A nanofibrous composite membrane of PLGA-chitosan/PVA prepared by electrospinning

Tissue engineering scaffolds produced by electrospinning feature a structural similarity to the natural extracellular matrix. In this study, poly(lactide-co-glycolide) (PLGA) and chitosan/poly(vinyl alcohol) (PVA) were simultaneously electrospun from two different syringes and mixed on the rotating drum to prepare the nanofibrous composite membrane. The composite membrane was crosslinked by glutaraldehyde vapor to maintain its mechanical properties and fiber morphology in wet stage. Morphology, shrinkage, absorption in phosphate buffered solution (PBS) and mechanical properties of the electrospun membranes were characterized. Fibroblast viability on electrospun membranes was discussed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and cell morphology after 7 days of culture. Results indicated that the PBS absorption of the composite membranes, no matter crosslinked or not, was higher than the electrospun PLGA membrane due to the introduction of hydrophilic components, chitosan and PVA. After crosslinking, the composite membrane had a little shrinkage after incubating in PBS. The crosslinked composite membrane also showed moderate tensile properties. Cell culture suggested that electrospun PLGA-chitosan/PVA membrane tended to promote fibroblast attachment and proliferation. It was assumed that the nanofibrous composite membrane of electrospun PLGA-chitosan/PVA could be potentially used for skin reconstruction.     

Preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization

Nanofibrous membrane with a fiber diameter of 80-150 nm was fabricated from mixed chitosan/poly(vinyl alcohol) (PVA) solution by an electrospinning process. Field emission scanning electron microscope and transmission electron microscope were used to characterize the morphology of the nanofibrous membrane. It was found that chitosan nanofibrous membrane with stabilized morphology could be prepared through removing most of PVA from the nascent one with 0.5 M NaOH aqueous solution. This treatment also resulted in an obvious decrease in fiber diameter. The stabilized chitosan nanofibrous membrane was explored as support for enzyme immobilization due to the characteristics of excellent biocompatibility, high surface/volume ratio, and large porosity. Lipase from Candida rugosa was immobilized on the nanofibrous membrane using glutaraldehyde (GA) as coupling reagent. The properties of the immobilized lipase were assayed and compared with the free one. Results showed that, the observed lipase loading on this nanofibrous membrane was up to 63.6 mg/g and the activity retention of the immobilized lipase was 49.8% under the optimum condition. The pH and thermal stabilities of lipase were improved after it was immobilized on the chitosan nanofibrous membrane. In addition, the experimental results of reusability and storage stability indicated that the residual activities of the immobilized lipase were 46% after 10 cycles and 56.2% after 30 days, which were obviously higher than that of the free one.     

In vitro biocompatibility of electrospun hexanoyl chitosan fibrous scaffolds towards human keratinocytes and fibroblasts

With the ability to form a submicron-sized fibrous structure with interconnected pores mimicking the extracellular matrix (ECM) for tissue formation, electrospinning was used to fabricate ultra-fine fiber mats of hexanoyl chitosan (H-chitosan) for potential use as skin tissue scaffolds. In the present communication, the in vitro biocompatibility of the electrospun fiber mats was evaluated. Indirect cytotoxicity evaluation of the fiber mats with mouse fibroblasts (L929) revealed that the materials were non-toxic and did not release substances harmful to living cells. The potential for use of the fiber mats as skin tissue scaffolds was further assessed in terms of the attachment and the proliferation of human keratinocytes (HaCaT) and human foreskin fibroblasts (HFF) that were seeded or cultured on the scaffolds at different times. The results showed that the electrospun fibrous scaffolds could support the attachment and the proliferation of both types of cells, especially for HaCaT. In addition, the cells cultured on the fibrous scaffolds exhibited normal cell shapes and integrated well with surrounding fibers. The obtained results confirmed the potential for use of the electrospun H-chitosan fiber mats as scaffolds for skin tissue engineering.     

