Graft copolymerization of acrylic acid onto guar gum initiated by vanadium (V)–mercaptosuccinic acid redox pair

Guar gum has been modified by graft copolymerization with acrylic acid in aqueous medium using vanadium (V)–mercaptosuccinic acid redox system. The optimum reaction conditions affording maximum grafting ratio, efficiency, add on and conversion have been determined. The grafting parameters have been found to increase with increase in vanadium (V) concentration upto 1.0 × 10⁻² mol dm⁻³, but these parameters decrease on further increasing the vanadium (V) concentration. On increasing the mercaptosuccinic acid concentration from 1.0 × 10⁻² to 4.0 × 10⁻² mol dm⁻³ grafting ratio, efficiency and add on increase up to 2.0 × 10⁻² mol dm⁻³ but decrease with further increase in mercaptosuccinic acid concentration. On varying the acrylic acid concentration from 5.0 × 10⁻² to 30.0 × 10⁻² mol dm⁻³, maximum grafting ratio, efficiency and add on have been obtained at 20.0 × 10⁻² mol dm⁻³. The grafting ratio, add on and conversion increase, on increasing the H⁺ ion concentration from 1.5 × 10⁻¹ to 6.0 × 10⁻¹ mol dm⁻³. On increasing the guar gum concentration the grafting parameters increase. The grafting ratio, add on and conversion have been found to increase with time period while efficiency started decreasing after 120 min. It has been observed that %G increases on increasing the temperature up to 35 °C. The graft copolymer has been characterized by IR spectroscopy and thermogravimetric analysis.     

Synthesis of graft copolymer (k-carrageenan-g-N,N-dimethylacrylamide) and studies of metal ion uptake, swelling capacity and flocculation properties

Graft copolymer of k-carrageenan and N,N-dimethylacrylamide has been synthesized by free radical polymerization using peroxymonosulphate/glycolic acid redox pair in an inert atmosphere. The grafting parameters i.e. grafting ratio, add on and efficiency decrease with increase in concentration of k-carrageenan from 0.6 to 1.4 g dm⁻³ and hydrogen ion from 3 × 10⁻³ to 7 × 10⁻³ mol dm⁻³, but these grafting parameters increase with increase in concentration of N,N-dimethylacrylamide from 16 × 10⁻² to 32 × 10⁻² mol dm⁻³, and peroxymonosulphate from 0.8 × 10⁻² to 2.4 × 10⁻² mol dm⁻³. The metal ion sorption, swelling behaviour and flocculation properties have been studied. The intrinsic viscosity of pure and grafted samples has been measured by using Ubbelohde capillary viscometer. Flocculation capability of k-carrageenan and k-carrageenan-g-N,N-dimethylacrylamide for both coking and non-coking coals has been studied for the treatment of coal mine waste water. The graft copolymer has been characterized by Infrared (IR) spectroscopy and thermogravimetric analysis.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

Comparison of the characterization on binding of alpinetin and cardamonin to lysozyme by spectroscopic methods

Studies on the binding affinity of protein to the active components of herbs are novel in biochemistry and are valuable for the information about speciation of drugs and exchange in biological systems. Alpinetin and cardamonin, two of the main constituents from the seeds of Alpinia katsumadai Hayata, have been used in traditional herbs as antibacterial, anti-inflammatory, and other important therapeutic activities of significant potency and low systemic toxicity. The interactions between two flavonoids analogs and lysozyme have been studied for the first time by spectroscopic method including Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) and UV-absorption spectroscopy in combination with Fluorescence quenching study. Both molecules showed high affinities to lysozyme under the experimental condition with drug concentrations from 3.33 × 10⁻⁶ to 2.67 × 10⁻⁵ mol L⁻¹ for alpinetin and 1.67 × 10⁻⁶ to 13.33 × 10⁻⁶ mol L⁻¹ for cardamonin. The alterations of protein secondary structure in the presence of drugs in aqueous solution were quantitatively estimated by the evidences from CD and FT-IR spectroscopy. The thermodynamic parameters obtained and the results of spectroscopic measurements suggest that hydrophobic and electrostatic interactions are the predominant intermolecular forces stabilizing two coordination compounds. The quenching mechanism and the number of binding site (n ≈ 1) were obtained by fluorescence titration data. The efficiency of energy transfer provided the binding distances of 4.04 and 5.90 nm for alpinetin-LYSO and cardamonin-LYSO systems, respectively.     

Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching

Genistein is an isoflavone and phytoestrogen that is a potent inhibitor of cell proliferation and angiogenesis. This study was designed to investigate the binding of genistein to human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 6.7 × 10⁻⁶ to 2.0 × 10⁻⁵ mol L⁻¹ and HSA concentration at 1.5 × 10⁻⁶ mol L⁻¹. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the binding mode, the binding constant and the protein structure changes in the presence of genistein in aqueous solution. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching change did significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be −22.24 kJ mol⁻¹and 19.60 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which indicated that hydrophobic and electrostatic interactions play the main role in the binding of genistein to HSA.     

CYP450 biosensors based on gold chips for antiepileptic drugs determination

Three-electrode configuration chips containing a Pt, Au and a screen-printed Ag/AgCl as counter, working and reference electrode, respectively, have been developed. Selective determination of Phenobarbital (PB) has been carried out by Cytochrome P450 2B4 (CYP450) immobilization into a polypyrrole matrix onto the gold working electrode. Chronoamperometric experiments show a PB diffusion coefficient of 2.42 × 10⁻⁶ cm² s⁻¹, a reproducibility and repeatability in terms of residual standard deviation (RSD) of 13% and 5.51%, respectively, and a limit of detection (LOD) of 0.289 μmol dm⁻³ ( = β = 0.05) for the developed CYP450-biosensor chip. Its performance has been showed by the determination of PB in pharmaceutical drugs. HPLC has been used as reference technique.     

Electrochemical quartz crystal impedance study on the overoxidation of polypyrrole-carbon nanotubes composite film for amperometric detection of dopamine

The electrochemical quartz crystal impedance (EQCI) method was used to study the overoxidation of polypyrrole (PPy)–multiwalled carbon nanotubes (MWCNT) nanocomposite film in neutral and alkaline solutions. The values of molar mass per electron transferred (M/n) obtained during the overoxidation of PPy in 0.10 mol L⁻¹ Na₂SO₄ and 0.20 mol L⁻¹ NaOH aqueous solutions were estimated to be ca. 17 and 22 g mol⁻¹, respectively, suggesting the nucleophilic attack of solution OH⁻ to the pyrrole units during the overoxidation, and the possible partial formation of carboxylic groups after the overoxidation in the NaOH solution. Also, the overoxidized PPy–MWCNT composite film prepared in the NaOH solution showed a notably larger affinity to dopamine (DA) dissolved in a neutral phosphate buffer than that prepared in the Na₂SO₄ solution. The modification of the overoxidized nanocomposite film improved substantially the sensitivity for DA assay in a neutral phosphate buffer, as compared with the modification of overoxidized PPy or MWCNT alone. At a −6 kHz (201-nm thickness) nanocomposite film prepared in a polymerization bath containing 1.0 mg mL⁻¹ MWCNT and overoxidized in 0.20 mol L⁻¹ aqueous NaOH, the peak current response from differential pulse voltammetric assay of DA was linear with DA concentration from 4.0 × 10⁻⁸ to 1.4 × 10⁻⁶ mol L⁻¹, with a lower limit of detection of 1.7 nmol L⁻¹, good anti-interferent ability, as well as good stability and reproducibility.     

A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H₂O₂ in a choline solution at +0.6 V. The other one modified with ChO/HRP utilized the reduction process of H₂O₂ in a choline solution at −0.2 V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0×10⁻⁶ to 8.0×10⁻⁵ M and the other based on ChO/CPME from 1.0×10⁻⁶ to 5.0×10⁻⁵ M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0×10⁻⁷ and 4.0×10⁻⁷ M, respectively. The response time of sensors was less than 5 s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.     

Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer--formed using a micro-contact imprinting method

Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities.

The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers.

With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03 ± 0.89 × 10⁻¹¹ mole/cm² of template from a 5.68 × 10⁻⁷ M myoglobin solution, compared to 2.58 ± 0.02 × 10⁻¹¹ mole/cm² for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2 wt.% SDS and 0.6 wt.% NaOH used at 80 °C for 30 min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal.

The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (K_d) for the specific binding phase to be 3.4 × 10⁻⁷ M and the binding site capacity to be 7.24 × 10⁻¹¹ mole/cm². For the non-specific binding phase, K_d was found to be 1.355 × 10⁻⁵ M and the binding site capacity was determined as 9.62 × 10⁻¹⁰ mole/cm².

Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68 × 10⁻⁷ M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.     

