Oxygen reduction on rotating porous cylinder of modified reticulated vitreous carbon

A rotating cylinder porous electrode (RCPE) of reticulated vitreous carbon (RVC) matrix was used for oxygen reduction reaction (ORR) in H₂SO₄ solutions. Cyclic voltammetry and hydrodynamic voltammetric techniques were used for electrochemical characterization of the ORR. Cyclic voltammograms in stationary solutions showed better performance of the anodically oxidized RVC (for periods of 1 and 5 min) for the ORR than the untreated RVC in which the first scan (ORR) after the surface treatment was of no utility, and the second scan was presented here. The hydrodynamic voltammograms obtained at the treated RCPE gave well-defined limiting current plateau with positively shifted onset potential as compared with the untreated (plain) RVC electrode. The analysis of the limiting current data on RCPE and the determination of a limiting current enhancement factor α enabled us to quantify the enhancement extent exerted by the anodic oxidation treatment. An enhancement factor of up to ∼3 was obtained at the RCPE electrode anodically oxidized for 5 min. It was found that the α slightly decreased with the rotation speed depending on the extent of anodic oxidation of RVC. This was attributed to the different mode of mass transfer (diffusion) to the interior of the micropores with different microstructure resulting from different extent of anodic oxidation. X-ray photoelectron spectroscopic and scanning electron microscopic measurements helped us to characterize the anodically oxidized RVC surface.     

Voltammetric determination of alkaline phosphatase and horseradish peroxidase activity using 3-indoxyl phosphate as substrate Application to enzyme immunoassay

The use of 3-indoxyl phosphate (3-IP) as an electrochemical substrate for ELISAs with voltammetric detection was investigated. Indirect measurements of alkaline phosphatase (AP) and horseradish peroxidase (HRP) activity in solution were carried out. Picomolar levels of both enzymes can be detected, which enables the design of electrochemical immunoassays using this substrate. The enzymatic turnover of the substrate gives indigo blue, insoluble in aqueous solutions. This product is easily converted into its soluble parent compound, indigo carmine (IC), by addition of fuming sulphuric acid to the reaction media. IC shows a reversible voltammetric peak at the formal potential of −0.15 V (versus Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. The peak current of this process constitutes the analytical signal. Using this approach an ELISA assay to quantify pneumolysin (PLY, a toxin related to respiratory infections) was carried out using AP or HRP as enzymatic label. Calibration plots obtained are reported. 3-IP is demonstrated to be the first suitable substrate for the two most common enzyme labels used in immunoassays.     

One-pot synthesis,structural elucidation,DNA binding and alkaline phosphatase inhibition studies of zinc(II) complexes with 4-nitrocinnamic acid and ethylenediamine

We report one-pot synthesis of zinc(II) complexes with 4-nitrocinnamic acid (HL), [ZnL₂(H₂O)₂] (1), [ZnL₂(DMSO)₂] (2) and [Zn(en)₂(H₂O)₂]L₂(H₂O)₂ (3), where DMSO?=?dimethylsulfoxide and en?=?ethylenediamine. The complexes were prepared by reacting sodium 4-nitrocinnamate with zinc acetate in aqueous medium and characterized by FT-IR, NMR and single crystal X-ray diffraction. The results have shown distorted octahedral geometry for 1 and 3 while tetrahedral for 2 where the carboxylate coordinated bidentate and monodentate in 1 and 2 is uncoordinated in 3, replaced completely by ethylenediamine. The complexes were also screened for their DNA interaction and alkaline phosphatase (ALP) inhibition using cyclic voltammetry, viscometry and UV–visible spectroscopy. All these techniques indicated strong electrostatic binding of the complexes with DNA. The diffusion coefficients of the complexes decreased on DNA addition indicating binding of the complexes with DNA. The binding constants were also calculated through CV and UV spectra. The complexes were screened for ALP inhibition activity as well, where 80% activity was found for 1.     

Multiwalled carbon nanotube modified screen-printed electrodes for the detection of p-aminophenol: optimisation and application in alkaline phosphatase-based assays

Carboxylated multiwalled carbon nanotubes (MWCNT-COOH) were used to modify the working electrode surface of different screen-printed electrodes. The effect of this modification on the electrodic characteristics (double layer capacitance, electroactive area and heterogeneous rate constants for the electron transfer) was evaluated and optimized for the cyclic voltammetric determination of p-aminophenol. The enzymatic hydrolysis of p-aminophenylphosphate was employed for the quantification of alkaline phosphatase, one of the most important label enzymes in immunoassays. Finally, ELISA assays were carried out to quantify pneumolysin using this enzymatic system. Results obtained indicated that low superficial densities of MWCNT-COOH (0.03-0.06 μg mm⁻²) yielded the same electrodic improvements but with better analytical properties.     

