Synthesis of hydroxyethyl-methacrylate-(L)-histidine methyl ester cryogels. Application on the separation of bovine immunoglobulin G.

In this study cryogels based 2-hydroxyethyl methacrylate (HEMA) functionalized with N-methacryloyl-L-histidine methyl ester (MAH) were synthesized and used for the adsorption and separation of bovine IgG. Two series of cryogels functionalized with 5 and 10 mg of MAH as pseudobioaffinity ligand were prepared and characterized by swelling test, FTIR and SEM analysis. The adsorption efficiency of the bovine immunoglobulin into cryogels is discussed with respect to the following chromatographic parameters: pH, flow rate, initial IgG concentration, adsorption time and ionic strength. Our results show good adsorption of bovine immunoglobulin under mild separation conditions at pH 7.4. The maximum binding capacity was determined (32.4 mg/g of cryogel) and demonstrates the efficiency of the used cryogels. This efficacy is clearly seen upon increasing the maximum binding capacity from 23.2 mg (obtained with cryogels with 5 mg MAH) to 32.4 mg/g (for cryogel with 10 mg MAH ligand concentration). The purity of separated fractions was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Together our observations highlights poly (HEMA-MAH) as an efficient adsorbent for bovine immunoglobulins G separation.

Protein C recognition by ion-coordinated imprinted monolithic cryogels.

The protein C imprinted monolithic cryogel was synthesized using 2-hydroxyethyl methacrylate by redox cryo-polymerization method. The prepared monolithic cryogel was characterized by Fourier transform infrared spectroscopy, swelling test, surface area measurements, and scanning electron microscopy. The nonimprinted cryogel was prepared as well for control. Adsorption of protein C from aqueous solutions was investigated in a continuous mode and several parameters affecting adsorption performance were optimized. The maximum protein C adsorption amount was 30.4 mg/g. The selectivity studies were performed by monolithic column studies and fast protein liquid chromatography, using hemoglobin and human serum albumin as competing proteins. The relative selectivity coefficients were 2.37 and 8.89 for hemoglobin and human serum albumin, respectively. Reusability was tested for ten consecutive adsorption-desorption cycles, and no significant change in adsorption capacity was recorded. A pseudo-second-order model was suitable to interpret kinetic data, and the Langmuir model suited the adsorption isotherms well.

Molecularly imprinted cryogels for human interferon-alpha purification from human gingival fibroblast culture.

Interferons are important proteins for the immune system because of their antiviral, anti-proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α-2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN-α-MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN-α-MIP cryogel was found as 254.8 × 10(4) IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption-desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α-2b on HeLa cell line was found between the values 3.45 × 10(8) IU/mg and 3.75 × 10(8) IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α-2b.

Hydrophobic cryogels for DNA adsorption: effect of embedding of monosize microbeads into cryogel network on their adsorptive performances.

As alternative hydrophobic adsorbent for DNA adsorption, supermacroporous cryogel disks were synthesized via free radical polymerization. In this study, we have prepared two kinds of cryogel disks: (i) poly(2-hydroxyethyl methacrylate-N-methacryloyl-l-tryptophan) [p(HEMA-MATrp)] cryogel containing specific hydrophobic ligand MATrp; and (ii) monosize p(HEMA-MATrp) particles synthesized via suspension polymerization embedded into p(HEMA) cryogel structure to obtain p(HEMA-MATrp)/p(HEMA) composite cryogel disks. These cryogel disks containing hydrophobic functional group were characterized via swelling studies, Fourier transform infrared spectroscopy, elemental analysis, surface area measurements and scanning electron microscopy. DNA adsorption onto both p(HEMA-MATrp) cryogel and p(HEMA-MATrp)/p(HEMA) composite cryogels was investigated. Maximum adsorption of DNA on p(HEMA-MATrp) cryogel was found to be 15 mg/g polymer. Otherwise, p(HEMA-MATrp)/p(HEMA) composite cryogels significantly increased the DNA adsorption capacity to 38 mg/g polymer. Composite cryogels could be used repeatedly without significant loss on adsorption capacity after 10 repetitive adsorption-desorption cycles.

