Biological sample analysis with immunoaffinity solid-phase microextraction.
A theophylline antiserum was covalently immobilized on the surface of a fused silica fiber, modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde, and used as a selective and sensitive extraction medium for the immunoaffinity solid-phase microextraction (SPME) determination of theophylline in serum samples. The specificity of the immunoaffinity SPME fiber was first investigated using a fixed concentration of [3H]theophylline together with various amounts of interference, possessing no cross-reactivity with the theophylline antibody. No significant non-specific binding was observed. The reproducibility of the fiber preparation and the immunoaffinity SPME analysis was also investigated, resulting in a relative standard deviation of 6.1% for five analyses of the same fiber. The antigen-antibody binding isotherm was obtained by analyzing theophylline standards of various concentrations (0.1-5 ng mL(-1)) until saturation values were reached. Initial binding of theophylline was linear with a r2 = 0.968. The cross-reactivity of the theophylline immunoaffinity SPME fiber for the structural analog caffeine was investigated by adding various amounts of caffeine in the presence of theophylline at a saturation concentration and produced a low cross-reactivity value of 0.1%. Finally. spiked serum samples (10 and 50 ng mL(-1)) were successfully analyzed with an excellent correlation with the standard binding isotherm, thus confirming the performance of the immunoaffinity SPME coating for improved bioanalysis.
Yuan
H
H
Department of Chemistry, University of Waterloo, Ontario, Canada.
Mullett
W M
WM
Pawliszyn
J
J
D000818:Animals; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D005976:Glutaral; D007106:Immune Sera; D008855:Microscopy, Electron, Scanning; D011817:Rabbits; D012822:Silicon Dioxide; D013806:Theophylline / Q000032:analysis*
D007106:Immune Sera; D012822:Silicon Dioxide; D013806:Theophylline; D005976:Glutaral