Recent Advances in Optical Sensing for the Detection of Microbial Contaminants.

Microbial contaminants are responsible for several infectious diseases, and they have been introduced as important potential food- and water-borne risk factors. They become a global burden due to their health and safety threats. In addition, their tendency to undergo mutations that result in antimicrobial resistance makes them difficult to treat. In this respect, rapid and reliable detection of microbial contaminants carries great significance, and this research area is explored as a rich subject within a dynamic state. Optical sensing serving as analytical devices enables simple usage, low-cost, rapid, and sensitive detection with the advantage of their miniaturization. From the point of view of microbial contaminants, on-site detection plays a crucial role, and portable, easy-applicable, and effective point-of-care (POC) devices offer high specificity and sensitivity. They serve as advanced on-site detection tools and are pioneers in next-generation sensing platforms. In this review, recent trends and advances in optical sensing to detect microbial contaminants were mainly discussed. The most innovative and popular optical sensing approaches were highlighted, and different optical sensing methodologies were explained by emphasizing their advantages and limitations. Consequently, the challenges and future perspectives were considered.

Preparation of Molecularly Imprinted Poly(N-Isopropylacrylamide) Thermosensitive Based Cryogels.

Cryogels are defined as polymeric gel matrices with interconnected macropores providing an advantage to be efficient carriers enabling unhindered diffusion of interested molecule. Cryogels could be easily prepared with the combination of molecular imprinting. Molecular imprinting technology provides selective and sensitive recognition for biomolecules. Immunoglobulin G (IgG) is a main effector component in human response. It is recently well recognized that the purification strategies for IgG have intensively gained attention to treat immune defects. Several methods including affinity chromatography have been applied for the purification of IgG from complex media. The purification of IgG plays a crucial role in medical applications using several materials. Among these, cryogels have been widely applied for the purification of several biomolecules. They offer to create low-cost affinity systems with high chemical and physical stability. Above all, temperature sensitive polymers enable a reversible phase transition against small temperature changes, by the way, reversible swelling and shrinking manner is observed.In this chapter, immunoglobulin G imprinted thermosensitive poly(N-isopropylacrylamide-N methacryloyl-(l)-histidine) [p(NIPA-MAH)/IgG-MIP] monolithic cryogel is explained. The preparation and characterization of cryogels are summarized. In addition, IgG binding studies with different parameters are briefly described. Herein, an effective design principle is presented to create imprinted temperature-sensitive cryogels for IgG purification. A p(NIPA-MAH)/IgG-MIP monolithic cryogel was synthesized for IgG purification. Afterward, IgG binding capacity of p(NIPA-MAH)/IgG-MIP cryogels was examined in different experimental conditions. Apart from these, selectivity of the p(NIPA-MAH)/IgG-MIP cryogel was shown by comparing IgG binding capacity of nonimprinted [p(NIPA-MAH)/NIP] one. Finally, the IgG purification ability of the p(NIPA-MAH)/IgG-MIP cryogel from human plasma was demonstrated proving its application in affinity chromatography using real sample.

Laccase bound to cryogel functionalized with phenylalanine for the decolorization of textile dyes.

In this study, amino acid functionalized poly(2-hydroxyethyl methacrylate-N-methacrylolyl-l-phenylalanine) [PHEMAPA] cryogel discs were prepared. In this respect, phenylalanine containing N-methacryloyl-(L)-phenylalanine methyl ester (MAPA) was polymerized with 2-hydroxyethyl methacrylate (HEMA) without requirement of any activation step. Laccase bound poly(2-hydroxyethyl methacrylate-N-methacryloyl-l-phenylalanine) [Lac-PHEMAPA] cryogel discs were applied for decolorization of Reactive Blue-247 (RB-247). The ability of Lac-PHEMAPA cryogel discs on dye decolorization was found to be as 90% in 2 h and even more within 4h. The decolorization activities of 86% and 73% were observed at relatively low (4°C) and high (60°C) temperatures, respectively. The effect of dye concentration on dye decolorization and 100% decolorization activity was achieved in dye concentration between 50-300 ppm. Lac-PHEMAPA cryogel discs maintained 80% of its decolorization activity after six cycles. Consequently, the PHEMAPA cryogel discs are promising materials for immobilizing laccase. The Lac-PHEMAPA has a rapid dye decolorization in a broad range of temperature. The preparation is furthermore very stable and activity is preserved during storage.

