Surface plasmon resonance aptasensor for Brucella detection in milk.

A Surface Plasmon Resonance (SPR) aptasensor was developed for the detection of Brucella melitensis (B. melitensis) in milk samples. Brucellosis is a bacterial zoonotic disease with global distribution caused mostly by contaminated milk or their products. Aptamers recognizing B. melitensis were selected following a whole bacteria-SELEX procedure. Two aptamers were chosen for high affinity and high specificity. The high affinity aptamer (B70 aptamer) was immobilized on the surface of magnetic silica core-shell nanoparticles for initial purification of the target bacteria cells from milk matrix. Another aptamer, highly specific for B. melitensis cells (B46 aptamer), was used to prepare SPR sensor chips for sensitive determination of Brucella in eluted samples from magnetic purification since direct injection of milk samples to SPR sensor chips is known for a high background unspecific signal. Thus, we integrated a quick and efficient magnetic isolation step for subsequent instant detection of B. melitensis contamination in one ml of milk sample by SPR with a LOD value as low as 27 ± 11 cells.

Fibrous polymer functionalized magnetic biocatalysts for improved performance.

Although enzymes are known for their excellent catalytic performance, industrial, medical or biotechnological applications should overcome some drawbacks like long-term stability under specific conditions of the application. Immobilized enzymes have offered advantages over soluble counterparts in many industrial and laboratory scale applications by increasing operational stability and reusability. When the immobilization matrix has magnetic properties, an additional advantage is obtained as simpler processing. Iron-based superparamagnetic nano-sized particles has large surface area for bio-compatible applications are especially in focus. Adding nanofibrous polymers to magnetic nanoparticles has been an excellent way to increase efficiency of biocatalyst immobilization by further increasing loading capacity. This chapter explains various magnetic enzyme-nanoparticles based preparations with potential for future industrial applications like invertase, lipase and as model studies and focus on the nanofibrous polymer brush grafting as a way to increase catalytic efficiency of magnetic nanoparticles.

Hydrophilic spacer-arm containing magnetic nanoparticles for immobilization of proteinase K: Employment for speciation of proteins for mass spectrometry-based analysis.

Proteinase K (ProK) is used for the degradation of proteins in cell lysates to isolate nucleic acids, and for the speciation of proteins for mass spectrometry analysis. In this work, a novel and sensitive immobilization process was developed for examination of protein mixtures by combining MALDI-ToF-MS and nLC-TIMS-ToF-MS/MS systems. To achieve these goals, magnetic nanoparticles (MPs) were prepared via thermal coprecipitation reaction under alkaline condition. The MPs were grafted with a silica layer (i.e., 3-(2,3-epoxypropoxy) propyltrimethoxysilane; EPTES) containing reactive epoxy groups. Then, the silica-grafted magnetic particles were coated with a long chain hydrophilic poly(ethylene glycol) diamine polymer (PEGDAP). The prepared materials were characterized by the Brunauer-Emmett-Teller (BET) method, X-ray diffraction (XRD), scanning electron microscopy (SEM) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The VSM data show that the MPs@EPTES@PEGDAP has paramagnetic performance with a saturation magnetization of approximately 32.3 emu g-1. Proteinase K (EC 3.4.21.64) was covalently immobilized on the MPs@EPTES by reaction of its epoxy groups with amine groups of the enzyme. On the other hand, the ProK was immobilized on the MPs@EPTES@PEGDAP after activation with glutaraldehyde and the immobilization reaction was realized by the coupling reaction between aldehyde groups of the support and amine groups of the enzyme. The amounts of immobilized ProK on the MPs@EPTES and MPs@EPTES@PEGDAP were found to be 27.4 and 19.6 mg g-1and the retained activities were determined to be 29 and 87%, respectively. For the first time, some important features such as thermal and storage stabilities, reusability and potential use in protein speciation for mass spectrometry-based techniques were also evaluated. For examples, after six weeks of storage at 4 °C, the immobilized ProK on the MPs@EPTES@PEGDAP-ProK still maintained 59% of its initial activity. However, at the end of the six-week storage period, its free counterpart had lost all of its initial activity. The immobilized ProK was also utilized for degradation and identification of model proteins (i.e., α-2-HS glycoprotein, ß-casein, bovine serum albumin and immunoglobulin). After enzymatic treatment, the digested peptides were analyzed and mapped by using nLC-TIMS-ToF-MS/MS systems.

