The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as...The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as formed through a simple reaction between amine immobilized surfaces and N-hydroxysucciimide groups at the end of biotin modifi ed PEG in anhydrous organic solutions（＂grafting to＂ technique）. The amount of the specifi cally adsorbed protein was measured as a function of spacer thickness between hard surface and biotin moieties. It has been shown that the amount of specifically adsorbed streptavidin decreases with the increase spacer thickness and the protein adsorbs onto the functionalized surfaces in a single molecular manner. It provides an interesting model system for studying single molecular interactions.展开更多
The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential land...The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential landscape, was evaluated by processing 22,500 force curves using two methods. One method is a conventional method, which is usually built in commercial AFM systems, i.e., difference between the baseline value and the minimum force value in the force curve. The other is a detection of rupture events based on a fuzzy logic algorithm to detect the rupture event from analyzing the shape of the force curves. Our statistical analysis revealed that the conventional method exhibited a significant artifact, which is the increase in the population of small forces comparable to thermal noise of cantilevers, resulting in a smaller MFOF. Based on this finding, we discuss the choice of a method and its effecton the illustrated potential landscapes of ligand-receptor complexes.展开更多
Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media.Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated...Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media.Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated with rabbit ami-Mycoplasma hyopneumoniae serum and goat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution (standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The standard calibration curve for biotin analysis was constructed in the range of 50-2000ng·L^-1. Results The detection limit for biotin was found to be 83 ng·L^-1 , which waa about 1000 times lower than the lowest determination concenlration in the reported ELISA for biotin analysis. The relative standard deviations for the spiked samples at biotin concerarations of 200 ng·L^-1, 500 ng·L^-1 , and 1000 ng·L^-1 were 24.87%, 6.15%, and 7.86%, respectively, with the average recovery of 101.13%. The wild yeast and its sixty-three transformed yeast culture media were applied to the developed ELISA for the determination of biotin. It was found that the biotin concentrations in more than 85 % of the tested samples were enhanced with different increase factors after transformation. Conclusion Utilization of Mycoplasma hyopnetunoniae as the coating protein improves the precision and accmacy oftbe ELISA assay, which might be used for the biotin assay in other media.展开更多
文摘The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as formed through a simple reaction between amine immobilized surfaces and N-hydroxysucciimide groups at the end of biotin modifi ed PEG in anhydrous organic solutions（＂grafting to＂ technique）. The amount of the specifi cally adsorbed protein was measured as a function of spacer thickness between hard surface and biotin moieties. It has been shown that the amount of specifically adsorbed streptavidin decreases with the increase spacer thickness and the protein adsorbs onto the functionalized surfaces in a single molecular manner. It provides an interesting model system for studying single molecular interactions.
文摘The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential landscape, was evaluated by processing 22,500 force curves using two methods. One method is a conventional method, which is usually built in commercial AFM systems, i.e., difference between the baseline value and the minimum force value in the force curve. The other is a detection of rupture events based on a fuzzy logic algorithm to detect the rupture event from analyzing the shape of the force curves. Our statistical analysis revealed that the conventional method exhibited a significant artifact, which is the increase in the population of small forces comparable to thermal noise of cantilevers, resulting in a smaller MFOF. Based on this finding, we discuss the choice of a method and its effecton the illustrated potential landscapes of ligand-receptor complexes.
文摘Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media.Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated with rabbit ami-Mycoplasma hyopneumoniae serum and goat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution (standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The standard calibration curve for biotin analysis was constructed in the range of 50-2000ng·L^-1. Results The detection limit for biotin was found to be 83 ng·L^-1 , which waa about 1000 times lower than the lowest determination concenlration in the reported ELISA for biotin analysis. The relative standard deviations for the spiked samples at biotin concerarations of 200 ng·L^-1, 500 ng·L^-1 , and 1000 ng·L^-1 were 24.87%, 6.15%, and 7.86%, respectively, with the average recovery of 101.13%. The wild yeast and its sixty-three transformed yeast culture media were applied to the developed ELISA for the determination of biotin. It was found that the biotin concentrations in more than 85 % of the tested samples were enhanced with different increase factors after transformation. Conclusion Utilization of Mycoplasma hyopnetunoniae as the coating protein improves the precision and accmacy oftbe ELISA assay, which might be used for the biotin assay in other media.
