Cholesterol imprinted composite membranes for selective cholesterol recognition from intestinal mimicking solution.

Molecularly imprinted polymers which have been extensively investigated as selective adsorbents were constructed using a template molecule during the polymerization to gain template-specific cavities. In this study, we prepared cholesterol imprinted poly(2-hydroxyethyl methacrylate-methacryloyamidotryptophan) (PHEMA-MTrp) particles embedded composite membranes. These membranes were characterized through elemental analysis, FTIR, SEM, swelling tests, and surface area measurements. Adsorption experiments were performed in a batch experimental set-up, and the adsorption medium was either a methanol or intestinal-mimicking solution. Stigmasterol and estradiol were used as competing molecules in selectivity tests. Some results are as follows: the specific surface areas of MIP particle-embedded membranes, NIP particle-embedded membranes, and membranes without particles were 36.5, 29.2 and 13.7 m2/g, respectively. The imprinted membranes were 1.96 and 2.13 times more selective for cholesterol when compared to stigmasterol and estradiol used as competitor agents, respectively. Cholesterol adsorption capacity increased up to 23.43 mg/g with increasing cholesterol concentration of 2 mg/mL. The MIP particle-embedded composite membranes showed a negligible loss in cholesterol adsorption capacity after ten consecutive adsorption cycles using the same adsorbent.

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.

Strategies for the development of an electrochemical bioassay for TNF-alpha detection by using a non-immunoglobulin bioreceptor.

TNF-α is an inflammatory cytokine produced by the immune system. Serum TNF-α level is elevated in some pathological states such as septic shock, graft rejection, HIV infection, neurodegenerative diseases, rheumatoid arthritis and cancer. Detecting trace amount of TNF-α is, also, very important for the understanding of tumor biological processes. Detection of this key biomarker is commonly achieved by use of ELISA or cytofluorimetric based methods. In this study the traditional optical detection was replaced by differential pulse voltammetry (DPV) and an affinity molecule, produced by evolutionary approaches, has been tested as capture bioreceptor. This molecule, namely a combinatorial non-immunoglobulin protein (Affibody®) interacts with TNF-α selectively and was here tested in a sandwich assay format. Moreover magnetic beads were used as support for bioreceptor immobilization and screen printed carbon electrodes were used as transducers. TNF-α calibration curve was performed, obtaining the detection limit of 38pg/mL, the quantification range of 76-5000pg/mL and RSD%=7. Preliminary results of serum samples analysis were also reported.

Preparation of cryogel columns for depletion of hemoglobin from human blood.

In this study, we aimed to prepare the metal chelate affinity cryogels for the hemoglobin (Hb) depletion. Poly(2-hydroxyethyl methacrylate) (PHEMA) cryogels were selected as base matrix because of their blood compatibility, osmotic, chemical, and mechanical stability. Cryogels are also useful when working with the viscous samples such as blood, because of their interconnected macroporous structure. Iminodiacetic acid (IDA), the chelating agent, was covalently coupled with PHEMA cryogels after activation with the epichlorohydrin and then the Ni(II) ions were chelated to the IDA-bound cryogels. The depletion of the Hb from hemolysate was shown by SDS-PAGE.

Molecularly imprinted cryogels for chondroitin sulfate recognition.

Chondroitin sulfate (Cs)-imprinted poly(hydroxyethyl methacrylate)-based macroporous cryogels (CsMIP) were prepared for selective recognition of Cs from an aqueous solution. The selective binding sites for Cs were maintained using vinyl imidazole-Cu(2+) functional groups, during the precomplexation step in the polymerization procedure. Newly synthesized CsMIP cryogel columns were characterized. The separation of Cs from aqueous solutions was studied, both in the continuous system and in the fast protein liquid chromatography (FPLC) system. According to the FPLC studies, the Rs value obtained was 14.72, which shows that the CsMIP cryogel column can successfully separate Cs from aqueous solutions of Cs in the presence of competitor molecules.

A novel chromatographic media: histidine-containing composite cryogels for HIgG separation from human serum.

Histidine-containing microspheres (HCM) with 2 µm in size were synthesized by suspension polymerization of poly(hydroxyethyl methacrylate) and N-methacryloyl-L-histidine methyl ester. Then, they were used to prepare composite cryogel columns by an embedding process for affinity depletion of immunoglobulin G (HIgG) from human serum via histidine groups on microspheres. Here, we describe HIgG adsorption performance of composite cryogel columns in both aqueous solution and human serum.

