Measuring the antioxidant capacity of blood plasma using potentiometry.

The use of potentiometry to measure plasma antioxidant capacity to contribute to oxidative stress evaluation is presented. In this assay, plasma (n=60) diluted (0.3 to 1 ml) in phosphate buffer, pH 7.4, NaCl 9%, was submitted to potentiometry. A platinum wire was the working electrode and saturated calomel the reference. The results are presented as the difference between sample and buffer potential (ΔE). ΔE presented a good inverse correlation with added increasing concentrations of ascorbate (2.5-75 µmol/L; R=-0.99), urate (9.0-150 µmol/L; R=-0.99), and bilirubin (0.78-13 µmol/L; R=-0.99). Increase in the antioxidant capacity decreased ΔE. Depletion of the antioxidant capacity by tert-butylhydroperoxide (6.5-50 µmol/L) presented a direct correlation (0.97) with ΔE. Furthermore, ΔE presented an inverse correlation (R=-0.99) with increased antioxidant capacity of plasma (FRAP) induced by the addition of ascorbate (2.5-75 µmol/L). The response of the potentiometric method proved be adequate for measuring the plasma antioxidant depletion induced by acute exhaustive exercise in rats (control, n=15; exercised, n=15). This exercise decreased the concentration of urate (p<0.05), decreased FRAP (p<0.5), increased TBARS (p<0.5), and decreased the potentiometer sensor response (p=6.5×10⁻³). These results demonstrate the adequacy of potentiometry for evaluating the antioxidant capacity of blood plasma samples.

In situ fabrication of ultrathin porous alumina and its application for nanopatterning Au nanocrystals on the surface of ion-sensitive field-effect transistors.

In situ fabrication in a single step of thin films of alumina exhibiting a thickness of less than 100 nm and nanopores with a highly regular diameter distribution in order to pattern nanostructures over field-effect devices is a critical issue and has not previously been demonstrated. Here we report the fabrication in situ of 50 nm thick ultrathin nanoporous alumina membranes with a regular pore size directly over metal-free gate ion-sensitive field-effect transistors. Depositing thin films of aluminum by an electron beam at a relatively low rate of deposition on top of chips containing the transistors and using a conventional single-step anodization process permits the production of a well-adhering nanoporous ultrathin layer of alumina on the surface of the devices. The anodization process does not substantially affect the electrical properties of the transistors. The small thickness and pore size of ultrathin alumina membranes allow them to be sequentially employed as masks for patterning Au nanocrystals grown by an electroless approach directly on the top of the transistors. The patterning process using a wet chemical approach enables the size of the patterned crystals to be controlled not only by the dimensions of the pores of alumina, but also by the concentration of the reactants employed. Surface modification of these nanocrystals with alkanethiol molecules demonstrates that the electrostatic charge of the functional groups of the molecules can modulate the electrical characteristics of the transistors. These results represent substantial progress towards the development of novel nanostructured arrays on top of field-effect devices that can be applied for chemical sensing or non-volatile memories.

Modulation of gut microbiota by antibiotics improves insulin signalling in high-fat fed mice.

AIMS/HYPOTHESIS: A high-fat dietary intake induces obesity and subclinical inflammation, which play important roles in insulin resistance. Recent studies have suggested that increased concentrations of circulating lipopolysaccharide (LPS), promoted by changes in intestinal permeability, may have a pivotal role in insulin resistance. Thus, we investigated the effect of gut microbiota modulation on insulin resistance and macrophage infiltration. METHODS: Swiss mice were submitted to a high-fat diet with antibiotics or pair-feeding for 8 weeks. Metagenome analyses were performed on DNA samples from mouse faeces. Blood was collected to determine levels of glucose, insulin, LPS, cytokines and acetate. Liver, muscle and adipose tissue proteins were analysed by western blotting. In addition, liver and adipose tissue were analysed, blinded, using histology and immunohistochemistry. RESULTS: Antibiotic treatment greatly modified the gut microbiota, reducing levels of Bacteroidetes and Firmicutes, overall bacterial count and circulating LPS levels. This modulation reduced levels of fasting glucose, insulin, TNF-α and IL-6; reduced activation of toll-like receptor 4 (TLR4), c-Jun N-terminal kinase (JNK), inhibitor of κ light polypeptide gene enhancer in B cells, kinase ß (IKKß) and phosphorylated IRS-1 Ser307; and consequently improved glucose tolerance and insulin tolerance and action in metabolically active tissues. In addition, there was an increase in portal levels of circulating acetate, which probably contributed to an increase in 5'-AMP-activated protein kinase (AMPK) phosphorylation in mice. We observed a striking reduction in crown-like structures (CLS) and F4/80(+) macrophage cells in the adipose tissue of antibiotic-treated mice. CONCLUSIONS/INTERPRETATION: These results suggest that modulation of gut microbiota in obesity can improve insulin signalling and glucose tolerance by reducing circulating LPS levels and inflammatory signalling. Modulation also appears to increase levels of circulating acetate, which activates AMPK and finally leads to reduced macrophage infiltration.

