Determination of carcinoembryonic antigen (CEA) by label-free electrochemical immunosensor using functionalized boron nitride nanosheets.

In this study, a simple, specific and sensitive immunosensor for CEA detection was prepared based on BN nanosheets. Lewis acid-base interaction was sufficient for the immobilization of anti-CEA used as an antibody on the electrode surface without an activation agent. This immunosensor could be used for CEA determination without the need to use any label or secondary antibody. With its epedance-based recognition mechanism, this immunosensor offered a low LOD value of 0.017 ng/mL and a wide measurement range of 0.1-500 ng/mL and could be used for a long time. The analytical performance of this immunosensor is higher than the biosensors prepared in the literature. Compared to commercially available kits, it is attractive because it is cheap, simple and analyzes in a short time. This immunosensor, which has high selectivity against CEA in the presence of competitive agents in commercial human serum, has a high potential for clinical applications.

Immobilization of Urokinase onto Magnetically Directed Micromotors.

In this presented work, a new micromotor was prepared for urokinase immobilization. A covalent bond was constructed between the urokinase and the carboxyl groups of the graphene oxide, which is located at the outer layer of micromotors by EDC/NHS chemistry. The inner nickel layer gave magnetic properties to the micromotors and enables them to be separated from the reaction medium with the help of a simple magnet. For promising in vivo applications in the future, these micromotors do not require any fuel for their movement. The structures of the synthesized micromotors were illuminated by SEM and EDX analysis, and the movements of the micromotors were observed under an optical microscope with camera equipment. The immobilization yield of urokinase was found to be 68.07% (0.073 mg/100 µL micromotor solution) using the Bradford protein assay. In addition, to compare the activities of the immobilized and free enzymes, Lineweaver-Burk plots were constructed, and the kinetic parameters were calculated. Km values for free urokinase and immobilized urokinase were 2.0964 mM and 0.5602 mM, respectively. The maximum velocities of free and immobilized urokinase were found to be 25.25 µmol/min and 30.12 µmol/min, respectively. Also, storage stability profiles of the immobilized and free urokinase were monitored for 40-day incubation at + 4 °C, and the immobilized enzyme has 88% of its initial activity, while the free urokinase demonstrated only 30% of its initial activity. As a result, the experiments were carried out in human commercial serum, and specific activity values for free urokinase and immobilized urokinase were found to be 38.06 and 169.84 µmolmg-1 min-1, respectively.

Boron nitride nanosheet modified label-free electrochemical immunosensor for cancer antigen 125 detection.

In this presented study, a new boron nitride nanosheets modified label-free electrochemical immunosensors were prepared for early detection of cancer antigen 125 (CA125). To aim for, boron nitride (BN) nanosheets were synthesized by conventional sonication-assisted method and then characterized. BN nanosheets were used for the surface modification of the working electrode of the screen-printed electrode (SPE). Anti CA125 antibody was then directly immobilized onto the electrode surface due to its natural affinity towards BN nanosheets. Modified electrodes were blocked with BSA and finally protected with Nafion. The newly synthesized label-free immunosensor demonstrated good detection properties to CA125 with a linear range of 5-100 U and a detection limit of 1.18 U/mL. The developed immunosensor also showed excellent reproducibility, selectivity, and stability profiles. Additionally, this immunosensor was successfully used for the detection of CA125 in artificial human serum samples along with the interfering agents. Also, it is expected that the prepared immunosensor should carry the good potential for point-of-care diagnosis in real cases.

Immobilization of L-Asparaginase on Magnetic Nanoparticles for Cancer Treatment.

