Determination of Homocysteine in Blood by Fluorescence Spectrophotometry Based on Magnetic Molecularly Imprinted Material

A method for the determination of homocysteine (Hcy) in blood by fluorescence spectrophotometry based on magnetic molecularly imprinted material was proposed. Fe₃O₄ nano magnetic core was prepared by chemical coprecipitation method, and modified with tetraethyl orthosilicatete. Then Hcy nano magnetic molecularly imprinted material was prepared in ethanol solution using Hcy as template molecule, ethylene glycol dimethacrylate as crosslinking agent and azodiisobutyronitrile as initiator. Hcy in blood was adsorbed by the material, the derivative reagent N-(1-pyrene) maleimide was used for derivation after releasing Hcy, and the fluorescence intensity of the system was determined by fluorescence spectrophotometer. It was shown that the particle size of Hcy nano magnetic molecularly imprinted material was about 40 nm. The adsorption time of Hcy was 10 min, with the adsorption pH of 3.0, the maximum apparent adsorption capacity of 32.4 mg·g^-1, and the release time of Hcy was 40 min, with the release pH of 8.0. N-(1-pyrene) maleimide was added and reacted for 10 min. The stable fluorescence was generated at 387 nm with 365 nm as excitation wavelength. The material had good selective adsorption for Hcy and could be reused 4 times at most. Linear relationship between the fluorescence intensity and the mass concentration of Hcy was in the range of 0.02-0.10 mg·L^-1, with detection limit of 0.008 mg·L^-1. Tests for precision (n=5) and stability (n=6) were made on Hcy standard solution, and RSDs of the determined values were not more than 4.0%. This method was applied to analysis of the blood samples from 5 adult rabbits, and the results showed that Hcy was not detected. Test for recovery was made on the above blank samples, giving results in the range of 91.2%-109%.