Catalytic Activity of Immobilized Chymotrypsin on Hybrid Silica-Magnetic Biocompatible Particles and Its Application in Peptide Synthesis.

In this work, novel silica hybrid magnetic particles with biocompatible surface were designed as a support for enzyme immobilization, and the immobilized chymotrypsin (CT) performance was clarified as a model biocatalyst. CT is used in food technology for drink clarification and protein hydrolysis. The enzyme was directly immobilized onto polydopamine-grafted magnetic silica particles (MNP@SiO2@PDA-CT) via the Schiff base reaction. Immobilized enzyme had broadened for both pH and temperature profiles compared with the native CT. The MNP@SiO2@PDA-CT system also improved in thermostability compared with the native enzyme. The immobilized CT was operated in a continuous enzyme reactor (CER) for the hydrolysis of different proteins (i.e., cytochrome c (Cyt c), human serum albumin (HSA), human immunoglobulin G (HIgG), and lysozyme (Lys)). The peptide synthesis rate was shown to decrease as a function of increasing flow rate. The catalytic activity of the CER remained stable for 6.0 h in a continuous operation period; thus, the presented method may increase applicability in the food technology area. The immobilized CT in the CER showed a good hydrolysis performance for all the tested model proteins. The peptides hydrolyzed from the tested proteins were analyzed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Results show that the MNP@SiO2@PDA-CT system also permits for the applicability in the area of proteomic research.

Rapid and label-free detection of Brucella melitensis in milk and milk products using an aptasensor.

In this work, a novel quartz crystal microbalance (QCM) aptasensor is designed for the diagnosis of Brucella melitensis bacteria, which affects the Mediterranean fever (brucellosis) from the zoonotic diseases that are very common in the Middle East Countries. The method is based on the selection of B. melitensis bacterium from solutions using B. melitensis specific binding aptamer (Apt) attached magnetic nanoparticles. The surface of the magnetic nanoparticles (i.e.,Fe3O4) was modified by 3-aminopropyltriethoxysilane (APTES) and then grafted with a hydrophilic macromonomer poly(ethyleneglycol)-methacrylate (PEG-MA) as a first block polymer and glycidylmethacrylate (GMA) as a second block functional polymer via atom transfer radical polymerization (ATRP) method [Fe3O4 @SiO2 @p(PEG-MA-GMA)], then, the specific binding aptamer was immobilized. The aptamer immobilized magnetic nanoparticles were used for the pre-concentration of the target bacterium, and the same aptamer sequence was also immobilized on the QCM chip and used for the quantitative detection of B. melitensis using QCM aptasensor. The detection limits of the QCM aptasensor were in the range 1.02-1.07 CFU mL-1, with recoveries up to 79%. The synthesized [Fe3O4 @SiO2 @p(PEGMA-GMA)] nanoparticles showed a good permanence and high isolation recoveries for the pull down of the target bacterium from food samples, after recycling eight times. The method was successfully applied to target bacterium determinations in milk and milk product samples.

Biodegradation of Cibacron Blue 3GA by insolubilized laccase and identification of enzymatic byproduct using MALDI-ToF-MS: Toxicity assessment studies by Daphnia magna and Chlorella vulgaris.

