Sequential robust design methodology and X-ray photoelectron spectroscopy to analyze the grafting of hyaluronic acid to glass substrates.

Sequential Robust Design experiments and X-ray photoelectron spectroscopic (XPS) studies were performed to examine the immobilization of hyaluronic acid (HA) on glass substrates chemisorbed with N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS). Numerous reaction conditions were investigated, including the concentrations of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), and HA, and the reaction buffer type, concentration, and pH. The elemental surface compositions of carbon and silicon (C/Si ratio) were used to assess the extent of HA immobilization, leading to the identification of critical HA-binding reaction conditions and the determination of an optimum surface chemistry. The optimum chemistry consisted of 200 mM EDC, 50 mM Sulfo-NHS, 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer at a pH of 7.0, and 3 mg/mL HA. This work emphasizes the advantages of using Robust Design methods over traditional statistical experimental design, particularly when large numbers of variables are examined and costly analytical techniques are employed.