p-aminophenyl phosphate: an improved substrate for electrochemical enzyme immnoassay

An alternative substrate is described for enzyme immunosaasay with electrochemical detection. Alkaline phosphatase (EC.3.1.3.1) activity is determined by using p-aminophenyl phosphate as the enzyme substrate. Enzyme-generated p-aminophenol is detected amperometrically at a glassy carbon electrody by liquid chromatography with electrochemical detection. The oxidation potential obtained for the detectionof p-aminophenol is lower than that for phenol, the previously used substrate product. The detection limit for p-aminophenol is 0.20pmol. A detection limit of 30 pg ml^-1 for digoxin and a 5-min incubationtime for the enzyme reaction were obtained with the new system.     

297 - Electrochemical Oxidation of Catechol and p-aminophenol esters in the presence of hydrolases

The half-wave d.c. oxidation potentials of the substrates of alkaline phosphatase: dihydrogen o-hydroxyphenyl phosphate (I) and dihydrogen p-aminophenyl phosphate (II) and that of aryl-β-glucosidase: β-D-glucoside p-aminophenol (III) on platinum electrodes are equal to 681, 516 and 696 mV vs. N.H.E. at pH S.o (I,II) and pH 4.7 (III) respectively. On the pyrographite electrode the substrate oxidation proceeds at nearly the same potentials. Cyclic voltammetry indicates that in the course of electrochemical oxidation o-benzoquinone and p-quinoneimine are formed from the substrate (I) and substrates (II,III), respectively.The products of enzymatic reactions: catechol and p-aminophenol are oxidized at the potentials shifted by 225–300 mV towards the cathodic region. The parameters of enzymatic reactions determined voltammetrically are the following: K_M(app) = 1.8 x 10⁻⁴M (I), 1.7 × 10⁻⁴M (II) and 5.1 × 10⁻⁴M (III); V_max of the substrates (I) and (III) is reduced to 1.6- and 5.3-fold in comparison with dihydrogen p-nitrophenyl phosphate or β-D-glucoside p-nitrophenol.The results obtained demonstrate the methods of conjugation of electrode processes with the reactions catalyzed by hydrolases.     

Preparation of electrochemically reduced graphene oxide-modified electrode and its application for determination of p-aminophenol

A simple and eco-friendly electrochemical method to reduce graphene oxide precursor was employed for fabrication of graphene sheets modified glassy carbon electrode, and then, the resulting electrode [electrochemically reduced graphene oxide (ERGO)/glassy carbon electrode (GCE)] was used to determine p-aminophenol. The experimental results demonstrated that the modified electrode exhibited excellent electrocatalytic activity toward the redox of p-aminophenol as evidenced by the significant enhancement of redox peak currents and the decreased peak-to-peak separation in comparison with a bare GCE. A highly sensitive and selective voltammetry determination of p-aminophenol is developed using the ERGO/GCE. This method has been applied for the direct determination of p-aminophenol in artificial wastewater.     

Poly(2,2′-(1,4-phenylenedivinylene) Bis-8-hydroxyquinaldine) Modified Glassy Carbon Electrode for the Simultaneous Determination of Paracetamol and p-Aminophenol

A novel assay is reported for the simultaneous determination of paracetamol and p-aminophenol using a poly(2,2′-(1,4-phenylenedivinylene)bis-8-hydroxyquinaldine) modified glassy carbon electrode. Poly(2,2′-(1,4-phenylenedivinylene)bis-8-hydroxyquinoline) modified electrodes were prepared by electrochemical polymerization. The electrode surface was characterized by scanning electron microscopy. The electrochemical behavior of the modified electrode was investigated by cyclic voltammetry, square wave voltammetry, and electrochemical impedance spectroscopy. The anodic peak potentials for paracetamol and p-aminophenol were at 580 and 337 millivolts, respectively, with a separation of 243 millivolts, adequate for their simultaneous determination. The results showed that the linear dynamic ranges for paracetamol and p-aminophenol were 0.5–200 micromolar and 3–150 micromolars, whereas the limits of detection were 0.075 and 0.45 micromolar, respectively. The novel poly(2,2′-(1,4-phenylenedivinylene)bis-8-hydroxyquinaldine) modified electrode provided excellent selectivity, sensitivity, and stability and was employed for the determination of paracetamol and p-aminophenol in pharmaceutical products and urine.     

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.     

