Colloids and Surfaces B: Biointerfaces 112 (2013) 1–8 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Simultaneous depletion of immunoglobulin G and albumin from human plasma using novel monolithic cryogel columns Lokman Uzun a,∗ , Canan Armutcu a , Özlem Bic ¸ en b , Arzu Ersöz b , Rıdvan Say b , Adil Denizli a a Hacettepe University, Department of Chemistry, Beytepe, Ankara, Turkey b Anadolu University, Department of Chemistry, Eskis ¸ ehir, Turkey article info Article history: Received 24 January 2013 Received in revised form 1 July 2013 Accepted 3 July 2013 Available online 18 July 2013 Keywords: Simultaneous depletion Albumin IgG Cryogel Cibacron blue F3GA Protein A abstract In this study, we aimed to develop an alternative matrix able to deplete the albumin (Alb) and immunoglobulin G (IgG) from blood plasma simultaneously to prepare plasma samples for large-scale applications of blood-related proteomics. As a first step, nano-protein A nanoparticles (nanoProA) were prepared and characterized. Subsequently, cibacron blue F3GA (CB) was immobilized onto the nanoProA’s to enhance their specific affinity for Alb molecules. Finally, both nanoparticles, specifically, nanoProA and CB-nanoProA, were separately embedded into cryogel structures to combine advantages of the nanoparti- cles with those of the cryogels. The protein adsorption was optimized using aqueous Alb and IgG solutions separately. Subsequently, competitive protein adsorption was performed using a protein mixture pre- pared with Alb and IgG adhering to their plasma protein ratios. Because of the CB-immobilization, the Alb depletion performance of the cryogels increased whereas the IgG depleting performance decreased. Using the nanoProA, embedded cryogel removed 99.3% of the IgG, while using the CB-nanoProA embed- ded cryogel removed 97.5% of the Alb content. The simultaneous depletion performances of the cryogels for Alb and IgG were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the monolithic cryogel-based adsorbents were classified as an alternative matrix to prepare plasma samples for proteomics applications at the preparative scale. © 2013 Elsevier B.V. All rights reserved. 1. Introduction A cryogel is a type of hydrogel prepared at temperatures below the melting point of the solvent. The solid crystals of solvent gen- erate large and interconnected macropores, while polymerization occurs in the thawed solution [1–6]. Water and water-soluble monomers are frequently selected to prepare cryogels with excellent water-contact properties. The crystalline structure of water molecules also creates random pores and highly intercon- nected flow-channels, creating a super-macroporous, sponge-like polymeric structure [2,3]. In addition to the cryogels’ favorable structural properties, their adsorptive properties are characterized by short diffusion times, thin surface films, tolerance toward convective transport, and low back-pressure, positioning them as promising adsorbent materials for various chromatographic tasks, including environmental, biotechnology and biochromatography applications [1,7]. The most favorable properties of cryogels during this study are that they can interact with whole blood without ∗ Corresponding author at: Hacettepe University, Department of Chemistry, Bio- chemistry Division, 06381 Beytepe/Ankara, Turkey. Tel.: +90 312 297 7337; fax: +90 312 299 2163. E-mail addresses: lokman@hacettepe.edu.tr, lokmanuzun@gmail.com (L. Uzun). causing damage to the blood cells, and they do not require any sample pre-treatment steps [8]. In addition, cryogels might be classified as economically favorable adsorbents that may be used in a disposable manner to avoid cross-contamination between successive batches of sample [5,9]. After the Human Genome Project completed in 2002, pro- teomics became a fast growing research area, gaining prominence because it offered potential explanations concerning events within biological systems [10]. However, the development of this area has been limited by the technical challenges caused by the complex- ity of the samples that are not easily handled, in addition to the difficulties posed even by repetitively sampled biological samples, such as blood serum or plasma [11]. In theory, by comparing the proteomes of healthy and diseased plasma samples, it should be possible to recognize biomarkers as the proteomic signatures of the disease of interest and/or to monitor the progress of the disease and applied treatment [12]. Although the proteomic analysis of plasma samples is promising approach for biomarker identification, the wide range of pro- tein concentration, which varies from highly abundant proteins at mgmL -1 to non-abundant proteins at pgmL -1 for a 10 10 –10 12 fold, is still major obstacle for the analysis of the human plasma pro- teome [13]. As stated in many proteomics approaches, the highly abundant proteins, such as albumin and the immunoglobulins, 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.07.010