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Efficient elution of purified proteins from polyvinylidene difluoride membranes (Immobilon) after transfer from SDS-PAGE and their use as immunogens.

Following separation of proteins by SDS-PAGE, they are electroblotted onto polyvinylidene difluoride membranes (Immobilon). Protein bands of interest are excised, and the proteins are eluted from the membrane with detergent-containing buffers at pH 9.5. The method routinely yields recovery of 70-90%, and this is independent of protein molecular weight.

Antigens↗

Shipboard analysis of picomolar levels of manganese in seawater by chelating resin concentration and chemiluminescence detection.

A new shipboard analytical method for determining picomolar levels of manganese in seawater has been developed. The method is based on a combination of chelating resin column extraction and improved chemiluminescence (CL) detection in a closed flow system. In this method, manganese in sample solution is selectively collected on newly-developed iminodiacetate-immobilized chelating resin, and then eluted with acidic solution containing hydrogen peroxide. The resulting eluent is mixed with luminol solution and aqueous ammonia after removal of iron ions by a chelating resin column, and then the mixture is introduced into the CL cell. The manganese concentration is obtained from the CL intensity. The detection limit (3SD) of manganese is 5 pmol L(-1) from 9 mL of seawater sample. The method was applied to seawater samples collected at the Okinawa Trough.

Adsorption↗

A novel amperometric sensor and chromatographic detector for determination of parathion.

A novel amperometric sensor and chromatographic detector for determination of parathion has been fabricated from a multi-wall carbon nano-tube (MWCNT)/Nafion film-modified glassy-carbon electrode (GCE). The electrochemical response to parathion at the MWCNT/Nafion film electrode was investigated by cyclic voltammetry and linear sweep voltammetry. The redox current of parathion at the MWCNT/Nafion film electrode was significantly higher than that at the bare GCE, the MWCNT-modified GCE, and the Nafion-modified GCE. The results indicated that the MWCNT/Nafion film had an efficient electrocatalytic effect on the electrochemical response to parathion. The peak current was proportional to the concentration of parathion in the range 5.0x10(-9)-2.0x10(-5) mol L(-1). The detection limit was 1.0x10(-9) mol L(-1) (after 120 s accumulation). In high-performance liquid chromatography with electrochemical detection (HPLC-ED) a stable and sensitive current response was obtained for parathion at the MWCNT/Nafion film electrode. The linear range for parathion was over four orders of magnitude and the detection limit was 6.0x10(-9) mol L(-1). Application of the method for determination of parathion in rice was satisfactory.

Biosensing Techniques↗

A sensitive determination of dopamine in the presence of ascorbic acid using a nafion-coated clinoptilolite-modified carbon paste electrode.

A selective dopamine determination using a nafion-coated clinoptilolite-modified carbon paste electrode in the presence of ascorbic acid was studied. Both cyclic voltammetry (CV) and differential pulse anodic stripping voltammetry (DPASV) were used for measurements of dopamine. To improve the selectivity of the clinoptilolite-modified carbon paste electrode in presence of a high concentration of ascorbic acid, the electrode surface was coated with nafion membrane. Experimental parameters affecting the determination of dopamine, including the clinoptilolite ratio, nafion membrane thickness, preconcentration time, preconcentration solution pH, stripping solution pH and interferences are discussed. The developed sensor has a wide linear range, a low detection limit, and good stability and reproducibility. The sensor offers a good alternative to existing analytical methods for dopamine, permits a relatively short analysis time, and is simple, selective and inexpensive.

Ascorbic Acid↗

Horseradish peroxidase-based organic-phase enzyme electrode.

