Topography of dipalmitoyl-phosphatidyl-choline monolayers penetrated by β-casein

In this work we have analyzed the topography by atomic force microscopy (AFM) of dipalmitoyl-phosphatidyl-choline (DPPC) monolayers previously spread at the air–water interface and penetrated by β-casein. AFM images of β-casein–DPPC monolayers were taken from Langmuir–Blodgett films deposited onto hydrophilic mica substrates at different initial surface pressures (π_i) and after the compression of the mixed films. The monolayer topography depends on the initial structure of the phospholipid:liquid expanded (LE) at 3 mN/m, coexistence between LE and liquid condensed (LC) structures at 7 mN/m, at the end of the LE–LC transition at 10 mN/m, and with a LC structure at 15 mN/m. The area occupied by DPPC domains in the mixed film increases with the π_i value, especially for DPPC with a LC structure at 15 mN/m. At this surface pressure the thickness of the film is at a maximum. After the film compression at 25 mN/m, which is above the equilibrium spreading pressure of β-casein (), this protein is displaced from the interface by DPPC and the topography of the mixed monolayer depends on the initial structure of the DPPC monolayer. A notable feature of the topography of these mixed monolayers is the presence of multilayers of β-casein and DPPC of high thickness (50–70 nm) at the lower π_i values. Although the film is dominated by DPPC at the highest surface pressures (at 25 mN/m), β-casein is not displaced totally from the interface and coexists as β-casein collapsed domains within the network of the DPPC structure.     

Stacking and determination of phenazine-1-carboxylic acid with low p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> in soil via moving reaction boundaryformed by alkaline and double acidic buffers in capillary electrophoresis

As shown herein, a normal moving reaction boundary (MRB) formed by an alkaline buffer and a single acidic buffer had poor stacking to the new important plant growth promoter of phenazine-1-carboxylic acid (PCA) in soil due to the leak induced by its low pK _a. To stack the PCA with low pK _a efficiently, a novel stacking system of MRB was developed, which was formed by an alkaline buffer and double acidic buffers (viz., acidic sample and blank buffers). With the novel system, the PCA leaking into the blank buffer from the sample buffer could be well stacked by the prolonged MRB formed between the alkaline buffer and blank buffer. The relevant mechanism of stacking was discussed briefly. The stacking system, coupled with sample pretreatment, could achieve a 214-fold increase of PCA sensitivity under the optimal conditions (15 mM (pH 11.5) Gly-NaOH as the alkaline buffer, 15 mM (pH 3.0) Gly-HCl-acetonitrile (20%, v/v) as the acidic sample buffer, 15 mM (pH 3.0) Gly-HCl as the blank buffer, 3 min 13 mbar injection of double acidic buffers, benzoic acid as the internal standard, 75 μm i.d. × 53 cm (44 cm effective length) capillary, 25 kV and 248 nm). The limit of detection of PCA in soil was decreased to 17 ng/g, the intra-day and inter-day precision values (expressed as relative standard deviations) were 3.17–4.24% and 4.17–4.87%, respectively, and the recoveries of PCA at three concentration levels changed from 52.20% to 102.61%. The developed method could be used for the detection of PCA in soil at trace level.     

New protocols for preparing dipalmitoylphosphatidylcholine dispersions and controlling surface tension and competitive adsorption with albumin at the air/aqueous interface