Whole Cell Recognition of Staphylococcus aureus Using Biomimetic SPR Sensors.

Over the past few decades, a significant increase in multi-drug-resistant pathogenic microorganisms has been of great concern and directed the research subject to the challenges that the distribution of resistance genes represent. Globally, high levels of multi-drug resistance represent a significant health threat and there is a growing requirement of rapid, accurate, real-time detection which plays a key role in tracking of measures for the infections caused by these bacterial strains. It is also important to reduce transfer of resistance genes to new organisms. The, World Health Organization has informed that millions of deaths have been reported each year recently. To detect the resistant organisms traditional detection approaches face limitations, therefore, newly developed technologies are needed that are suitable to be used in large-scale applications. In the present study, the aim was to design a surface plasmon resonance (SPR) sensor with micro-contact imprinted sensor chips for the detection of Staphylococcus aureus. Whole cell imprinting was performed by N-methacryloyl-L-histidine methyl ester (MAH) under UV polymerization. Sensing experiments were done within a concentration range of 1.0 × 102-2.0 × 105 CFU/mL. The recognition of S. aureus was accomplished by the involvement of microcontact imprinting and optical sensor technology with a detection limit of 1.5 × 103 CFU/mL. Selectivity of the generated sensor was evaluated through injections of competing bacterial strains. The responses for the different strains were compared to that of S. aureus. Besides, real experiments were performed with milk samples spiked with S. aureus and it was demonstrated that the prepared sensor platform was applicable for real samples.

Evaluation of hyaluronic acid nanoparticle embedded chitosan-gelatin hydrogels for antibiotic release.

The development of chitosan-gelatin (CS-G) hydrogels embedded with ampicillin-loaded hyaluronic acid nanoparticles (HA-NPs) for wound dressing is proposed. It was aimed to provide controlled ampicillin delivery by incorporation of HA-NPs into biocompatible CS-G hydrogel structure. According to in vitro ampicillin release studies, 55% of ampicillin was released from CS-G/HA-NPs hydrogels after 5 days. Antibacterial performance of CS-G/HA-NPs hydrogels was proven with agar disc diffusion test. For cytotoxicity assay, fibroblast cell viability increased in CS-G/HA-NPs hydrogels compared with CS-G group after 24 hr incubation. Consequently, the potential ability of CS-G/HA-NPs hydrogels as a controlled drug delivery system has been verified.

RNA purification from Escherichia coli cells using boronated nanoparticles.

Boronate affinity chromatography is a common purification method used for isolation and purification of cis-diol containing biomolecules. Poly (hydroxyethyl methacrylate-co-vinyl phenyl boronic acid) [P(HEMA-VPBA)] nanoparticles were prepared by miniemulsion polymerization to use in RNA purification methods. The P(HEMA-VPBA) nanoparticles were characterized by particle size distribution, surface area, Fourier transform infrared spectroscopy and atomic force microscopy. The effects of temperature, pH, RNA concentration and different salt types on RNA binding on the P(HEMA-VPBA) nanoparticles were examined. It was observed that RNA binding was increased with the increasing of pH and max RNA binding was obtained at pH 9.0. RNA binding capacity of the P(HEMA-VPBA) nanoparticles was increased from 167mg/g to 601mg/g with addition of BaCl2 to the binding medium. Maximum RNA binding capacity of the P(HEMA-VPBA) nanoparticles was 172mg/g at 1.0mg/mL initial RNA concentration. The P(HEMA-VPBA) nanoparticles were reusable for RNA binding. RNA was also extracted from Escherichia coli cells and purified successfully using the P(HEMA-VPBA) nanoparticles.