Oriented immobilized anti-hIgG via F(c) fragment-imprinted PHEMA cryogel for IgG purification.

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F(c) fragment-imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti-hIgG for IgG purification from human plasma. Non-imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti-hIgG to compare the adsorption capacities of oriented (MIP/anti-hIgG) and random (NIP/anti-hIgG) cryogel columns. The amount of immobilized anti-hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti-hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti-hIgG column (29.7 mg/g) for the MIP/anti-hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti-hIgG cryogel column. Adsorbed IgG was eluted using 1.0 M NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti-hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification.

Molecular imprinted based microcryogels for thrombin purification.

In addition to understanding and explaining the functions of proteins, the need for low-cost, easy and efficient purification methods has been increasing in the field of protein purification, which is also important for enzyme production. In this context, an alternative approach has been developed for the purification of thrombin, which has a crucial role in the hemostatic process, via thrombin imprinted microcryogels that allow reuse and have high selectivity. The characterization studies of the microcryogels were accomplished with micro-computed tomography (µCT), scanning electron microscopy (SEM), optical microscope, surface area measurements (BET analyses) and swelling test measurements. By scanning various parameters affecting thrombin adsorption, the maximum thrombin adsorption capacity (Qmax) was found to be 55.86 mg/g. Also, the selectivity of microcryogels was investigated with the competitive agents and reusability studies were performed. The purity of thrombin was evaluated by Fast Performance Liquid Chromatography (FPLC) method. Experimental results indicated that adsorption of thrombin by the developed microcryogels fit the Langmuir isotherm model (Qmax: 55.86 mg/g, R2: 0.9505) and pseudo-second order for three different thrombin concentrations (R2: 0.9978, R2: 0.9998, R2: 0.9999).

Human serum albumin depletion based on dye ligand affinity chromatography via magnetic microcryogels.

One of the primary purposes of proteomic studies is to analyze the proteins in the blood to be considered as biomarkers. Albumin, which constitutes the majority of total serum proteins, complicates the discovery of low-density proteins that are important for the diagnosis of diseases. Based on this, an alternative approach for albumin depletion was developed in this study by covalently attached Cibacron Blue 3GA (CB) to magnetic microcryogels. After detailed characterization of CB attached magnetic microcryogels synthesized via a microstencil array chip, albumin adsorption studies were performed to examine the optimum depletion conditions. In the presented study, the maximum albumin adsorption capacity (Qmax) was calculated as 149.25 mg/mL in pH 5.0 acetate buffer solution, which is the optimum pH value for albumin. Experimental studies have demonstrated that CB-attached magnetic microcryogels can be reused without loss of performance for albumin depletion after 10 adsorption-desorption cycles.

Development of Optical-Based Molecularly Imprinted Nanosensors for Adenosine Detection.

Adenosine nucleoside is an important molecule in human physiology. The levels of adenosine nucleoside in urine and plasma are directly or indirectly related to diseases such as neurodegenerative diseases and cancer. In the present study, adenosine-imprinted and non-imprinted poly(2-hydroxyethyl methacrylate-methacrylic acid) (poly(HEMA-MAA)) surface plasmon resonance (SPR) nanosensors were prepared for the determination of adenosine nucleoside. First, MAA/adenosine pre-polymerization complexes were prepared at different molar ratios using adenosine as a template molecule and methacrylic acid (MAA) as a monomer, and SPR nanosensor surfaces were optimized by determining the highest imprinting factor of the chip surfaces. The surfaces of adenosine-imprinted and non-imprinted SPR nanosensors were characterized by using atomic force microscopy, ellipsometry, and contact angle measurements. Kinetic analyses were made with different concentrations in the range of 0.5-400.0 nM for the detection range with a pH 7.4 phosphate buffer solution. The limit of detection in adenosine aqueous solutions, artificial plasma, and artificial urine was determined to be 0.018, 0.015, and 0.013 nM, respectively. In the selectivity analysis of the developed nanosensors, the selectivity of adenosine SPR nanosensors in solutions at different concentrations was determined by using guanosine and cytidine nucleosides. The relative selectivity coefficients of adenosine-imprinted SPR nanosensors for adenosine/cytidine and adenosine/guanosine are 3.836 and 3.427, respectively. Since adenosine-imprinted SPR nanosensors are intended to be used in medical analysis and research, adenosine analysis has also been studied in artificial urine and artificial plasma samples.