The adsorption behavior of dipalmitoylphosphatidylcholine (DPPC), which is the major component of lung surfactant, at the air/aqueous interface and the competitive adsorption with bovine serum albumin (BSA) were studied with tensiometry, infrared reflection absorption spectroscopy (IRRAS), and ellipsometry. Dynamic surface tensions lower than 1 mN/m were observed for DPPC dispersions, with mostly vesicles, prepared with new protocols, involving extensive sonication above 50 °C. The lipid adsorbs faster and more extensively for DPPC dispersions with vesicles than with liposomes. For DPPC dispersions by a certain preparation procedure at T > T_c, when lipid particles were observed on the surface, dynamic surface tensions as low as 1 mN/m were measured. Moreover, IRRAS intensities and ellipsometric δΔ values were found to be much higher than the values for other DPPC dispersions or spread DPPC monolayers, suggesting that a larger amount of liposomes or vesicles adsorb on the surface. For DPPC/BSA mixtures, the tension behavior is controlled primarily by BSA, which prevents the formation of a dense DPPC monolayer. When BSA is injected into the subphase with a spread DPPC monolayer or into a DPPC dispersion with preadsorbed layers, little or no BSA adsorbs and the DPPC layer remains on the surface. When a DPPC monolayer is spread on a BSA solution at 0.1 wt% at 25 °C, then DPPC lipid can displace the adsorbed BSA molecules. The lack of BSA adsorption, and the expulsion of BSA by DPPC monolayer is probably due to the strong hydrophilicity of the lipid polar headgroup. When a DPPC dispersion is introduced with Trurnit's method or when dispersion drops are sprayed onto the surface of a DPPC/BSA mixture, the surface tension becomes lower and is controlled by DPPC, which can prevent the adsorption of BSA. The results may be important in understanding inhibition of lung surfactants by serum proteins and in designing efficient protocols of surfactant preparation and administration.     

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δπ_max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC₅₀ concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δπ_max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.     

Separation of Anthraquinones by Capillary Electrophoresis and High‐Performance Liquid Chromatography

The separation and determination of twelve anthraquinones, viz. anthraquinone 1, chrysphanol 2, aloe‐emodin 3, alizarin 4, anthraquinone‐2‐carboxylic acid 5, purpurin 6, sennoside B 7, sennoside A 8, emodin 9, quinalizarin 10, rhein 11, and anthraflavic acid 12, were achieved by capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). Detection at 260 nm with a buffer solution containing 30 mM sodium borate (adjusted to pH = 10.56 with 0.05N NaOH) and acetonitrile (9 : 1) in CE or with a linear gradient elution containing 20 mM KH₂PO₄ with 0.05% phosphoric acid (pH = 2.91) and methanol in HPLC was found to be the most suitable approach for this separation. Contents of six components (2, 3, 7, 8, 9, 11) in crude Rhei Rhizoma extract could easily be determined within 39 min by CE or 63 min by HPLC. The effects of buffers on this separation and the validation of the two methods were studied.     

Effect of the Buffer Concentration on the Separation of Lactate Dehydrogenase Isoenzymes from Different Sources by Electrophoresis

Abstract

The separation of lactate dehydrogenase isoenzymes by zone electrophoresis using cellulose acetate strips as support was dependent on the concentration of the buffer used (5 mM and 50 mM, pH 7.4) and on the source of the material (chicken liver or guinea-pig liver).

In three different 5 mM buffer systems, pH 7.4 (phosphate, veronal and Tris-HC1) the four lactate dehydrogenase isoenzymes present in chicken liver cytosol: M₃H, M₂H₂, MH₃ and H₄ were resolved into four separated bands. M₃H and M₂H₂ isoenzymes migrated towards the cathode whereas the other two isoenzymes showed anodic mobilities. In 50 mM buffers, pH 7.4 all enzyme activity appeared as a single band with anodic mobility similar to that of H₄. Guinea-pig liver isoenzymes were well resolved in both buffer conditions and appeared as five bands with anodic mobilities.

The different behaviour of the lactate dehydrogenase isoenzymes in 5 mM and 50 mM buffers can not be assigned to ionic strength effects but it may explained by assuming the binding of buffer anions to the different isoenzymes. The binding would increase with the molar concentration of the buffer and reduce charge differences among the isoenzymes to different extents depending on the source of the enzyme, chicken or guinea-pig liver.     

