Biotinylated Heparin Synthesis and Characterization

Biotinylated heparin was synthesized by adapting a previously reported procedure (Thakar et al., 2014 (link)). First, a solution containing heparin (4 mM, Sigma-Aldrich), 10 mM acetate buffer (made from glacial acetic acid (Carl Roth, Karlsruhe, Germany) and sodium acetate (Sigma-Aldrich) at pH 4.5) and aniline (100 mM, Sigma-Aldrich) was prepared. Biotin-PEG₃-oxyamine (3.4 mM, Conju-Probe, San Diego, United States) was added to the heparin solution and allowed to react for 48 h at 37°C. The final product was dialyzed against water for 48 h by using a dialysis membrane with a 3.5 kDa cutoff. The final solution was then lyophilized and stored at −20°C. For further use, the conjugates were diluted to the desired concentrations in buffer. The obtained biotinylated heparin was characterized by biotin-streptavidin binding assays using QCM-D. The average mass of the heparin isolate, when anchored to the surface, was estimated at 9 kDa (∼18 disaccharide units) by QCM-D analysis (Srimasorn et al., 2022 (link)).