The presented paper describes a detailed study on the use of immobilized laccase for effective degradation of Cibacron Blue 3GA dye. The amount of laccase loading on the cyclic carbonate groups containing poly(hydroxyethyl methacrylate-co-vinylene carbonate), p(HEMA-co-VC), microbeads was 27.8 mg g-1, and the retained immobilized enzyme activity was 73% compared to free enzyme. The toxicity of the dye and its byproducts were studied using Daphnia magna as test organism. The micro-algal growth inhibition was also studied using a green micro algae "Chlorella vulgaris". MALDI-ToF-MS was used to verify dye degradation byproducts. After 60 min of incubation period, Cibacron Blue 3GA (CB3GA) and its byproducts disappeared from the medium. After 60-min enzymatic treatment, the non-toxic nature of medium was confirmed by toxicity studies. On the other hand, the initial byproducts of the dye seemed to be more toxic than the later formed dye products. It should be noted that the information obtained from this study can be beneficial for understanding the initial degradation byproducts toxicities of the enzymatically treated dyes to provide information about environmental protection.

Design of an aptamer-based magnetic adsorbent and biosensor systems for selective and sensitive separation and detection of thrombin.

An aptasensor was designed for sensitive detection of thrombin using in biological fluids by integrating a magnetic aptamer-microbeads. To achieve this goal, the surface of gold plated QCM crystals was coated with L-cysteine and a thrombin binding DNA aptamer was immobilized on the L-cysteine coated QCM crystals surface via glutaraldehyde coupling. The binding interactions of thrombin to QCM crystals were characterized. Magnetic poly(2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate-vinylene carbonate), Mp(HEMA-EGDMA-VC) microbeads were synthesized and thrombin binding aptamer (TBA) was immobilized. The Mp(HEMA-EGDMA-VC)-TBA microbeads were effectively adsorbed thrombin from serum in a relatively short contact time (ca. 5.0 min), and the eluted protein from Mp(HEMA-EGDMA-VC)-TBA was transferred to the QCM aptasensor that showed a specific detection of thrombin from serum. The detection limit of thrombin using aptasensor was 1.00 nmol L-1. The calculation dissociation constant of the aptasensor was 68.5 nmol L-1. The selectivity of the aptasensor system was tested with three different proteins (i.e., elastin, immunoglobulin G (IgG) and human serum albumin (HSA)) and showed high specificity to thrombin. The aptasensor was regenerated by washing with NaOH solution, and repeatedly used until 20 cycles without a change in the performance.

Fast and Sensitive Detection of Salmonella in Milk Samples Using Aptamer-Functionalized Magnetic Silica Solid Phase and MCM-41-Aptamer Gate System.

General detection methods for S. enterica include PCR analysis, immunologic methods, solid culturing techniques, and various microscopic studies. Milk and other food samples demonstrate an especially difficult challenge for direct detection, resulting from high biological contents. In this report, we aimed for fast detection of pathogen cells through an efficient magnetic capture and subsequent quick detection based on aptamer affinity. The Fe3O4@SiO2@pGMA and MCM-41 particles were prepared separately and used for preconcentration and detection, respectively. Aptamer oligonucleotide sequences against S. enterica were fixed on both amine-functionalized MCM-41 and Fe3O4@SiO2@pGMA particles via glutaraldehyde coupling. The captured Salmonella cells were determined by a fluorescent homogeneous assay in the samples by aptamer-gated MCM-41 silica particles. Our method achieved a sensitive assay to detect Salmonella cells in milk samples as low as 103 CFU/ml without any culturing. Hence, the proposed sensing strategy might be an efficient platform for pathogen detection in a food matrix.

Improvement stability and performance of invertase via immobilization on to silanized and polymer brush grafted magnetic nanoparticles.

In this study, magnetic nanoparticles (Fe3O4) were modified sequentially with silica (Fe3O4@SiO2), glycidyl methacrylate (GMA) by surface initiated atom transfer radical polymerization (SI-ATRP) and hexamethylene diamine (as a spacer arm). The p(GMA) grafted and SA modified form (i.e., Fe3O4@SiO2@pGMA-SA-3) was used for covalent immobilization of invertase (EC 3.2.1.26). The amount of immobilized enzyme on Fe3O4@SiO2@p(GMA) and Fe3O4@SiO2@p(GMA)-SA-3 was 36.1±0.9 and 33.4±1.3mg/g, respectively. The Km and Vmax values of immobilized invertase were found to be 39.4mmol/L and 349.5mmol/L min, and not significantly changed compared with free form (34.3mmol/L and 387.2mmol/Lmin), respectively, revealed that the applied protocol did not have any detrimental effect on the retained activity of immobilized invertase.