Bruch’s-Mimetic Nanofibrous Membranes Functionalized with the Integrin-Binding Peptides as a Promising Approach for Human Retinal Pigment Epithelium Cell Transplantation

Background: This study aimed to develop an ultrathin nanofibrous membrane able to, firstly, mimic the natural fibrous architecture of human Bruch’s membrane (BM) and, secondly, promote survival of retinal pigment epithelial (RPE) cells after surface functionalization of fibrous membranes. Methods: Integrin-binding peptides (IBPs) that specifically interact with appropriate adhesion receptors on RPEs were immobilized on Bruch’s-mimetic membranes to promote coverage of RPEs. Surface morphologies, Fourier-transform infrared spectroscopy spectra, contact angle analysis, Alamar Blue assay, live/dead assay, immunofluorescence staining, and scanning electron microscopy were used to evaluate the outcome. Results: Results showed that coated membranes maintained the original morphology of nanofibers. After coating with IBPs, the water contact angle of the membrane surfaces varied from 92.38 ± 0.67 degrees to 20.16 ± 0.81 degrees. RPE cells seeded on IBP-coated membranes showed the highest viability at all time points (Day 1, p < 0.05; Day 3, p < 0.01; Days 7 and 14, p < 0.001). The proliferation rate of RPE cells on uncoated poly(ε-caprolactone) (PCL) membranes was significantly lower than that of IBP-coated membranes (p < 0.001). SEM images showed a well-organized hexa/polygonal monolayer of RPE cells on IBP-coated membranes. RPE cells proliferated rapidly, contacted, and became confluent. RPE cells formed a tight adhesion with nanofibers under high-magnification SEM. Our findings confirmed that the IBP-coated PCL membrane improved the attachment, proliferation, and viability of RPE cells. In addition, in this study, we used serum-free culture for RPE cells and short IBPs without immunogenicity to prevent graft rejection and immunogenicity during transplantation. Conclusions: These results indicated that the biomimic BM-IBP-RPE nanofibrous graft might be a new, practicable approach to increase the success rate of RPE cell transplantation.     

Epidermal cellular response to poly(vinyl alcohol) nanofibers containing silver nanoparticles

A heat-treated PVA nanofibrous matrix containing silver (Ag) was prepared by electrospinning an aqueous 10 wt% PVA solution and followed by heat treatment at 150 °C for 10 min. The average diameter of the as-spun and heat-treated PVA nanofibers was 330 nm. The heat-treated PVA nanofibrous matrix containing Ag was irradiated with UV light to transform the Ag ions in the nanofibrous matrix into Ag nanoparticles. The in vitro cytotoxicity of the Ag ions and/or nanoparticles on normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) cultures was examined. The PVA nanofibrous matrix containing Ag showed slightly higher level of attachment and spreading in the early stage culture (1 h) than the PVA nanofibers without Ag (control). However, compared with the PVA nanofibers without Ag, the heat-treated and UV-irradiated PVA nanofibers, containing mainly Ag ions and nanoparticles, respectively, showed reduced cell attachment and spreading. This shows that both Ag ions and Ag nanoparticles are cytotoxic to NHEK and NHEF. There was no significant difference in cytotoxicity to NHEK and NHEF between Ag ions and Ag nanoparticles. NHEF appeared to be more sensitive to Ag ions or particles than NHEK. In addition, the residual nitrate ions (NO₃⁻) in the PVA nanofibers had an adverse effect on the culture of both cells.     

Importance of dynamic culture for evaluating osteoblast activity on dense silicon-substituted hydroxyapatite

This paper reports an investigation on human osteoblast-like cells (SaOs-2) seeded onto pure hydroxyapatite (HA) and silicon-substituted HA (SiHA) tablets under static and dynamic culture conditions. The biological characterizations were conducted in classical static conditions in multi-wells plates, and in a perfusion bioreactor that permits continuous circulation of culture medium at 2 mL/h. The morphology, proliferation and differentiation of osteoblastic cells were examined for the two types of samples in the both culture conditions after 1, 3 and 8 days. Under dynamic conditions, cells cultured on SiHA surfaces showed a faster adhesion process and the formation of longer and thinner focal adhesions than in static conditions. The number of cells grown onto both ceramic surfaces was higher in dynamic conditions when compared with static conditions. Moreover, a higher activity of alkaline phosphatase was found for cells seeded under dynamic conditions. Our findings suggest that the application of perfusion culture system on cells cultured on dense substrates is valuable for predicting in vivo behaviour of cells on biomaterials.     

