l-histidine functionalized multi-walled carbon nanotubes for on-line affinity separation and purification of immunoglobulin G in serum

In this work, the multi-walled carbon nanotubes were covalently functionalized with l-histidine (His–MWNTs) as online pseudospecific affinity adsorbent for immunoglobulin G (IgG) separation and purification with a simple surface modification method, using 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimde (NHS). The affinity of the His–MWNTs toward IgG was investigated in a microcolumn incorporated into a sequential injection system, which also involves an UV spectrometer with a flow cell for online real-time detection. The incorporation of histidine as affinity groups noticeably increased the selectivity and binding capacity of MWNTs for IgG and the His–MWNTs exhibited high retention and recovery rate of nearly 100% under optimized conditions. This separation and enrichment process made it possible to determine a lower concentration range of IgG in serum from 1.0–33 μg/mL with a detection limit of 0.3 μg/mL with a sampling volume of 4.0 mL. The static and dynamic adsorption capacities obtained were 267 mg of IgG/g His–MWNTs and 35 mg/g in aqueous solution, respectively, which are among the highest reported results in literatures employing affinity separation methods. Desorption of IgG from His–MWNTs could be accomplished by lowering the pH to 1.5 with glycine–HCl buffer. The practical application of His–MWNTs for separation of IgG in serum was evaluated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis which confirmed that the purity of recovered IgG from human serum was over 85% and better than a commercial product.