Heparin removal from human plasma using molecular imprinted cryogels.

In this study, heparin-imprinted poly(hydroxyethyl methacrylate-N-[(3-dimethylamino)-propyl] methacrylamide) cryogel column (HpMIP) was synthesized for removal of Hp from human plasma using molecular imprinting technique. Hp removal studies were performed from both aqueous solution and human plasma. Selectivity studies were performed using fast protein liquid chromatography (FPLC) system. The obtained results showed that the HpMIP column can remove Hp from both aqueous solutions and human plasma samples selectively. Ninety per cent of Hp was removed from 28 U/mL of human plasma samples successfully. Non-imprinted cryogel column (NIP) and plane PHEMA column were also synthesized to compare selectivity and non-specific adsorption properties.

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.

Affinity composite cryogel discs functionalized with Reactive Red 120 and Green HE 4BD dye ligands: application on the separation of human immunoglobulin G subclasses.

Naturally produced by the human immune system, immunoglobulin nowadays is widely used for in vivo and in vitro purposes. The increased needs for pure immunoglobulin have prompted researchers to find new immunoglobulin chromatographic separation processes. Cryogels as chromatographic adsorbents, congregate several mechanical features including good compatibility, large pore structure, flexibility, short diffusion pathway and stability. These different characteristics make them a good alternative to conventional chromatographic methods and allowing their potential use in separation technology. In the present study, two sets of poly(2-hydroxyethyl methacrylate) (PHEMA) based beads were prepared and functionalized with Reactive Red 120 (RR) and Reactive Green HE 4BD (RG) dyes, and then embedded into supermacroporous cryogels. The morphology, physical and chemical features of the prepared bead embedded composite cryogel discs (CCDs) were performed by scanning electron microscopy (SEM), swelling test, elemental analysis and Fourier transform infrared spectroscopy (FTIR). The results showed that the embedded composite cryogel discs have a specific surface area of 192.0 m(2)/g with maximum adsorption capacity of HIgG 239.8 mg/g for the RR functionalized CCD and 170 mg/g for RG functionalized CCD columns, both at pH 6.2.

Molecularly imprinted composite cryogels for hemoglobin depletion from human blood.

A molecularly imprinted composite cryogel (MICC) was prepared for depletion of hemoglobin from human blood prior to use in proteome applications. Poly(hydroxyethyl methacrylate) based MICC was prepared with high gel fraction yields up to 90%, and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, swelling studies, flow dynamics and surface area measurements. MICC exhibited a high binding capacity and selectivity for hemoglobin in the presence of immunoglobulin G, albumin and myoglobin. MICC column was successfully applied in fast protein liquid chromatography system for selective depletion of hemoglobin for human blood. The depletion ratio was highly increased by embedding microspheres into the cryogel (93.2%). Finally, MICC can be reused many times with no apparent decrease in hemoglobin adsorption capacity.

Cholesterol removal from various samples by cholesterol-imprinted monosize microsphere-embedded cryogels.

Cholesterol-imprinted monosize poly(glycidyl methacrylate-N-methacryloyl-(L)-tyrosine methylester) microspheres were embedded into the poly(hydroxyethyl methacrylate) (PHEMA) cryogels and the resulting composite cryogel was used for the selective removal of cholesterol. Composite cryogels were characterized by swelling tests, multipoint BET apparatus, SEM, FTIR and elemental analysis studies. Specific surface area of the PHEMA cryogel was increased from 13 to 72.7 m(2)/g by embedding of microspheres. Composite cryogels removed 80% of cholesterol from homogenized milk. The maximum adsorption capacity was found as 42.7 mg/g for intestinal mimicking solution. After 20 adsorption-desorption cycles, there was no remarkable decrease in the adsorption capacity.

Microsphere-embedded cryogel for selective and efficient depletion of immunoglobulin G from human serum.

In this study a novel composite cryogel column was synthesized for affinity depletion of immunoglobulin G (IgG) from human serum. The microsphere-embedding technique combines the large surface area of microspheres with excellent properties of cryogels. The poly(hydroxyethyl methacrylate- N-methacryloyl-L-histidine methyl ester) microsphere (2 µm size)- embedded composite cryogel (MECC) column was synthesized and characterized by various methods. The specific surface area of the composite cryogel was found as 92 m(2)/g by using BET measurement. Maximum HIgG adsorption on MECC column was found as 50.5 mg/g. MECC column can be reused many times with no apparent decrease in HIgG adsorption capacity.