壳聚糖修饰纳米纤维膜表面对氧化还原酶行为的影响

采用静电纺丝法制备了丙烯腈/丙烯酸共聚物(PANCAA)纳米纤维膜, 研究了纺丝液浓度对纤维形态的影响, 以扫描电子显微镜观察纤维形貌, 遴选得到最佳纺丝条件. 以1-乙基-3-(N,N-二甲基氨基丙基)碳二亚胺/N-羟基丁二酰亚胺(EDC/NHS)为偶联剂, 在纤维膜表面引入壳聚糖修饰层, 采用衰减全反射傅里叶变换红外光谱(ATR/FTTIR)、水接触角和称重法考察了修饰前后膜的变化. 通过戊二醛将过氧化氢酶固定到壳聚糖修饰的PANCAA纳米纤维膜上, 研究了壳聚糖及戊二醛浓度对固定化过氧化氢酶的影响, 结果表明, 在壳聚糖浓度为25 mg/mL及戊二醛质量分数为5%条件下, 壳聚糖修饰膜的固定化酶活性比空白膜提高了41.7%, 稳定性也得到了不同程度的提高.     

Evaluation of retention and release processes of two antibiotics from the biocompatible core-shell microparticles

By dropwise addition of a chitosan solution into different non-solvent, such as: 1 N and 2 N NaOH as well as 1 N NaOH: C₂H₅OH mixture (2:1, v/v) at temperature of 25 °C and 50 °C under stirring, the spherical pure chitosan microparticles were performed. As solvents for chitosan was used 0.1 N acetic acid or 0.1 N HCl. The immersion of the pure chitosan microparticles in hyaluronan solution led to complex microparticles, namely chitosan microparticles covered by a hyaluronan layer. For all the microparticles performed the behaviours in the retention process of two antibiotics: chloramphenicol succinate sodium salt and cefotaxime sodium salt were analyzed. Also, the study shows the release behaviour of cefotaxime sodium salt by the microparticles loaded with this drug. Among the microparticles performed a type of complex microparticles can be considered a suitable drug delivery system for cefotaxime. These microparticles were performed by dropwise addition of chitosan solution in 0.1 N acetic acid into the 1 N NaOH: C₂H₅OH (2:1, v/v) non-solvent at 20 °C for 3 h, followed by their washing up to alkalinity loss and the immersion in hyaluronan solution of 10 g/L concentration for 24 h.     

Compatibility of mammalian cells on surfaces of poly(dimethylsiloxane)

This paper describes the influence of the composition of poly(dimethylsiloxane) (PDMS) on the attachment and growth of several different types of mammalian cells: primary human umbilical artery endothelial cells (HUAECs), transformed 3T3 fibroblasts (3T3s), transformed osteoblast-like MC3T3-E1 cells, and HeLa (transformed epithelial) cells. Cells grew on PDMS having different ratios of base to curing agent: 10:1 (normal PDMS, PDMSN), 10:3 (PDMSCA), and 10:0.5 (PDMSB). They were also grown on "extracted PDMS" (normal PDMS that has reduced quantities of low molecular-weight oligomers, PDMSN,EX) and normal PDMS that had been extracted and then oxidized (PDMSN,EX,OX); all surfaces were exposed to a solution of fibronectin prior to cell attachment. Generally, fibronectin-coated PDMS is a suitable substrate for culturing mammalian cells. Compatibility of cells on some surfaces, however, was dependent on the cell type: PDMSN,EX,OX caused cell detachment of 3T3 fibroblasts and MC3T3-E1 cells, and PDMSCA caused detachment of HUAECs and HeLa cells. Growth of cells on PDMSN, PDMSN,EX, and PDMSB was comparable to growth on tissue culture-treated polystyrene for most of the cell types. All cells grew at similar rates on PDMS substrates regardless of the stiffness of the substrate, for substrates having Young's moduli ranging from E=0.60 +/- 0.04 to 2.6 +/- 0.2 MPa (for PDMSB and PDMSN,EX, respectively).     