Microcontact Imprinted Plasmonic Nanosensors: Powerful Tools in the Detection of Salmonella paratyphi.

Identification of pathogenic microorganisms by traditional methods is slow and cumbersome. Therefore, the focus today is on developing new and quicker analytical methods. In this study, a Surface Plasmon Resonance (SPR) sensor with a microcontact imprinted sensor chip was developed for detecting Salmonella paratyphi. For this purpose, the stamps of the target microorganism were prepared and then, microcontact S. paratyphi-imprinted SPR chips were prepared with the functional monomer N-methacryloyl-L-histidine methyl ester (MAH). Characterization studies of the SPR chips were carried out with ellipsometry and scanning electron microscopy (SEM). The real-time Salmonella paratyphi detection was performed within the range of 2.5 × 106-15 × 106 CFU/mL. Selectivity of the prepared sensors was examined by using competing bacterial strains such as Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The imprinting efficiency of the prepared sensor system was determined by evaluating the responses of the SPR chips prepared with both molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs). Real sample experiments were performed with apple juice. The recognition of Salmonella paratyphi was achieved using these SPR sensor with a detection limit of 1.4 × 106 CFU/mL. In conclusion, SPR sensor has the potential to serve as an excellent candidate for monitoring Salmonella paratyphi in food supplies or contaminated water and clearly makes it possible to develop rapid and appropriate control strategies.

Tentacle-type immobilized metal affinity cryogel for invertase purification from Saccharomyces cerevisiae.

The aim of this study is to investigate the usability of cryogel columns for the purification of invertase from Saccharomyces cerevisiae. Poly(2-hydroxyethyl methacrylate) monolithic columns were produced via cryogelation. Ester groups of the poly(2-hydroxyethyl methacrylate) structure were then converted to imine groups by the reaction with poly(ethylene imine) in the presence of NaHCO3. Transition metal ions, Cu(II), Co(II), and Ni(II), were chelated on the PEI-modified cryogel columns. Purification of invertase from natural source namely S. cerevisiae was also studied, and the purification fold values were obtained as 41.350, 44.714, and 30.302 for Cu(II)-chelated, Co(II)-chelated, and Ni(II)-chelated PHEMA/PEI columns, respectively.

Catalase purification from rat liver with iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) cryogel discs.

In this study, iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) (PHEMAGA/Fe(3+)) cryogel discs were prepared. The PHEMAGA/Fe(3+) cryogel discs were characterized by elemental analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, swelling tests, and surface area measurements. The PHEMAGA/Fe(3+) cryogel discs had large pores ranging from 10 to 100 µm with a swelling degree of 9.36 g H2O/g cryogel. Effects of pH, temperature, initial catalase concentration, and flow rate on adsorption capacity of the PHEMAGA/Fe(3+) cryogel discs were investigated. Maximum catalase adsorption capacity (62.6 mg/g) was obtained at pH 7.0, 25°C, and 3 mg/ml initial catalase concentration. The PHEMAGA/Fe(3+) cryogel discs were also tested for the purification of catalase from rat liver. After tissue homogenization, purification of catalase was performed using the PHEMAGA/Fe(3+) cryogel discs and catalase was obtained with a yield of 54.34 and 16.67 purification fold.

Gelatin-loaded p(HEMA-GMA) cryogel for high-capacity immobilization of horseradish peroxidase.

Poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) [p(HEMA-GMA)] cryogel discs were prepared under sub-zero temperatures. Gelatin was attached covalently on the p(HEMA-GMA) cryogel discs and reversible immobilization of horseradish peroxidase (HRP) was performed. The p(HEMA-GMA) cryogel discs were characterized by swelling tests, scanning electron microscopy, and surface area measurements. HRP immobilization capacity of p(HEMA-GMA)/gelatin cryogel discs was 24.8 mg/g. Removal of phenol from aqueous solutions was performed using HRP immobilized p(HEMA-GMA)/gelatin cryogel. It was observed that within 2 h of contact time, the percentage of phenol removal reaches up to 91% in the presence of H2O2.

