Electrochemical genosensor for Klotho detection based on aliphatic and aromatic thiols self-assembled monolayers.

Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10-10 to 5·10-8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.

Electrochemical detection of magnetically-entrapped DNA sequences from complex samples by multiplexed enzymatic labelling: Application to a transgenic food/feed quantitative survey.

Monitoring of genetically modified organisms in food and feed demands molecular techniques that deliver accurate quantitative results. Electrochemical DNA detection has been widely described in this field, yet most reports convey qualitative data and application in processed food and feed samples is limited. Herein, the applicability of an electrochemical multiplex assay for DNA quantification in complex samples is assessed. The method consists of the simultaneous magnetic entrapment via sandwich hybridisation of two DNA sequences (event-specific and taxon-specific) onto the surface of magnetic microparticles, followed by bienzymatic labelling. As proof-of-concept, we report its application in a transgenic food/feed survey where relative quantification (two-target approach) of Roundup Ready Soybean® (RRS) was performed in food and feed. Quantitative coupling to end-point PCR was performed and calibration was achieved from 22 and 243 DNA copies spanning two orders of magnitude for the event and taxon-specific sequences, respectively. We collected a total of 33 soybean-containing samples acquired in local supermarkets, four out of which were found to contain undeclared presence of genetically modified soybean. A real-time PCR method was used to verify these findings. High correlation was found between results, indicating the suitability of the proposed multiplex method for food and feed monitoring.

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.

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.

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.

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.

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.

Biosensors based on acrylic microgels: a comparative study of immobilized glucose oxidase and tyrosinase.

Acrylic microgels are proposed as enzyme immobilizing support in amperometric biosensors. Two enzymes, glucose oxidase and tyrosinase, were entrapped in this matrix and their behaviour is compared. The optimum cross-linking of the polymeric matrix required to retain the enzyme, and to allow the diffusion of the substrate is different for each enzyme, 3.2% for glucose oxidase and 4.5% for tyrosinase. The effect of pH and temperature on the biosensor responses has been studied by experimental design methodology and predictions have been compared with independently performed experimental measurements. A quadratic effect of the variables studied (pH and T) on the biosensor response and the small or null interaction between them was confirmed. The pH results obtained with both methods are coincident revealing an reversible effect on the enzyme. However, the temperature optimum value obtained by experimental design was 10 degrees C lower as a result of an activity decay due to irreversible thermal denaturation of both enzymes.

Design of an amperometric biosensor using polypyrrole-microgel composites containing glucose oxidase.

A new material consisting of a water-dispersed complex of polypyrrole-polystyrensulfonate (PPy) embedded in polyacrylamide (PA) has been prepared and tested as enzyme immobilizing system for its use in amperometric biosensors. Glucose oxidase (GOx) and the water-dispersed polypyrrole complex were entrapped within polyacrylamide microgels by polymerization of acrylamide in the dispersed phase of concentrated emulsions containing GOx and PPy. Polymerization of the dispersed phase provides microparticles whose size lies between 3.5 and 7 microm. The aim of incorporating polypyrrole into the polyacrylamide microparticles was to facilitate the direct transfer of the electrons released in the enzymatic reaction from the catalytic site to the platinum electrode surface. The conductivity of the microparticles was measured by a four-point probe method and confirmed by the successful anaerobic detection of glucose by the biosensor. Thus, the polyacrylamide-polypyrrole (PAPPy) microparticles combine the conductivity of polypyrrole and the pore size control of polyacrylamide. The effects of the polyacrylamide-polypyrrole ratio and cross-linking on the biosensor response have been investigated, as well as the influence of analytical parameters such as pH and enzymatic loading. The PAPPy biosensor is free of interferences arising from ascorbic and uric acids, which allows its use for quantitative analysis in human blood serum.

Microstructural modifications induced by the entrapped glucose oxidase in cross-linked polyacrylamide microgels used as glucose sensors.

Glucose oxidase (GO(x)) has been immobilized in cross-linked polyacrylamide microgels using the concentrated emulsion polymerization method. The immobilization of the enzyme is effective when the number of cross-links is equal or greater than 0.021mol/g. The swelling of the microgels is not affected by the entrapped enzyme but decreases with increasing the number of cross-links. The glass transition temperature of microgels with GO(x) presents considerable differences with that of the empty microgels, which are explained on base of the competition between the plasticizer effect of the encapsulated enzyme and the chain mobility restrictions imposed by the microgel cross-linking content. Immobilization of GO(x) slightly modifies the X-ray diffraction patterns of the microgels; however, the diffraction profiles of cross-linked microgels suggest the formation of bis(acrylamide) aggregates in the outer layers of the microgel particles.