Development of a disposable amperometric biosensor for salicylate based on a plastic electrochemical microcell.

The use of an amperometric biosensor for rapid salicylate determination in blood is described. Photolitography was used to make gold electrodes on a polyester film. The plastic microcell was characterized using cyclic voltammetry to demonstrate the electrochemical performance of the system. The biosensor was constructed by immobilizing salicylate hydroxylase onto the working electrode of the plastic electrochemical microcell. The optimized working conditions were 0.1 mol L(-1) phosphate buffer at pH 7.6 with 0.5 mmol L(-1) of NADH and 300 mV vs. Au as the applied potential. The resulting biosensor exhibited a high sensitivity (97.4 nA/mmol L(-1) salicylate) and an adequate linear response range (1.2x10(-4) to 1.0x10(-3)mol(-1)). The biosensor performance was verified by determining salicylate in spiked blood samples and the results were statistically equivalent to the values obtained from the standard Trinder spectrophotometric method, with a 95% confidence level. This study shows the potential development of a portable, inexpensive and disposable device for point-of-care monitoring.

The effects of dimensionality on electrochemical sensors based on carbon nanotubes and metallic nanowires.

Electrochemistry based unidimensional nanoelectrodes, such as carbon nanotubes and metallic nanowires, is now a reality that is leaving the academy and reaching a broad range of high-tech companies around the world. The intrinsic properties of electron-electron interactions, sizes and geometries significantly differentiate those nanostructures from conventional macroelectrodes or electrodes with sizes in a millimetric range. The inherent properties as well as the configuration of carbon nanotubes and metallic nanowires as electrochemical sensors are the main focus of this review. Applications and some future trends on developing of a clear pathway for assembly and integration of these into functional materials, are summarized as well.

Surface plasmon resonance immunosensor for early diagnosis of Asian rust on soybean leaves.

Soybean rust (Asian rust) is a disease that occurs in soy cultures, negatively affecting pod formation and final grain weight and reducing value and product quality. Early identification of fungus in the plants prevents severe farming losses and spread to neighboring cultures. In this paper, a fast response sensor was developed based on surface plasmon resonance to detect Asian rust in soybean leaf extract at early stages of the disease. The antibody anti-Phakopsora pachyrhizi (pathogen) was covalently immobilized on a gold substrate via a self-assembled monolayer (SAM) of thiols using cysteamine-coupling chemistry. This immunosensor presented a linear response range for the antigen from 3.5 to 28.0 microg mL(-1) (r(2)=0.996). The effects of the antibody amount and the surface blocking to minimize non-specific adsorption on immunosensor response were evaluated. These studies provide new perspectives on using SPR technology for the development of a highly sensitive sensor for agricultural applications.

Biosensors as a tool for the antioxidant status evaluation.

Antioxidants are one of the main ingredients that protect food attributes by preventing oxidation that occurs during processing, distribution and end preparation of food. Physiological antioxidant protection involves a variety of chemical system of endogenous and exogenous origin in a multiplicity of pathways. Associate to this, researches have been directed in the development of methods as biosensors that can characterize antioxidants capable of removing harmful radicals in living organisms in an adequate way. Biosensors have represented a broad area of technology useful for environmental, food monitoring, clinical applications and can represent a good alternative method to evaluate the antioxidant status. The demonstration of the highlighted current application of biosensor as a potential tool to evaluate the antioxidant status is the main aim of this review.

Exploiting micellar environment for simultaneous electrochemical determination of ascorbic acid and dopamine.