Development of a microbioreactor with ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) immobilized on a polyacrylamide-coated capillary at the outlet

A simple and fast method of immobilization of cell membrane suspension containing human ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) on a polyacrylamide-coated capillary was developed. The enzyme microbioreactor was prepared by hydrodynamic injection of a small plug of the polycationic electrolyte hexadimethrine bromide (HDB) followed by a suspension of an enzyme-containing membrane preparation. In order to shorten the enzyme assay time and to increase the throughput of the assay, the capillary was coated from the outlet end and all injections were performed from the outlet end of the capillary. For the monitoring of the enzymatic reaction, the substrate ATP dissolved in reaction buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM Hepes, pH 7.4, internal standard: 10 μM UMP) in the absence or presence of inhibitor was injected electrokinetically and incubated in the microbioreactor for 1 min with 1 kV of applied voltage. Then, the electrophoretic separation of the reaction products was initiated by applying a constant current of 60 μA. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by UV absorbance at 260 nm. The new method was compared with an at-capillary-inlet method without immobilization of the enzyme. The results (K_m values, K_i values for inhibitor) obtained with both methods were similar and comparable with literature data. The developed outlet immobilized enzyme microreactor using a coated capillary is very fast, simple and most economic allowing multiple use of the enzyme.     

Molecular origin for rheological characteristics of native gellan gum

The ¹H-nuclear magnetic resonance spectrum showed that the l-rhamnosyl residues of native gellan gum were coinvolved in both a small number of ⁴C₁-pyranose conformations and a large number of ¹C₄-pyranose conformations, whereas for deacylated polymer, almost of the residues were involved in ⁴C₁-pyranose conformation. The flow curves of native gellan gum showed plastic behavior above 0.2%. The elastic modulus stayed at a constant value with increase in temperature up to 40 °C, then decreased rapidly. The elastic modulus increased with addition of CaCl₂ (6.8 mM) and stayed constant value with increase in temperature up to 65 °C, then decreased rapidly. The stronger elastic modulus was observed in deacylated gellan gum with addition of CaCl₂. The elastic modulus of native gellan gum showed larger value than that in aqueous solution in the presence of urea (4.0 M). Intra- and intermolecular associations of native gellan gum molecules in the presence of Ca⁺² were proposed.     

Electrochemical behaviour of isatin at a glassy carbon electrode

A rapid, reliable and simple capillary zone electrophoresis method for the determination of organic acids in beverages was developed. The complete separation of oxalic, formic, tartaric, malic, succinic, maleic, glutaric, pyruvic, acetic, lactic, citric, butyric, benzoic, sorbic, ascorbic and gluconic acids can be achieved in less than 3.5 min with a simple electrolyte composed by phosphate as the carrier buffer (7.5 mM NaH₂PO₄ and 2.5 mM Na₂HPO₄), 2.5 mM TTAOH as electroosmotic flow modifier and 0.24 mM CaCl₂ as selectivity modifier, adjusting the pH at 6.40 constant value. Injection was performed in hydrodynamic mode (30 s) and the detection mode was UV direct at 185 nm. The running voltage was −25 kV at thermostated temperature of 25 °C. The method developed has been applied to several beverage samples with only a simple dilution and filtration treatment of the sample. The proposed method is fast because the separation time decrease two, four or, even, six times the separation times of the previous reported CZE methods. It is also simple and cheap due to a low consumption of chemicals and samples. These reasons permit it to be considered adequate for routine analysis of organic acids in beverage samples.     

Voltammetry of immobilized cytochrome c on novel binary self-assembled monolayers of thioctic acid and thioctic amide modified gold electrodes

Horse heart cytochrome c (cyt c) was adsorbed on the binary self-assembled monolayers (SAMs) composed of thioctic acid (T-COOH) and thioctic amide (T-NH₂) at gold electrodes via electrostatic interaction. The cyt c adsorbed on the modified gold electrode exhibited well-defined reversible electrochemical behavior in 10 mM phosphate buffer solution (PBS, pH 7.0). The surface concentration (Γ) of electroactive species, cyt c, on the binary SAMs was higher than that in single-component SAMs of T-COOH, and reached a maximum value of 9.2 × 10⁻¹² mol cm⁻² when the ratio of T-COOH to T-NH₂ in adsorption solution was of 3:2, and the formal potential (E^0′=(E_pa+E_pc)/2) of cyt c was −0.032 V (vs. Ag|AgCl (3 M NaCl)) in a 10 mM PBS. The interaction between cyt c and the binary SAMs made the E^0′ shift negatively when compared with that of cyt c in solution (+0.258 V vs. NHE, i.e., +0.058 V vs. Ag|AgCl (3 M NaCl)). The fractional coverage of bound cyt c was a 0.64 theoretical monolayer. The standard electron transfer rate constant of cyt c immobilized on the binary SAMs was also higher than that on single-component SAMs of T-COOH, and the maximum value of 15.8 ± 0.6 s⁻¹ was obtained when the ratio of T-COOH to T-NH₂ in adsorption solution was at 3:2. The results suggest that the electrode modified with the binary SAMs functions better than the electrode modified with single-component SAMs of T-COOH.     

