Amperometric assay for the ketone body 3-hydroxybutyrate

A stable dry-strip electrochemical sensor for the direct measurement of 3-hydroxybutyrate in blood is described. The sensor utilizes the electrocatalytic oxidation of enzymically generated NADH by the redox mediator 4-methyl-o-quinone. The enzyme 3-hydroxybutyrate dehydrogenase, cofactor NAD⁺ and 4-methyl-o-quinone were incorporated into single-use disposable strip electrodes.     

Simultaneous kinetic determination of 3-hydroxybutyrate and 3-hydroxyvalerate in biopolymer degradation processes

A new kinetic method is proposed for the simultaneous determination of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) based on the different rate of the 3-hydroxybutyrate dehydrogenase-catalysed reactions of these compounds with coenzyme NAD⁺. A flow injection system with two reactors of immobilised 3-hydroxybutyrate dehydrogenase and dual detection is used. The concentrations of NADH produced after two different reaction times are measured by fluorometry or spectrophotometry and multivariate linear calibration is applied for quantification. Concentrations of 3HB and 3HV between 1 × 10⁻⁶ and 1 × 10⁻⁴ M can be determined at an average sampling frequency of 20 h⁻¹. In contrast to usual methods, the proposed here makes possible the discrimination of 3HB and 3HV without previous separation so that usual extraction with chlorinated solvents and/or chromatographic separation is not required. The method is of interest in a wide variety of fields concerning PHAs, as it can provide information on the degradation rate and mechanism, composition and structure of these polymers. Its applicability has been proved through the determination of 3HB and 3HV in the digests of some chemically degraded commercial PHAs.     

Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10^?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l^?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.     

New whole blood methods and instruments: glucose measurement and test menus for critical care.

A glucose oxidase-H 2O 2 substrate-selective electrode (SSE) along with eight other electrodes allows the Stat Profile 5 (SP5) to measure glucose, electrolytes, blood gases, pH, and hematocrit simultaneously in whole blood. For heparinized blood gas syringe samples (n=178), orthogonal (Deming) regression showed y=0.43 + 1.00x (mmol/L), where y is whole blood glucose, and x is plasma glucose measured with the Ektachem 400 glucose oxidase slide method. For heparinized plasma (n=197), y=0.25 + 1.02x. Least squares linear regression showed y=0.25 + 1.00x (s yxx=0.49) for whole blood versus plasma, y = 0.30 + 1.01x (s yxx=0.49) for plasma versus plasma, and r=0.994 for both cases. Whole blood and plasma glucose in the same samples measured by SSE differed by less than 1%. The SP5 combines the largest number of whole blood tests currently available in a rapid response instrument well-suited for care of the critically ill patient.     

Characterization of surface modified flax fibers and their biocomposites with PHB

Flax fiber was treated with acetic anhydride or with ethylene plasma to improve adhesion with poly(3-hydroxybutyrate) (PHB). The flax fiber surface became hydrophobic by the surface treatments. The ethylene plasma treated flax fiber has better thermal resistance and shows higher interfacial adhesion strength in flax/PHB biocomposite than the chemically modified flax fiber with acetic anhydride. Optimum conditions of the ethylene plasma treatment were found to be 0.5 cm³/sec of the ethylene flow rate and 50 W of the plasma power with 5 min of the treatment time.     

Amperometric probe for 3-hydroxybutyrate with immobilized 3-hydroxybutyrate dehydrogenase

The enzyme 3-hydroxybutyrate dehydrogenase is immobilized on a graphite electrode suitable for determination of NADH. Of the several procedures tested for the immobilization, direct adsorption on the electrode surface was most satisfactory, with useful lifetimes of up to 3 days. The best calibration graphs for the NADH, and for 3-hydroxybutyrate were obtained at pH 7.5 (phosphate buffer). 3-Hydroxybutyrate was determined in the range 5–100 μmol l^?1 at pH 7.5 with good precision. Interferences are discussed.     

Determination of selenium in human body fluids by hydride-generation atomic absorption spectrometry : Optimization of sample decomposition

The accuracy of the determination of selenium in human body fluids by hydride-generation a.a.s. depends critically upon the sample decomposition-method used. Digestion with HNO₃ alone gave low selenium recoveries, but with nitric, sulfuric and perchloric acids at a final temperature of 310°C gave results that agreed with those obtained by other techniques. The recovery of selenomethionine added to whole blood and of trimethylselenonium iodide added to urine was 97–104%. The average selenium values found for 6 healthy individuals were 88 μg l^?1 in whole blood, 75 μg l^?1 in blood plasma and 307 μg (kg Hb)^?1 in erythrocytes. A detection limit of 5 μg l^?1 Se in body fluids was found under routine conditions.     