Chitosan/poly(tetrafluoroethylene) composite membranes using in pervaporation dehydration processes

Chitosan/PTFE composite membranes were prepared from casting a γ-(glycidyloxypropyl)trimethoxysilane (GPTMS)-containing chitosan solution on poly(styrene sulfuric acid) grafted expended poly(tetrafluoroethylene) film surface. The adhesion between the chitosan skin layer and the PTFE substrate was pretty good to warrant the high performance of chitosan/PTFE composite membranes using in pervaporation dehydration processes on isopropanol. The chitosan/PTFE membrane exhibited a permeation flux of 1730 g/m² h and a separation factor of 775 at 70 °C on pervaporation dehydration of a 70 wt% isopropanol aqueous solution. The membrane also survived after a long-term operation test in 45 days.     

Temperature‐sensitive chitosan membranes as a substrate for cell adhesion and cell sheet detachment

A series of temperature‐sensitive poly(CSA‐co‐NIPAAm) membranes that were suitable for cell culture and confluent cell sheets detachment were prepared. The membranes with thermo‐responsive surface properties were synthesized by the copolymerization of acrylic acid‐derivatized chitosan (CSA) and N‐isopropylacrylamide (NIPAAm) in aqueous solution. Characterization of the membranes were carried out by means of the Fourier transform infrared (FTIR), X‐ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), and water contact‐angle (WCA) measurements. The adhesion and detachment of mouse fibroblast (L929) cells on these membranes have been investigated. The study showed that poly(CSA‐co‐NIPAAm) membranes could not only enhance fibroblasts attachment but also harvest confluent cell sheets by simply lowering the temperature. Furthermore, the detached cells retained high viability and could proliferate again after transferred to a new culture surface. Copyright © 2011 John Wiley & Sons, Ltd.     

Structural and dynamical properties of aqueous mixtures of pectin and chitosan

In this study phase separation, structure, and dynamics of aqueous pectin-chitosan mixtures of different ratios and a pure aqueous pectin sample have been investigated under various conditions by turbidimetry, SANS and dynamic light scattering (DLS). Only the mixture with r = 0.75 gelled upon decreasing the temperature ((r ≡ m_pectin/(m_pectin + m_chitosan), where m denotes the mass of the considered component). The pure pectin sample (r = 1) did not gel and the decrease in temperature seemed to promote phase separation. The addition of chitosan reduced the tendency of pectin to phase separate in the mixtures of pectin and chitosan. The general trend when cooling the samples was that the turbidity and the growth of the turbidity became more pronounced as the amount of pectin in the mixture was increased. The wavelength dependence of the turbidity indicated a change of the conformation of pectin chains from an extended form to a more compact structure in pectin solutions without chitosan as the temperature decreased. This was not observed for the mixture of pectin and chitosan. SANS measurements revealed excess scattered intensity in the low wave vector area with the strongest upturn for the pure pectin sample (r = 1). DLS experiments showed longer slow relaxation times after a temperature quench for all samples, with the most pronounced effect for the mixture of pectin and chitosan with r = 0.75. The synergism between pectin and chitosan at high pectin contents (r = 0.75) generated large association complexes over time.     

Physico-chemical and thermodynamic aspects of fibroblastic attachment on RGDS-modified chitosan membranes

In the present study, the cell attachment/spreading behaviour of L929 mouse fibroblasts on chitosan membranes was evaluated by using physico-chemical properties. For this purpose chitosan membranes were prepared and then photochemically modified with the cell adhesive peptide RGDS (Arg-Gly-Asp-Ser). The physico-chemical properties of unmodified (CHI) and RGDS-modified chitosan (CHI-RGDS) membranes were evaluated by calculating surface free energy (γ_sv) and interfacial free energy (γ_sw) values using captive bubble contact angle measurements and harmonic mean equation. The cell attachment experiments were performed both in 10% FBS containing and serum-free media with CHI and CHI-RGDS membranes. Eventually, it was not possible to predict a direct relationship between the change in physico-chemical properties and L929 cell attachment behaviour. The experimental results obtained from cell attachment agree with the theoretical prediction for the free energy of adhesion except for the cell attachment on CHI membrane in serum-free medium. Although a negative interfacial free energy of adhesion was calculated for CHI membrane in serum-free medium (ΔF_adh = −2.19 ergs/cm²), the cell attachment was poor (70%) compared to CHI-RGDS (90%) and none of the cells were spread on CHI surface to gain a fibroblastic morphology. Negative energy of adhesion was calculated for CHI and CHI-RGDS in 10% FBS medium, in which 100% of cells were attached on the membranes correlating with the thermodynamic approach. It can be suggested that, adsorption of serum proteins strongly affected the cell attachment meanwhile the presence of biosignal RGDS molecules triggered the cell spreading in serum medium.     