Determination of digoxin in serum samples using a flow-through fluorosensor based on a molecularly imprinted polymer

This work describes the development of a competitive flow-through FIA assay for digoxin using a molecularly imprinted polymer (MIP) as the recognition phase. In previous work, a number of non-covalent imprinted polymers were synthesised by “bulk” polymerisation. The digoxin binding and elution characteristics of these MIPs were then evaluated to obtain a highly selective material for integration into a sensor. The optimum MIP was synthesised by photo-initiated polymerisation of a mixture containing digoxin, MAA, EDGMA and AIBN in acetonitrile. The bulk polymer was ground and sieved and the template removed by Soxhlet extraction in MeOH/ACN. The MIP was packed into a flow cell and placed in a spectrofluorimeter to integrate the reaction and detection systems. The physical and chemical variables involved in digoxin determination by the sensor (nature and concentration of solution, flow rates, etc.) were optimised. Binding with the non-imprinted polymer (NIP) was also analysed. The new fluorosensor showed high selectivity and sensitivity, a detection limit of 1.7 × 10⁻² μg l⁻¹, and high reproducibility (R.S.D. of 1.03% and 1.77% for concentrations of 1.0 × 10⁻³ and 4.0 × 10⁻³ mg l⁻¹, respectively). Selectivity was tested by determining the cross-reactivity of several compounds with structures analogous to digoxin. Under the assay conditions used, in which the potential interfering compounds were in concentrations 100 times higher than that of the analyte, no interference was recorded. The proposed fluorosensor was successfully used to determine digoxin concentration of human serum samples.     

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nM to 30 nM. The association and dissociation rate constants of the immobilized aptamer–thrombin complex were 6.7 × 10³ M⁻¹ s⁻¹ and 1.0 × 10⁻⁴ s⁻¹, respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Bioelectrocatalytic application of titania nanotube array for molecule detection

A bioelectrocatalysis system based on titania nanotube electrode has been developed for the quantitative detection application. Highly ordered titania nanotube array with inner diameter of 60 nm and total length of 540 nm was formed by anodizing titanium foils. The functionalization modification was achieved by embedding glucose oxidases inside tubule channels and electropolymerizing pyrrole for interfacial immobilization. Morphology and microstructure characterization, electrochemical properties and bioelectrocatalytic reactivities of this composite were fully investigated. The direct detection of hydrogen peroxide by electrocatalytic reduction reaction was fulfilled on pure titania nanotube array with a detection limit up to 2.0 × 10⁻⁴ mM. A biosensor based on the glucose oxidase–titania/titanium electrode was constructed for amperometric detection and quantitative determination of glucose in a phosphate buffer solution (pH 6.8) under a potentiostatic condition (−0.4 V versus SCE). The resulting glucose biosensor showed an excellent performance with a response time below 5.6 s and a detection limit of 2.0 × 10⁻³ mM. The corresponding detection sensitivity was 45.5 μA mM⁻¹ cm⁻². A good operational reliability was also achieved with relative standard deviations below 3.0%. This novel biosensor exhibited quite high response sensitivity and low detection limit for potential applications.     

Direct glucose determination in blood using a reagentless optical biosensor

This paper demonstrates that glucose determination in blood can be done directly (without sample pretreatment) using a reagentless reversible biosensor based on the intrinsic spectroscopic properties of peroxidase (HRP). The biosensor, prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix, works in continuous mode, presents a linear response range from 1.5 × 10⁻⁶ up to 5.5 × 10⁻⁵ M and can be used for at least 750 measurements; in the best conditions (0.1 M pH 6 phosphate buffer, HRP and GOx amounts in the polymersation mixture for the sensor film preparation 0.0165 and 0.0010 g, respectively) the minimum samples rate is 30 h⁻¹. For glucose determination, blood is simply diluted in water (until haemolysis is completed) and fed into the sensor without a cleaning step between samples; the blood absorption is corrected in a simple way by working at a proper reference wavelength. The biosensor signals have been mathematically modeled in order to facilitate the design of sensors based on the same idea for other biochemical compounds.     

Fabrication of sucrose biosensor based on single mode planar optical waveguide using co-immobilized plant invertase and GOD