A simple and reliable method for simultaneous electrochemical determination of ascorbic acid (AA) and dopamine (DA) is presented in this work. It was based on the use of the cationic surfactant cetylpyridinium chloride (CPC) that enables the separation of the oxidation peaks potential of AA and DA. Cyclic voltammetry (CV) as well as pulse differential voltammetry (PDV) were used in order to verify the voltammetric behaviour in micellar media. In the cationic surfactant CPC, a remarkable electrostatic interaction is established with negatively charged AA, as a consequence, the oxidation peak potential shifted toward less positive potential and the peak current increased. On the other hand, the positively charged DA is repelled from the electrode surface and the oxidation peak potential shifts toward more positive potential in comparison to the bare electrode. Therefore, the common overlapped oxidation peaks of AA and DA can be circumventing by using CPC. Parameter that affects the E(pa) and I(pa) such as CPC concentration and pH were studied. Under optimised conditions, the method presented a linear response to AA and DA in the concentration range from 5 to 75 micromol L(-1) and 10 to 100 micromol L(-1), respectively. The proposed method was successfully applied to the simultaneous determination of AA and DA in dopamine hydrochloride injection (DHI) samples spiked with AA.

Amperometric sensor for glutathione reductase activity determination in erythrocyte hemolysate.

The development of an amperometric sensor for glutathione reductase (GR) activity in erythrocyte hemolysate to contribute to oxidative stress evaluation is presented. In this assay, the reduced form of glutathione, the product of the GR reaction, reacts with 5,5(')-dithiobis(2-nitrobenzoic acid), producing GSTNB, which is easily reduced in the electrode surface. The current was recorded during 180 s after the sample addition, applying a potential of -300 mV. The sensor presented a suitable sensitivity, a good operational range, and precision. The effects of pH variations and specific uncompetitive inhibitor (safranin-O) in the enzyme activity were also evaluated. The GR activity determination in human erythrocyte hemolysate using this method has provided results that are statistically equal to those obtained by the classical spectrophotometric method, with 95% of confidence. The advantages of this method are the saved time, reagents, and samples and the possibility of its use in the field.

Use of silica gel chemically modified with zirconium phosphate for preconcentration and determination of lead and copper by flame atomic absorption spectrometry.

An analytical method using silica gel chemically modified with zirconium (IV) phosphate for preconcentration of lead and copper, in a column system, and their sequential determination by flame atomic absorption spectrometry (FAAS), was developed. Sample solutions are passed through a glass column packed with 100 mg of the sorbent material, at pH 4.5, and lead and copper are eluted with 1.0 mol l(-1) HNO(3) at a flow rate of 2.0 ml min(-1). The extraction of copper is affected by Fe(II), Mn(II), Zn(II), Ni(II) and Co(II) while only Fe(II) interferes in the lead determination. These interferences may be overcome with an appropriate addition of a KI or NaF solution. An enrichment factor of 30 was obtained for both metals. While the limits of detection (3sigma) were 6.1 and 1.1 microg l(-1), for Pb and Cu, respectively, the limits of determination were 16.7 and 3.3 microg l(-1). The precision expressed as relative standard deviation (R.S.D.) obtained for 3.3 microg l(-1) of Cu and 16.7 microg l(-1) of Pb were 4.3 and 4.7%, respectively, calculated from ten measurements. The proposed method was evaluated with reference material and was applied for the determination of lead and copper in industrial and river waters.

Remediation and toxicity removal from Kraft E1 paper mill effluent by ozonization.

The degradation of Kraft E1 pulp mill effluent was studied by four different ozonization oxidation systems (O3/pH3, O3/pH11, O3/pH11/H2O2, O3/pH11/UV). The investigation was focused on the reduction of total organic carbon (TOC), total phenols, color and acute toxicity (monitoring by inhibition of Escherichia coli respiration). For a reaction time of 90 minutes, the O3/pH11/UV was the most effective process for decoloration (45%). The O3/pH11/H2O2, O3/pH11/UV and O3/pH11 processes showed the best results for total phenols reduction (approximately/= 90%). None of the studied processes showed a significant TOC reduction. The O3/pH11/UV and O3/pH11 processes were effective for the acute toxicity reduction. Different kinetic parameters were also determined in order to quantify the reactivity of the effluent towards the applied oxidation systems.

Influence of gamma irradiation on a natural source of peroxidase and its effect in the reagentless amperometric biosensor for hydrogen peroxide.

Peroxidase was extracted from a natural source (turnip) and irradiated with 60Co (0.1 kGy). It was then used in the construction of biosensors for hydrogen peroxide determination, in order to study the effect of the gamma irradiation on the performance of the biosensors. The biosensors were constructed using two immobilization procedures: cross-linking with glutaraldehyde and covalent binding through carbodiimide. The biosensor prepared using covalent binding through carbodiimide showed a higher sensitivity for H2O2. A good enhancement in stability and sensitivity was obtained for the biosensors from irradiated material, when compared to biosensors prepared with non-irradiated enzyme. However, the initial linear response range (1.0 to 10.0 mmol dm-3) and response time (0.5 s) were equal with or without irradiation.

