S-citalopram imprinted monolithic columns for capillary electrochromatography enantioseparations.

In this study, the molecular imprinting method was used to separate enantiomeric forms of chiral antidepressant drug, R,S-citalopram (R,S-CIT) in aqueous solution by CEC system combining the advantages of capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). For that, an amino acid-based molecularly imprinted monolithic capillary column was designed and used as a stationary phase for selective separation of S-citalopram (S-CIT) for the first time. S-CIT was selectively separated from the aqueous solution containing the other enantiomeric form of R-CIT, which is the same in size and shape as the template molecule. Morphology of the molecularly imprinted (MIP S-CIT) and non-imprinted (NIP S-CIT) monolithic capillary columns was observed by scanning electron microscopy. Imprinting efficiency of MIP S-CIT monolithic capillary column used for selective S-CIT separation was verified by comparing with NIP S-CIT and calculated imprinting factor (I.F:1.81) proved the high selectivity of the MIP S-CIT for S-CIT. Cavities formed for S-CIT form enabled selective (α = 2.08) separation of the target molecule from the other enantiomeric R-CIT form. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 7.68 × 10-6 m2 /Vs for R,S-CIT at pH 7.0 10 mM PB and 50% ACN ratio. The performance of both MIP S-CIT and NIP S-CIT columns was estimated by repeating the R,S-CIT separations with intra-batch and inter-batch studies for reproducibility of retention times of R,S-CITs. Estimated RSD values that are lower than 2% suggest that the monolithic columns separate R,S-CIT enantiomers without losing separation efficiency.

Phenol removal from wastewater by surface imprinted bacterial cellulose nanofibres.

In this study, we have reported a novel wastewater treatment technique by phenol imprinted bacterial cellulose (BC-MIP) nanofibres with high specificity and adsorption capacity. N-methacryloyl-(L) phenylalanine methyl ester (MAPA) functional monomer was used to create specific binding sites for the template molecule phenol via electrostatic and hydrophobic interactions. BC-MIP nanofibres were synthesized by surface imprinting approach in the presence of different amounts of total monomer (% weight), monomer/template ratio and polymerization time. Then, the nanofibres were characterized by FTIR-ATR, surface area analysis (BET), elemental analysis, scanning electron microscopy (SEM) and contact angle measurements. Adsorption studies were performed with respect to pH, temperature and ionic strength, and the adsorption capacity was calculated by using the spectrophotometer. In order to desorb the adsorbed phenol from BC-MIP nanofibres, 0.1 M NaCl solution was used. Besides, BC-MIP nanofibres were applied to real wastewater samples from Ergene basin in Turkey. The suitable equilibrium isotherm was determined as Langmuir isotherm. To evaluate the selectivity of the BC-MIP nanofibres, similar molecules were utilized as competitor molecules, which were 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Electrostatic interactions were found to contribute to the generation of specific recognition binding sites. The results have shown that imprinting of phenol was achieved successfully with high adsorption capacity. The phenol removal efficiency was reported up to 97%. BC-MIP nanofibres were used 10 times with a negligible decrease in adsorption capacity.

Polymethacryloyl-L-Phenylalanine [PMAPA]-Based Monolithic Column for Capillary Electrochromatography.

The ability to detect catecholamines (CAs) and their metabolites is vital to understand the mechanism behind the neuronal diseases. Neurochemistry aims to provide an improved pharmacological, molecular and physiological understanding of complex brain chemistries by analytical techniques. Capillary electrophoresis (CE) is one such analytical technique that enables the study of various chemical species ranging from amino acids and peptides to natural products and drugs. CE can easily adapt the changes in research focus and in recent years remains an applicable technique for investigating neuroscience and single cell neurobiology. The prepared phenylalanine-based hydrophobic monolithic column, Polymethacryloyl-L-phenylalanine [PMAPA], was used as a stationary phase in capillary electrochromatography to separate CAs that are similar in size and shape to each other including dopamine (DA) and norepinephrine (NE) via hydrophobic interactions. Separation carried out in a short period of 17 min was performed with the electrophoretic mobility of 5.54 × 10-6 m2 V-1 s-1 and 7.60 × 10-6 m2 V-1 s-1 for DA and NE, respectively, at pH 7.0, 65% acetonitrile ratio with 100 mbar applied pressure by the developed hydrophobic monolithic column without needing any extra process such as imprinting or spacer arms to immobilize ligands used in separation.

Dopamine-imprinted monolithic column for capillary electrochromatography.

A dopamine-imprinted monolithic column was prepared and used in capillary electrochromatography as stationary phase for the first time. Dopamine was selectively separated from aqueous solution containing the competitor molecule norepinephrine, which is similar in size and shape to the template molecule. Morphology of the dopamine-imprinted column was observed by scanning electron microscopy. The influence of the organic solvent content of mobile phase, applied pressure and pH of the mobile phase on the recognition of dopamine by the imprinted monolithic column has been evaluated, and the imprinting effect in the dopamine-imprinted monolithic polymer was verified. Developed dopamine-imprinted monolithic column resulted in excellent separation of dopamine from structurally related competitor molecule, norepinephrine. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 5.81 × 10-5  m2 V-1 s-1 at pH 5.0 and 500 mbar pressure.

Molecular Imprinting of Macromolecules for Sensor Applications.

Molecular recognition has an important role in numerous living systems. One of the most important molecular recognition methods is molecular imprinting, which allows host compounds to recognize and detect several molecules rapidly, sensitively and selectively. Compared to natural systems, molecular imprinting methods have some important features such as low cost, robustness, high recognition ability and long term durability which allows molecularly imprinted polymers to be used in various biotechnological applications, such as chromatography, drug delivery, nanotechnology, and sensor technology. Sensors are important tools because of their ability to figure out a potentially large number of analytical difficulties in various areas with different macromolecular targets. Proteins, enzymes, nucleic acids, antibodies, viruses and cells are defined as macromolecules that have wide range of functions are very important. Thus, macromolecules detection has gained great attention in concerning the improvement in most of the studies. The applications of macromolecule imprinted sensors will have a spacious exploration according to the low cost, high specificity and stability. In this review, macromolecules for molecularly imprinted sensor applications are structured according to the definition of molecular imprinting methods, developments in macromolecular imprinting methods, macromolecular imprinted sensors, and conclusions and future perspectives. This chapter follows the latter strategies and focuses on the applications of macromolecular imprinted sensors. This allows discussion on how sensor strategy is brought to solve the macromolecules imprinting.

Synthesis of hydrophobic nanoparticles for real-time lysozyme detection using surface plasmon resonance sensor.

Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR-based sensor is developed to detect lysozyme with hydrophobic poly (N-methacryloyl-(L)-phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection-Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM-1 , and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real-time, label-free, and a low-detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.

[PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma.

The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

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.

Triazine herbicide imprinted monolithic column for capillary electrochromatography.

Trietazine was selectively separated from aqueous solution containing the competitor molecule cyanazine, which is similar in size and shape to the template molecule. Structural features of the molecularly imprinted column were figured out by SEM. The influence of the mobile-phase composition, applied electrical field, and pH of the mobile phase on the recognition of trietazine by the imprinted monolithic polymer has been evaluated, and the imprint effect in the trietazine-imprinted monolithic polymer was demonstrated by an imprinting factor. The optimized monolithic column resulted in separation of trietazine from a structurally related competitor molecule, cyanazine. In addition, fast separation was obtained within 6 min by applying higher electrical field, with the electrophoretic mobility of 2.97 × 10(-8) m(2) V(-1) s(-1) at pH 11.0.

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.

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.

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.