Synthesis and characterization of microparticles based on poly-methacrylic acid with glucose oxidase for biosensor applications.

In the line of the applicability of biocompatible monomers pH and temperature dependent, we assayed poly-methacrylic acid (p-MAA) microparticles as immobilization system in the design of enzymatic biosensors. Glucose oxidase was used as enzyme model for the study of microparticles as immobilization matrices and as biological material in the performance of glucose biosensors. The enzyme immobilization method was optimized by investigating the influence of monomer concentration and cross-linker content (N',N'-methylenebisacrylamide), used in the preparation of the microparticles in the response of the biosensors. The kinetics of the polymerization and the effects of the temperature were studied, also the conversion of the polymerization was determinates by a weight method. The structure of the obtained p-MAA microparticles were studied through scanning electron microscopy (SEM) and differential scanning microscopy (DSC). The particle size measurements were performed with a Galai-Cis 1 particle analyzer system. Furthermore, the influence of the swelling behavior of hydrogel matrix as a function of pH and temperature were studied. Analytical properties such as sensitivity, linear range, response time and detection limit were studied for the glucose biosensors. The sensitivity for glucose detection obtained with poly-methacrylic acid (p-MAA) microparticles was 11.98mAM(-1)cm(-2) and 10µM of detection limit. A Nafion® layer was used to eliminate common interferents of the human serum such as uric and ascorbic acids. The biosensors were used to determine glucose in human serum samples with satisfactory results. When stored in a frozen phosphate buffer solution (pH 6.0) at -4°C, the useful lifetime of all biosensors was at least 550 days.

Highly Sensitive DNA Sensor Based on Upconversion Nanoparticles and Graphene Oxide.

In this work we demonstrate a DNA biosensor based on fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er nanoparticles and graphene oxide (GO). Monodisperse NaYF4:Yb,Er nanoparticles with a mean diameter of 29.1 ± 2.2 nm were synthesized and coated with a SiO2 shell of 11 nm, which allowed the attachment of single strands of DNA. When these DNA-functionalized NaYF4:Yb,Er@SiO2 nanoparticles were in the proximity of the GO surface, the π-π stacking interaction between the nucleobases of the DNA and the sp(2) carbons of the GO induced a FRET fluorescence quenching due to the overlap of the fluorescence emission of the NaYF4:Yb,Er@SiO2 and the absorption spectrum of GO. By contrast, in the presence of the complementary DNA strands, the hybridization leads to double-stranded DNA that does not interact with the GO surface, and thus the NaYF4:Yb,Er@SiO2 nanoparticles remain unquenched and fluorescent. The high sensitivity and specificity of this sensor introduces a new method for the detection of DNA with a detection limit of 5 pM.

Silicon calcium phosphate ceramic as novel biomaterial to simulate the bone regenerative properties of autologous bone.

This study was conducted to develop novel ceramic bone substitute that resembles the autologous bone behavior when used as graft material. Solid-state reaction at 1100°C was performed to synthesize ß-tricalcium phosphate (ß-TCP) and biphasic calcium phosphate (BCP). The ceramics were further analyzed to characterize phase composition, microstructural properties, cytocompatability and then challenged to regenerate critical bone defects in the parietal bone of rabbits. X-ray diffraction analysis confirmed the production of ß-TCP and indicated the synthesis of novel BCP composed of ß-TCP and silicocarnotite (calcium phosphate silicate mineral). The cytocompatibility test with human osteoblast cell line revealed enhanced cell proliferation on the BCP ceramic. The novel BCP induced the filling of about 73% of the bone defect with a newly formed bone tissue and an almost complete degradation after 12 weeks of healing. This novel ceramic resembles the autologous bone properties of complete degradation and efficient enhancement of bone formation, making it promising as bone graft material.

Synthesis and characterization of biocatalytic γ-Fe2O3@SiO2 particles as recoverable bioreactors.