In this presented work, magnetic poly(HEMA-GMA) nanoparticles were synthesized, characterized, and used for immobilization of an anti-leukemic enzyme L-asparaginase. The average particle size of the synthesized magnetic nanoparticles was found to be as 117.5 nm. L-asparaginase was successfully immobilized onto the synthesized magnetic nanoparticles, and attached amount of L-asparaginase was found to be as 66.43 mg/g nanoparticle. The effects of the medium pH, temperature, and substrate concentration on the L-asparaginase activity were also tested. Optimum pH of the free and immobilized L-asparaginase was found to be as 7.5 and 6.5, respectively. Optimum temperature of the free L-asparaginase was 45 °C, while optimum temperature was shifted to 55 °C after immobilization onto the magnetic nanoparticles. Also, kcat value of free L-asparaginase (47,356 min-1) was calculated to be higher than that of immobilized L-asparaginase (497 min-1). Thermal stability of both asparaginase preparation was followed for 10 h, and at the end of the incubation time, free asparaginase almost lost its activity, while immobilized asparaginase protected 50% of its initial activity. Storage stabilities of free and immobilized asparaginase were also tested, and at the end of the 40 days storage, free asparaginase lost all of its activity, while immobilized asparaginase still showed 30% activity. Operational stability of immobilized asparaginase was tested for 8 successive usage, and immobilized asparaginase lost only 15% of its initial activity. In present study, activities of free and immobilized L-asparaginase were tested in artificial human serum medium, to foresee the in vivo efficiency, and it was demonstrated here that immobilized L-asparaginase protected its 74.74% of its initial activity in artificial serum medium.

Peroxidase Immobilized Cryogels for Phenolic Compounds Removal.

In this presented work, preparation of poly(AAm) cryogel, peroxidase immobilization onto the poly(AAm) cryogel, and usability of these enzyme modified cryogels for phenolic compounds removal were described. For this purpose, poly(AAm) cryogels were synthesized by using cryocopolymerization technique at sub-zero temperatures, and covalently functionalized with peroxidase enzyme by EDC/NHS chemistry. Characterization of the cryogels was carried out by FTIR, SEM, and EDX analysis. Maximum peroxidase loading onto poly(AAm) cryogel was found to be as 127.30 mg/g cryogel. Kinetic parameters of free and immobilized peroxidases were also investigated along with the stability tests. Finally, phenolic compounds removal efficiency of the peroxidase immobilized poly(AAm) cryogel was studied towards model phenolics such as phenol, bisphenol A, guaiacol, pyrogallol, and catechol; and very high phenolic removal efficiency was recorded.

Quercetin adsorption with imprinted polymeric materials.

In this study, molecularly imprinted polymer membranes were synthesized for the recognition and adsorption of quercetin. For this, quercetin imprinted polymeric membranes [p(HEMA-MAH)] (Poly(2-hydroxyethyl methacrylate-co-N-methacryloly-l-histidinemethylester) were synthesized by UV polymerization technique using HEMA and MAH as monomers. Synthesized polymeric membranes were characterized with SEM, FTIR and swelling test. Characterized membranes were used for the direct adsorption of quercetin in a batch system. Quercetin adsorption conditions were optimized by using the quercetin imprinted polymeric membrane by altering the pH, temperature and initial quercetin concentration of the adsorption medium. Effect of adsorption time was also studied for up to 180 min. The optimum pH and temperature was determined between 4.0 and 45 °C. Maximum adsorbed amount of quercetin onto quercetin imprinted poly(HEMA-MAH) membrane was found to be as 299.6 mg/g membrane using the initial quercetin concentration of 2.0 mg/ml. Adsorbed quercetin was desorbed from the polymeric membranes with isopropyl alcohol with desorption yield of 98.3%. and repeated usability of the quercetin imprinted polymeric membranes was fallowed for 7 adsorption/desorption cycles. At the end of the 7th reuse, quercetin adsorption capacity of the quercetin imprinted poly(HEMA-MAH) membranes decreased only about 10%.

Antibody separation using lectin modified poly(HEMA-EDMA) hydrogel membranes.