Aminopyridine modified Spirulina platensis biomass for chromium(VI) adsorption in aqueous solution.

Chemical modification of Spirulina platensis biomass was realized by sequential treatment of algal surface with epichlorohydrin and aminopyridine. Adsorptive properties of Cr(VI) ions on native and aminopyridine modified algal biomass were investigated by varying pH, contact time, ionic strength, initial Cr(VI) concentration, and temperature. FTIR and analytical analysis indicated that carboxyl and amino groups were the major functional groups for Cr(VI) ions adsorption. The optimum adsorption was observed at pH 3.0 for native and modified algal biomasses. The adsorption capacity was found to be 79.6 and 158.7 mg g(-1), for native and modified algal biomasses, respectively. For continuous system studies, the experiments were conducted to study the effect of important design parameters such as flow rate and initial concentration of metal ions, and the maximum sorption capacity was observed at a flow rate of 50 mL h(-1), and Cr(VI) ions concentration 200 mg L(-1) with modified biomass. Experimental data fitted a pseudo-second-order equation. The regeneration performance was observed to be 89.6% and 94.3% for native and modified algal biomass, respectively.

Staphylococcus aureus detection in blood samples by silica nanoparticle-oligonucleotides conjugates.

A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood.

Removal of bisphenol A from aqueous medium using molecularly surface imprinted microbeads.

The aim of this study is to prepare bisphenol A (BPA) imprinted polymers, which can be used for the selective removal of BPA from aqueous medium. The BPA-imprinted (MIP) and non-imprinted (NIP) microbeads were synthesized, and characterized by Zeta-sizer, FTIR, SEM and BET method. Bisphenol A was determined in solutions using liquid chromatography-mass spectroscopy (LC-MS). The effect of initial concentration of BPA, the adsorption rate and the pH of the medium on the capacity of BPA-imprinting polymer were studied. Adsorption capacity of BPA was affected by the amount of the incorporated functional monomer in the polymer network. BPA adsorption capacity of MIP-3 and NIP microbeads from aqueous medium was estimated as 76.7 and 59.9 mg g(-1), respectively. The binding efficiencies of BPA-MIP-3 microbeads for different phenolic compounds (i.e., BPA with p-toluidine, 4-aminophenol or 2-naphthol) were explored at binary solutions, and the binding capacities of BPA-imprinted microbeads were found to be 2.79 × 10(-1), 2.39 × 10(-1), 7.59 × 10(-2) and 5.48 × 10(-2) mmol g(-1) microbeads, respectively. The satisfactory results demonstrated that the obtained BPA-MIP microbeads showed an appreciable binding specificity toward BPA than similar structural compounds in the aqueous medium. Moreover, the reusability of BPA-MIP-3 microbeads was tested for several times and no significant loss in adsorption capacity was observed. Finally, the binary and multi-component systems results show that MIP-3 microbeads have special recognition selectivity and excellent binding affinity for template molecule "BPA".

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor.

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity.

Study of polyethyleneimine- and amidoxime-functionalized hybrid biomass of Spirulina (Arthrospira) platensis for adsorption of uranium (VI) ion.