In this work, we present a suitable methodology to produce magnetically recoverable bioreactors based on enzymes, which are covalently attached on the surface of iron oxide@silica nanoparticles. In order to produce this system, iron oxide clusters with a mean diameter of 68 nm were covered with silica. This strategy yields spherical γ-Fe2O3@SiO2 cluster@shell nanoparticles with a mean diameter of 200 nm which present magnetic responsiveness and enhanced stability. The surface of these nanoparticles was modified into two steps with the aim to obtain carboxylic functional groups, which were activated to react with the enzyme glucose oxidase (GOx) that was thus immobilized on the surface of the nanoparticles. The objective of this chemistry at the nanoparticles interface is to produce magnetic-responsive bioreactors. The enzymatic activity was evaluated by using the recoverable bioreactors as part of an amperometric biosensor. These measurements allowed determining the stability, catalytic activity and the amount of enzyme immobilized on the surface of the nanoparticles. Furthermore, the functionalized nanoparticles can be recovered by applying an external magnetic field, which allows them to be employed in chemical processes where the recovery of the biocatalyst is important.

Fluorescence decrease of conjugated polymers by the catalytic activity of horseradish peroxidase and its application in phenolic compounds detection.

We report the fluorescence decrease of the water-soluble π-π-conjugated polymer poly(2-methoxy-5-propyloxy sulfonate phenylene vinylene, MPS-PPV) by the catalytic activity of horseradish peroxidase in the presence of H(2)O(2). MPS-PPV acts as a donor substrate in the catalytic cycle of horseradish peroxidase where the electron-deficient enzymatic intermediates compounds I and II can subtract electrons from the polymer leading to its fluorescence decrease. The addition of phenolic drug acetaminophen to the former solution favors the decrease of the polymer fluorescence, which indicates the peroxidase-catalyzed co-oxidation of MPS-PPV and acetaminophen. The encapsulation of horseradish peroxidase within polyacrylamide microgels allows the isolation of intermediates compound I and compound II from the polymer, leading to a fluorescence decrease that is only due to the product of biocatalytic acetaminophen oxidation. This system could be used to develop a new device for phenolic compounds detection.

Development of an acetaminophen amperometric biosensor based on peroxidase entrapped in polyacrylamide microgels.

In this work, horseradish peroxidase (HRP) has been entrapped in cross-linked polyacrylamide microparticles using the concentrated emulsion polymerization method. The feasibility of amperometric detection of acetaminophen (APAP) in a biosensor using this HRP immobilized system as the biological material in the presence of hydrogen peroxide was investigated. We found that the optimum microgel cross-linking degree required to retain the protein and to allow the diffusion of the phenolic drug onto the microparticles was 8%. The apparent diffusion coefficients of APAP across the different microparticles have been calculated using the Cottrell equation. The diffusion coefficients decrease as the microgel cross-linking increases, and the data fit an uniexponential equation well. Those microparticles with a cross-linking degree lower than 5% operated under kinetic control, whereas those whose cross-linking degree was above this value operated under diffusion control. Biosensor response was also optimized to investigate the effect of H(2)O(2) concentration and enzyme loading on the current intensity. Under optimal conditions, the sensitivity of this biosensor for APAP was 74.9 mA M(-1) cm(-2), the detection limit was 3.1×10(-6) M based on S/N=3 and the response time was 135 s. The linear range goes from 1.0×10(-5) to 4.9×10(-4) M APAP, and can be extended using the Hill equation to 5.7×10(-3) M. The biosensor is selective for APAP and was applied to determine the APAP concentration in three commercial pharmaceutical formulations.

Amperometric glucose biosensor based on biocompatible poly(dimethylaminoethyl) methacrylate microparticles.

Glucose oxidase (GOx) has been immobilized within poly(dimethylamino) ethyl methacrylate (p-DMAEM) microparticles which were subsequently used as biological material in the fabrication of a glucose biosensor. The enzyme immobilization method was optimized in relation with the monomer concentration and cross-linker content. It was found that the best biosensor response corresponds to microparticles synthesized with 1.19M monomer and 0.37% cross-linking content. Furthermore, the influence on the biosensor response of parameters such as working potential, pH, temperature, and loaded enzyme were investigated. In addition, analytical properties such as sensitivity, linear range, response time, and detection limit were determined. The biosensor was used to analyze glucose in human serum samples with satisfactory results. The useful lifetime of the biosensor is at least 520 days.

Investigation of the relationship between hydrogen bonds and macroscopic properties in hybrid core-shell gamma-Fe2O3-P(NIPAM-AAS) microgels.

