Surface interaction of well‐defined,concentrated poly(2‐hydroxyethyl methacrylate) brushes with proteins

The interaction of concentrated polymer brushes with proteins was chromatographically investigated. By the use of surface‐initiated atom transfer radical polymerization, a low‐polydispersity poly(2‐hydroxyethyl methacrylate) (PHEMA) was densely grafted onto the inner surfaces of silica monoliths with mesopores of about 50 and 80 nm in mean size. The graft density reached 0.4–0.5 chains/nm². The 80‐nm‐mesopore monolithic column with the concentrated PHEMA brush was characterized through the elution of low‐polydispersity pullulans with different molecular weights, clearly showing two modes of size exclusion, that is, one by the mesopores and the other by the brush phase. The latter mode gave a sharp separation with a critical molecular weight (size‐exclusion limit) of about 1000. This molecular size of pullulan was comparable to the distance between the nearest‐neighbor graft points. The elution behaviors of five proteins of different sizes (bovine serum thyroglobulin, bovine serum immunoglobulin G, bovine serum albumin, horse heart myoglobin, and bovine serum aprotinin) were studied with this PHEMA‐grafted column. The smallest protein, aprotinin, with a pullulan‐reduced molecular weight slightly larger than the critical value of 1000, was eluted much behind the corresponding pullulan, and this indicated that it barely got into the brush layer, suffering from a strong affinity interaction within the brush. On the other hand, the other four larger proteins were eluted at the same elution volumes as the equivalent pullulans, and this meant that they were perfectly excluded from the brush layer and separated only in the size‐exclusion mode by the mesopores without an affinity interaction with the brush surface. This excellent inertness of the concentrated brush in the interaction with the large proteins should afford the system long‐term stability against biofouling. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4795–4803, 2007     

Ferritin immobilization on patterned poly(2-hydroxyethyl methacrylate) brushes on silicon surfaces from colloid system

In this paper, we describe a graft polymerization/solvent immersion method for generating poly(2-hydroxyethyl methacrylate) (PHEMA) brushes in various patterns. We used a novel fabrication process, involving very-large-scale integration and oxygen plasma treatment, to generate well-defined patterns of polymerized PHEMA on patterned Si(100) surfaces. We observed brush- and mushroom-like regions for the PHEMA brushes, with various pattern resolutions, after immersing wafers presenting lines of these polymers in MeOH and n-hexane, respectively. The interaction between PHEMA and ferritin protein sheaths in MeOH and n-hexane (good and poor solvent for PHEMA, respectively) was used to capture and release ferritins from fluidic system. The “tentacles” behaver for PHEMA brushes was found through various solvents in fluidic system. Using high-resolution scanning electron microscopy, we observed patterned ferritin Fe cores on the Si surface after pyrolysis of the patterned PHEMA brushes and ferritin protein sheaths, which verify the “tentacles” behaver for PHEMA brushes.     

Raft polymerization of N,N‐dimethylacrylamide from magnetic poly(2‐hydroxyethyl methacrylate) microspheres to suppress nonspecific protein adsorption

Nonspecific interaction is a key parameter affecting the efficiency of proteins, nucleic acids or cell separation. Currently, many approaches to introduce antifouling properties to materials have been developed. Among these, surface modification with polymer brushes plays a prominent role. The aim of this study was to synthesize new magnetic microspheres grafted with poly(N,N‐dimethylacrylamide) (PDMA) that resist nonspecific protein adsorption. Monodisperse macroporous poly(2‐hydroxyethyl methacrylate) (PHEMA) microspheres, 4 μm in size, were synthesized by a multiple swelling polymerization method. To render the microspheres magnetic, iron oxide was precipitated inside the microsphere pores. Functional carboxyl groups, introduced by the hydrolysis of the 2‐(methacryloyl)oxyethyl acetate (HEMA‐Ac) comonomer, were used to react with propargylamine, followed by coupling of a chain transfer agent via an azide‐alkyne click reaction. PDMA was grafted from the PHEMA microspheres using reversible addition‐fragmentation chain transfer polymerization (RAFT), resulting in surfaces with more than 81 wt % PDMA attached. The successful modification of the microspheres was confirmed by XPS. The magnetic microspheres grafted with PDMA showed excellent antifouling properties as tested in bovine serum protein solutions. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1036–1043     

