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Published on: January 16, 2015
Pseudo-homogeneous immunoextraction of epitestosterone from human urine samples based on gold-coated magnetic
Shuang Qiu1, Li Xu, Yi-Ran Cui
1Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Biochemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry, Peking University, Beijing, China.
Abstract:
A pseudo-homogeneous immunoextraction method based on gold-coated magnetic nanoparticles (MNPs) for the specific extraction and quantitative analysis of epitestosterone (17alpha-hydroxy-4-androsten-3-one, abbreviated as "ET") from human urine samples by high-performance liquid chromatography (HPLC) has been developed. Half-IgG of anti-ET monoclonal antibodies were covalently immobilized onto (Fe(3)O(4))(core)-Au(shell) (Fe(3)O(4)@Au) MNPs. An external magnetic field was applied to collect the MNPs which were then rinsed with distilled water followed by elution with absolute methanol to obtain ET as the analyte. The obtained extraction solution was analyzed by HPLC with UV detection (244nm) within 12min. The standard calibration curve for ET showed good linearity in the range of 20-200ngmL(-1) in phosphate-buffered saline (PBS) solutions with acceptable accuracy and precision. Limit of detection for ET was 0.06ngmL(-1) due to an enrichment factor of 100-fold was achieved. The results obtained by the present method for spiked human urine samples were in agreement with those from indirect competitive enzyme-linked immunoadsorbent assays (ELISAs). The antibody-conjugated Fe(3)O(4)@Au MNPs are novel materials for immunoaffinity extraction. Compared with the conventional technique using immunoaffinity column, the method described here for sample pretreatment was fast, highly specific, and easy to operate.