Chitosan macroporous asymmetric membranes—Preparation,characterization and transport of drugs

Chitosan macroporous membranes with asymmetric morphology were obtained by using an inorganic porogen agent (SiO₂). Chitosan/silica ratios used were 1:1, 1:3, and 1:5 w/w. A methodology to obtain asymmetric membranes with control of porosity and average pore size was proposed. The porous membranes were obtained taking advantage of the opposite solubility characteristics of chitosan and silica (4–20 μm). The membranes were characterized by SEM and water sorption capacity. The porosity was calculated by the relationship between dense and macroporous membranes. The SEM images of both surfaces and cross-section of the membranes confirmed their asymmetric morphology. Using a double-cell method, the permeability coefficients of two model drugs (sodium sulfamerazine and sulfametoxipyridazine) were determined. The effects of porous layer, drug type, concentration and temperature were evaluated. The results revealed that the increase in porosity results in significant differences in permeability and that the effects of drug concentration and bath temperature become less pronounced as porosity increases. The mass transport was analyzed in terms of pore-flow mechanism and the solution-diffusion mechanism. The results showed that the methodology was very efficient to yield asymmetric membranes with good mechanical resistance, control of porous size and dense layer thickness and that these membranes can potentially be used to the transport of drugs.     

Phytochemical Analysis,Antioxidant, and Wound Healing Activity of Pluchea indica L. (Less) Branch Extract Nanoparticles

Proliferation and migration of keratinocytes and fibroblasts play an important role in cutaneous wound healing, while oral mucosal squamous cell proliferation and migration are crucial for oral wound healing. In this study, the phytochemical profile of Pluchea indica branch ethanolic extract was characterized. The bioactive compound of Pluchea indica branch ethanolic extract was identified and analyzed by the validated HPLC method. The nanoparticles of P. indica branch extract were formulated by solvent displacement method to increase the solubility and the colloidal stability of the extract. The stability of the nanoparticles was investigated by using the dynamic light scattering technique. Effects of P. indica crude extract and nanoparticles on cell viability, proliferation and migration of primary epidermal keratinocytes, human dermal fibroblasts, and oral mucosal keratinocyte cells were investigated by MTT assay and scratch assay, respectively. The results showed that P. indica branch extract contained a high content of total phenolic and total flavonoids. The HPLC analysis revealed that the main compound in the extract was 4,5-O-dicaffeoylquinic acid. The cell viability of the extract and nanoparticles decreased when cells were exposed to a high concentration of extract and nanoparticles. These results demonstrate that P. indica branch extract and extract nanoparticles at specific concentrations possess in vitro wound healing activity and they may be possibly used to treat different types of wounds including dermal and oral mucosal wounds.     

Controlled assembly of superparamagnetic iron oxide nanoparticles on electrospun PU nanofibrous membrane: A novel heat-generating substrate for magnetic hyperthermia application