In present studies, the new optical sensing platform based on optical planar waveguide (OPWG) for sucrose estimation was reported. An evanescent-wave biosensor was designed by using novel agarose–guar gum (AG) biopolymer composite sol–gel with entrapped enzymes (acid invertase (INV) and glucose oxidase (GOD)). Partially purified watermelon invertase isolated from Citrullus vulgaris fruit (specific activity 832 units mg⁻¹) in combination with GOD was physically entrapped in AG sol–gel and cladded on the surface of optical planar waveguide. Na⁺–K⁺ ion-exchanged glass optical waveguides were prepared and employed for the fabrication of sucrose biosensor. By addressing the enzyme modified waveguide structure with, the optogeometric properties of adsorbed enzyme layer (12 μm) at the sensor solid–liquid interface were studied. The OPWG sensor with short response time (110 s) was characterized using the 0.2 M acetate buffer, pH 5.5. The fabricated sucrose sensor showed concentration dependent linear response in the range 1 × 10⁻¹⁰ to 1 × 10⁻⁶ M of sucrose. Lower limit of detection of this novel AG–INV–GOD cladded OPWG sensor was found to be 2.5 × 10⁻¹¹ M sucrose, which indicates that the developed biosensor has higher sensitivity towards sucrose as compared to earlier reported sensors using various transducer systems. Biochips when stored at room temperature, showed high stability for 81 days with 80% retention of original sensitivity. These sucrose sensing biochips showed good operational efficiency for 10 cycles. The proper confinement of acid invertase and glucose oxidase in hydrogel composite was confirmed by scanning electron microscopy (SEM) images. The constructed OPWG sensor is versatile, easy to fabricate and can be used for sucrose measurements with very high sensitivity.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

SEC of mono-carboxymethyl cellulose (CMC) in a wide range of pH; Mark–Houwink constants

A method for determination of carboxymethyl cellulose (CMC) molecular weight (M_W) and chemical heterogeneity (degree of oxidation (DO)) using a bi-detector HPSEC (UV-detector online with refractometer) has been developed. It has been found that the use of 0.5 N NaOH or 0.4 M acetate buffer as the eluent ensures CMC separation according to M_W. It has been revealed that the universal calibration for the polyelectrolyte CMC and the neutral polymer dextran is valid under the conditions applied. The Mark–Houwink equations for CMC in 0.5 N NaOH and 0.4 M acetate buffer have been estimated to be [η]=5.37×10⁻⁴ M_W^0.73 and [η] =2.24×10⁻⁴ M_W^0.83 dl g⁻¹, respectively. The equation log K=1.64−4.00 ml g⁻¹ for CMC has been estimated. An approach for determining DO from adsorption at 290 or 313 nm has been developed.     

Template synthesis of highly ordered Prussian blue array and its application to the glucose biosensing

In this paper, we propose a strategy to form nanoelectrode arrays by electrochemical deposition of the Prussian blue (PB) through highly ordered porous anodic alumina (PAA) membrane. The structure and morphology of the nanoarrays were characterized by scanning electron microscopy (SEM). As the highly ordered PB arrays can behave as an ensemble of closely spaced but isolated nanoelectrodes, the nanostructured PB arrays are successfully applied to improve the analytical performances of glucose by electrocatalytic reduction enzymatically liberated H₂O₂. The resulting PB based nanoelectrode arrays show a wide linear calibration range over three orders of magnitude of glucose concentrations (5.0 × 10⁻⁶ to 8.0 × 10⁻³ M) and a low detection limit of 1 μM. Moreover, the biosensor exhibits other good characteristics, such as short response time, high selectivity, excellent operation stability. In addition, effects of the glucose oxidase (GOx) loading, applied potential and pH on the biosensor performance were also discussed.     

The development of an amperometric microbial biosensor using Acetobacter pasteurianus for lactic acid

A microbial biosensor, using Acetobacter pasteurianus cells and an oxygen electrode, was developed for the determination of lactic acid. The bacterial cells were retained on a nylon membrane and attached to the surface of the oxygen electrode. In view of response time, stability and sensitivity, the biosensor performed best at 26°C and in pH 6 phthalate buffer containing magnesium sulfate. The activity of the retained cells was stable for approximately 170 h and was regenerable. The biosensor exhibited a hyperbolic response to both D- and L-lactic acid in the range of 10⁻⁴ M to 25 × 10⁻³ M. However, in the range 10⁻⁴ M to 15 × 10⁻⁴ M the response was linear. The microbial biosensor was applicable for detecting lactate concentration in yogurt and milk, since it was not sensitive to lactose, sucrose and glucose — three major components of such dairy products.     

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues

It is well accepted that estradiol (E₂) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E₁S) ‘via sulfatase’ is a much more likely precursor for E₂ than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E₂ was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [³H]-E₁S (5 × 10⁻⁹ M) alone or in the presence of E₂ (5 × 10⁻⁵ to 5 × 10⁻⁷ M) during 30 min or 3 h. E₁S, E₁ and E₂ were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E₁ was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10⁻⁷ M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10⁻⁵ M after 30 min incubation. The values were 24% and 18% for 5 × 10⁻⁷ M and 49% and 42% for 5 × 10⁻⁵ M, respectively after 3 h incubation. It was observed that [³H]-E₁S is only converted to [³H]-E₁ and not to [³H]-E₂ in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.