Metal-immobilized magnetic nanoparticles for cytochrome C purification from rat liver.

Cu(2+) -immobilized magnetic poly(hydroxyethylmethacrylate-N-methacryloyl-(l)-histidinemethylester) (mPHEMAH) nanoparticles were prepared by surfactant-free emulsion polymerization for cytochrome C (cyt C) purification from rat liver. Elemental analysis, atomic force microscopy, zeta sizer, and vibrating sample magnetometer were used to characterize mPHEMAH nanoparticles. In addition to these characterization steps, surface area, average particle size, and size distribution of mPHEMAH nanoparticles were determined. Quantity of immobilized Cu(2+) was measured using atomic absorption spectrophotometry. N-Methacryloyl-(l)-histidinemethylester and Cu(2+) content of mPHEMAH nanoparticles were 0.18 mmol/g polymer and 0.11 mmol/g polymer, respectively. Specific surface area of Cu(2+) -immobilized mPHEMAH nanoparticles was 1180 m(2) /g. Effect of initial cyt C concentration, pH, temperature, and ionic strength on cyt C adsorption onto Cu(2+) -immobilized mPHEMAH nanoparticles was investigated. Maximum cyt C adsorption capacity of Cu(2+) -immobilized mPHEMAH nanoparticles was 311.9 mg/g polymer. Maximum adsorption was obtained at pH 8.0 and 4 °C. Cu(2+) -immobilized mPHEMAH nanoparticles were used ten times with 4.1% decrease in adsorption capacity. In the last stage, Cu(2+) -immobilized mPHEMAH nanoparticles were used to purify cyt C from rat liver tissue, and the purity of desorbed fractions was controlled by SDS-PAGE.

Dye affinity cryogels for plasmid DNA purification.

The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9µmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA.

Concanavalin A immobilized magnetic poly(glycidyl methacrylate) beads for prostate specific antigen binding.

The aim of this study was to prepare Concanavalin A (Con A) immobilized magnetic poly(glycidyl methacrylate) (mPGMA) beads for prostate specific antigen (PSA) binding and to study binding capacities of the beads using lectin-glycoprotein interactions. Firstly, iron oxide nanoparticles were synthesized by co-precipitation method and then, beads were synthesized by dispersion polymerization in the presence of iron oxide nanoparticles. Con A molecules were both covalently immobilized onto the beads directly and through the spacer arm (1,6-diaminohexane-HDMA). The total PSA and free PSA binding onto the mPGMA-HDMA-Con A beads were higher than that of the mPGMA-Con A beads. Maximum PSA binding capacity was observed as 91.2 ng/g. Approximately 45% of the bound PSA was eluted by using 0.1 M mannose as elution agent. The mPGMA-HDMA-Con A beads could be reused without a remarkable decrease in the binding capacities after 5 binding-desorption cycles. Serum fractions were analyzed using SDS-PAGE. The mPGMA-HDMA-Con A beads could be useful for the detection of PSA and suggested as a model system for other glycoprotein biomarkers.

Megaporous poly(hydroxy ethylmethacrylate) based poly(glycidylmethacrylate-N-methacryloly-(L)-tryptophan) embedded composite cryogel.