Single component and selective competitive protein adsorption in a patchy polymer brush: opposition between steric repulsions and electrostatic attractions

This work explores the use of "patchy" polymer brushes to control protein adsorption rates on engineered surfaces and to bind targeted species from protein mixtures with high selectivity but without invoking molecular recognition. The brushes of interest contain embedded cationic "patches" composed of isolated adsorbed poly(l-lysine) coils (PLL) that are about 10 nm in diameter and are randomly arranged on a silica substrate. Around these patches is a protein-resistant poly(ethylene glycol) (PEG) brush that is formed from the adsorption of a PLL-g-PEG graft copolymer on the remaining silica surface. Electrostatic attractions between individual cationic patches and the negative regions of approaching proteins may be energetically insufficient to bind proteins. Furthermore, protein-patch attractions are reduced by steric repulsions between proteins and the PEG brush. We show that protein adsorption, gauged by ultimate short-term coverages and by the initial protein adsorption rate, exhibits an adhesion threshold: pure PEG brushes of the architectures employed here and brushes containing sparse loadings of PLL patches do not adsorb protein. Above a critical PLL patch loading or threshold, protein adsorption proceeds, often dramatically. The PLL patch thresholds are specific to the protein of interest, allowing surfaces to be engineered to adhesively discriminate different proteins within a mixture. The separation achieved is remarkably sharp: one protein adsorbs, but the second is completely rejected from the interface. The surfaces in this study, by virtue of their well-controlled and well-characterized patchy nature, distinguish themselves from multicomponent brushes or brushes used to end-tether peptide sequences and nucleotides.     

Fabrication of high-aspect-ratio poly(2-hydroxyethyl methacrylate) brushes patterned on silica surfaces by very-large-scale integration process

We used a novel fabricated process including electron beam and isotropic oxygen plasma to generate signal line patterns of polymerized 2-hydroxyethyl methacrylate (HEMA) on patterned Si(1 0 0) surfaces. Isotropic oxygen plasma was introduced to enhance the resolutions of the line and dots patterns of the PHEMA brush are approached to 350 nm and 2 μm, respectively. We established the surface grafting polymerization kinetics of the PHEMA chains on silicon surface by to fit the thickness and number-average molecular weight (M(n)). The propagation rate (k(p)) and active grafting specie deactivation rate (k(d)) lies in the range of ~3.6 × 10(-2) s(-1) M(-1) and 4.8 × 10(-5) s(-1), respectively. The measured thicknesses by ellipsometer and analyzed M(n) of "free" PHEMA by gel permeation chromatography (GPC) are fitted well by the polymerization kinetic model. In addition, aspect-ratios (height/width) are used to define the shape of patterned PHEMA brushes. The high-aspect-ratio of the PHEMA brush line with width of 350 nm is 0.27. We use a graft polymerization/solvent immersion method for generating various patterns of polymer brushes to investigate the deformation of the PHEMA brush through aspect-ratios.     

Water Content of Hydrated Polymer Brushes Measured by an In Situ Combination of a Quartz Crystal Microbalance with Dissipation Monitoring and Spectroscopic Ellipsometry

Poly(methacryloyloxy ethyltrimethylammonium chloride) (PMETAC), poly(sulfopropylmethacrylate potassium salt), or poly(N‐isopropyl acrylamide) (PNIPAM) brushes are synthesized by means of the atom transfer radical polymerization technique from gold surfaces coated with a monolayer of the initiator ω‐mercaptoundecyl bromo isobutyrate. The brush growth is followed in situ and in real time by the combination of quartz crystal microbalance with dissipation technique (QCM‐D) and spectroscopic ellipsometry in a single device. The combination of QCM‐D and ellipsometry allows for the simultaneous determination of both the acoustic mass, m_acous, comprising the mass of the polymer and the solvent, and the optical mass, m_opt, which corresponds to the polymer mass alone. Brush hydration is calculated from the difference between the values obtained for m_acous and m_opt for each polymer synthesized. Brush hydration is then used to quantify the percentage of water released in the brush during collapse; a 30–40% release of water for PMETAC and PSPM brushes in 1 M NaCl and 80% for PNIPAM brushes when the temperature is increased to values above the lower critical solution temperature is observed.     