Preparation of magnetic poly(ethylene glycol dimethacrylate-N-Methacryloyl-(L)-glutamic acid) particles for thrombin purification.

In this study, magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-glutamic acid) (mPEGDMA-MAGA) particles were prepared by the dispersion polymerization in order to purify thrombin effectively. mPEGDMA-MAGA particles were synthesized by adding different ratios of magnetite (Fe3O4) to the medium in addition to the monomer phases EGDMA and MAGA. The characterization studies of mPEGDMA-MAGA particles were used by fourier transform infrared spectroscopy, zeta size measurement, scanning electron microscopy and electron spin resonance. mPEGDMA-MAGA particles were used in thrombin adsorption studies from aqueous thrombin solutions in both batch and magnetically stabilized fluidized bed (MSFB) system. Maximum adsorption capacity in pH 7.4 phosphate buffer solution is 964 IU/g polymer and 134 IU/g polymer in MSFB system and batch system, respectively. The developed magnetic affinity particles enabled the separation of thrombin from different patient serum samples in one step. It has also been observed that magnetic particles can be used repeatedly without significant reduction in adsorption capacity.

Fab fragment immobilized immunoaffinity cryogels as a tool for human serum albumin purification: Characterization of Fab immobilized cryogels.

As a general approach Fab immobilized immunoaffinity cryogels for human serum albumin purification is presented in this article. The directed immobilization of antibodies, which have been used as diagnostic and therapeutic agents in applications recently, has brought a different perspective to the purification method. In our study to shed light on this method, anti-HSA Fab fragment immobilized poly(hydroxylethyl methacrylate-N-methacryloyl-(l)-cysteine) (PHEMAC-Fab) and PHEMAC cryogels were prepared for the purification of human serum albumin (HSA). The specific surface areas of the PHEMAC-Fab and PHEMAC cryogels were calculated as 38.6 m2/g, and 20.0 m2/g, respectively. In comparison, the maximum swelling ratio was observed in the PHEMAC-Fab cryogel and more water was absorbed than the PHEMAC cryogel. While the macropore ratio in the PHEMAC-Fab cryogel is 54.3%, the macropore ratio of the PHEMAC cryogels is 41.6%. The results obtained here showed that anti-HSA cryogel prepared by immobilization of Fab fragments can be an alternative to traditional immunoaffinity techniques for HSA purification.

Metal-chelated polyamide hollow fiber membranes for ovalbumin purification from egg white.

In the study, purification of ovalbumin was performed by modifying polyamide hollow fiber membranes using immobilized metal affinity chromatography technique. For this purpose, firstly polyethyleneimine (PEI) solutions of different concentrations were attached to hollow fiber membranes. Then, Cu(II), Ni(II) and Zn(II) metal ions were chelated separately to polyethyleneimine attached hollow fiber membranes. Characterization studies of modified hollow fiber membranes were performed with scanning electron microscopy (SEM). Also, the surface area was measured with the Brunner Emmet Teller (BET) method and the porosity was measured with mercury porosimeter. pH, ionic strength, initial ovalbumin concentration, temperature and reusability parameters affecting adsorption capacity were investigated. The maximum ovalbumin adsorption capacities of hollow fiber membranes were found to be 317 mg/g for Cu(II), 169 mg/g for Ni(II) and 101 mg/g for Zn(II), respectively. Desorption ratio of metal ions were calculated as 91.6% for Cu(II), 92.9% for Ni(II) and 91.8% for Zn(II), which are quite high and suitable. When examined in terms of adsorption isotherm models, it was concluded that the Langmuir model is suitable. Purification of ovalbumin from egg white was carried out by fast performance liquid chromatography (FPLC), and the purity of ovalbumin was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method.