Development of an amperometric biosensor based on glutathione peroxidase immobilized in a carbodiimide matrix for the analysis of reduced glutathione from serum.

The development of an amperometric biosensor for the reduced glutathione determination in serum is described. The biosensor is based on glutathione peroxidase (GSH-Px, EC 1.11.1.9) immobilized onto a pyrolytic graphite-working electrode using carbodiimide as enzymatic condensing reagent. This resulted in an amperometric biosensor with good sensitivity and stability. The reduced glutathione (GSH) was enzymatically converted to glutathione disulfide (GSSG) in the presence of hydroperoxide, which was monitored amperometrically by its electrooxidation at +0.65 V vs. SCE (saturated calomel electrode). Glutathione measurement was carried out by maintaining the ratio between GSH and hydrogen peroxide at 2:1 (25 degrees C). The amperometric response of the biosensor was linearly proportional to the GSH concentration between 1.9x10(-5) and 1.4x10(-4) mol/l, in 0.1 mol/l phosphate buffer (pH=7.8), containing 0.1 mol/l KCl and 0.5 mmol/l Na(2)H(2)EDTA, as the supporting electrolyte. In presence of interfering compounds, the recoveries ranged between 97.2% and 110.7%. The biosensor useful lifetime was at least 2 months when it was evaluated after continuous use. Serum samples analyzed by this biosensor showed a good correlation with the results from the spectrophotometric method (Ellman's reagent) used as reference, presenting relative deviations lower than 7.0%. The low apparent Michaelis-Menten constant value, K(M)(app)=1.6 mmol/l, demonstrated that GSH-Px immobilized on pyrolytic graphite exhibited a high affinity to GSH, without loss of enzymatic activity.

Reagentless biosensor for isocitrate using one step modified Pt-Ir microelectrode.

The Pt-Ir microelectrode modified through one step electropolymerization is proposed for the isocitrate amperometric biosensor construction. The enzyme (isocitrate dehydrogenase-ICDH), coenzyme (NADP(+)) and mediator (Meldola's Blue) were immobilized onto the microelectrode surface in one step from a PIPES buffer solution containing pyrrole. The optimized experimental conditions were 25 cycles of cyclic voltammetric in a solution containing 3.58 10(-5) mol l(-1) of mediator, 3.51 10(-4) mol l(-1) of coenzyme and 2.68 U ml(-1) of enzyme. In contrast to the biosensor for isocitrate reported in literature, just one enzyme was immobilized and no coenzyme addition in the solution of analysis was necessary. Catalytic currents were proportional to the isocitrate concentration between 7.7 10(-6) and 1.04 10(-4) mol l(-1), showing good repeatability. The detection limit of the proposed biosensor was 3.50 10(-6) mol l(-1), the response time was lower than 20 s, the lifetime was about 30 determinations and no significant interference of sugars and citric acid was verified. Orange juice samples were analysed by both methodology biosensor and spectrophotometric commercial kit, and the obtained results presented a good correlation. The data demonstrated that the developed biosensor is suitable for isocitrate determination in orange juice without matrix interferences.

Use of self-plasticizing EVA membrane for potentiometric anion detection.

In this work, five different types of membranes were developed responsible to anion-selective electrodes. The membranes were based on tri-caprylyl-trimethyl-ammonium chloride (Aliquat-336S) supported on poly(ethylene-co-vinyl-acetate) copolymer (EVA). The sensors were prepared by solubilizing the copolymer with the appropriate exchanger in chloroform, without using of any plasticizers. The ion-selective electrodes prepared using these membranes show the utility for anions determinations in the concentration range between 10(-5) and 10(-1) mol l(-1) in the steady-state. The membrane performance was also evaluated in FIA system using tubular electrode for salicylate and iodide. In FIA system, the electrode exhibited nernstian response for salicylate in the concentration range of 2.5x10(-3) and 1.0x10(-1), while for iodide from 5.0x10(-4) up to 1.0x10(-1) mol l(-1). The systems were employed for the salicylate and iodide determination in pharmaceutical samples obtaining a relative deviation of 1.6%, when compared to the reference method.

Effects of fungal laccase immobilization procedures for the development of a biosensor for phenol compounds.

