The Detection Of Prion Protein Based On Au Nanoparticle Probes

Au nanoparticles due to the unique optical properties received extensive attention in recent years. Surface plasmon resonance absorption and scattering spectra become hot topic and widely used in many fields. The color and plasmon resonance absorption peak of Au nanoparticles are associated with the shape, size, and dispersion of Au nanoparticles. The well monodisperse Au nonoparticle solution was in wine red color and have absorption peak in the visible spectral range of 510-550 nm; while in the state of aggregation, surface plasmon resonance absorption appears red shift and the color turns red to blue or purple. Due to the simple approach, low experiment cost and direct operation, there have been some new Au nanoparticle sensors based on colorimetric analysis in recent years. Use surface plasmon resonance absorption change that caused by Au nanoparticles disperse state change can detect metal ions and DNA, protein or other biological molecules.Prions that cause a number of fatal neurodegenerative diseases in mammalians are special class of pathogens. The diseases include scrapie of sheep, bovine spongiform encephalopathy (BSE), and humans Creutzfeldt-Jakob disease (CJD) and other neurodegenerative syndromes as Kuru disease. These diseases are caused by infection, genetic, or sporadic disorders. Host-encoded noninfection cellular prion proteins (PrPc) converted to conformation altered infectious forms PrPsc was proved to be the pathogenesis of these diseases.The experimental results indicate that the PrPc abound in the majority of the organization of the nervous system and play a role in the cellular metabolism, antioxidant, the nervous system in normal development and signal transduction. The conversion of PrPc to PrPsc alter their biochemical and physiochemical properties, such as insolubility, aggregation,β-sheet-rich secondary structure, and portease digestion resistance.The fibrils have a diameter of 7-10 nm, high-resolution methods which studies the structural of amyloids found a cross-β-pleated sheet conformation. Although the species barrier provided important protection for prion protein pass throuth among species, the outbreak of human variability Creutzfeldt-Jakob disease (vCJD) caused by BSE infected cattle confirmed that prion protein possess potential influence for human. Even the current state-of-the-art method, such as fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or protein misfolding cyclic amplification are difficult to detect its presence in cattle or diagnosis in human or blood banks. For this reason, the confirmation of scrapie, BSE, or CJD still relies on the postmortem imaging analysis of degenerated tissues for the suspected individuals. This analysis may lead to late response. Pandemic outbreak may occur and with important economic consequences. So it is very important to develop the benefical detection technology for accurately diagnosing the presence of prion in the blood or serum of infected but not clinically sick animals, which could avoid the dissemination of the disease. The main works of this thesis are summarized as follows:(1) Using dihydrolipoic acid modified Au nanoparticles as probe lable-free detect prion protein. Utilizing recombinant human prion protein (rPrP) as model protein, investigate prion protein induced the aggregation of DHLA-AuNPs and DHLA-AuNPs effected the fibrosis of prion protein. DHLA-AuNPs as a probe intereacted with prion protein, leading to the DHLA-AuNPs absorption decrease linear with the increase of prion protein concentration, which could be used to quantitatively detect prion protein. Adjust experimental conditions, add NaCl, can significantly increased the sensitivity of detection. This probe was used to detect the concentration of prion protein in fetal bovine serum and E.coli lysates which expressed prion protein. Transmission electron microscopy imaging (TEM) results showed that the precipitate produced by the interaction between DHLA-AuNPs and rPrP existed amyloid fibrils, proved that the aggregation of DHLA-AuNPs concerned with the fibrosis of prion protein.(2) We obtained APT-AuNPs through modified Au nanoparticle with prion protein aptamer. Base on the special binding between aptamer and prion protein, using APT-AuNPs as a probe, we acquired a new method to detect prion protein. The selectivity and coexistence interference results proved that this method with excellent selectivity.(3) Using two aptamer which could specitic binding to prion protein to modify sodium citrate coated Au nanoparticles. As the two kinds of aptamers are special bind to prion protein, we can quantitative detect prion protein. Under the same sensitivity we significantly improve the linear rang of detection, and get orderly assembly Au nanoparticles complex.