Molecular imprinted nanoparticle assisted surface plasmon resonance biosensors for detection of thrombin.

In this study, we designed a surface plasmon resonance (SPR) biosensors based on molecular imprinting to detect low amounts of thrombin directly in a short time, both in thrombin aqueous solutions and in patient serum samples. For this purpose, the thrombin imprinted and non-imprinted SPR biosensors were prepared by integrating the synthesized thrombin imprinted and non-imprinted nanoparticles on the allyl mercaptan modified gold SPR chip surface. The kinetic studies were performed with a thrombin concentration range from 0.1 to 400 pM with a detection limit of approximately 0.017 pM in thrombin aqueous solution. Morever, the limit of detection was found to be 0.033 pM in patient serum samples. When the selectivity of the thrombin imprinted SPR biosensors was compared with the competing molecules as bovine serum albumin and lysozyme, it was showed that the thrombin was 4.58 times and 3.99 times more selective than the bovine serum albumin and lysozyme molecules, respectively. The shelf life and reusability of the designed SPR biosensor showed that there was a 12.93% decrease in the performance according to the kinetic analyzes that performed after 8 months. In addition, thrombin detection from the patient serum samples was performed using both SPR biosensor and the enzyme-linked immunosorbent assay methods, and were calculated recoveries.

Preparation of surface plasmon resonance-based nanosensor for curcumin detection.

In this study, the curcumin imprinted and the non-imprinted poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-tryptophan) (poly(HEMA-MATrp)) nanoparticle based surface plasmon resonance (SPR) nanosensors were prepared for the detection of curcumin and characterized by zeta-size analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. After, the curcumin imprinted and the non-imprinted nanoparticles are attached on the surface of SPR chips. The curcumin imprinted and the non-imprinted SPR nanosensors are characterized by using atomic force microscope, ellipsometer, and contact angle measurements. Kinetic studies were carried out with curcumin aqueous solution at a concentration range of 0.01-150 mg/L using the curcumin imprinted and the non-imprinted SPR nanosensors. In all kinetic analysis, the response time is 14 min for equilibration, adsorption, and desorption cycles. The limit of detection and limit of quantification for the curcumin imprinted SPR nanosensors was 0.0012 mg/L and 0.0040 mg/L, respectively. The validity of the curcumin imprinted SPR nanosensors in real samples was carried out using liquid chromatography-tandem mass spectrometry (LC-MS).

Molecularly imprinted supermacroporous cryogels for cytochrome c recognition.

Molecular imprinting is an attractive biomimetic approach that creates specific recognition sites for the shape and functional group arrangement to template molecules. The purpose of this study is to prepare cytochrome c-imprinted poly(hydroxyethyl methacrylate) (PHEMA)-based supermacroporous cryogel which can be used for the separation of cytochrome c from protein mixtures. N-Methacryloyl-(L)-histidinemethylester (MAH) was used as the metal-coordinating monomer. In the first step, Cu(2+) was complexed with MAH, and the cytochrome c imprinted PHEMA (MIP) cryogel was prepared by free radical cryopolymerization initiated by N,N,N',N'-tetramethylene diamine at -12°C. After polymerization is completed, the template cytochrome c molecules were removed from the MIP cryogel using 0.5 M NaCl solution. The maximum cytochrome c binding amount was 126 mg/g polymer. Selective binding studies were performed in the presence of lysozyme and bovine serum albumin. The relative selectivity coefficients of MIP cryogel for cytochrome c/lysozyme and cytochrome c/bovine serum albumin were 1.7 and 5.2 times greater than those of the non-imprinted PHEMA cryogel, respectively. The selectivity of MIP cryogel for cytochrome c was also confirmed with fast protein liquid chromatography. The MIP cryogel could be used many times with no remarkable decrease in cytochrome c binding capacity.