We investigate in a hybrid material the interactions existing between magnetic nanoparticles of gamma-Fe(2)O(3) and the polymer matrix constituted by core-shell poly(N-isopropylacrylamide-sodium acrylate) microgels. These interactions provoke the shifting of the microgel volume phase transition to higher temperatures when the amount of gamma-Fe(2)O(3) increases. The study was performed using different techniques such as incoherent quasi-elastic neutron scattering (IQNS), infrared spectroscopy (FTIR-ATR), and dynamic light scattering (DLS). Below the low critical solution temperature (LCST) of the polymer, the IQNS data confirm that the presence of inorganic nanoparticles affects the PNIPAM chain motions. Thus, in the swollen state both the mean-square displacement of the polymer segments and the diffusive motion of the polymer chains decrease as the iron oxide content increases. The FTIR-ATR study indicates that the reduction of vibrational and diffusional motions of the polymer chains is due to the formation of hydrogen bonds between the amide groups of the polymer matrix and the OH groups of the magnetic nanoparticles. The creation of this hybrid complex would explain the reduction of the swelling capacity with increasing the iron content in the microgels. Furthermore, this interaction could also explain the shift of the polymer LCST to higher temperatures as due to the extra energy required by the system to break the hydrogen bonds prior to the PNIPAM collapse.

Interpenetrated PNIPAM-polythiophene microgels for nitro aromatic compound detection.

In this work, we present a facile and reproducible method to obtain thermally responsive, monodisperse, fluorescent microgels with diameters smaller than 700 nm based on poly(N-isopropyl acrylamide) (PNIPAM) interpenetrated with poly(thiophene-ethyl buthyl sulfonate) (PTEBS). Changing the temperature and inducing the microgel volume phase transition, it is possible to modify the photoluminescence (PL) properties of the microgels. Thus, when the temperature was below the low critical solution temperature (LCST) of PNIPAM, the PL intensity was higher than that above the LCST. Time-resolved fluorescence measurements indicate that, in the swollen state, the increment of cross-linking increases the fluorescence decay time of PTEBS. By contrast, in the collapsed state, variations in the decay time were attributed to higher rigidity of the PNIPAM-PTEBS system, which was confirmed by neutron scattering measurements. Moreover, the shift in the wavelength of the fluorescence emission peak observed above the LCST indicates that the collapsed PNIPAM matrix was able to interact with the PTEBS chains hindering the formation of pi-pi interactions. This property is envisaged for developing a picric acid microsensor based on the formation of pi-pi interactions with the pi-conjugated polymer, thus quenching its PL emission. Above the LCST of PNIPAM-PTEBS microgels, the interactions would be broken and the initial PL emission would be recovered. This property could render reusable microsensors for detection of nitro aromatic compounds.

Highly sensitive amperometric biosensor based on a biocompatible calcium phosphate cement.

Brushite is a biocompatible calcium phosphate mineral with properties of solid electrolyte. In this study we take advantage of this characteristic to develop an enzymatic amperometric biosensor based on brushite cement. The biosensor was prepared by immobilizing tyrosinase (PPO) on a brushite cement layer which was subsequently cross-linked with glutaraldehyde (GA) on the surface of a glassy carbon electrode. The system was optimized for the detection of phenolic compounds in both aqueous and non-aqueous solutions. Several variables involved in the enzyme immobilization method such as glutaraldehyde cross-linking time, PPO/brushite ratio and thickness of the brushite film were investigated. Furthermore, the effects of the pH, temperature and applied potential on the biosensor performance were also optimized. On the other hand, the biosensor analytical properties were studied in presence of different organic solvents: dioxane, acetonitrile and ethanol. In both, phosphate buffer solution (PBS) and acetonitrile/PBS solution, the biosensor exhibits a rapid response (12 s); a wide linear range (0.001-3 microM and 0.007-2 microM respectively); low detection limit (1 and 2 nM respectively); and high sensitivity (46.6 and 28.6 A M(-1) cm(-2) respectively). The performance of the biosensor in the analysis of phenols in real samples was successful.

Switchable photoluminescence of CdTe nanocrystals by temperature-responsive microgels.