4-Amino-1-naphthylphosphate as a substrate for the amperometric detection of alkaline phosphatase activity and its application for immunoassay

Immunosensors and biochemical array detection systems based on electrochemical transducers have many advantages such as low detection limit, fast response, simple design and ease of miniaturization. However, further development of such sensors will depend on the availability of suitable substrates that can be converted by a labeling enzyme to an electrochemically active product. Here, we report the synthesis of 4-amino-1-naphthylphosphate and it’s application as a new substrate for alkaline phosphatase. The electrochemical and enzymatic properties of this compound were investigated and compared with the properties of other aromatic 1,4-dihydroxy and 1,4-hydroxy-amine derivatives. The product of the enzyme reaction was 4-aminonaphthol, which was rapidly converted in the presences of air to 1,4-iminonaphthoquinone. This compound could then be detected in an amperometric flow injection assay (AFIA) with −200 mV versus Ag/AgCl potential application. The analytical range for mouse IgG, in an alkaline phosphatase amplified sandwich immuoassay with amperometric detection, was 0.01-100 μg ml⁻¹.     

Development of an immunosensor for the determination of rabbit IgG using streptavidin modified screen-printed carbon electrodes

Voltammetric enzyme immunosensors based on the employment of streptavidin modified screen-printed carbon electrodes (SPCEs) for the detection of rabbit IgG, as a model analyte, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate (3-IP) were used as the enzymatic label and substrate, respectively. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 °C on the pre-oxidized surface of the SPCEs. The analytical characteristics of these sensors were evaluated using biotin conjugated to AP.The immunosensor devices were based on a specific reaction of rabbit IgG with its biotinylated antibodies, which were immobilised on the modified screen-printed carbon electrodes through the streptavidin:biotin reaction. The immunosensors were used for a direct determination of AP labelled rabbit IgG, and for free rabbit IgG detection using a sequential competitive immunoassay. A calibration curve in the range of 5 × 10⁻¹¹ to 1 × 10⁻⁹ M of rabbit IgG was obtained with a estimated detection limit of 5 × 10⁻¹¹ M (7.0 ng/ml). These immunosensors were stable for 5 months if they were stored at 4 °C.     

Oxygen reduction at the surface of glassy carbon electrodes modified with anthraquinone derivatives and dyes

The preparation, electrochemical and catalytic behaviour of glassy carbon electrodes modified by anthra-9,10-quinone, its amino derivatives and dyes were investigated. The stability of the modified electrodes was studied by cyclic voltammetry in acidic and neutral media. The electrocatalytic ability of the modified electrodes for the reduction of dioxygen to hydrogen peroxide was examined by cyclic voltammetry, chronoamperometry and chronocoulometry techniques. The influence of pH on the electrochemical and catalytic behaviour was studied and pH 5.0–8.0 was chosen as the optimum working pH by comparing the shift in oxygen reduction potential. The anthraquinone-adsorbed glassy carbon electrodes possess excellent electrocatalytic abilities for dioxygen reduction with overpotential ranging from 280 to 560 mV lower than that at a plain glassy carbon electrode. Hydrodynamic voltammetric studies were performed to determine the heterogeneous rate constants for the reduction of O₂ at the surface of the modified electrodes, mass specific activity of the anthraquinones used and the apparent diffusion coefficient of O₂ in buffered aqueous O₂-saturated solutions. Studies showed the involvement of two electrons in dioxygen reduction.     

Pulsed amperometric detection at glassy carbon electrodes: A new waveform for sensitive and reproducible determination of electroactive compounds

In this work, the application of a new pulsed amperometric detection (PAD) waveform at a glassy carbon electrode, operating in typical chromatographic mobile phases, is proposed for the sensitive and reproducible determination of arylethanolaminic and phenolic moiety based compounds (e.g. beta-agonists and polyphenols). Preliminary experiments by cyclic voltammetry were carried out to investigate the electrochemical behaviour and to select the detection and cleaning electrode potentials. The proposed potential-time profile was designed to prevent the carbon electrode fouling under repeated analyses, thus ensuring a reproducible and sensitive quantitative determination, without the need of any mechanical or chemical electrode cleaning procedure. The waveform electrochemical parameters, including detection and delay times, were optimized in terms of sensitivity, limit of detection and response stability. The optimized waveform allowed the sensitive and stable detection of model compounds, such as clenbuterol and caffeic acid, that showed detection limits of 0.1 μg L⁻¹ and 14 μg L⁻¹, quantification limits of 0.4 μg L⁻¹ and 46 μg L⁻¹, and linearity up to 100 μg L⁻¹ (r = 0.9993) and 10 mg L⁻¹ (r = 0.9998), respectively. Similar results were obtained for other compounds of the same classes, with precision values under repeatability conditions ranging from 3.0 to 5.9%. The proposed method can be then considered as an excellent alternative to the post-column detection of beta-agonists, phenols and polyphenols.     

Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases

A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)_n-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)_n-DMA complex containing several FITC molecules. F-ol-(FITC)_n-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)_n-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot⁻¹ for F-ol and 0.097 ag AP spot⁻¹ for FITC in F-ol-(FITC)_n-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot⁻¹ for F-ol-DMA and 0.22 ag AP spot⁻¹ for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)_n-DMA labelling of WGA was discussed.