Molecularly Imprinted Polymer-Based Quartz Crystal Microbalance Sensor for the Clinical Detection of Insulin.

In this study, we reported the design of a quartz crystal microbalance (QCM) sensors for selective insulin detection. In the first step, N-methacryloyl-(L) 3-histidine methyl ester (MAH) monomer was formed a complex with insulin. Then, 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate were mixed with MAH:insulin complex. Insulin-imprinted and non-imprinted QCM sensors were synthesized by ultraviolet polymerization for the insulin detection. Insulin-imprinted QCM sensors was characterized by the contact angle measurements, atomic force microscopy and ellipsometry. Limit of detection (LOD) was found as 0.00158 ng/mL for the insulin-imprinted QCM sensors. Selectivity of insulin-imprinted and non-imprinted QCM sensors was carried in the presence of glucagon and aprotinin. Insulin-imprinted QCM sensor for insulin detection was also examined in the artificial plasma.

Surface Plasmon Resonance Based on Molecularly Imprinted Polymeric Film for l-Phenylalanine Detection.

In this study, we designed a simple, rapid, sensitive and selective surface plasmon resonance (SPR) sensor for detection of L-phenylalaine by utilizing molecular imprinting technology. l-phenylalanine imprinted and non-imprinted poly(2-hydroxyethyl methacrylate-methacryloyl-l-phenylalanine) polymeric films were synthesized onto SPR chip surfaces using ultraviolet polymerization. l-phenyalanine imprinted and non-imprinted SPR sensors were characterized by using contact angle, atomic force microscopy and ellipsometry. After characterization studies, kinetic studies were carried out in the concentration range of 5.0-400.0 µM. The limit of detection and quantification were obtained as 0.0085 and 0.0285 µM, respectively. The response time for the test including equilibration, adsorption and desorption was approximately 9 min. The selectivity studies of the l-phenylalanine imprinted SPR sensor was performed in the presence of d-phenylalanine and l-tryptophan. Validation studies were carried out via enzyme-linked immunosorbent analysis technique in order to demonstrate the applicability and superiority of the l-phenylalanine imprinted SPR sensor.

Selective Detection of Penicillin G Antibiotic in Milk by Molecularly Imprinted Polymer-Based Plasmonic SPR Sensor.

Molecularly imprinted polymer-based surface plasmon resonance sensor prepared using silver nanoparticles was designed for the selective recognition of Penicillin G (PEN-G) antibiotic from both aqueous solution and milk sample. PEN-G imprinted sensors (NpMIPs) SPR sensor was fabricated using poly (2-hydroxyethyl methacrylate-N-methacroyl-(L)-cysteine methyl ester)-silver nanoparticles-N-methacryloyl-L-phenylalanine methyl ester polymer by embedding silver nanoparticles (AgNPs) into the polymeric film structure. In addition, a non-imprinted (NpNIPs) SPR sensor was prepared by utilizing the same polymerization recipe without addition of the PEN-G template molecule to evaluate the imprinting effect. FTIR-ATR spectrophotometer, ellipsometer, contact angle measurements were used for the characterization of NpMIPs SPR sensors. The linear concentration range of 0.01-10 ng/mL PEN-G was studied for kinetic analyses. The augmenting effect of AgNPs used to increase the surface plasmon resonance signal response was examined using polymer-based PEN-G imprinted (MIPs) sensor without the addition of AgNPs. The antibiotic amount present in milk chosen as a real sample was measured by spiking PEN-G into the milk. According to the Scatchard, Langmuir, Freundlich and Langmuir-Freundlich adsorption models, the interaction mechanism was estimated to be compatible with the Langmuir model.