Completely aqueous procedure for the growth of polymer brushes on polymeric substrates

The growth of polymer brushes on polymer substrates is often challenging because of substrate incompatibility with the organic solvents used for initiator attachment. This letter reports the use of layer-by-layer adsorption of macroinitiators and subsequent aqueous ATRP from these immobilized initiators to prepare polymer brushes on polymeric substrates. Polyethersulfone (PES) films and porous membranes were modified with polyelectrolyte multilayer films, and a previously developed polycationic initiator, poly(2-(trimethylammonium iodide)ethyl methacrylate-co-2-(2-bromoisobutyryloxy)ethyl acrylate), was then electrostatically adsorbed onto these polyelectrolyte films. The immobilized macroinitiator is very efficient in initiating the growth of polymer brushes on PES, as demonstrated by aqueous syntheses of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) films. PHEMA (250 nm thick) and PDMAEMA (40 nm thick) brushes were grown in 2 h from surfaces modified with polycationic initiators. Moreover, this procedure is effective for growing brushes in the pores of PES membranes.     

Complex pH- and temperature-sensitive swelling behavior of mixed polymer brushes

The ability of a mixed polymer brush consisting of poly(N-isopropyl acrylamide) (PNIPAAM) and poly(acrylic acid) (PAA) to modify physicochemical interfacial properties is presented. The answer of the binary brush toward changes of environmental conditions like temperature, salt concentration, and pH value was investigated by in situ spectroscopic VIS-ellipsometry as well as AFM and contact angle mesurements in the dry state and compared with the behavior of the corresponding homopolymer brushes. A coupled swelling of PNIPAAm and PAA could be found, leading to a complex pH-, salt-, and temperature-sensitive swelling behavior of these mixed brushes, also depending on the composition of the brush. The complex interaction of the two polymers resulted in new properties of the mixed system. Although the temperature sensitivity of the mixed system was decreased compared with that of the corresponding PNIPAAM brushes, the sensitivity toward pH and salt concentration was amplified compared with that of pure PAA brushes. Additionally it is shown that in spite of the decreased temperature sensitivity of the mixed brush, a temperature-dependent adsorption of human serum albumin was observed whereas an increased adsorption affinity was found that is not predictable from the adsorption affinity of the corresponding homopolymer brushes. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1606–1615, 2010     

Electrostatic repulsion between two parallel plates covered with polymer brush layers

An expression for the electrostatic repulsive force is obtained for two parallel similar plates immersed in an electrolyte solution at separation h covered with a uniformly charged polymer brush layer of intact thickness d _o under compression (h<2d _o) after the two brushes come into contact. It is assumed that when the two brushes come into contact, they are squeezed against each other but do not interdigitate. The electrostatic repulsive force is found to increase with decreasing h as 1/h for highly charged brushes and as 1/h ² for weakly charged brushes. This is in contrast to the interaction force between the brush layers before contact (h≥2d _o), which is essentially proportional to exp[−κ(h−2d _o)] (where κ is the Debye–Hückel parameter). It is also shown that the interaction force for highly charged brushes, which becomes independent of the electrolyte concentration, can be comparable in magnitude to the steric repulsive forces between the brushes resulting from osmotic repulsion and the elastic energy of the brushes. Received: 21 October 1998 Accepted in revised form: 25 December 1998     

Primary versus ternary adsorption of proteins onto PEG brushes

Polyethylene glycol (PEG) brushes are used to reduce protein adsorption at surfaces. Their design needs to allow for two leading adsorption modes at the brush-coated surface. One is primary adsorption at the surface itself. The second is ternary adsorption within the brush as a result of weak PEG-protein attraction. We present a scaling theory of the equilibrium adsorption isotherms allowing for concurrent primary and ternary adsorption. The analysis concerns the weak adsorption limit when individual PEG chains do not bind proteins. It also addresses two issues of special relevance to brushes of short PEGs: the consequences of large proteins at the surface protruding out of a shallow brush and the possibility of marginal solvent conditions leading to mean-field behavior. The simple expressions for the adsorption isotherms are in semiquantitative agreement with experiments.     