Electrochemical Nano-Roughening of Gold Microstructured Electrodes for Enhanced Sensing in Biofluids**

A key challenge for sensor miniaturization is to create electrodes with smaller footprints, while maintaining or increasing sensitivity. In this work, the electroactive surface of gold electrodes was enhanced 30-fold by wrinkling followed by chronoamperometric (CA) pulsing. Electron microscopy showed increased surface roughness in response to an increased number of CA pulses. The nanoroughened electrodes also showed excellent fouling resistance when submerged in solutions containing bovine serum albumin. The nanoroughened electrodes were used for electrochemical detection of Cu²⁺ in tap water and of glucose in human blood plasma. In the latter case, the nanoroughened electrodes allowed highly sensitive enzyme-free sensing of glucose, with responses comparable to those of two commercial enzyme-based sensors. We anticipate that this methodology to fabricate nanostructured electrodes can accelerate the development of simple, cost-effective, and high sensitivity electrochemical platforms.     

Determination of copper, lead and cadmium in whole blood by stripping voltammetry with the use of graphite electrodes

The problem with toxic metal ion determination in blood is the adsorption of organic compounds on the electrode surface and the formation of complexes between metal ions and organic constituents of blood. This is the reason why usually preliminary acid digestion or other sample pretreatment is used. Two kinds of electrodes have been used: “Ultra-Trace Electrode”, made from impregnated graphite (I), and thick film graphite disposable electrodes (II). The analysis of whole blood with different sample preparation methods shows, that chemical digestion is not necessary for the analysis. Electrochemical two-stage sample preparation provides the possibility for analysing whole blood with the mentioned electrodes. Thick film disposable electrodes are less sensitive to the interference of organic constituents of blood. These electrodes give the possibility to determine total cadmium, lead and copper concentration in whole blood without special sample pretreatment. The application of “Ultra-Trace Electrode” for blood analysis is possible only after preliminary pretreatment of blood by chemical digestion or electrochemical sample preparation.     

Specific structural features of crystalline regions in biodegradable composites of poly-3-hydroxybutyrate with chitosan

Differential scanning calorimetry and X-ray diffraction at wide and small angles were used to examine the biodegradable composites of poly-3-hydroxybutyrate with chitosan, produced by mixing of these polymers in a rotor disperser at 150°C. Samples of individual polymers and composites with 80, 40, and 20 wt % poly- 3-hydroxybutyrate were studied. It was found that the presence of chitosan in the composites leads to a change in the crystallite size of poly-3-hydroxybutyrate and to an increase in the large period in this polymer. Mixing of poly-3-hydroxybutyrate with chitosan affects the structural rearrangement in crystalline regions of poly-3-hydroxybutyrate under a high-temperature treatment. The effect of a high-temperature treatment of the composites via alternation of melting–crystallization cycles in the nonisothermal mode, when a sample is heated and cooled at the same constant rate of 8 deg min–1 in the range from 20 to 200°C and is annealed at a temperature of 150°C, was analyzed. This analysis suggests that, in composites of this kind, the intermolecular interaction between the components is a factor strongly affecting the structure of the crystalline regions and the mechanism of their rearrangement in the course of annealing. The mechanism of this interaction is discussed.     

Reduction of hexavalent chromium at solid electrodes in acidic media: reaction mechanism and analytical applications

The electrochemical detection of hexavalent chromium species was investigated. It was found that Cr(VI) can undergo chemically irreversible reduction in acidic solutions at gold, glassy carbon and boron-doped diamond electrodes. The process was found to be diffusionally controlled at all three electrodes studied. The response obtained at a gold electrode towards the reduction of chromium(VI) produced an electrochemically reversible wave in contrast to those recorded at glassy carbon and boron-doped diamond electrodes. The analytical response of the hexavalent species was studied at gold electrodes in the presence of common environmental interferences: Ni²⁺, Cu²⁺, Fe³⁺, Cr³⁺ and Triton X-100 (surfactant), with an LoD of 4.3 μM obtained in the presence of 5 mM Cr(III).     