Herein we describe the synthesis of Concanavalin A-poly(2-hydroxyethyl methacrylate-ethylene dimethacrylate) hydrogel membranes (via photopolymerization technique) for antibody separation from aqueous solutions. Different characterization techniques including Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy, Elemental Analysis and swelling tests revealed the highly rough morphology and spherical shape of the synthetized membranes. Attached amount of IMEO (salinization agent) onto polymeric structure and Con A binding capacity were found to be 10.85 mol/g and 3.52 mg/g, respectively. Optimum conditions for IgG adsorption such as adsorption capacity, pH and reusability profile of HMs were judiciously characterized. Maximum IgG adsorption capacity of hydrogel membrane was found to be as 26.81 mg/g. Adsorbed IgG was eluted successfully by using 2.0 M of NaCl solution. Reusability profiles of hydrogel membrane in five adsorption-desorption cycles revealed that there was no significant decrease in IgG adsorption capacity at the end of the 5th reuse. The hydrogel membranes reported here hold considerable promise as an effective sorbent system for IgG adsorption with good stability and efficient repeated usage.

Synthesis and characterization of albumin imprinted polymeric hydrogel membranes for proteomic studies.

In this presented study, a novel molecularly imprinted polymeric hydrogel membranes (PHMs) were developed to use for the albumin depletion studies. For this, albumin imprinted poly(2-hydroxyethyl methacrylate-N-methacryloyl-(L)-phenylalanine methyl ester) polymeric hydrogel membranes [p(HEMA-MAP) PHMs] were synthesized by the photopolymerization technique, and then characterized by SEM, EDX, FT-IR and swelling studies. Synthesized PHMs had spherical structure and the MAP monomer incorporation onto the PHMs was determined by EDX analysis by using nitrogen stoichiometry. Also, the swelling ratio of the albumin imprinted p(HEMA-MAP) PHMs was determined as 215%. The optimum albumin adsorption condition (adsorption capacity, medium pH, adsorption rate, temperature, ionic strength) were studied and the maximum albumin adsorption capacity was found to be as 34.28 mg/g PHMs. Selectivity experiments were also carried out with the presence of the competitive proteins such as lysozyme and amylase, and the results demonstrated that the albumin imprinted p(HEMA-MAP) PHMs showed high affinity towards the BSA molecules than the competitive proteins of lysozyme and amylase. Adsorbed albumin was desorbed from the PHMs by 1.0 M of NaCl, and the reusability of the imprinted PHMs was also demonstrated for five successive adsorption-desorption cycles without any significant loss in the albumin adsorption capacity. As an application, sodium-dodecyl sulfate polyacrylamide gel electrophoresis was used to indicate the albumin depletion efficiency of albumin imprinted p(HEMA-MAP) PHMs. This presented study showed that, these imprinted membranes are promising for proteomic studies and applications, and can be used for the investigations for human diagnostics.

A new support material for IgG adsorption: Syntrichia papillosissima (Copp.) Loeske.

In this presented work, Syntrichia papillosissima (Copp.) Loeske (S. papillosissima) was used as a natural phytosorbent for IgG purification. These moss species were collected for the natural habitat and prepared for IgG adsorption studies by cleaning, drying, and grinding to uniform size. Syntrichia papillosissima samples were characterized by using FTIR and SEM studies. Functional groups of S. papillosissima were identified by FTIR analysis, while surface characteristics were determined by SEM studies. A batch system was used for the adsorption of IgG onto S. papillosissima surface and physical conditions of the IgG adsorption medium were investigated by modifying the pH, IgG concentration and temperature. Maximum IgG adsorption onto S. papillosissima was found to be 68.01 mg/g moss by using pH 5.0 buffer system. Adsorption kinetic isotherms were also studied and it was found that, Langmuir adsorption model was appropriate for this adsorption study. Reusability profile of S. papillosissima was also investigated and IgG adsorption capacity did not decrease significantly after 5 reuse studies. Results indicated that S. papillosissima species have the capacity to be used as biosorbent for IgG purification, with its low cost, natural and biodegradable structure.

Controlled release of curcumin from poly(HEMA-MAPA) membrane.

In this work, poly(HEMA-MAPA) membranes were prepared by UV-polymerization technique. These membranes were characterized by SEM, FTIR, and swelling studies. Synthesized membranes had high porous structure. These membranes were used for controlled release of curcumin which is already used as folk remedy and used as drug for some certain diseases and cancers. Curcumin release was investigated for various pHs and temperatures. Optimum drug release yield was found to be as 70% at pH 7.4 and 37 °C within 2 h period. Time-depended release of curcumin was also investigated and its slow release from the membrane demonstrated within 48 h.

