Study Of Nanoparticle Linear Light Scattering Probes On Immunoassay And DNA Hybridization

In this thesis, the history and development of DNA hybridization and immunoassay has first been stated. In the second part of the thesis, a simple, rapid, sensitive and high-specific universal platform of homogeneous noncompetitive immunoassay using human immunoglobulin (IgG) as a model analyte has first been developed. The assay is based on aggregation of antibody-functionalized gold nanoparticles directed by the immunoreaction coupled with light scattering detection with a common spectrofluorimeter. In phosphate buffer (pH=7.0) solution, the light scattering intensity of the gold nanoparticles functionalized with goat-anti-human IgG can been greatly enhanced by addition of the human IgG. Based on this phenomenon, a wide dynamic range of 0.05～10μg/mL for determination of human IgG can be obtained, and the detection limit can reach 10 ng/mL. The proposed immunoassay can be accomplished in homogeneous solution with one-step operation within 10 min. The proposed method has been successfully applied to the determination of human IgG in serum samples and the results are well consistent with those of the enzyme-linked immunosorbent assay (ELISA), indicating its high selectivity and practicality. Therefore, the gold nanoparticle-based light scattering method can be used as a model to establish the general methods for protein assay in the fields of molecular biology and clinical diagnostics.In the third part of the thesis, A simple,sensitive,and specific light scattering assay for DNA hybridization in homogeneous solution has been developed by using gold nanopartical as the labels of oligonucleotide probes.The assay relies on the observation of greatly enhanced light scattering originated form the aggregation of oligonucleotide-functionalized gold nanoparticles directed by DNA hybridization.The light scattering can be easily measured by using a common spectrofluorimeter through simultaneously scanning the excitation and emission monochromators with the same wavelength.With the proposed light scattering assay, as low as 0.1 pmol of target DNA can be detected and no washing steps is needed.The sensitivity increase four orders of magnitude greater than that of the gold nanopartical-based colorimetric method and is more favorable to that of fluorescent-based homogeneous detection of DNA hybrisization.Moreover, the light scattering assay exhibits a high degree of discrimination between perfectly matched target DNA and targets with single base-pair mismatch.A simple, sensitive, and specific linear light scarring (LLS) assay for detection of single nucleotide polymorphisms (SNPs) in homogeneous solution has also been developed. The assay relies on the extraordinary selectivity of non-cross-linking aggregation of oligonucleotide-modified gold nanoparticals(GNPs) against terminal mismatch of the GNP surface-bound DNA duplex. It has been demonstrated that the LLS is a very sensitive and powerful technique for detection of the non-crossing-linking aggregation of the GNPs. With this assay, as low as 1 pmol of target DNA can be detected.The sensitivity increases about 3 orders of magnitude greater than that of fluorescent-based methods for SNP detection. By combination with a one base extension protocol, this LLS assay can be directly used to identify SNPs using the extension reaction mixture without requirement of separating steps and controlling temperature.