Submerged Arc Breakdown of Methylene Blue in Aqueous Solutions

Low voltage, low energy submerged pulsed arcs between a pair of carbon or iron electrodes with a pulse repetition rate of 100?Hz, energies of 2.6?C192?mJ and durations of 20, 50 and 100???s were used to remove methylene blue (MB) contamination from 30?ml aqueous solutions. The MB concentration decreased exponentially with rates of 0.0006?C0.0143?s^?1 during processing with the carbon electrode pair. With the iron electrodes, the MB concentration initially decreased faster (0.030?s^?1) than with the carbon electrodes, but later saturated. However when microparticles produced with the iron electrodes were periodically filtered, the high removal rate was maintained. Under these conditions, the volume of water which can be treated per unit energy expenditure was much higher with the submerged arc than with other plasma processes. A kinetic model based on MB degradation by OH· radicals formed by the discharge was formulated. The higher initial MB removal rate with iron electrodes is explained by additional OH· production from Fenton??s reaction between Fe⁺⁺ and H₂O₂ produced by the discharge. This rate is maintained if the eroded iron particles are filtered, but if eroded iron particles accumulate, degradation slows down and stops, possibly because the iron particles catalytically decompose H₂O₂ and hence stops Fenton??s reaction, and either directly or via increased Fe⁺⁺ dissolved from the particles, scavenge the OH· radicals.     

Trace determination of nickel by microwave-induced plasma atomic emission spectrometry after preconcentration of nickel tetracarbonyl on Chromosorb

A method is presented for the determination of ultra-trace nickel concentrations in various samples. Ni²⁺ is reduced in aqueous solution by tetrahydroborate to Ni⁰, which reacts readily with carbon monoxide to give gaseous Ni(CO)₄, the latter being preconcentrated on Chromosorb, cooled by liquid nitrogen. After desorption of the carbonyl by electrically heating the Chromosorb trap, it is swept by argon to the microwave-induced plasma (hollow-cylinder O₂-Ar MIP with Beenakker resonator). Using this technique, nickel detection is possible without any interferences, because other carbonyl-forming reactions are too slow. The detection limit is 5 pg (3σ), corresponding to 5 ng l^?1, and the relative standard deviation is 3% at 100 pg. The method was applied to sea water and urine without digestion and whole blood, blood serum and human hair after decomposition by HNO₃-HClO₄.     

Differential-pulse anodic stripping voltammetry of mercury with gold-film micro-electrodes

Cylindrical gold film micro-electrodes are easily produced by plasma-sputtering of gold onto carbon fiber electrodes. The micro-electrodes produced were found to maintain their cylindrical geometry indefinitely, unlike gold wire electrodes of similar dimensions. Application of these electrodes in differential-pulse anodic stripping voltammetry provides a method for quantifying trace levels of mercury(II). Up to 100 μg l^?1 Hg(II) the area of the mercury stripping peak varied linearly with mercury concentration; the detection limit was 3.7 μg l^?1. With more than 100 μg l^?1 Hg(II) a new mercury stripping peak grows in at less positive potentials; its peak height is linear with Hg(II) concentration.     

Polarography of disodium pentacyanonitrosylferrate(II): Part 2. Determination at Therapeutic Levels in Serum,Plasma, and Whole Blood

Disodium pentacyanonitrosylferrat(II) (sodium nitroprusside) is determined at therapeutic (ng ml^?1) levels in plasma, serum and blood with conventional and high-performance differential pulse polarography (d.p.p. and h.p.d.p.p.) at a dropping mercury electrode or a static mercury drop electrode. Serum or plasma (3 ml) is treated with perchloric acid containing 1 mg ml^?1 potassium hexacyanoferrate(II), centrifuged for 10 min and subjected to polarography. For spiked serum, calibration graphs are linear over the range 30–1000 ng ml^?1 sodium nitroprusside, regardless of the polarographic technique; the estimated detection limit is 15 ng ml^?1 (5 × 10^?8 M). Calculated therapeutic levels range from 100 to 1000 ng ml^?1. Similar results were obtained for spiked plasma. A similar procedure is suitable for whole blood and was used to study the in-vitro degradation of sodium nitroprusside (200 ng ml^?1) on incubation at 37°C. The in-vitro loss is rapid (t_{$\text{1}\text{2}$} ≈ 6 min) but meaningful in-vivo levels can be obtained when the blood is collected in a 0.9% sodium chloride solution at 0°C. Thiocyanate, the main metabolite of nitroprusside, and thiosulphate, which is a potential antidote for cyanide, do not interfere.     