Injectable Cryogels in Biomedicine.

Cryogels are interconnected macroporous materials that are synthesized from a monomer solution at sub-zero temperatures. Cryogels, which are used in various applications in many research areas, are frequently used in biomedicine applications due to their excellent properties, such as biocompatibility, physical resistance and sensitivity. Cryogels can also be prepared in powder, column, bead, sphere, membrane, monolithic, and injectable forms. In this review, various examples of recent developments in biomedical applications of injectable cryogels, which are currently scarce in the literature, made from synthetic and natural polymers are discussed. In the present review, several biomedical applications of injectable cryogels, such as tissue engineering, drug delivery, therapeutic, therapy, cell transplantation, and immunotherapy, are emphasized. Moreover, it aims to provide a different perspective on the studies to be conducted on injectable cryogels, which are newly emerging trend.

Surface Plasmon Resonance-Based Immunosensor for Igm Detection with Gold Nanoparticles.

In this work, a surface plasmon resonance (SPR) based immunosensor was prepared by the immobilization of the amine-functionalized gold nanoparticles (N-AuNPs) on the sensing surface to sense immunoglobulin M (IgM) antibodies in the aqueous solution and artificial plasma. The characterization studies of SPR based immunosensor for IgM detection were performed with scanning electron microscope (SEM), contact angle measurements, and ellipsometry. Kinetic studies for the IgM immunosensor were carried out in the range of 1.0 to 200 ng/mL IgM concentrations in an aqueous solution. The total IgM analysis time including adsorption, desorption, and regeneration cycles was nearly 10 min for the prepared immunosensor. The limit of detection (LOD) and limit of quantification (LOQ) were found as 0.08 and 0.26 ng/mL, respectively. The reusability of the proposed immunosensor was tested with 6 consecutive adsorption-desorption, and regeneration cycles. Also, enzyme-linked immunosorbent assay (ELISA) method was utilized in the validation of the immunosensor.

Antibacterial effect against both Gram-positive and Gram-negative bacteria via lysozyme imprinted cryogel membranes.

The development of novel biocompatible and cost effective cryogel membrane which shows enhanced antimicrobial properties in order to use for several approaches such as wound dressing, scaffold or food packaging was aimed in this study. A super macro porous lysozyme imprinted cryogel membranes showing antibacterial effect against both Gram-positive and Gram-negative bacteria were prepared by using molecular imprinting technique. N-methacryloyl-(L)-histidine methyl ester (MAH) was used as the pseudo specific ligand and complexed with Cu++ in order to provide metal ion coordination between MAH and template molecule (lysozyme). Comparing the antibacterial activity of different lysozyme concentrations, cryogel membranes were prepared in three different concentrations. To synthesize Poly (hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methylester) P(HEMA-MAH) cryogel membrane, free radical polymerization initiated by N, N, N', N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) was carried out at -12 °C. The characterization of the lysozyme imprinted cryogel membrane was accomplished by using scanning electron microscopy (SEM), swelling degree measurements and Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) spectroscopy. The cytotoxicity test of produced membrane was performed by using mouse fibroblast cell line L929. The antibacterial activity of P(HEMA-MAH) lysozyme molecular imprinted [P(HEMA-MAH) Lyz-MIP] cryogel membranes against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were determined by Kirby-Bauer membranes diffusion and viable cell counting methods. When the antibacterial effect of P(HEMA-MAH) Lyz-MIP cryogel membranes were evaluated, it was found that P(HEMA-MAH) Lyz-MIP cryogel membranes had stronger antibacterial effects against Gram-negative E. coli bacteria even in low lysozyme concentrations. In addition, 100% bacterial inhibition was detected for both of two bacteria at increasing lysozyme concentrations.