Condromatosis sinovial de hombro / Synovial chondromatosis of the shoulder

Se presenta el caso de una condromatosis sinovial de hombro en una paciente, analizando las bases fisiopatológicas del proceso, la gradación histológica del mismo, la definición de la posible degeneración sarcomatosa y su coexistencia con zonas de condromatosis sinovial primitiva. Desde el punto de vista terapéutico, se prefiere la técnica artroscópica y se remarca la necesidad de un control radiológico intraoperatorio para confirmar la exéresis de todos los condromas, reservando la cirugía abierta para permitir la sinovectomía en las recidivas. Se revisa la bibliografía y se hace referencia a las últimas aportaciones sobre el tema en el campo de la genética. (AU)

Advances in the determination of inorganic anions by ion chromatography.

The time period covered for this review includes articles published from 1997 to 1999, with the addition of a few classic references. The purpose of the review is to include the most relevant works from each topic area of the determination of inorganic anions by ion chromatography, including new sample pretreatments, new separation methods, new detection systems and the latest applications in the field of environmental, water, foods, etc. samples. Experimental conditions such as stationary phase, eluent, detection mode, as well as matrix are summarized in a table.

Ion chromatographic determination of chloride in mustard sauces.

A new, simple, precise, and rapid ion chromatography (IC) method has been developed to determine chloride in mustard sauces using a mixture of phthalic acid, acetone, and water adjusted to pH 5.0 as eluent. Conductometric detection was carried out. The retention time for chloride was 1.5 min. Linearity was obtained up to a concentration level of 100 mg/L NaCl. The method was statistically evaluated for accuracy and precision after being used to assay the chloride from mustard sauces. Within the same samples, the chloride levels obtained by IC were compared with the sodium concentrations quantified by atomic absorption spectrophotometry.

Enzymic determinations with rotating bioreactors and continuous-flow-stopped-flow processing. Determination of choline esters in pharmaceuticals.

The neuromuscular blocking agent, succinylcholine, and the neurotransmitter, acetylcholine, have been determined in pharmaceutical preparations utilizing a sensing unit comprised of a rotating bioreactor and an amperometric detector. The bioreactor consisted of a rotating disk made of Teflon (1 cm id) with cholinesterase (EC 3.1.1.8) and choline oxidase (EC 1.1.3.17) co-immobilized on the top surface of the disk. Amperometric detection of H2O2 and initial rate measurements were employed for quantification.

Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode.

A yeast alcohol dehydrogenase amperometric carbon paste-based biosensor, with Meldola Blue as a mediator and a dialysis membrane with a very small molecular weight cut-off for protection, is described. The influence of membrane pore size on the stability and overall kinetics of the biosensor is shown using cyclic voltammetry and stationary potential measurements. The operating potential is + 50 mV vs. Ag/AgCl, KCl sat. reference electrode. Application of this device to the determination of ethanol in alcoholic beverages was achieved successfully. In these kinds of samples and at this working potential no interferences were found.

Application of the quadratic response surface in the polarographic determination of lead: biological samples.

Differential-pulse polarography was applied to lead determination in blood, urine and hair samples after pressurized acid digestion. Quantitative determination was carried out using a single analytical measurement of the experimental sample (without the need for addition or calibration methods), using the equation calculated as a result of the study of the quadratic response surface. The elimination of the possible interferences has been considered. The reliability of the analytical procedure was evaluated using relative standard deviations (2.34-11.93%) and recovery (97.28-103.92%) measurements.

Electrochemical behaviour of tizanidine at solid electrodes.

The electrochemical behaviour of tizanidine [5-chloro(delta-2-imidazolinyl-2-amino)-4-benzothiadiazole-2,1,3], a centrally-active skeletal muscle relaxant has been investigated in aqueous media at the carbon paste electrode (CPE). Cyclic voltammetry at different pH values, controlled potential coulometry and comparative studies on three structurally related molecules have permitted identification of the oxidation site of tizanidine and suggest possible oxidation products in acidic media. The electrochemical reduction at the CPE occurred in one irreversible step and the reduction product (diamine derivative) was detected and characterized on the positive going scan in cyclic voltammetry. Quantitative measurements of tizanidine within the range 2 x 10(-5) M and 1 x 10(-4) M have been realized at the CPE using the differential pulse technique.