Preparation&Characterization Of Nano-immunomicrosphere And Its Application In Immunochromatographic Detection

In recent years, nanospheres colloidal gold and latex as the functional carrier materialare widely used in cell biology, life sciences, clinical medicine, immune diagnosis and otherfields. The immunochromatographic diagnostic technology and related products came out forthe combination of nanospheres, target substance immunoassay and lateral flow microfluidictechnology. It has the advantage of quick response, simple operation, high sensitivity andgood stability, and has widely used in targeted research and clinical, home-version diagnostickits. However, the deficiencies such as low sensitivity(false negative), specific problems(falsepositive), the problems of how to balance the relationship between sensitivity and specificity,and what was the root cause of the appearance of false negative and false positivephenomenon, which showed adverse impact in the application of immunochromatographicassay. Therefore, in order to promote the further application of nanospheres andimmunochromatographic assay, it is very important to research the preparation ofnano-immunomicrosphere, to study various changes in the process of application and itsinfluential factors, to explore the nature of false negative and false positive phenomenon.In this paper, two kinds of nanospheres material, colloidal gold and latex, were selectedto react with target protein. Immune colloidal gold and immunolatex were prepared by themethods of physical adsorption and covalent coupling, respectively. The stability ofimmunomicrospheres were also studied by transmission electron microscope, Zeta potentialand dynamic light scattering. Results showed that immune colloidal gold (the optimum pHvalue5.0and the optimal protein concentration20μg/mL) and immunolatex (activatorEDC/NHS, active time15min, the reaction buffer pH value6.0MES, coupling time2.5h,and the antibody concentration800μg/mL) complex could be clearly observed by electronmicroscopy. Zeta potential results showed that the colloidal gold and latex microspheres werein a relatively stable state after they were coupled with antibody protein. Dynamic lightscattering showed that the hydrated particle size of immune colloidal gold graduallydecreased as the pH increased, and the hydrated particle size of immunolatex increased as theantibody protein concentration gradually increased.The secondary structure of antibody protein on nano-immunomicrospheres, prepared in different conditions, was detailed using FTIR combining with computer aided analysistechnology. Results showed that the content of ordered structure of antibody protein increasedalong with the increase of pH value, microspheres concentration and antibody proteinconcentration.The interaction mechanism between nanospheres and antibody protein was revealed byfluorescence spectrum technology. Results showed that colloidal gold and latex nanosphereshad a significant quenching effect on the intrinsic fluorescence of antibody protein. Thequenching mechanisms were all static quenching. However, the interaction force between thetwo kinds of nanospheres and antibody protein were different. The binding process ofcolloidal gold and antibody protein was a physical spontaneous adsorption process, in whichhydrophobic force and hydrogen bonds played a major role. The main interaction betweenlatex nanospheres and antibody protein was electrostatic interaction. These interaction forceschanged the tertiary structure of antibody protein to some extent, the hydrophobicity of themicroenvironment around the amino acids residues increased, and made a significant changein the hydrophobicity of antibody protein.The colloidal gold and latex immunochromatographic assay strips were prepared by thetwo kinds of nanospheres, and the relationship between the structure of antibody protein andsensitivity and specificity were detailed. Results showed that the structure of antibody proteincombined with nanospheres had a significant relationship with the sensitivity and specificityof the test strips, which were enhanced when the content of ordered structure of antibodyprotein increased.