Synthesis and characterization of amino acid containing Cu(II) chelated nanoparticles for lysozyme adsorption.

Immobilized metal ion affinity chromatography (IMAC) is a useful method for adsorption of proteins that have an affinity for transition metal ions. In this study, poly(hydroxyethyl methacrylate-methacryloyl-L-tryptophan) (PHEMATrp) nanoparticles were prepared by surfactant free emulsion polymerization. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles to be used in lysozyme adsorption studies in batch system. The maximum lysozyme adsorption capacity of the PHEMATrp nanoparticles was found to be 326.9 mg/g polymer at pH 7.0. The nonspecific lysozyme adsorption onto the PHEMA nanoparticles was negligible. In terms of protein desorption, it was observed that adsorbed lysozyme was readily desorbed in medium containing 1.0 M NaCl. The results showed that the metal-chelated PHEMATrp nanoparticles can be considered as a good adsorbent for lysozyme purification.

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.

Molecularly imprinted composite cryogel for albumin depletion from human serum.

A new composite protein-imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl-methacylate-based molecularly imprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform infrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity.

Selective removal of 17ß-estradiol with molecularly imprinted particle-embedded cryogel systems.

The selective removal of 17ß-estradiol (E2) was investigated by using molecularly E2 imprinted (MIP) particle embedded poly(hydroxyethyl methacrylate) (PHEMA) cryogel. PHEMA/MIP composite cryogel was characterized by FTIR, SEM, swelling studies, and surface area measurements. E2 adsorption studies were performed by using aqueous solutions which contain various amounts of E2. The specificity of PHEMA/MIP cryogel to recognition of E2 was performed by using cholesterol and stigmasterol. PHEMA/MIP cryogel exhibited a high binding capacity (5.32 mg/gpolymer) and high selectivity for E2 in the presence of competitive molecules, cholesterol (k(E2/cholesterol) = 7.6) and stigmasterol (k(E2/Stigmasterol) = 85.8). There is no significant decrease in adsorption capacity after several adsorption-desorption cycles.

Bilirubin recognition via molecularly imprinted supermacroporous cryogels.

Recent years molecular imprinting has received considerable attention as an excellent and simple approach to recognize small molecules and bioactive substances. The aim of this study is to prepare the bilirubin-imprinted supermacroporous cryogels which can be used for the adsorption of bilirubin from human plasma. N-methacryloyl-(L)-tyrosinemethylester (MAT) was chosen as the pre-organization monomer. In the first step, bilirubin was complexed with MAT and the bilirubin-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-tyrosine methylester) [BR-MIP] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After that, the template molecules (i.e., bilirubin) were removed from the polymeric structure using sodium carbonate and sodium hydroxide. The maximum bilirubin adsorption amount was 3.6 mg/g polymer. The relative selectivity coefficients of the BR-MIP cryogel for bilirubin/cholesterol and bilirubin/testosterone mixtures were 7.3 and 3.2 times greater than non-imprinted poly(HEMA-MAT) [NIP] cryogel, respectively. The BR-MIP cryogel could be used many times without decreasing bilirubin adsorption amount significantly. Therefore, as a reusable carrier possessing high selectivity, BR-MIP cryogel has a potential candidate as a clinical hemoperfusion material.

Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels.

Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. The aim of this study is to prepare lysozyme-imprinted supermacroporous cryogels which can be used for the purification of lysozyme (Lyz) from egg white. N-Methacryloyl-(L)-histidinemethylester (MAH) was chosen as the metal-coordinating monomer. In the first step, Cu2+ was complexed with MAH and the lysozyme-imprinted poly(HEMA-MAH) [Lyz-MIP] cryogel were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) in an ice bath. After that, the template (i.e., lysozyme) was removed using 0.05 M phosphate buffer containing 1M NaCl (pH 8.0). The maximum lysozyme adsorption capacity was 22.9 mg/g polymer. The relative selectivity coefficients of Lyz-MIP cryogel for lysozyme/bovine serum albumin and lysozyme/cytochrome c were 4.6 and 3.2 times greater than non-imprinted poly(HEMA-MAH) (NIP) cryogel, respectively. Purification of lysozyme from egg white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 94% with recovery about 86%. The Lyz-MIP cryogel could be used many times without decreasing the adsorption capacity significantly.