Analysis of amino acid neurotransmitters by capillary electrophoresis and laser-induced fluorescence using a new fluorescein-derived label

A capillary electrophoresis laser-induced fluorescence detection method (CE-LIF) was developed for the separation of eight neurotransmitters tagged on their amino function with 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl) fluorescein (SAMF), a new fluorescent reagent synthesized in our lab. Derivatization was performed in boric acid buffer (pH = 7.75) at 37 °C over 15 min. The pH-independent fluorescence of SAMF (pH 4–9) permits background buffers over a wide range of pH. It was demonstrated that an acidic running buffer offers a better resolution compared to basic medium in terms of resolution and peak shapes. Employing Cu²⁺ as the additive, the molecules were baseline-separated using a running buffer consisting of 40 mM sodium acetate and 2 mM Cu²⁺ (pH 6.0). The detection limits ranged from 1 to 2 × 10⁻¹⁰ M. The method has been validated for the characterization of lymphocyte samples. The results obtained illustrate the advantages of combining SAMF derivatization with CE-LIF for determining neurotransmitters.     

Excess fibrinogen adsorption to monolayers of mixed lipids

Adsorption of fibrinogen to the monolayers of mixed lipids, dipalmitoyl phosphatidyl choline (DPPC) and eicosylamine (EA) was measured at a surface pressure of 20 mN/m by an in situ surface plasmon resonance technique. Pressure–area isotherms of DPPC + EA mixtures on water and buffer subphases indicated good lipid miscibility and some contraction of the monolayers at intermediate and higher surface pressures. Surface electric potential of the DPPC + EA monolayers showed excess values for intermediate DPPC:EA ratios. Fibrinogen adsorption and its adsorption rates from a dilute solution (0.03 mg/ml) were proportional to the fraction of EA in the monolayer indicating that protein binding was primarily driven by electrostatic interactions between positive EA charges in the monolayer and a net negative protein charge. At a higher protein concentration (0.06 mg/ml) both the fibrinogen adsorbed amount and its maximum adsorption rate showed excess values relative to the pure EA for 1:1, 2:1 and 3:1 DPPC + EA monolayers. This excess adsorption could be explained, in part, by the contraction of the monolayers with intermediate DPPC:EA ratios which resulted in an excess surface electric potential.     

Dispersion stability and aggregation behavior of TEMPO-oxidized cellulose nanofibrils in water as a function of salt addition

Dispersion stability of TEMPO-oxidized cellulose nanofibrils (TOCNs) in water was investigated through both experimental and theoretical analyses to elucidate the critical aggregation concentration of different salts. The 0.1 wt% TOCN/water dispersions with various NaCl concentrations were evaluated by measuring light transmittance, viscosity under steady-shear flow, and the weight fraction of TOCN that had aggregated. Homogeneous TOCN/water dispersion turned to gel as the NaCl concentration increased. The TOCN dispersion maintained its homogeneous state up to 50 mM NaCl, but aggregated gel particles were formed at 100 mM NaCl. The mixture became separated into two phases (gel and supernatant) at ≥200 mM NaCl. Theoretical analysis using ζ-potentials of TOCN elements in the dispersions revealed that the aggregation behavior upon NaCl addition could be explained well in terms of the interaction potential energy between two cylindrical rods based on the Derjaguin–Landau–Verwey–Overbeek theory. The experiments were extended to analyze critical aggregation concentrations of MgCl₂ and CaCl₂ for the 0.1 wt% TOCN dispersion. In the case of divalent electrolytes, TOCN elements began to form aggregated gel particles at salt concentrations of 2–4 mM, corresponding to the critical aggregation concentration predicted by the empirical Schultz-Hardy rule.     

