Preliminary Study On Multiplex Bioanalytical Method Based On Atomic Spectrometric Detection And Metal Nanoparticle Labeling

Atomic spectrometry has been widely used in many fields of analytical chemistry. Electro-thermal atomic absorption spectrometry(ETAAS) is a widely used element detector, thanks to its low cost, high sensitivity, multiple element detectability, and low sample consumption. Inductively coupled plasma mass spectrometry(ICP-MS) is another powerful analytical tool, which has the feature of high sensitivity and multiplex detection. Combining these two analytical technologies with metal nanoparticle labeling can potentially built a highly sensitive and highly multiplex method for biomacromolecules. In this thesis, we propose two analytical methods to detect DNA and protein biomarkers. The one is based on the gold nanoparticle labeling strategy for single component analysis; and the other is based on rare earth nanoparticle labeling strategy for multiplex protein biomarkers analysis.1. An electro-thermal atomic absorption spectrometry-based assay for single component disease-related DNA.In this work, a simple and sensitive DNA assay utilizing ETAAS detection was demonstrated with Au nanoparticles(NPs) tag. After the thiolated report DNA was immobilized on Au NPs and biotinylated DNA was immobilized on magnetic microparticles, and then the capture DNA, target DNA, report DNA is hybridization reaction. After the reaction, aqua regia was added to dissolve Au NPs and release Au ions, which were subsequently detected by ETAAS. Under the optimize sandwich hybridization reaction conditions, we success to detect different concentration of DNA with a good linearity was obtained between the concentration of target DNA and the atomic absorbance signal intensity. The limit of detection(LOD) was 3 pmol/L(LOD, 3?), with the linear range of 10–200 pmol/L. This method established a foundation for the further exploration of multiplex bioanalysis.2. An ICP-MS-based multiplex protein assay after rare earth nanoparticle labeling.After the successful demonstration of single component analysis, we aimed to identify and quantify multiple tumor protein biomarkers. By chemical modification of nanoparticles' surface, the rare earth nanoparticles can be connected with tumor marker's antibody. Through quantitative analysis of rare earth elements by ICP-MS, tumor marker levels were indirectly obtained. In this work, we synthesized four kinds of earth nanoparticles by solvothermal method. We attempted three bioconjugation methods to couple antibody with rare earth nanoparticles. The nanoparticles and bioconjugates were characterized by transmission electron microscope(TEM), Fourier transform infrared spectrum(FT-IR), Zeta potential and UV-Vis spectrophotometry(UV).