One-step activation, purification, and stabilization of lipase enzyme were performed by using composite hydrophobic support at low ionic strength with increased surface area during embedding process. A novel hydrophobic poly(hydroxyethylmethacrylate) [PHEMA] based, poly(glycidyl methacrylate-N-methacryloly-(L)-tryptophan) [PGMATrp] bead embedded composite cryogel membrane having specific surface area of 195m(2)/g was used as hydrophobic matrix for adsorption of commercial Candida Rugosa lipase in a continuous system. PGMATrp embedded PHEMA cryogel membrane with 60-100 µm pore size was obtained by dispersion polymerization of GMA and MATrp to form PGMATrp beads followed by embedding of PGMATrp to HEMA via APS and TEMED redox pair. The introduction of hydrophobic MATrp monomer into bead structure aiming to increase interaction between lipase and composite membrane was estimated using nitrogen stoichiometry of elemental analysis and found to be 239 µmol/g of polymer. Hydophobicity increment due to embedding process was confirmed by measuring contact angle, it was found 42° and 48.4° for the PHEMA and PHEMA/PGMATrp composite cryogel respectively. Some parameters i.e. pH, flow-rate, protein concentration, temperature, salt type and ionic intensity were evaluated on the adsorption capacity in a continuous system. Fast protein liquid chromatography (FPLC) studies were performed for specific adsorption of lipase onto the PHEMA/PGMATrp embedded composite cryogel membrane.

Strong cation-exchange chromatography of proteins on a sulfoalkylated monolithic cryogel.

A new strong cation exchanger (SCX) monolithic column was synthesized by at-line surface modification of a cryogel prepared by copolymerization of 2-hydroxyethylmethacrylate (HEMA) and glycidylmethacrylate (GMA). Sodium salt of 3-Mercaptopropane sulfonic acid (3-MPS) was used as the ligand to transform the surface of the monolith into a strong cation exchanger. The obtained material was characterized in terms of elemental analysis, infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Brunauer-Emmett-Teller (BET) N2 adsorption, and used as a stationary phase for strong-cation exchange chromatography of some proteins, such as α-chymotrypsinogen, cytochrome c and lysozyme. Water permeability of the column was calculated according to Darcy's law (2.66×10(-13)m(2)). The performance of the monolithic cryogel column was evaluated on the basis of Height Equivalent to a Theoretical Plate (HETP). Retention behavior of the studied proteins was modeled on the basis of Yamamoto model to understand the role of the ion-exchange mechanism in retention behaviors. The considered proteins were successfully separated, and the obtained chromatogram was compared with that obtained with a non-functionalized cryogel column.

Comparison of two different reactive dye immobilized poly(hydroxyethyl methacrylate) cryogel discs for purification of lysozyme.

In this study, cibacron blue F3GA and alkali blue 6B immobilized poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were prepared. The cryogel discs were characterised by swelling tests, elemental analysis and scanning electron microscopy. Each cryogel disc was used for lysozyme adsorption from aqueous solutions. Maximum adsorption capacities were 103.3 and 106.7 mg/g for cibacron blue F3GA and alkali blue 6B immobilized cryogel discs, respectively. Equilibrium lysozyme concentration, pH, ionic strength and temperature were the factors of which effect on lysozyme adsorption was investigated. Reusability of the cryogel discs was tested and less than 5% decrease in adsorption capacity was reported. In the last stage of this work, the cryogel discs were used for lysozyme purification from chicken egg white. Molecular weight and purity of the eluted lysozyme from cryogel discs were controlled by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In conclusion, cibacron blue F3GA and alkali blue 6B immobilized cryogel discs present a cheap and fast way for purification of lysozyme with a high purity.

Composite cryogels for lysozyme purification.

Beads-embedded novel composite cryogel was synthesized to purify lysozyme (Lyz) from chicken egg white. The poly(hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) (PHEMAPA) beads of smaller than 5 µm size were synthesized by suspension polymerization and then embedded into a poly(hydroxyethyl methacrylate) (PHEMA)-based cryogel column. The PHEMAPA bead-embedded cryogel (BEC) column was characterized by swelling tests, scanning electron microscopy (SEM), surface area measurements by the Brunauer-Emmett-Teller (BET) method, elemental analysis, and flow dynamics. The specific surface area of the PHEMAPA BEC was found as 41.2 m(2) /g using BET measurements. Lyz-binding experiments were performed using aqueous solutions in different conditions such as initial Lyz concentration, pH, flow rate, temperature, and NaCl concentration of an aqueous medium. The PHEMAPA BEC column could be used after 10 adsorption-desorption studies without any significant loss in adsorption capacity of Lyz. The PHEMAPA BEC column was used to purify Lyz from chicken egg white, and gel electrophoresis was used to estimate the purity of Lyz. The chromatographic application of the PHEMAPA BEC column was also performed using fast protein liquid chromatography.