Mixed layers of DPPC and a linear poly(ethylene glycol)-b-hyperbranched poly(glycerol) block copolymer having a cholesteryl end group

Interactions of the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with the amphiphilic diblock copolymer Ch-lPEG₃₀-b-hbPG₂₄ (ChP) are studied at the air–water interface by surface pressure–mean molecular area (π–mmA) measurements of mixed Langmuir films and adsorption measurements of ChP to the air–water interface covered with DPPC monolayers at different initial surface pressure values π ₀. ChP is composed of a single hydrophobic cholesteryl (Ch) moiety covalently bound to a diblock copolymer consisting of a hydrophilic linear poly(ethylene glycol) (lPEG) block and a hydrophilic hyperbranched poly(glycerol) (hbPG) block. Langmuir isotherms and compression moduli of the mixed Langmuir films of different molar ratios reveal distinct interactions between DPPC and ChP during compression. It is demonstrated that the behavior of the DPPC/ChP mixtures is dominated by DPPC up to a molar ratio of 10:1, whereas the behavior is predominantly governed by ChP in mixtures with lower DPPC content (molar ratios of 5:1, 2:1, and 1:1). In adsorption measurements, a strong affinity of ChP to DPPC is observed after injection into the water subphase. The surface pressure value π _in up to which ChP is able to penetrate into DPPC monolayers is determined to the remarkably high value of 48.2 mN/m which attests the favorable interactions between DPPC and the Ch moiety of ChP. Atomic force microscopy on LB films of DPPC/ChP mixtures of different molar ratios transferred onto hydrophilic substrates confirms the presence of two different phases, a DPPC-rich phase and a ChP-rich phase.     

Capillary electrophoresis-chemiluminescence determination of norfloxacin and prulifloxacin

A capillary electrophoresis (CE)–chemiluminescence (CL) method for determining norfloxacin (NFLX) and prulifloxacin (PFLX) was developed based on the enhanced CL intensity of the cerium(IV)–sulfite–fluoroquinolone (FQ) reaction sensitized by terbium(III). The separation was conducted in buffer composed of 20 mM sodium citrate, 4 mM citric acid and 10 mM sodium sulfite at pH 6.1. The CL reagent solution consisted of 2 mM cerium(IV), 4 mM terbium(III) and 1.1 mM hydrochloric acid. NFLX and PFLX were baseline separated within 11 min with detection limits (S/N = 3) of 0.057 and 0.084 μg mL⁻¹, respectively. The maximum intra- and inter-day relative standard deviations (R.S.D.s) of migration time of the analytes were less than 4.0% and 4.2%, respectively. The proposed method was applied to detect NFLX and PFLX in fortified urine sample and the results were comparable to high-performance liquid chromatography (HPLC)–UV method. Moreover, the high selectivity of the CL detection and the high-separation efficiency of CE render the method the potential of quick analyzing fluoroquinolones in real complex matrix.     

A simple isocratic LC method for quantification of trace-level inorganic degradation impurities (ferricyanide,ferrocyanide, nitrite,and nitrate) in sodium nitroprusside injection and robustness by quality using design approach