Fungal laccase was immobilized on carbon-fiber electrodes using classical methods: physical adsorption, glutaraldehyde, carbodiimide and carbodiimide/glutaraldehyde. The highest biosensor response was obtained using carbodiimide/glutaraldehyde for coupling laccase to carboxyl groups on the carbon fibers. In this method, different percentages of glutaraldehyde had important effects on the sensitivity of the biosensor, the best percentage of glutaraldehyde being 10% (m/v). The behavior of the obtained biosensor was investigated in terms of sensitivity, operational range, pH and applied potential. The developed biosensor showed an optimum response at pH 5.0 and at an applied potential of -100 mV. The immobilized laccase retained a good activity for over 2 months.

Electrochemical properties of Doyle catalyst immobilized on carbon paste in the presence of DNA.

The electrochemical behaviour of Doyle catalyst, dirhodium(II) tetrakis [methyl-2-oxopyrrolidine-5(S)-carboxylate] (Rh2(5S-MEPY)4), immobilised in graphite powder was evaluated preparing the carbon paste electrode, as well as its electrochemical properties in the presence (DCDE) and absence (DCE) of DNA. In both cases, one redox couple at 0.35 V vs. SCE in 0.5 mol l(-1) KCl solution at pH 7 and 10 mV s(-1) was observed. The resolution of the peak current in the voltammetric studies and other electrochemical properties were improved when the Doyle catalyst was immobilised in the presence of DNA. The estimated rate constants were of 17 and 26 s(-1) for a scan rate of 1 V s(-1) for DCE and DCDE, respectively. Furthermore, the interaction between rhodium carboxylates and electrolytes become more evident, suggesting a good hydrophilic and conductor character of this biopolymer.

Determination of acetylsalicylic acid by FIA-potentiometric system in drugs after on-line hydrolysis.

A potentiometric flow injection (FI) system was developed for the acetylsalicylic acid (ASA) determination in drugs, without previous treatment. The tubular potentiometric electrode for salicylate (SA) was based on tricaprylyl-trimethyl-ammonium-salicylate (aliquat-salicylate) as the ion-exchanger, supported on poly(ethylene-co-vinyl-acetate) (EVA) matrix and applied directly onto a conducting support. The standards and samples were freshly prepared in ethanol solution (0.10 mol l(-1) Tris-SO(4) buffer, pH 8.0, containing 0.25 mol l(-1) Na(2)SO(4) and 8.0% v/v ethanol) to facilitate the dissolution of ASA and were injected directly into the system. The SA formed due to the on-line alkaline hydrolysis of alcoholic ASA solution, with 0.50 mol l(-1) NaOH (coil, 50 cm length), was monitored by the tubular electrode after neutralization with 0.25 mol l(-1) H(2)SO(4). A solution of 0.10 mol l(-1) Tris-SO(4) buffer (pH 8.0), containing 0.25 mol l(-1) Na(2)SO(4) was employed as carrier. In optimized conditions (flow rate of 2.1 ml min(-1) and volume of injection of 150 mul), the tubular electrode showed a linear response to ASA in the concentration range between 4.0x10(-3) and 4.0x10(-2) mol l(-1). A conversion factor of ASA to SA of 85% occurs in these conditions with an increase of about 130% in the signal to the system with on-line hydrolysis (three-channel) in comparison to the system without (one-channel). The response time of the electrode was about 5 s with an analytical frequency of 28 samples per h and a relative standard deviation (R.S.D.) of 2.1% for 30 successive injections. Determinations of ASA in tablet samples by the proposed method exhibited relative differences of 1.0-3.5%, compared to the official method of the British Pharmacopoeia. The useful lifetime of the sensor was greater than 1 month, in continuous use.

Polishable and renewable DNA hybridization biosensors.

Routine applications of DNA hybridization biosensors are often restricted by the need for regenerating the single-stranded (ss) probe for subsequent reuse. This note reports on a viable alternative to prolonged thermal or chemical regeneration schemes through the mechanical polishing of oligonucleotide-bulk-modified carbon composite electrodes. The surface of these biocomposite hybridization biosensors can be renewed rapidly and reproducibly by a simple extrusion/polishing protocol. The immobilized probe retains its hybridization activity on confinement in the interior of the carbon paste matrix, with the use of fresh surfaces erasing memory effects and restoring the original target response, to allow numerous hybridization/measurement cycles. We expect that such reusable nucleic acid modified composite electrodes can be designed for a wide variety of biosensing applications.