Evaluating the Binding Capacity of Streptavidin Magnetic Particles Using HPLC: Implications for Biotechnology and Nanomedicine

• Date: 2025
• Main Authors: Sereda, O.V., Melnychuk, M.D., Yu, W., Spyrydonov, V.G., Середа, О.В., Мельничук, М.Д., Ю, В., Спиридонов, В.Г.
• Format: Article
• Language: English
• Published: PH "Akademperiodyka" of the NAS of Ukraine 2025
• Online Access: https://ojs.microbiolj.org.ua/index.php/mj/article/view/331
• Journal Title: Microbiological Journal

Streptavidin-functionalized magnetic particles (SAMP) are an indispensable tool in biotechnology and biomedical research due to their unique physicochemical properties and highly specific biochemical interactions. These particles enhance biomolecule analysis, immunoassays, diagnostics, and purification processes. Streptavidin, known for its high affinity for biotin, acts as an ideal biocompatible linker, facilitating precise biochemical manipulations. This study aimed to synthesize and characterize SAMP and evaluate their binding activity toward biotin using high-performance liquid chromatography (HPLC). Methods. Recombinant streptavidin was produced in E. coli, and magnetic particles with an iron oxide core and a silica shell were functionalized with amino and carboxyl groups for streptavidin conjugation. The synthesis involved activating carboxyl groups with N-hydroxysuccinimide (NHS) and N,N'-dicyclohexylcarbodiimide (DCC), followed by covalent coupling with streptavidin. Results. The resulting SAMP exhibited a hydrodynamic diameter of approximately 1 µm and a polydispersity index of around 26%, indicating a uniform size distribution. A previously undescribed direct method for evaluating streptavidin-biotin interactions by HPLC was used to determine the activity of SAMP. This method proved to be reliable and overcomes the limitations of indirect methods, such as the spectrophotometric assay using 4-hydroxyazobenzene-2′-carboxylic acid (HABA) dye. The binding capacity was estimated to be approximately 0.21 µg of free biotin per mg of particles. Comparative analysis showed that SAMP prepared with in-house streptavidin had higher activity (852 pmol/mg) compared to commercial streptavidin (683 pmol/mg). Conclusios. This research underscores the potential of SAMP for biomedical applications, including early diagnostics and personalized therapeutic approaches. The developed HPLC method offers a robust tool for assessing streptavidin-functionalized magnetic particle activity, advancing biotechnology and nanomedicine.