Sensitivity of protein adsorption to architectural variations in a protein-resistant polymer brush containing engineered nanoscale adhesive sites

Patchy polymer brushes contain nanoscale (5-15 nm) adhesive elements, such as polymer coils or nanoparticles, embedded at their base at random positions on the surface. The competition between the brush's steric (protein resistant) repulsions and the attractions from the discrete adhesive elements provides a precise means to control bioadhesion. This differs from the classical approach, where functionality is placed on the brush's periphery. The current study demonstrates the impact of poly(etheylene glycol) (PEG) brush architecture and ionic strength on fibrinogen adsorption on brushes containing embedded poly-l-lysine (PLL, 20K MW) coils or "patches". The consistent appearance of a fibrinogen adsorption threshold, a minimum loading of patches on the surface, below which protein adsorption does not occur, suggests multivalent protein capture: Adsorbing proteins simultaneously engage several patches. The surface composition (patch loading) at the threshold is extremely sensitive to the brush height and ionic strength, varying up to a factor of 5 in the surface loading of the PLL patches (~50% of the range of possible surfaces). Variations in ionic strength have a similar effect, with the smallest thresholds seen for the largest Debye lengths. While trends with brush height were the clearest and most dominant, consideration of the PEG loading within the brush or its persistence length did not reveal a critical brush parameter for the onset of adsorption. The lack of straightforward correlation on brush physics was likely a result of multivalent binding, (producing an additional dependence on patch loading), and might be resolved for univalent adsorption onto more strongly binding patches. While studies with similar brushes placed uniformly on a surface revealed that the PEG loading within the brush is the best indicator of protein resistance, the current results suggest that brush height is more important for patchy brushes. Likely the interactions producing brush extension normal to the interface act similarly to drive lateral tether extension to obstruct patches.     

Thermo-responsive polymer brush-grafted porous polystyrene beads for all-aqueous chromatography

Poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads with variable grafted polymer densities were prepared using surface-initiated atom transfer radical polymerization (ATRP) for applications in thermo-responsive chromatography. Utilization of these grafted beads as a stationary phase in aqueous chromatographic analysis of insulin provides a graft density-dependent analyte retention behavior. The separations calibration curve on PIPAAm-grafted polystyrene was obtained using pullulan standards and exhibited inflection points attributed to analyte diffusion into bead pores and partitioning into grafted PIPAAm brush surfaces. Presence of these inflection points supports a separation mechanism where insulin penetrates pores in polystyrene beads and hydrophobically interacts with PIPAAm brushes grafted within the pores. Control of PIPAAm brush graft density on polystyrene facilitates effective aqueous phase separation of peptides based on thermally modulated hydrophobic interactions with grafted PIPAAm within stationary phase pores. These results indicated that PIPAAm brush-grafted porous polystyrene beads prepared by surface-initiated ATRP was effective stationary phase of thermo-responsive chromatography for aqueous phase peptide separations.     

High capacity, charge-selective protein uptake by polyelectrolyte brushes

Surface plasmon resonance was used to measure binding of proteins from solution to poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes end-grafted from gold surfaces by atom transfer radical polymerization (ATRP). PDMAEMA brushes were prepared with a variety of grafting densities and degrees of polymerization. These brushes displayed charge selective protein uptake. The extent of uptake for net negatively charged bovine serum albumin (BSA) scaled linearly with the surface mass concentration of grafted PDMAEMA, regardless of grafting density. BSA was bound at a constant ratio of 120 DMAEMA monomer units per protein molecule for all brushes examined. The equivalent three-dimensional concentration of BSA bound in the brush (i.e., the bound BSA surface excess concentration divided by the brush thickness) decreased monotonically with decreasing grafting density. The concentration of BSA bound within brushes prepared at higher grafting densities was comparable with the aqueous protein solubility limit. BSA desorption from the brush required changes in solution pH and/or ionic strength to eliminate its net electrostatic attraction to PDMAEMA. Net positively charged lysozyme was completely rejected by the PDMAEMA brushes.     