This study developed and validated a trace-level quantification inorganic impurities method using reversed-phase HPLC and performed the robustness check using quality-by-design approach by varying the multiple factors simultaneously. This method is economical and simple and exhibits its stability-indicating nature [for the determination of ferrocyanide ([Fe(CN)₆]^4–), ferricyanide ([Fe(CN)₆]³⁻), nitrate (NO₃^–), and nitrite (NO₂^–)] in sodium nitroprusside (SNP) drug substance and liquid dosage form. Chromatographic separation was achieved using a USP L43 column (ACE PFP, 150 × 4.6 mm, 3 μm) with a simple isocratic elution. The buffer consists of potassium dihydrogen phosphate (50 mM), tetrabutylammonium hydrogen sulfate (9 mM), and tetrabutylammonium hydroxide (25 mM). The buffer pH was adjusted to 7.2 with tetrabutylammonium hydroxide. The mobile phase was mixed with the buffer and acetonitrile (68:32 v/v). The flow rate was 0.8 mL/min, column temperature was maintained at 30°C, and injection volume was 5.0 μL. The SNP impurities were monitored at 225 nm using a UV detector. Further, the method was validated per the International Council for Harmonisation (ICH) guidelines, and forced degradation studies were carried out under different stress conditions. The detector responses were plotted against concentrations, and correlation was linear (r > 0.999) over the range of 0.8–7.5 μg/mL for ferricyanide; 1.0–37.5 μg/mL for SNP; and 0.2–7.5 μg/mL for ferrocyanide, nitrite, and nitrate. The method repeatability was established for all the impurities with relative standard deviation (%), and the results were found to be less than 2.0.     

Analysis of methotrexate and its eight metabolites in cerebrospinal fluid by solid-phase extraction and triple-stacking capillary electrophoresis

We establish a triple-stacking capillary electrophoresis (CE) separation method to monitor methotrexate (MTX) and its eight metabolites in cerebrospinal fluid (CSF). Three stacking methods with different mechanisms were combined and incorporated into CE separation. Complete stacking and sharp peaks were achieved. Firstly, the optimized buffer (60 mM phosphate containing 15% THF and 100 mM SDS) was filled into the capillary, which was followed by the higher conductivity buffer (100 mM phosphate, 2 psi for 45 s). The analytes extracted from CSF were injected at 2 psi for 99.9 s, which provided long sample zones and pH junction for focusing. Finally, the stacking step was performed by sweeping, and separation was achieved by micellar electrokinetic chromatography. The results of the linear regression equations indicated high linearity (r ≥ 0.9981) over the range of 0.5–7 μM. In intra- and inter-batch results, all data of RSD and RE were below 11%, indicating good precision and accuracy of this method. The LODs (S/N = 3) were 0.1 μM for MTX, 7-hydroxymethotrexate (7-OHMTX) and MTX-polyglutamates (MTX-(Glu) _{n, n = 2–5}), 0.2 μM for MTX-(Glu)₆, and 0.3 μM for 2,4-diamino-N ¹⁰-methylpteroic acid (DAMPA) and MTX-(Glu)₇. Our method was implemented for analysis of MTX and its metabolites in the CSF, and could be used for evaluation of its curative effects of acute lymphoblastic leukemia patients. The data were also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed good coincidence.     

An enzymatic glucose/O2 biofuel cell: Preparation,characterization and performance in serum

This study demonstrates a new kind of single-walled carbon nanotubes (SWNT)-based compartment-less glucose/O₂ biofuel cell (BFC) with glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) as the anodic and cathodic biocatalysts, respectively, and with poly(brilliant creysl blue) (BCB) adsorbed onto SWNT nanocomposite as the electrocatalyst for the oxidation of NADH. The prepared GDH-polyBCB-SWNT bioanode exhibits an excellent electrocatalytic activity toward the oxidation of glucose biofuel; in 0.10 M phosphate buffer containing 20 mM NAD⁺ and 100 mM glucose, the oxidation of glucose commences at −0.25 V and the current reaches its maximum of 310 μA/cm² at −0.05 V vs. Ag/AgCl. At the BOD-SWNT biocathode, a high potential output is achieved for the reduction of O₂ due to the direct electron transfer property of BOD at the SWNTs. In 0.10 M phosphate buffer, the electrocatalytic reduction of O₂ is observed at a high potential of 0.53 V vs. Ag/AgCl with an electrocatalytic current plateau of ca. 28 μA/cm² at 0.45 V under ambient air and ca. 102 μA/cm² under O₂-saturated atmosphere. In 0.10 M phosphate buffer containing 10 mM NAD⁺ and 40 mM glucose under O₂-saturated atmosphere, the power density of the assembled SWNT-based glucose/O₂ BFC reaches 53.9 μW/cm² at 0.50 V. The performance and the stability of the glucose/O₂ BFC are also evaluated in serum. This study could offer a new route to the development of new kinds of enzymatic BFCs with a high performance and provide useful information on future studies on the enzymatic BFCs as in vivo power sources.     

