Biological sample analysis with immunoaffinity solid-phase microextraction

Abstract

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.