DNA isolation by galactoacrylate-based nano-poly(HEMA-co-Gal-OPA) nanopolymers.

Isolation of DNA is one of the important processes for biotechnological applications such as investigation of DNA structures and functions, recombinant DNA preparations, identification of genetic factors and diagnosis and treatment of genetic disorders. The aim of this study was to synthesis and characterizes the galactoacrylate based nanopolymers with high surface area and to investigate the usability of these synthesized nanopolymers for DNA isolation studies. Nanopolymers were synthesized by the surfactant free emulsion polymerization technique by using the monomers of 2-hydroxyl ethylmethacrylate and 6-O-(2'-hydroxy-3'-acryloyloxypropyl)-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose. Galactoacrylate origin of these newly synthesized nanopolymers increased the interaction between DNA and nanopolymers. Prepared nanopolymers were characterized by SEM, FT-IR and ZETA sizer analysis. Synthesized nanopolymers were spherical, and their average particle size was about 246.8 nm. Adsorption of DNA onto galactoacrylate based nanopolymers was investigated by using different pHs, temperatures, ionic strength, DNA concentrations and desorption studies and maximum DNA adsorption was found to be as 567.12 mg/g polymer at 25 °C, in pH 5.0 acetate buffer. Reusability was investigated for 5 successive reuse and DNA adsorption capacity decreased only about 10% at the end of the 5th reuse.

Boronate affinity nanoparticles for nucleoside separation.

Boronate affinity systems have been recently used for the specific isolation of cis-diol group carrying biomolecules such as glycoproteins, nucleosides, carbohydrates. Nanosized materials have been extremely used for the biotechnological purposes due to their unique properties and their high surface areas. The objective of this presented work was to develop a new boronate affinity system for the nucleoside adsorption. For this purpose, poly(HEMA) nanoparticles were synthesized by using surfactant free emulsion polymerization technique and then functionalized with phenylboronic acid. Synthesized nanoparticles were characterized with FTIR, SEM, and Zeta size analysis. Nucleic acid adsorption experiments were repeated for different medium pH values, for various nucleosides concentrations, for different temperatures and ionic strengths, in order to determine the optimum adsorption conditions. In the light of these studies, it can be concluded that this boronate ligand carrying nanoparticles were very valuable for the separation of nucleosides.

Reversible adsorption of catalase onto Fe(3+) chelated poly(AAm-GMA)-IDA cryogels.

In this presented study, poly(acrylamide-glycidyl methacrylate) [poly(AAm-GMA)] cryogels were synthesized by cryopolymerization technique at sub-zero temperature. Prepared cryogels were then functionalized with iminodiacetic acid (IDA) and chelated with Fe(3+) ions in order produce the metal chelate affinity matrix. Synthesized cryogels were characterized with FTIR, ESEM and EDX analysis, and it was found that the cryogel had sponge like structure with interconnected pores and their pore diameter was about 200 µm. Fe(3+) chelated poly(AAm-GMA)-IDA cryogels were used for the adsorption of catalase and optimum adsorption conditions were determined by varying the medium pH, initial catalase concentration, temperature and ionic strength. Maximum catalase adsorption onto Fe(3+) chelated poly(AAm-GMA)-IDA cryogel was found to be 12.99 mg/g cryogel at 25 °C, by using pH 5.0 acetate buffer. Adsorbed catalase was removed from the cryogel by using 1.0M of NaCl solution and desorption yield was found to be 96%. Additionally, reusability profile of the Fe(3+) chelated poly(AAm-GMA)-IDA cryogel was also investigated and it was found that, adsorption capacity of the cryogels didn't decrease significantly at the end of the 40 reuses. Catalase activity studies were also tested and it was demonstrated that desorbed catalase retained 70% of its initial activity.

Immobilization of amyloglucosidase onto macroporous cryogels for continuous glucose production from starch.