A facile method of fabricating novel heat-generating membranes composed of electrospun polyurethane (PU) nanofibers decorated with superparamagnetic iron oxide nanoparticles (NPs) is reported. Electrospinning was used to produce polymeric nanofibrous matrix, whereas polyol immersion technique allowed in situ assembly of well-dispersed Fe₃O₄ NPs on the nanofibrous membranes without any surfactant, and without sensitizing and stabilizing reagent. The assembly phenomena can be explained by the hydrogen-bonding interactions between the amide groups in the PU matrix and the hydroxyl groups capped on the surface of the Fe₃O₄ NPs. The prepared nanocomposite fibers showed acceptable magnetization value of 33.12 emu/g, after measuring the magnetic hysteresis loops using SQUID. Moreover, the inductive heating property of electrospun magnetic nanofibrous membranes under an alternating current (AC) magnetic field was investigated. We observed a progressive increase in the heating rate with the increase in the amount of magnetic Fe₃O₄ NPs in/on the membranes. The present electrospun magnetic nanofibrous membrane may be a potential candidate as a novel heat-generating substrate for localized hyperthermia cancer therapy.     

Electrochemical attachment of motile bacterial cells to gold

Selective attachment of Escherichia coli K-12 bacterial cells to charged gold surfaces was demonstrated. Electrostatic binding of E. coli K-12 bacterial cells to positively charged surfaces was observed starting at +750 mV. The binding of E. coli K-12 cells to positively charged gold surfaces is proposed to occur due to long-range electrostatic interactions between the negatively charged O-chain of lipopolysaccharide (LPS) molecules protruding the bacterial cell body and the electrode surface. Removing LPS alters the cellular surface charge and results in cellular attachment to negatively charged surfaces. Thus, applying an electrical potential allows for the direct, real time detection of live, dead or damaged bacterial cells. The attachment of E. coli K-12 bacterial cells to surfaces with an applied potential substantiates the hypothesis that an electrostatic interaction is responsible for the binding of bacterial cells to positively charged molecular assemblies on surfaces used for building bacterial microarrays.     

Construction of a Microstructured Collagen Membrane Mimicking the Papillary Dermis Architecture and Guiding Keratinocyte Morphology and Gene Expression

A papillary‐structured collagen fibril membrane is created, mimicking the 3D‐architecture of the human papillary dermis. Primary human keratinocytes cultured to confluency on papillar‐structured films are compared to keratinocytes cultured on flat membranes. Microscopical evaluation reveals the presence of morphologically distinct cells at the base of the papillar structures that are not observed on flat membranes. Gene expression microarrays and RT‐qPCR indicate that these cells are in a more proliferative/migrational state, whereas cells on flat membranes have a more differentiated expression profile. Immunohistochemical stainings confirm these results. In conclusion, specific collagen architecture can direct keratinocyte behavior, and this may be used to further improve skin regeneration.

     

A comparison of solid-phase microextraction and stir bar sorptive extraction coupled to liquid chromatography for the rapid analysis of resveratrol isomers in wines, musts and fruit juices

A comparison of direct immersion solid-phase microextraction (DI-SPME) and stir bar sorptive extraction (SBSE) coupled to liquid chromatography (HPLC) with fluorimetric detection for the rapid analysis of resveratrol isomers is described. For DI-SPME, a polar Carbowax-template resin (CW/TPR) 50 μm fiber was the most efficient and optimum extraction conditions were 40 °C and an extraction time of 30 min, stirring in the presence of 5% (m/v) sodium chloride and 0.07 M acetate/acetic acid buffer (pH 6). Desorption was carried out using the static mode for 10 min. Linearity was obtained in the 5-150 and 2-150 ng mL⁻¹ ranges for trans- and cis-resveratrol, with detection limits of 2 and 0.5 ng mL⁻¹, respectively. When using SBSE, a polydimethylsiloxane (PDMS) twister provided best extraction by means of a derivatization reaction in the presence of acetic anhydride and potassium carbonate. The same time and temperature were used for the extraction step in the presence of 2.5% (m/v) sodium chloride, and liquid desorption was performed with 150 μL of a 50/50 (v/v) acetonitrile/1% (v/v) acetic acid solution in a desorption time of 15 min. Linearity was now between 0.5 and 50 ng mL⁻¹ for trans-resveratrol with a detection limit of 0.1 ng mL⁻¹, while cis-resveratrol could not be extracted. The proposed methods were successfully applied to determining the resveratrol isomer content of wine, must and fruit juices.     

Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection

In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10 mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K_m =2.0745 mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K_m =0.549 mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K_m =0.1698 mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications.