are complementary to DNA and can occur hybridization chain reaction.3) chemiluminescence detection process:achieving beads magnetic separation through adding avidin-modified HRP. Conducting chemiluminescence detection with the Luminol-H2O2-HRP-PIP system.It can achieve the high-sensitivity detection of DNA methylase with the detection limit up to0.52UmL.