In the present study, we report a method for preparing a fluorescent thermosensitive hybrid material based on monodisperse, thermosensitive poly( N-isopropyl acrylamide) (PNIPAM) microgels covered with CdTe nanocrystals of 3.2 nm diameter. The CdTe nanocrystals were covalently immobilized on the surface of PNIPAM microgels. The chemical environment around the CdTe nanocrystals was modified by changing the temperature and inducing the microgel volume-phase transition. This change provoked a steep variation in the nanocrystal photoluminescence (PL) intensity in such a way that when the temperature was under the low critical solution temperature (LCST) of the polymer (36 degrees C) the PL of the nanocrystals was strongly quenched, whereas above the LCST the PL intensity was restored.

Encapsulation of glucose oxidase within poly(ethylene glycol) methyl ether methacrylate microparticles for developing an amperometric glucose biosensor.

Poly(ethylene glycol) methyl ether methacrylate (PEGMEM) microparticles were synthesized and glucose oxidase (GOx) was immobilized within the microparticles. An amperometric biosensor was fabricated using the microparticles with GOx as biological component. The enzyme immobilization method was optimized by investigating the influence of monomer concentration and cross-linker content used in the preparation of the microparticles in the response of the biosensor. The best analytical results were obtained with the microparticles prepared with 0.21 M PEGMEM and 0.74% cross-linking. Furthermore, we have investigated the influence on the biosensor behaviour of parameters such as working potential, pH, temperature and enzymatic load. In addition, analytical properties such as sensitivity, linear range, response time and detection limit were determined. The biosensor was used to determine glucose in human serum samples and to avoid common interferents present in human serum such as uric and ascorbic acids. A Nafion layer was deposited on the electrode surface with satisfactory results. The useful lifetime of the biosensor was at least 520 days.

Beta-tricalcium phosphate release from brushite cement surface.

Different in vivo studies demonstrated that brushite cements are biocompatible, bioresorbable, and osteoconductive. However, the decay of brushite cements has been scarcely studied even though it may be of great concern for clinical applications in highly blood-perfused regions. This work was elaborated to elucidate factors that determine brushite cement surface disintegration. For that, brushite cements were modified using in their preparation different aqueous solutions of phosphoric, glycolic, tartaric, and citric acids in concentrations that were reported to improve the cement properties. Two-viscosity enhancing polysaccharides, chondroitin-4 sulfate and hyaluronic acid, were also assayed. Thereafter, pre- and set cement samples were immersed in distilled water for 24 h. The cement-solid weight loss, microstructure, liquid phase viscosity, mean size of the released particles, and zeta potential were analyzed using X-ray diffraction, FTIR spectroscopy, light scattering, scanning electron microscopy and optical microscopy. It was found that the particles released from the cement surface were beta-TCP, and their amount depends on the carboxylic acid used in the preparation of the cement. The addition of hyaluronic acid and chondroitin-4 sulfate decreased the amount of released particles from the surface of the set brushite cement made with citric acid. Furthermore, the hyaluronic acid increased significantly the viscosity of the citric acid solution and the cement paste prepared with this liquid phase showed a pronounced step down in particle release. In this study, we showed that the water solubility of calcium carboxylate and the viscosity of mixing liquid may dictate the superficial disintegration of brushite cements.

Synthesis and characterization of poly(magnesium acrylate) microgels.

In this work, we present the synthesis of a novel poly(magnesium acrylate) microgel, its microstructural characterization, and its application as an enzyme immobilization system. The variation of the monomer concentration employed in the synthesis permitted to tune up the shape of the microgels in such a way that using 1.5 mol L(-1) we produced microgels of average size 40 microm formed by smaller subunits of around 1 microm. This fact confers the microgels a pomegranate-like structure that increases the specific surface of the system. Glucose oxidase (GOx) from Aspergillus niger was immobilized within the microgels with the aim of using them as bioreactors. The microgels were characterized by scanning electron microscopy and by neutron scattering. The incorporation of the enzyme results in an increment in the network mesh size and the appearance of a new correlation length in the neutron scattering pattern. Finally, the enzymatic activity of the microgels with GOx entrapped was studied as a function of the microgel cross-linking content.

Increase of the final setting time of brushite cements by using chondroitin 4-sulfate and silica gel.