An organic-phase enzyme electrode (OPEE) based on horseradish peroxidase (HRP) immobilized within Nafion on spectroscopic graphite was investigated in acetonitrile. The amperometric electrode response to hydrogen peroxide and cumene hydroperoxide present was found to be the result of the reduction of oxygen, produced upon enzymatic decomposition of both hydroperoxides (i.e., by the catalase-like activity of HRP). The electrode response was found to depend linearly on the hydroperoxide concentration up to 700 microM within the range of potentials from -200 to -400 mV (versus Ag|AgCl). Detection limits of approximately 45 microM for H2O2 and 100 microM for cumene hydroperoxide were determined under the selected experimental conditions. Nernstian dependence (the open circuit voltage of HRP-based electrode versus logarithm of H2O2 concentration) was obtained between 0.2 and 2.0 mM, with a slope of approximately 23 mV per logarithmic unit, suggesting a catalase-like, two-electron disproportionation of the substrate in acetonitrile.

Benzene Derivatives↗

Stacked films immobilization of MBTH in nafion/sol-gel silicate and horseradish peroxidase in chitosan for the determination of phenolic compounds.

The stacked-film immobilization of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in hybrid nafion/sol-gel silicate film and horseradish peroxidase (HRP) in chitosan, performed in order to allow the determination of phenolic compounds, was investigated via an optical method. The stacked films were deposited onto a microscope glass slide by a spin-coating technique. The quinone or free radical product formed by the enzymatic reactions of phenolic compounds interacts with MBTH to form azo-dye products, which can be measured spectrophotometrically at a wavelength of 500 nm. The color intensity of the product was found to increase in proportion to the phenolic concentration after 5 min of exposure. The response of the biosensor was linear over concentration ranges of 0.025-0.500, 0.010-0.070 and 0.050-0.300 mM for guaiacol, resorcinol and o-cresol, respectively, and gave detection limits of 0.010, 0.005 and 0.012 mM. The sensor exhibited good sensitivity and stability for at least two months.

Azo Compounds↗

An improved method for the analysis of volatile polyfluorinated alkyl substances in environmental air samples.

This article describes the optimisation and validation of an analytical method for the determination of volatile polyfluorinated alkyl substances (PFAS) in environmental air samples. Airborne fluorinated telomer alcohols (FTOHs) as well as fluorinated sulfonamides and sulfonamidoethanols (FOSAs/FOSEs) were enriched on glass-fibre filters (GFFs), polyurethane foams (PUFs) and XAD-2 resin by means of high-volume air samplers. Sensitive and selective determination was performed using gas chromatography/chemical ionisation-mass spectrometry (GC/CI-MS). Five mass-labelled internal standard (IS) compounds were applied to ensure the accuracy of the analytical results. No major blank problems were encountered. Recovery experiments were performed, showing losses of the most volatile compounds during extraction and extract concentration as well as strong signal enhancement for FOSEs due to matrix effects. Breakthrough experiments revealed losses of the most volatile FTOHs during sampling, while FOSAs/FOSEs were quantitatively retained. Both analyte losses and matrix effects could be remediated by application of adequate mass-labelled IS. Method quantification limits (MQLs) of the optimised method ranged from 0.2 to 2.5 pg/m(3) for individual target compounds. As part of the method validation, an interlaboratory comparison of instrumental quantification methods was conducted. The applicability of the method was demonstrated by means of environmental air samples from an urban and a rural location in Northern Germany.

Air Pollutants↗

Amperometric detection studies of Nafion/indium hexacyanoferrate film for the determination of electroinactive cations in ion chromatography.

An amperometric detector based on the chemical modification of Nafion and indium (III) hexacyanoferrate (II, III) thin film (Nafion/In-CN-Fe) onto a glassy carbon (GC) electrode, was first successfully used for the determination of electroinactive cations (Li+, Na+, K+, Rb+, Cs+, NH4+) in single column ion chromatography (IC). A set of well-defined peaks of electroinactive cations was obtained. The detection limits of the cations are 8.9 x 10(-6) mol/L for Li+, 2.3 x 10(-6) mol/L for Na+, 5.2 x 10(-6) mol/L for K+, 4.8 x 10(-6) mol/L for Rb+, 4.0 x 10(-6) mol/L for Cs+ and 5.3 x 10(-6) mol/L for NH4+ at a single-to-noise ratio of 3. The proposed method was quick, sensitive and simple. The cations in rainwater and mineral water were successfully analyzed by this method.

