Establishment And Primary Application Of A Novel Homogeneous Fluorescence Immunoassay Based On Quantum Dots And Luminescent Oxygen Channel

Background and Purpose:Labelling immunology is a major about the combination of molecule marking and immunological techniques for analysis and determination. Activity and concentration of molecule can be detected with fluorescence, enzyme, radionuclide or Chemiluminescence substances marked antigens or antibodies based on the high specificity of immune reaction and the high sensitivity of the label. The most commonly used immunolabelling techniques include immunofluorescence (IF), enzyme immunoassay (EIA), immunohistochemistry technique, radioimmuno assay (RIA), Chemiluminescence immunoassay (CLIA), time-resolved fluoroimmunoassay (TRFIA), etc. With the development of molecular biotechnology, labelling immunology has been widely used in many fields such as medicine, agriculture, food and environment supervising. Immunoassay also can be divided into homogeneous immunoassay and heterogeneous immunoassay according to the analyte suspended in the liquid phase homogeneously or not. The most commonly used homogeneous immunoassay include AlphaScreen, TRACE, LANCE, etc. Currently, conventional heterogeneous immunoassays, ELISA and dipstick assay for example, are apply to clinical diagnosis oftenly. Generally, heterogeneous immunoassay has many drawbacks. For example, they usually include multi-steps and the immune response are slow so that heterogeneous immunoassays are time-consuming. Wash steps are necessary in heterogeneous immunoassay as separation of bounded and free antigens or antibodies. So it is hard to automate. Homogeneous immunoassay, by contrast, reduces the number of steps and errors in the assay and provide a higher sensitivity and signal-noise ratio. Furthermore, Homogeneous immunoassay usually has a lower quantification limit and wider working range and can be run in miniaturized formats and provide a higher throughput.Alpha-fetoprotein (AFP), also sometimes called alpha-fetoglobulin, is a single-chain glycoprotein that encoded by AFP gene which is located on the q arm of chromosome 4 in humans. AFP is secreted by the yolk sac and liver mainly in the fetal development process and can be detected in amnion liquid or maternal serum. Normally, concentration of AFP in plasma usually peaks at the age of 8 to 12 months. AFP levels of pregnant women can be monitored. When it is out of the normal range, which indicates the fetal abnormality, such as neural tube defect (NTD), intestinal atresia, congenital renal disease, fetal distress, stillborn foetus, etc. AFP detection in gravida serves as prenatal screening test for a subset of heteroplasia. Moreover, AFP can also be used as a biomarker to detect a subset of many kinds of tumor, because patients’ liver cells could produce AFP continually. High AFP levels is also related to Hodgkin’s disease, lymphoma, brain tumors, renal cell cancer and etc. AFP detection could be widely used in diagnose and prognostic evaluation.Quantum Dot (QDs) is a nanocrystal made of semiconductor materials which is small enough to limit its excitons in all three dimensions. Due to its unique properties in optical, such as size-dependent emission, symmetrical and narrow spectra, wide range of excitation and more stable, it plays a more and more important role in biological and medical research field. Quite a few applications have been established with regard to cell imaging, molecular and nucleic acid research, drug screening and discovery.Luminescent oxygen channeling assay (LOCI) is a type of homogeneous immunoassay, which is characterized by using an excited and reactive form of oxygen that is converted from ambient O2 in solutions, singlet oxygen (1O2), as energy and signal transmitter. Comparing with non-homogeneous methods, ELISA, for example, the wash steps are avoided, as separation of bounded and free antigens or antibodies. It reduces the number of steps in the assay, in some case, it also reduces the hours of incubation.384-well plates are optional in LOCI. In other words, LOCI is easy to automate, robust in routine testing, can be run in miniaturized formats and provide a higher throughput. Furthermore, LOCI usually has a higher sensitivity, lower quantification limit and wider working range than other immunoassay methods.Method:The purpose of this paper is to establish a homogeneous immunoassay by using semiconductor Quantum dots to encode nanoparticles which combines with the special optical characteristic of QDs and the high sensitivity of LOCI, and which could be available to quantitative determination of antigen (AFP).The main contributions are as follow:1. Construction and surface modification of acceptor-beads. We synthesis the core-shell quantum dots, CdTe/ZnS QDs, by self-assembling method and thioxene (N,N-dimethyl-4-(2-phenyl-5,6-dihydro-1,4-oxathiin-3-yl)benzenamine).Then, we encoded the carboxyl-modified polystyrene microspheres with QDs, thioxene and BPEA(9,10-Bis(phenylethynyl)anthracene) by swelling in organic solvent and modified its surface in order to lower its unspecific absorption. Conjugation of antibody to beads be applied. 2. Construction and surface modification of donor-beads. We synthesis the BTHS which can convert oxygen into singlet form and encode the carboxyl-modified polystyrene microspheres by heat swelling. Surface modification and streptavidin conjugation are applied.3. Immunology study. Label the antibody with Biotin and AFP antigen is diluted as 0 IU/mL,1 IU/mL,10 IU/mL,100 IU/mL,500 IU/mL and 1000 IU/mL. Donor-beads, acceptor-beads, biotin-antibody should be diluted into working concentration with assay buffer. Add 25 μL of acceptor-beads,25 μL of biotin-antibody and 25 μL of antigen into each well and incubate for 15min with shaking at 37℃. Add 175 μL of Donor-beads per well and incubate for 15min with shaking at dark,37℃. The fluorescence intensity was measured on EnSpire(?) multimode plate reader. Linearized standard curve was obtained on a log-log plot with six different concentrations.4. Optimization. It includes the optimizations about bead concentration, embedding conditions, materials, incubation time and etc.Results:With a two-antibody sandwich system, the immune complex shorten the distance between donor-beads and acceptor-beads and singlet oxygen pass the energy to thioxene. Due to fluorescence resonance energy transfer, QDs is lighten. The results indicated that the concentration of AFP has a close relationship with the fluorescence intensity of acceptor-beads. It can be described as LogY=2.401+1.052-LogX, R2=0.959.Here X is the concentration of AFP and Y is the fluorescence intensity. The sensitivity for analysis is calculated as 2.65 IU/mL.Conclusion:We have successfully prepared functional quantum dots polystyrene microspheres which coated with anti-AFP monoclonal antibody and donor-bead covered with streptavidin. With the biotin-streptavidin system and two-antibody sandwich system, the paper was studied to develop a novel homogeneous immunoassay based on quantum dots and luminescent oxygen for channel detection of AFP.