This study investigates the potential application of the polyethyleneimine- (PEI) and amidoxime-modified Spirulina (Arthrospira) platensis biomasses for the removal of uranium ion in batch mode using the native biomass as a control system. The uranium ion adsorption was also characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra, zeta potential analysis, and surface area measurement studies. The effects of pH, biomass amount, contact time, initial uranium ion concentration, and ionic strength were evaluated by using native and modified algal biomass preparations. The uranium ion removal was rapid, with more than 70% of total adsorption taking place in 40 min, and equilibrium was established within 60 min. From the experimental data, it was found that the amount of adsorption uranium ion on the algal preparations decreased in the following series: amidoxime-modified algal biomass > PEI-modified algal biomass > native algal biomass. Maximum adsorption capacities of amidoxime- and PEI-modified, and native algal biomasses were found to be 366.8, 279.5, and 194.6 mg/g, respectively, in batchwise studies. The adsorption rate of U(VI) ion by amidoxime-modified algal biomass was higher than those of the native and PEI-modified counterparts. The adsorption processes on all the algal biomass preparations followed by the Dubinin-Radushkevitch (D-R) and Temkin isotherms and pseudo-second-order kinetic models. The thermodynamic parameters were determined at four different temperatures (i.e., 15, 25, 35, and 45 °C) using the thermodynamics constant of the Temkin isotherm model. The ΔH° and ΔG° values of U(VI) ion adsorption on algal preparations show endothermic heat of adsorption; higher temperatures favor the process. The native and modified algal biomass preparations were regenerated using 10 mM HNO3. These results show that amidoxime-modified algal biomass can be a potential candidate for effective removal of U(VI) ion from aqueous solution.

Fibrous polymer grafted magnetic chitosan beads with strong poly(cation-exchange) groups for single step purification of lysozyme.

Lysozyme is an important polypetide used in medical and food applications. We report a novel magnetic strong cation exchange beads for efficient purification of lysozyme from chicken egg white. Magnetic chitosan (MCHT) beads were synthesized via phase inversion method, and then grafted with poly(glycidyl methacrylate) (p(GMA)) via the surface-initiated atom transfer radical polymerization (SI-ATRP). Epoxy groups of the grafted polymer, were modified into strong cation-exchange groups (i.e., sulfonate groups) in the presence of sodium sulfite. The MCTH and MCTH-g-p(GMA)-SO3H beads were characterized by ATR-FTIR, SEM, and VSM. The sulphonate groups content of the modified MCTH-g-p(GMA)-4 beads was found to be 0.53mmolg(-1) of beads by the potentiometric titration method. The MCTH-g-p(GMA)-SO3H beads were first used as an ion-exchange support for adsorption of lysozyme from aqueous solution. The influence of different experimental parameters such as pH, contact time, and temperature on the adsorption process was evaluated. The maximum adsorption capacity was found to be 208.7mgg(-1) beads. Adsorption of lysozyme on the MCTH-g-p(GMA)-SO3H beads fitted to Langmuir isotherm model and followed the pseudo second-order kinetic. More than 93% of the adsorbed lysozyme was desorbed using Na2CO3 solution (pH 11.0). The purity of the lysozyme was checked by HPLC and SDS gel electrophoresis. In addition, the MCTH-g-p(GMA)-SO3H beads prepared in this work showed promising potential for separation of various anionic molecules.

Pathogen detection in complex samples by quartz crystal microbalance sensor coupled to aptamer functionalized core-shell type magnetic separation.

A quartz crystal microbalance sensor (QCM) was developed for sensitive and specific detection of Salmonella enterica serovar typhimurium cells in food samples by integrating a magnetic bead purification system. Although many sensor formats based on bioaffinity agents have been developed for sensitive and specific detection of bacterial cells, the development of robust sensor applications for food samples remained a challenging issue. A viable strategy would be to integrate QCM to a pre-purification system. Here, we report a novel and sensitive high throughput strategy which combines an aptamer-based magnetic separation system for rapid enrichment of target pathogens and a QCM analysis for specific and real-time monitoring. As a proof-of-concept study, the integration of Salmonella binding aptamer immobilized magnetic beads to the aptamer-based QCM system was reported in order to develop a method for selective detection of Salmonella. Since our magnetic separation system can efficiently capture cells in a relatively short processing time (less than 10 min), feeding captured bacteria to a QCM flow cell system showed specific detection of Salmonella cells at 100 CFU mL(-1) from model food sample (i.e., milk). Subsequent treatment of the QCM crystal surface with NaOH solution regenerated the aptamer-sensor allowing each crystal to be used several times.