Stability and state of aggregation of aqueous fibrinogen and dipalmitoylphosphatidylcholine lipid vesicles

The stability and state of aggregation of aqueous fibrinogen (FB) and dipalmitoylphosphatidylcholine (DPPC) vesicles in water or buffer at 25 degrees C were studied with dynamic light scattering (DLS), UV-vis spectroturbidimetry (ST), and cryo-transmission electron microscopy (cryo-TEM). In water, when 1000 ppm (0.10 wt %) DPPC dispersions were prepared with a protocol including extensive sonication, they contained mostly vesicles and were quite clear, transparent, and stable for at least 30 days. FB mixtures with water (0.075 wt %) were quite unstable and biphasic. They formed large aggregates which eventually precipitated. The addition of DPPC vesicles into these unstable FB dispersions reversed FB aggregation and precipitation and produced stable translucent microdispersions. The inferred lipid/protein aggregates were limited in size, with average diameters ranging from 200 to 300 nm. In buffer, DPPC dispersions were also clear and quite stable, with average dispersed particles diameter of ca. 90 nm. FB dissolved in aqueous buffer and formed transparent and stable solutions. Adding salt to an aggregated FB dispersion in water reversed the aggregation. FB aggregated and redissolved in the presence of the citrate and after the citrate was removed. There was no effect of citrate (present in FB initially) in the FB aggregation or redissolution. FB molecules in buffer form dimers or higher aggregates. Their average aggregation number is 2, determined with Rayleigh scattering analysis of turbidity data. The average hydrodynamic diameter of FB solutions from DLS was 30 nm. Mixing a stable FB solution in buffer and a stable DPPC dispersion in buffer produced highly unstable mixtures, in which large aggregates precipitated. These results have implications in understanding the interactions of lipids and proteins in many biological applications and food processing applications.     

Evaluation of antitubercular drug insertion into preformed dipalmitoylphosphatidylcholine monolayers

Insertion profiles of antitubercular drugs isoniazid (INH), rifampicin (RFM) and ethambutol (ETH) into dipalmitoylphosphatidylcholine (DPPC) membrane models were evaluated by Langmuir monolayer technique. Maximum drug insertion into DPPC monolayer was observed with rifampicin with a surface pressure increase (Δπ_max) in the range of 21–33 mN/m depending upon rifampicin concentration. Isoniazid had minimal insertion resulting in a lower Δπ_max of about 2–3 mN/m, suggestive of minimal interactions between INH and DPPC. Ethambutol surface pressure increment on insertion resulted in an intermediate rise in the Δπ_max (6–10 mN/m). Antitubercular drug combination in the ratio of 2 mM:0.7 mM:4.5 mM for INH:RFM:ETH, attained Δπ_max between 25 and 33 mN/m. Insertion profiles similar to rifampicin were exhibited by the antitubercular drug mixture suggestive of predominant rifampicin insertion into the DPPC monolayer. The extent of drug insertion into the DPPC monolayer is suggestive of the drug penetration potential into biological membranes in vivo. Higher RFM Δπ_max is suggestive of excellent cell membrane penetration, which explains broad reach of the drug to all the organs including the cerebrospinal fluid while lower Δπ_max of INH suggests poor membrane penetration restricting the entry of the drug in different biological membranes. DPPC membrane destabilization was observed at higher antitubercular drug concentrations indicated by the negative slopes of the surface pressure–time curves. This may correlate with the dose related toxic effects observed in tuberculosis affected patients. Drug insertion studies offer a potential tool in understanding the pharmacotoxicological behavior of the various pharmacological agents.