Gelatin-immobilised poly(hydroxyethyl methacrylate) cryogel for affinity purification of fibronectin.

In this work, fibronectin purification from human plasma with the gelatin-immobilised poly(hydroxyethyl methacrylate) (PHEMA) cryogel has been evaluated. The PHEMA cryogel was prepared by cryo-polymerisation which proceeds in an aqueous solution of monomer frozen inside a plastic syringe. The PHEMA cryogel contained interconnected macrochannels of 10-200 µm in diameter. Gelatin molecules were covalently immobilised onto the PHEMA cryogel via carbodiimide activation. The gelatin-immobilised PHEMA cryogel was used to purify fibronectin from human plasma. Fibronectin adsorption from human plasma on the PHEMA cryogel was 0.30 mg/ml, while much higher adsorption values, up to 38 mg/ml, was obtained with the gelatin-immobilised PHEMA cryogel. The fibronectin adsorption capacity of the gelatin-immobilised PHEMA cryogel did not change with an increase in the flow rate of plasma. Up to 92 % of the adsorbed fibronectin was eluted using 2 M urea containing 1 M NaCl as elution agent. The adsorption-elution cycle was repeated ten times using the same PHEMA cryogel. No remarkable decrease was detected in the adsorption capacity of the gelatin-immobilised PHEMA cryogel.

Macroporous PHEMA-based cryogel discs for bilirubin removal.

A novel N-methacryloyl-L-tryptophan methyl ester (MATrp) containing poly (hydroxyethyl methacrylate) cryogel (PHEMATrp) disc was prepared for removal of bilirubin (BR) out of human plasma. PHEMATrp cryogel disc was produced by bulk polymerization, with high gelation yield up to 92% and characterized by swelling tests, scanning electron microscopy (SEM), elemental analysis, Brunauer- Emmett-Teller (BET) analysis, contact angle measurements and surface energy calculations. BR adsorption studies were performed in a batch system, and the maximum BR adsorption capacity was found as 22.2 mg/g cryogel disc.

Purification of urease from jack bean (Canavalia ensiformis) with copper (II) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) cryogels.

Jack bean (Canavalia ensiformis) is the source of interesting proteins that contribute to modern biochemistry, and urease is the primary of these proteins. Owing to its role and occurrence in nature, urease has become a part of extensive studies. In this study, jack bean urease (JBU) was purified by immobilized metal affinity chromatography using Cu(2+) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) [PHEMAH-Cu(2+)]-based cryogels. PHEMAH-Cu(2+) cryogel was synthesized and characterized for swelling degree, morphology (by SEM), N-methacryloyl-(L)-histidine methyl ester and Cu(2+) incorporation (by elemental analysis and atomic absorption spectrophotometry). The binding of JBU to PHEMAH-Cu(2+) cryogel was optimized by examining the effect of pH, flow rate and JBU concentration on binding. The maximal binding of JBU was 23.2 mg/dry gram of adsorbent. The maximal binding of JBU extracted from jack bean meal was 67.8 mg/dry gram of adsorbent. The elution of JBU from cryogel column was accomplished by 1.0 M NaCl in 20 mM phosphate buffer (pH 8.0). Molecular weight and purity of JBU from jack bean meal was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was observed that JBU could be repeatedly bound and eluted from (PHEMAH)-Cu(2+) cryogel with less than 10% loss in column capacity.