Preparation of poly(hydroxyethyl methacrylate) cryogels containing l-histidine for insulin recognition

In the present study, affinity adsorption technique was studied for insulin adsorption. Firstly, insulin-imprinted supermacroporous cryogel was prepared for the insulin adsorption. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the monomer. Insulin was complexed with MAH, and insulin-imprinted p(HEMA–MAH) [insulin-(MIP)] cryogel was prepared by free radical polymerization with 2-hydroxyethyl methacrylate (HEMA), N,N,N′,N′-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) in an ice bath. Then, insulin was removed from the cryogel by using 0.1 M glycine–HCl buffer (pH: 3.5). The characterization of the cryogel was carried out by using scanning electron microscopy (SEM) and swelling test. The equilibrium swelling ratios of the cryogels were found to be 8.56 ± 0.42 g H2O/g polymer for p(HEMA) and 7.20 ± 0.36 g H2O/g polymer for insulin-p(HEMA–MAH). Insulin adsorption experiments were performed under different conditions, such as flow rate, medium pH, initial insulin concentration and ionic strength. It was observed that insulin could be repeatedly adsorbed and desorbed with MIP cryogel without any significant decrease in the adsorption capacity.

Insulin-imprinted p(HEMA–MAH) supermacroporous cryogel has large continuous interconnected pores (50–100 μm in diameter) that provide channels for the mobile phase to flow through. The selectivity of insulin-MIP cryogel with respect to the competitive protein was applied by utilizing FPLC. Because the Rs value of competitive protein should be higher than 1.0 for a good resolution of two peaks in such a chromatography system, the results for the resolution of insulin/aprotinin can be accepted as good resolution values.Figure optionsDownload as PowerPoint slideHighlights
► Insulin imprinted p(HEMA–MAH) cryogel was produced by free radical polymerization.
► The insulin imprinted cryogel can be used for selective and efficient separation of insulin.
► The p(HEMA–MAH) cryogel can be used for insulin separation many times without any significant decrease in its capacity.