Protein adsorption on novel acrylamido-based polymeric ion-exchangers. I. Morphology and equilibrium adsorption

The protein uptake equilibrium and particle morphology are determined for novel polymeric ion-exchange media based on acrylamido monomers with a high density of functional groups and a variety of morphological characteristics. The study considers two anion-exchangers and a cation-exchanger. Physical properties determined experimentally include particle density, ion-exchange capacity, particle size distribution, and equilibrium isotherms for model proteins. The pore structure was evaluated using size exclusion chromatography with neutral probe molecules and transmission electron microscopy. For the anion-exchangers, two types of structures were inferred. The first is comprised of particles that contain a low-density gel supported by denser polymer aggregates. This material had a very low size-exclusion limit for neutral probes, but exhibited an extremely high and reversible protein adsorption capacity (280-290 mg BSA/ml). The second structure is comprised of particles with large, open macropores. While the size-exclusion limit was very high, the protein adsorption capacity was low (60 mg BSA/ml). Moreover, the adsorption was nearly irreversible. The physical structure of the cation-exchanger appeared to be intermediate between those of the anion-exchangers, containing both large pores and smaller pores yielding an intermediate, but reversible, protein uptake capacity (120-130 mg alphaCHY/ml). The different behavior of these materials with regards to protein adsorption correlates well with their physical structure. For these ion-exchangers, high protein adsorption capacities are attained when a low-density polymer gel with a high concentration of functional groups is present.     

In-situ investigation of the adsorption of globular model proteins on stimuli-responsive binary polyelectrolyte brushes

Binary brushes constituted from two incompatible polymers can be used in the form of ultrathin polymeric layers as a versatile tool for surface engineering to tune physicochemical surface characteristics such as wettability, surface charge, chemical composition, and morphology and furthermore to create responsive surface properties. Mixed brushes of oppositely charged weak polyelectrolytes represent a special case of responding surfaces that are sensitive to changes in the pH value of the aqueous environment and therefore represent interesting tools for biosurface engineering. The polyelectrolyte brushes used for this study were composed of two oppositely charged polyelelctrolytes poly(2-vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). The in-situ properties and surface characteristics such as as surface charge, surface tension, and extent of swelling of these brush layers are functions of the pH value of the surrounding aqueous solution. To test the behavior of the mixed polylelctrolyte brushes in contact with biosystems, protein adsorption experiments with globular model proteins were performed at different pH values and salt concentrations (confinement of counterions) of the buffer solutions. The influence of the pH value, buffer salt concentration, and isoelectric points (IEP) of the brush and protein on the adsorbed amount and the interfacial tension during protein adsorption as well as the protein adsorption mechanism postulated in reference to recently developed theories of protein adsorption on polyelectrolyte brushes is discussed. In the salted regime, protein adsorption was found to be similar to the often-described adsorption at hydrophobic surfaces. However, in the osmotic regime the balance of electrostatic repulsion and a strong entropic driving force, "counterion release", was found to be the main influence on protein adsorption.     

Preparation of thermo-responsive polymer brushes on hydrophilic polymeric beads by surface-initiated atom transfer radical polymerization for a highly resolutive separation of peptides

Poly(N-isopropylacrylamide-co-N-tert-butylacrylamide) [P(IPAAm-co-tBAAm)] brushes were prepared on poly(hydroxy methacrylate) (PHMA) [hydrolyzed poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)] beads having large pores by surface-initiated atom transfer radical polymerization (ATRP) and applied to the stationary phases of thermo-responsive chromatography. Optimized amount of copolymer brushes grafted PHMA beads were able to separate peptides and proteins with narrow peaks and a high resolution. The beads were found to have a specific surface area of 43.0 m²/g by nitrogen gas adsorption method. Copolymer brush of P(IPAAm-co-tBAAm) grafted PHMA beads improved the stationary phase of thermo-responsive chromatography for the all-aqueous separation of peptides and proteins.     