Acid Rain↗

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry.

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2-10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 +/- 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.

Calibration↗

Development of a new membrane-type heated humidifier for laparoscopic surgery.

BACKGROUND: Recently, several animal studies showed that the core body temperature falls during pneumoperitoneum, and this hypothermia could be prevented by using heated and humidified gas insufflation. However, there are no satisfactory heated humidifiers to meet this purpose. Therefore, we developed a new membrane-type heated humidifier. MATERIALS AND METHODS: The newly developed heated humidifier employs an ion-exchange membrane (Nafion: Du Pont Co. Ltd) tube that passes water selectively in molecular form and a gas compartment is completely separated from distilled water to prevent infection. This humidifier consists of a Nafion tube assembly and a case that includes the heater. A perforated protecting tube is located outside the Nafion tube to prevent direct contact with the Nafion tube when it is assembled. The Nafion tube assembly is inserted in the case, and dry gas flows inside of the Nafion tube. The space between the case and the Nafion tube assembly is filled with distilled water. A heater raises the temperature of the distilled water, and heat and water vapor are transferred to cold and dry gas through the Nafion membrane. Four different types of insufflators were involved in this performance comparison study: a Nafion-based heated and humidified insufflator, a conventional heated insufflator, a conventional heated and humidified insufflator, and a conventional cold and dry insufflator. Temperature and relative humidity were measured once each minute for 15 min, which was repeated four times. Each insufflator was operated at two rates of flow: 5 L/min and 10 L/min. RESULTS: Temperature and humidity of the conventional cold and dry insufflator, the Nafion membrane-type heated and humidified insufflator, the conventional heated insufflator, and the conventional heated and humidified insufflator measured at the distal end of circuit reached 22.0 +/- 0.2 degrees C and 0%, 36.7 +/- 1.1 degrees C and 100%, 29.0 +/- 0.4 degrees C and 0%, and 31.3 +/- 0.4 degrees C and 70.5 +/- 5.3% in 15 min at 5 L/min flow rates. At 10 L/min flow rates, temperature and humidity were almost the same as those at 5 L/min. The membrane-type heated humidifier demonstrated statistically significant improvement in both the temperature ( p < 0.0001) and relative humidity ( p < 0.0001) parameters in comparison to the conventional normal, heated, or heated and humidified insufflators at 5 L/min and 10 L/min continuous flow rate in statistics using repeated-measure ANOVA. CONCLUSION: This newly developed heated humidifier offers the great advantages of maintaining intraabdominal temperature and humidity in comparison to conventional insufflators for laparoscopic surgery.

Equipment Design↗

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots.

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV-vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0-10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H2O2 without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H2O2 in the range 5x10(-7)-3x10(-4) M with a detection limit of 6x10(-8) M. The response shows Michaelis-Menten behavior at higher H2O2 concentrations and the apparent Michaelis-Menten constant is estimated to be 112 microM.

Biosensing Techniques↗

Theoretical studies of the conformational behavior of chain molecules containing polar groups: simulations of a poly(vinylidene fluoride) model.

Atomistic Monte Carlo simulations have been conducted to elucidate the conformational behavior of a single chain molecule containing polar functional groups. Here, we resort to an atomistic poly(vinylidene fluoride) (PVDF) chain model as a representative example. The model is modified in such a way that bond lengths and bond angles are fixed, aiming to manifest the role of dipolar interactions. For a given chain length, chain conformation is sensitive to two environmental parameters, temperature and dielectric constant. The mean chain size increases when temperature and/or dielectric constant are increased. The conformational behavior is further characterized by chain size distribution function, and our findings show that temperature induced conformational transition for a chain molecule can be discrete or continuous, depending on its chain length. Also, the dipolar interactions in PVDF are effectively attractive, and enhance chain contraction. As a result, when the strength of dipolar interactions is increased, the discrete conformational transition shifts toward longer chains; and for a given chain length, such a transition occurs at higher temperatures.