Examination of fabrication conditions of acrylate-based hydrogel formulations for doxorubicin release and efficacy test for hepatocellular carcinoma cell.

The objective of the present study was to develop 2-hydroxypropyl methacrylate-co-polyethylene methacrylate [p(HPMA-co-PEG-MEMA)] hydrogels that are able to efficiently entrap doxorubicin for the application of loco-regional control of the cancer disease. Systemic chemotherapy provides low clinical benefit while localized chemotherapy might provide a therapeutic advantage. In this study, effects of hydrogel properties such as PEG chains length, cross-linking density, biocompatibility, drug loading efficiency, and drug release kinetics were evaluated in vitro for targeted and controlled drug delivery. In addition, the characterization of the hydrogel formulations was conducted with swelling experiments, permeability tests, Fourier transform infrared, SEM, and contact angle studies. In these drug-hydrogel systems, doxorubicin contains amine group that can be expected a strong Lewis acid-base interaction between drug and polar groups of PEG chains, thus the drug was released in a timely fashion with an electrostatic interaction mechanism. It was observed that doxorubicin release from the hydrogel formulations decreased when the density of cross-linking, and drug/polymer ratio were increased while an increase in the PEG chains length of the macro-monomer (i.e. PEG-MEMA) in the hydrogel system was associated with an increase in water content and doxorubicin release. The biocompatibility of the hydrogel formulations has been investigated using two measures: cytotoxicity test (using lactate dehydrogenase assay) and major serum proteins adsorption studies. Antitumor activity of the released doxorubicin was assessed using a human SNU398 human hepatocellular carcinoma cell line. It was observed that doxorubicin released from all of our hydrogel formulations which remained biologically active and had the capability to kill the tested cancer cells.

Pathogen detection by core-shell type aptamer-magnetic preconcentration coupled to real-time PCR.

The presence of pathogenic bacteria is a major health risk factor in food samples and the commercial food supply chain is susceptible to bacterial contamination. Thus, rapid and sensitive identification methods are in demand for the food industry. Quantitative polymerase chain reaction (PCR) is one of the reliable specific methods with reasonably fast assay times. However, many constituents in food samples interfere with PCR, resulting in false results and thus hindering the usability of the method. Therefore, we aimed to develop an aptamer-based magnetic separation system as a sample preparation method for subsequent identification and quantification of the contaminant bacteria by real-time PCR. To achieve this goal, magnetic beads were prepared via suspension polymerization and grafted with glycidylmethacrylate (GMA) brushes that were modified into high quantities of amino groups. The magnetic beads were decorated with two different aptamer sequences binding specifically to Escherichia coli or Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR, but our magnetic affinity system capture of bacteria from 100% milk samples allowed accurate determination of bacterial contamination at less than 2.0 h with limit of detection around 100 CFU/mL for both bacteria in spiked-milk samples.

Activity and stability of urease entrapped in thermosensitive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate) hydrogel.

Urease was entrapped in thermally responsive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate), p[NIPAM-p(PEG)-MA], copolymer hydrogels. The copolymer membrane shows temperature-responsive properties similar to conventional p(NIPAM) hydrogels, which reversibly swell below and de-swell above the lower critical solution temperature of p(NIPAM) hydrogel at around 32 °C. The retained activities of the entrapped urease (in p[NIPAM-p(PEG)-MA]-4 hydrogels) were between 83 and 53% compared to that of the same quantity of free enzyme. Due to the thermo-responsive character of the hydrogel matrix, the maximum activity was achieved at around 25 °C with the immobilized urease. Optimum pH was the same for both free and entrapped enzyme. Operational, thermal and storage stabilities of the enzyme were found to increase with entrapment of urease in the thermoresponsive hydrogel matrixes. As for reusability, the immobilized urease retained 89% of its activity after ten repeated uses.