Poly(methyl methacrylate-glycidyl methacrylate) [Poly(MMA-GMA)] cryogels were synthesized using monomers of methylmethacrylic acid and epoxy group bearing GMA via radical cryopolymerization technique. Synthesized cryogels were used for the immobilization of amyloglucosidase to the cryogel surface using epoxy chemistry. Characterizations of the free and immobilized amyloglucosidase were carried out by comparing the optimum and kinetic parameters of enzymes. For this, pH and temperature profiles of free and immobilized preparation were studied and, it was found that, optimum pH of enzyme was not change upon immobilization (pH 5.0), while optimum temperature of the enzyme shifted 10 °C to warmer region after immobilization (optimum temperatures for free and immobilized enzyme were 55 and 65 °C, respectively). Kinetic parameters of free and immobilized enzyme were also investigated and Km values of free and immobilized amyloglucosidase were found to be 2.743 and 0.865 mg/mL, respectively. Vmax of immobilized amyloglucosidase was found to be (0.496 µmol/min) about four times less than that of free enzyme (2.020 µmol/min). Storage and operational stabilities of immobilized amyloglucosidase were also studied and it was showed that immobilized preparation had much more stability than free preparation. In the present work, amyloglucosidase immobilized poly(MMA-GMA) cryogels were used for continuous glucose syrup production from starch for the first time. Efficiency of immobilized enzyme was investigated and released amount of glucose was found to be 2.54 mg/mL at the end of the 5 min of hydrolysis. The results indicate that the epoxy functionalized cryogels offer a good alternative for amyloglucosidase immobilization applications with increased operational and thermal stability, and reusability. Also, these cryogels can be used for immobilization of other industrially valuable enzymes beyond amyloglucosidase.

Lysozyme-Based Antibacterial Nanomotors.

An effective and rapid bacterial killing nanotechnology strategy based on lysozyme-modified fuel-free nanomotors is demonstrated. The efficient antibacterial property of lysozyme, associated with the cleavage of glycosidic bonds of peptidoglycans present in the bacteria cell wall, has been combined with ultrasound (US)-propelled porous gold nanowire (p-AuNW) motors as biocompatible dynamic bacteria nanofighters. Coupling the antibacterial activity of the enzyme with the rapid movement of these p-AuNWs, along with the corresponding fluid dynamics, promotes enzyme-bacteria interactions and prevents surface aggregation of dead bacteria, resulting in a greatly enhanced bacteria-killing capability. The large active surface area of these nanoporous motors offers a significantly higher enzyme loading capacity compared to nonporous AuNWs, which results in a higher antimicrobial activity against Gram-positive and Gram-negative bacteria. Detailed characterization studies and control experiments provide useful insights into the underlying factors controlling the antibacterial performance of the new dynamic bacteria nanofighters. Rapid and effective killing of the Gram-positive Micrococcus lysodeikticus bacteria (69-84% within 1-5 min) is demonstrated.

Purification of alcohol dehydrogenase from Saccharomyces cerevisiae using magnetic dye-ligand affinity nanostructures.

Reactive Green 19 was covalently immobilized onto magnetic nanostructures for purification of alcohol dehydrogenase from Saccharomyces cerevisiae. The Reactive Green 19 immobilized magnetic nanostructures were characterized by Fourier transform infrared spectroscopy, electron spin resonance, atomic force microscope, and energy dispersive X-ray analysis. Particle size of nanostructures was found to be roughly 70 nm. Alcohol dehydrogenase adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, alcohol dehydrogenase concentration, temperature, and ionic strength). Maximum alcohol dehydrogenase adsorption capacity was found to be 176.09 mg/g polymer while nonspecific alcohol dehydrogenase adsorption onto plain magnetic nanostructures was negligible (19.4 mg/g polymer). Alcohol dehydrogenase molecules were desorbed by using 1.0 M NaCl with 98.4 % recovery. Alcohol dehydrogenase from S. cerevisiae was purified 45.63-fold in single step with dye-immobilized magnetic nanostructures, and purity of alcohol dehydrogenase was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.