Chondroitin 4-sulfate (C4S) is a bioactive glycosaminoglycan with inductive properties in bone and tissue regeneration. Dicalcium phosphate dehydrate cements (known as brushite) are biocompatible and resorbable materials used in bone and dental surgery. In this study we analyzed the effect of C4S on the setting of a calcium phosphate cement and the properties of the resulting material. Brushite based cement powder was synthesised by mixing monocalcium phosphate with beta-tricalcium phosphate and sodium pyrophosphate. When the concentration of C4S, in the liquid added to the cement powder, was between 1 and 8% the cement final setting time increases. Furthermore, the cement diametral tensile strength remains unaffected when solutions with concentrations of C4S below 5% were used, but decreases at higher C4S concentrations. Calorimetric analysis showed that the cements prepared with C4S alone and in combination with silica gel have a greater content of hydrated water. We concluded from our study that the addition of small amounts of C4S increases the cement setting time without affecting its diametral tensile strength and at the same time improves the cement's hydrophilicity.

Enzymatic biodegradable micro-reactors for therapeutic applications.

Poly(epsilon-caprolactone) is a well known biocompatible polymer, widely used as drug immobilization systems. In this work poly(epsilon-caprolactone) microparticles with average size between 5 and 25 microm have been prepared by O/W emulsion evaporation method. Inside the microparticles, we have encapsulated Glucose Oxidase with the aim of preparing micro-reactors for enzymatic therapy. These microparticles were structurally characterized and its enzymatic activity analyzed in order to improve the enzyme entrapment. Thus, at the optimum synthesis conditions the enzyme entrapped in the microparticles showed an enzymatic activity of (29.9 +/- 2.1)% comparing with the same amount of free enzyme. Moreover the microparticles maintained a (70.4 +/- 3.2)% of their initial enzymatic activity after placing them in buffer solution for two weeks.

The application of methacrylate-based polymers to enzyme biosensors.

Enzyme electrodes based on methacrylates have received significant attention in the development of biosensors. This article reviews the use and application of methacrylate and its derivatives as an immobilization system for the preparation of enzyme electrodes. Resent examples, extracted from the literature, illustrate the superior performance of such materials in the fabrication of biosensors and bioreactors.

Amperometric tyrosinase biosensor based on polyacrylamide microgels.

An amperometric enzyme sensor using tyrosinase (PPO) entrapped in polyacrylamide microgels has been developed for determination of phenolic compounds. Polyacrylamide microgels were obtained by the concentrated emulsion polymerization method. The crosslinking of the polymer matrix optimum to retain the enzyme and to allow the diffusion of the compounds involved in the enzyme reaction has been studied (4.0%) as well as the influence on the response of analytical parameters such as pH, temperature, enzyme load and working potential. The useful lifetime of the biosensor was 27 days and it was useful to determine monophenolics compounds (e.g. cresol, chlorophenol) and diphenolics compounds (e.g. catechol and dopamine) by amperometric measurements at -100mV (versus SCE) in a batch system. The results showed that the substrate structures have a great influence on the sensor response.

Organic phase enzyme electrodes.

In the development of biosensors, organic phase enzyme electrodes (OPEEs) have received considerable attention for the detection of substrates in organic media. This article reviews different enzymes, transductors and immobilization methods used for the preparation of OPEEs in the last decade.

Amperometric glucose biosensor based on polymerized ionic liquid microparticles.

A glucose amperometric biosensor based on the immobilization of glucose oxidase (GOx) in microparticles prepared by polymerization of the ionic liquid 1-vinyl-3-ethyl-imidazolium bromide (ViEtIm+ Br-) using the concentrated emulsion polymerization method has been developed. The polymerization of the emulsion dispersed phase, in which the enzyme was dissolved together with the ionic liquid monomer, provides poly(ViEtIm+ Br-) microparticles with entrapped GOx. An anion-exchange reaction was carried out for synthesizing new microparticles of poly(ViEtIm+ (CF3SO2)2N-) and poly(ViEtIm+ BF4-). The enzyme immobilization method was optimized for biosensor applications and the following optimal values were determined: pH 4.0 for the synthesis medium, 1.23 M monomer concentration and 3.2% (w/w) cross-linking content. The performance of the biosensor as a function of some analytical parameters such as pH and temperature of the measuring medium, and enzymatic load of the microparticles was also investigated. The effect of the substances which are present in serum samples such as uric and ascorbic acid was eliminated by using a thin Nafion layer covering the electrode surface. The biosensor thus prepared can be employed in aqueous and in non-aqueous media with satisfactory results for glucose determination in human serum samples. The useful lifetime of this biosensor was 150 days.