Identification of imprinted sites by fluorescence detection method based on reversible dynamic bond modified template protein

Abstract With the development of protein imprinting technique, the certification about the existence of imprinted sites, the arrival of template molecules and target molecules into the same cavity, and the specific separation process based on imprinted sites have seldom been studied so far. To promote the development in the field of protein imprinting, a method for proving the existence of the imprinting sites based on the fluorescence signal changes of the fluorescent materials labeling at the imprinted sites is first proposed in this work. Simultaneously, a novel protein imprinted material with ability of binding signal transduction is developed. The core of the imprinted materials is that bovine serum albumin (BSA) modified by a designed and synthesized functional monomer containing a polymerizable double bond and a cleavable dynamic disulfide bond is selected as a template. And the imprinted polymers supported on the surface of the tubular carbon nanofibers are constructed by thermally initiated polymerization. Fluorescein 4-(N, N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) is used to label the sulfhydryl groups generated by the cleavage of disulfide bonds at the imprinting sites. The identification of the imprinting sites is achieved by the change in the fluorescent signal generated when BSA is recombined to the imprinting sites. The results of fluorescence detection and adsorption experiments show that the imprinting sites exhibit specific binding and excellent reusability for BSA. In addition, another fluorescein N-(9-acridinyl) maleimide is utilized to label the imprinting sites, which produced a similar fluorescence signal response. Therefore, the experimental results are verified.