Tuning the pH sensitivity of poly(methacrylic acid) brushes

The pH-induced swelling and collapse of surface-tethered, weak polyelectrolyte brushes is of interest for the development of actuators or to allow pH controlled transport or adsorption. This contribution discusses results of an extensive series of quartz crystal microbalance (QCM) experiments that aimed at (i) further understanding the influence of brush thickness and density on the pH responsiveness of poly(methacrylic acid) (PMAA) brushes and (ii) developing strategies that allow one to engineer the pH responsiveness and dynamic response range of PMAA based brushes. It was observed that, due to their high grafting density, the apparent pK(a) of surface-tethered PMAA differs from that of the corresponding free polymer in solution and also covers a broader pH range. The pK(a) of the PMAA brushes was found to depend on both brush thickness and density; thicker brushes showed a higher pK(a) value, and brushes of higher density started to swell at higher pH. The second part of the paper demonstrates the feasibility of the N-hydroxysuccinimide-mediated post-polymerization modification to engineer the pH responsiveness of the PMAA brushes. By using appropriate amine functionalized acids, it was possible to tune both the pH of maximum response as well as the dynamic response range of these PMAA based polyelectrolyte brushes.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Comprehensive study of the macropore and mesopore size distributions in polymer monoliths using complementary physical characterization techniques and liquid chromatography

Poly(styrene‐co‐divinylbenzene) monolithic stationary phases with two different domain sizes were synthesized by a thermally initiated free‐radical copolymerization in capillary columns. The morphology was investigated at the meso‐ and macroscopic level using complementary physical characterization techniques aiming at better understanding the effect of column structure on separation performance. Varying the porogenic solvent ratio yielded materials with a mode pore size of 200 nm and 1.5 μm, respectively. Subsequently, nano‐liquid chromatography experiments were performed on 200 μm id × 200 mm columns using unretained markers, linking structure inhomogeneity to eddy dispersion. Although small‐domain‐size monoliths feature a relatively narrow macropore‐size distribution, their homogeneity is compromised by the presence of a small number of large macropores, which induces a significant eddy‐dispersion contribution to band broadening. The small‐domain size monolith also has a relatively steep mass‐transfer term, compared to a monolith containing larger globules and macropores. Structural inhomogeneity was also studied at the mesoscopic level using gas‐adsorption techniques combined with the non‐local‐density‐function‐theory. This model allows to accurately determine the mesopore properties in the dry state. The styrene‐based monolith with small domain size has a distinctive trimodal mesopore distribution with pores of 5, 15, and 25 nm, whereas the monolith with larger feature sizes only contains mesopores around 5 nm in size.     

A novel adsorbent for protein chromatography: supermacroporous monolithic cryogel embedded with Cu2+-attached sporopollenin particles

The aim of this study is to prepare supermacroporous cryogels embedded with Cu(2+)-attached sporopollenin particles (Cu(2+)-ASP) having large surface area for high protein adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Cu(2+)-ASP was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for affinity purification of human serum albumin (HSA). Firstly, Cu(2+) ions were attached to sporopollenin particles (SP), then the supermacroporous PHEMA cryogel with embedded Cu(2+)-ASP was produced by free radical polymerization using N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Embedded particles (10 mg) in PHEMA-based cryogel column were used in the adsorption/desorption of HSA from aqueous solutions. Optimum conditions of adsorption experiments were performed at pH 8.0 phosphate buffer, with flow rate of 0.5 mL/min, and at 5°C. The maximum amount of HSA adsorption from aqueous solution was very high (677.4 mg/g SP) with initial concentration 6 mg/mL. It was observed that HSA could be repeatedly adsorbed and desorbed to the embedded Cu(2+)-ASP in PHEMA cryogel without significant loss of adsorption capacity.