Fluorescence Detection Of Biological Molecules Based On Nanoparticle Material

Gold nanoparticles(AuNPs) and graphene oxide(GO) are two important nanomaterials, which were widely applied in various fields, especially the application in life science and pharmaceutical analysis. Both of them have excellent features and provide the conditions for the construction of biosensor. On the other hand, aptamer was selected for the recognition of target analytes with high affinity and specificity, so the interference test material can be eliminated. Moreover, aptamer can be functionalized modification with gold nanoparticles and grapheme oxide in the preparation of biosensors. Fluorescent probes was based on the principle that the intensity of fluorescence was relate with the concentration of the target and target materials can be observed by monitored the change of the intensity of fluorescence. In this study, we developed some fluorescent probe with different mechanism to detect biological molecules such as adenosine, dopamine and kanamycin and all of these methods are simple, sensitive, selective.(1) The first chapter presents the following contents: the characteristics, preparation and application of gold nanoparticles and grapheme oxide; the preparation and application of nucleic acid aptamers; analysis of the mechanism of the fluorescence biosensor; analysis of the significance and main content of this study.(2) A novel strategy was developed for fluorescent detection of analytes in aqueous solution based on the participation of aptamer-functionalized gold nanoparticles(AuNPs) and graphene oxide(GO). In this system, high selective single-stranded oligonucleotides(ssDNA) aptamer of adenosine was modified on AuNPs through the strong bond of Au-S. In the absence of adenosine, the aptamer-functionalized AuNPs can be adsorbed onto the surface of GO due to the π-π stacking interaction between the ring structure in the nucleobases and nucleosides, and the hexagonal cells of GO, and then aptamer-functionalized AuNPs-GO precipitation would be formed. In the presence of adenosine, adenosine-aptamer-functionalized AuNPs complexes were produced and dispersed in aqueous solution, rather than the aptamer-functionalized AuNPs-GO precipitation. With the addition of fluorescein, the supernatant of the solution without adenosine presented strong fluorescence, while the supernatant with adenosine showed weak fluorescence. By measuring the difference fluorescent intenstiy of fluorescein, the concentration of adenosine was readily determined. The linear response range was obtained over the concentration range from 5?10-8 M to 5?10-7 M and with a detection limit of 5.8?10-9 M. Moreover, this fluorescence biosensor shows high selectivity toward adenosine against its analogs due to the specific recognition ability of the aptamer for the target and the biosensor was applied to the determination of adenosine in urine samples by using standard adding method with satisfactory results.(3) In the present work, a novel fluorescence strategy based on amino pyrene(AP) and grapheme oxide(GO) was proposed for the determination of dopamine(DA). In the absence of DA, the fluorescence of AP would be quenched by GO for the ?-? stacking between AP and GO. While in the presence of DA, DA was firstly attached to GO sheets and lead the fluorescence of AP turn on. Due to the strong adsorption between GO and DA, the proposed fluorescence sensor exhibited high selectivity and sensitivity toward DA. The linear response ranges for DA was 4×10-9 M to 4×10-7 M and corresponding detection limit was 7.53 nM. Furthermore, we developed a series assays to compare the adsorption of DA to two different components(GO and its aptamer). The results of fluorescence spectrum, Fourier Transform infrared spectroscopy(FTIR) and adsorption kinetic demonstrated that DA tend to adsorb onto GO sheet in the mixed solution with GO and its aptamer(DBA). The superior adsorption between GO and DA make it very promising for applications in direct detection of DA in biological samples and potential applications in various areas.(4) This experiment is based on the interaction between cationic fluorescent polymers product, aptamer, graphene oxide and DNA enzyme to detect target kanamycin. The fluorescence of cationic product was quenched when joined with the aptamer, and fluorescence quenched further if joined with graphene oxide. While kanamycin and DNA enzyme were added, the aptamer would left from the surface of graphene oxide. Due to the catalysis of DNA enzyme to aptamer, kanamycin would released from aptamer and interacted for the next circulation with fluorescence enhanced and amplified. The linear response ranges for kanamycin was 2×10-8 M to 10×10-8 M and corresponding detection limit was 8.67 nM. Moreover, this probe with high selectivity and could be used to detect kanamycin in real sample.