The Establishment And Primary Application Of Novel Homogeneous Quantum-dots-based Time-Resolved Fluoroimmunoassay

The research background and purpose:Immunoassay is based on highly selective immune reation of antigen with antibody, and can be used for the qualitative detection or quantitative analysis of the antigen or antibody. Currently, immunoassay becomes one of very widely used analytical technologies in food and environmental analyses, biological and biomedical studies and clinical diagnosis and other fields. According to the analyte suspended in the sample or not, immunoassay techniques is divided into homogeneous and heterogenous (or solid phase). The conventional heterogeneous immunoassays mainly use a microwell plate as an assay platform. In general, entire operation process of the heterogenous assay (eg. ELISA, TRFIA) has many deficiencies, such as multi-steps, slow-phase immune response system, time-consuming due to repeated washing steps to separate the unlabeled antibody before signal measurement and not easy automation. On the contrary, homogeneous assay is to directly determine analytes in immune reaction mixture. This method is usually fast and amenable to miniaturization and automation. Homogeneous immunoassay could avoid instability and low sensitivity as a result of repeated washing steps, greatly improve the detection efficiency, and it is suitable for the development of rapid high-throughput and automated detection technologies.Alpha-fetoprotein (a-fetoprotein, AFP) is a single polypeptide chain glycoprotein, and a major plasma protein produced by the yolk sac and liver during fetal development. The human fetus has the highest AFP concentrations, with lower adult concentrations realized by the age of8-12months, and AFP detection in pregnant women serves as a screening test for a subset of developmental abnormalities. AFP in adult blood can indicate the presence of certain types of cancer, particularly of the liver, stomach, pancreas, ovaries, or testicles; high AFP levels may also be found in Hodgkin’s disease, lymphoma, brain tumors, and renal cell cancer. More importantly, some patients with early cancer are asymptomatic, which often results in late diagnosis and a high death rate. Therefore, AFP is also used as a biomarker to detect a subset of tumors in men, nonpregnant women, and children. Moreover, it is very important to detect AFP in high-risk but asymptomatic groups to identify early curable tumors, decrease disease-related mortality, and to improve survival.The research content and method:The purpose of this paper was to use semiconductor Quantum dots (Quantum dots, QDs) as a tag probe, and combine with the time-resolved fluorescence from rare earth metal chelate terbium (Luminescent terbium chelates, LTCs).Based on the Fluorescence Resonance Energy Transfer (FRET) principle, the donor (LTCs) and the receptor (QDs), specifically combined with the antigen (AFP). Homogeneous immunoassay could be available to quantitative determination of AFP.The main contributions are as follow:1. The preparation of quantum dot microspheres. We used high-throughput encoding quantum dots preferred method, in order to bring cadmium selenide/zinc sulfide (CdSe/ZnS) quantum dots to this project. Firstly, carboxylated polystyrene microspheres (Carboxyl-Functionalized Polystyrene Microspheres, CPs) swelled completely into chloroform/isopropanol. After that, quantum dots were embedded into the above carboxylated polystyrene microspheres to make it with carboxyl on the surface of the quantum dots. Meanwhile, the surface-functionalized composite microspheres with high fluorescence intensity can be stably stored in a PBS buffer solution at4℃.2. Synthesis of terbium chelates. The terbium chelates (LTC-DTPA-Tb3+) was synthesized with the materials of diethylenetriamine pentaacetic acid dianhydride (DTPA, A), TbCl3and CS124.3. The surface chemical modification of fluorescence resonance energy transfer acceptor (Donor) and receptors (Receptor) was help to eliminate or prevent the accumulation of non-specific adsorption of precipitation. On the one hand, polystyrene microspheres (CPs) was a highly non-specific substance, despite its surface had been carboxyl-modified in the water soluble system. However, the hydrophilic surface had been partially damaged after repeated swelling chloroform/isopropanol to adsorb fat-soluble quantum dots. In this case, the nonspecific adsorption capacity was great, especially in the detection of antigen or antibody, and it made a great impact on the experimental results. On the other hand, terbium chelate donor was a multi carboxylic acid and ester group, synthesized by terbium (Tb34) and special chelator. It had the nature of the aqueous sol and its rich anionic surface electrostatic repulsion force with charge terbium chelates to biological molecular receptor or non-specific adsorption or gathered themselves together, and its surface had ruled out the nonspecific adsorption. Therefore, this study also surface chemical modification of quantum dots nanometer microspheres, make it as much as possible with a hydrophilic group, so as to avoid nonspecific adsorption.4. Taking advantages of the double antibody sandwich method, with the help of the fluorescence resonance energy transfer (FRET) principle, the quantum dot carboxy polystyrene microspheres (QPs) and terbium chelates (LTCs) labeled monoclonal antibodies, we established the homogeneous immunoassay to quantitative determination of AFP.The research results:The activities of these labeled antibodies were analyzed by fluorescence resonance energy transfer(FRET). The concentrations of AFP were measured by FRET between LTCs and QPs with a two-antibody sandwich system. The results indicated that quantum dots could be applied in homogeneous fluoroimmunoassay. The signals of FRET are proportional to the concentrations of AFP with LogY=3.65786+0.43863·log X (R=0.996, P<0.01), and the sensitivity for analysis is calculated as0.4ng/mL. The novel homogeneous fluorescence immunoassay is no need to wash away the free fluorescent conjugates with high sensitivity. It provides a scientific basis for the development of rapid and convenient biomedical sensor technology and modern immunoassay technology with multi-channel, high sensitivity, and high-throughput.The research conclusion:We have successfully prepared Quantum dots polystyrene microspheres (QPs) coated with anti-AFP monoclonal antibody (E014), and LTCs labeled with another anti-AFP monoclonal antibody (E010), respectively. The paper was studied to develop a novel quantum dot-based homogeneous fluoroimmunoassay technology for detection of AFP.