Effects of dielectrophoresis on thrombogenesis in human whole blood

Thrombogenesis (blood clot formation) is a major barrier to the development of biomedical devices that interface with blood. Although state‐of‐the‐art chemically and pharmacologically mediated clot mitigation strategies are effective, some limitations of such approaches include depletion of active agents, or adverse reactions in patients. Increased clotting protein adsorption and platelet adhesion, which occur when artificial surfaces are exposed to blood result in enhanced clot formation on artificial surfaces. It is hypothesized that repelling proteins and platelets using dielectrophoresis (DEP), a contact‐free particle manipulation technique, will reduce clot formation in biomedical devices. In this paper, the effect of DEP on thrombogenesis in human blood is investigated. Undiluted whole blood from human donors is pumped through microchannels at a physiological shear rate (400 s ⁻¹). Experiments are performed by applying 0 V, 0.5 V_rms , 2 V_rms , and 3 V_rms to electrodes in the channel. Clot formation is observed to decrease in experiments in which DEP electrodes are active (average of 6% coverage @ 0V reduced to 0.08% coverage @ 3 V_rms ). Repulsion is more effective at higher voltages. DEP causes a quantifiable reduction in microscopic and macroscopic clot formation in PDMS microchannels.     

Polyester-based Copolymers for Biomaterials Fabrication

Summary: Results on synthesis of poly(3-hydroxybutyrate)s possessing one or two hydroxyl groups at one terminus of the chain and carboxylic group at the other chain end are reported. These polymers were further functionalised via transesterification with dimethyl H-phosphonate thus incorporating a reactive/biodegradable center in the polyester backbone. Block/star-like copolymers composed of hydrophilic PEG and hydrophobic poly(3-hydroxybutyrate) segments linked by phosphoester moiety were also obtained. Chemical structure and composition of the reaction products were analysed applying different spectroscopic techniques (¹H, and ³¹P NMR, IR and ESI-MS) and size exclusion chromatography was applied to describe molecular weight averages and distribution.     

Performance of a highly lipophilic Li+ lonophore in solvent polymeric membranes

Ionophores based on non-cyclic triamides exhibiting Li⁺ selectivity in solvent polymeric membranes are described. Their lipophilicity (water/1-n-octanol partition coefficient) is up to 10^13.8 so that they can be used in ion-selective electrodes with a lifetime of up to 40 000 h even in permanent contact with whole blood or undiluted blood serum.On leave from the Department of General Chemistry, Technical University of Gdansk, PL-80-952 Gdansk, Poland     

Characterization of a distributed plasma ionization source (DPIS) for ion mobility spectrometry and mass spectrometry

A recently developed atmospheric pressure ionization source, a distributed plasma ionization source (DPIS), was characterized and compared to commonly used atmospheric pressure ionization sources with both mass spectrometry (MS) and ion mobility spectrometry (IMS). The source consisted of two electrodes of different sizes separated by a thin dielectric. Application of a high RF voltage across the electrodes generated plasma in air yielding both positive and negative ions. These reactant ions subsequently ionized the analyte vapors. The reactant ions generated were similar to those created in a conventional point-to-plane corona discharge ion source. The positive reactant ions generated by the source were mass identified as being solvated protons of general formula (H₂O)_nH⁺ with (H₂O)₂H⁺ as the most abundant reactant ion. The negative reactant ions produced were mass identified primarily as CO₃⁻, NO₃⁻, NO₂⁻, O₃⁻ and O₂⁻ of various relative intensities. The predominant ion and relative ion ratios varied depending upon source construction and supporting gas flow rates. A few compounds including drugs, explosives and amines were selected to evaluate the new ionization source. The source was operated continuously for 3 months and although surface deterioration was observed visually, the source continued to produce ions at a rate similar that of the initial conditions.     

Sub-Minute Analysis of Lactate from a Single Blood Drop Using Capillary Electrophoresis with Contactless Conductivity Detection in Monitoring of Athlete Performance

A simple and fast method for the analysis of lactate from a single drop of blood was developed. The finger-prick whole blood sample (10 µL) was diluted (1:20) with a 7% (w/v) solution of [tris(hydroxymethyl)methylamino] propanesulfonic acid and applied to a blood plasma separation device. The device accommodates a membrane sandwich composed of an asymmetric polysulfone membrane and a supporting textile membrane that allows the collection of blood plasma into a narrow glass capillary in less than 20 s. Separated and simultaneously diluted blood plasma was directly injected into a capillary electrophoresis instrument with a contactless conductivity detector (CE-C⁴D) and analyzed in less than one minute. A separation electrolyte consisted of 10 mmol/L l-histidine, 15 mmol/L dl-glutamic acid, and 30 µmol/L cetyltrimethylammonium bromide. The whole procedure starting from the finger-prick sampling until the CE-C⁴D analysis was finished, took less than 5 min and was suitable for monitoring lactate increase in blood plasma during incremental cycling exercise. The observed lactate increase during the experiments measured by the developed CE-C⁴D method correlated well with the results from a hand-held lactate analyzer (R = 0.9882). The advantage of the developed CE method is the speed, significant savings per analysis, and the possibility to analyze other compounds from blood plasma.