Algorithms↗

Infragenicular bypass graft for limb salvage using polytetrafluoroethylene and distal vein cuff as the first alternative in patients without ipsilateral greater saphenous vein.

The objectives of this study were to evaluate the results of polytetrafluoroethylene infragenicular bypass grafts with a distal interposition vein cuff in patients with critical limb ischemia in the absence of ipsilateral greater saphenous vein. From January 1997 to June 2002, 58 consecutive below-knee bypass grafts with PTFE and distal interposition vein cuff were performed in 57 patients with a median age of 70.8 years. The distal anastomosis was located at the infragenicular popliteal artery in 18 cases and at tibial vessels in 40. Primary patency, secondary patency, and limb salvage were analyzed using the Kaplan-Meier method. During a median follow-up of 14.4 months (range, 1-50) 26 cases of graft occlusion and 19 major amputations were registered. The primary and secondary patency rates at 12, 24, and 36 months were 57%, 54%, and 47% and 61%, 58%, and 50%, respectively. Limb salvage rates reached 69%, 69%, and 59% at 12, 24, and 36 months. When below-knee revasculanzation is required in patients with limb-threatening ischemia, in the absence ipsilateral greater saphenous vein, PTFE grafts with a distal vein cuff are a reasonable substitute with acceptable long-term patency and limb salvage rates.

Aged↗

Development of a fluorinated polyimide hollow fiber for medical devices.

We fabricated an asymmetric polyimide hollow fiber for medical devices. A dry/wet phase inversion process was applied to a spinning process to prepare the hollow fiber. The outer diameter was 330 microm with a wall thickness of 70 microm. Transfer rates of O2 and CO2 in the asymmetric polyimide fiber were 6.9 x 10(-3) and 5.5 x 10(-3) cm3 (STP)/(cm2 s cmHg), respectively, which are approximately 10 times higher than those measured in the Menox and Si-polypropylene fibers of the presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber was evaluated in vivo, indicating that polyimide had excellent blood compatibility when compared with silicone-coated fiber. Additionally, we fabricated a novel porous membrane with three-dimensional fine structure from cylindrical microscale pores and examined possibility of a porous membrane for use in hemodialysis.

Animals↗

Development of an oxygen-rich biosensor using enzymatic reaction.

This work reports a novel strategy for the development of an O2-rich biosensor. The principle is based on an enzymatic reaction between catalase and H2O2 to release O2, thus to increase the O2 amount in the enzyme matrix. This method improves the determination reliability by alleviating the O2 dependence.

Animals↗

A wideband PVDF-on-silicon ultrasonic transducer array with microspheres embedded low melting temperature alloy backing.

A PVDF-based 8-element ultrasound transducer array (1 mm x 1 mm element size with an inter-element spacing of 1 mm) on a silicon carrier substrate is fabricated and characterized. To improve the performance of the transducer, new CMOS-compatible fabrication technologies are introduced. These include: (1) adhesive micro-contact printing on non-radiating areas, and (2) glass microspheres (7-20 microm in diameter) embedded low melting temperature alloy (LMA) for backside electrical connection. The first improvement removes the adverse effects of adhesive layer (e.g., lower sensitivity) between the PVDF and backside contact while the second one improves the pulse-echo signal quality by eliminating reflections at the backing/water interface. The fabricated array elements are tested in a water tank and their pulse-echo response are recorded. The central frequency of each element is 25 MHz with a 100% measured 6-dB bandwidth (60% 3-dB bandwidth).

Alloys↗