Nafion-graphene composite film modified glassy carbon electrode for voltammetric determination of p-aminophenol

A Nafion-graphene (Nafion-GR) nanocomposite film modified glassy carbon electrode was fabricated by a simple drop-casting method, and used in the electrochemical detection of p-aminophenol (4-AP). Owing to the large surface area, good conductivity of GR and good affinity of Nafion, the sensor exhibited excellent electrocatalytic activity for the oxidation of 4-AP. The electrochemical behaviors of 4-AP on Nafion/GR film modified glassy carbon electrodes were investigated by cyclic voltammetry and differential pulse voltammetry. A calibration curve is constructed in the same matrix, urine, as the unknown samples to be analyzed. The Nafion-GR film modified electrode was linearly dependent on the 4-AP concentration and the linear analytical curve was obtained in the ranges of 0.5–200 μM with differential pulse voltammetry (DPV) and the detection limit was 0.051 μM. The Nafion-graphene nanocomposite modified electrode exhibited good reusability than pure graphene modified GCE. This procedure can be used for the determination of p-aminophenol in the presence of its degradation products and paracetamol. Finally, the proposed method was successfully used to determine p-aminophenol in local tap water samples in urine samples and pharmaceutical preparations.     

Simultaneous Spectrophotometric Determination of Paracetamol and <Emphasis Type="Italic">p</Emphasis>-Aminophenol in Pharmaceutical Products with Tiron Using Dissolved Oxygen as Oxidant

A simple, rapid, and specific method was developed for the determination of paracetamol and p-aminophenol. The method is based on the hydrolysis of paracetamol to p-aminophenol, which, using dissolved oxygen as an oxidant in the alkaline region, was further transformed into benzoquinoneimine, capable of reacting with tiron to produce a green indophenol dye. The stabilization of indophenol dye was achieved by the addition of copper(II) solution. The absorbance was measured at 601 nm in alkaline medium, and the molar absorptivity was found to be 1.1 × 10⁴ L/(mol cm). Paracetamol (PCT) and p-aminophenol (PAP) were determined in pharmaceutical products in the 1.5–15 mg/L PAP concentration range with a detection limit of 1.2 × 10⁻⁶ M or 0.13 μg/mL PAP. The developed method can be applied to the determination of p-aminophenol in the presence of paracetamol without prior separation. The proposed method is successfully employed for determination of paracetamol and p-aminophenol in various synthetic mixtures and pharmaceutical preparations. The obtained results were statistically compared with those given by the official method and the procedures evaluated as regards to both figures of merit and ease of applicability. __________ From Zhurnal Analiticheskoi Khimii, Vol. 60, No. 11, 2005, pp. 1147–1151. Original English Text Copyright ? 2005 by Cekic, Filik, Apak. This article was submitted by the authors in English.     

Redox cycling amplified electrochemical detection of DNA hybridization: application to pathogen E. coli bacterial RNA

An electrochemical genosensor in which signal amplification is achieved using p-aminophenol (p-AP) redox cycling by nicotinamide adenine dinucleotide (NADH) is presented. An immobilized thiolated capture probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the detection probe, and streptavidin-alkaline phosphatase as reporter enzyme. The phosphatase liberates the electrochemical mediator p-AP from its electrically inactive phosphate derivative. This generated p-AP is electrooxidized at an Au electrode modified self-assembled monolayer to p-quinone imine (p-QI). In the presence of NADH, p-QI is reduced back to p-AP, which can be re-oxidized on the electrode and produce amplified signal. A detection limit of 1 pM DNA target is offered by this simple one-electrode, one-enzyme format redox cycling strategy. The redox cycling design is applied successfully to the monitoring of the 16S rRNA of E. coli pathogenic bacteria, and provides a detection limit of 250 CFU μL⁻¹.     

Electrocatalytic performance of cobalt microparticles film-modified platinum disk electrode for amperometric detection of ascorbic acid

Deposited cobalt microparticales (Co-MPs) film onto the platinum disk electrode has been successfully used as a new amperometric sensor for the determination of ascorbic acid (AA). AA is detected by surface catalyzed oxidation involving cobalt(III) oxyhydroxides in alkaline solution. The Co-MPs/Pt electrode exhibits a high electrocatalytic activity toward the AA oxidation. The diffusion coefficient of AA (6.09 × 10⁵ cm²/s) and the catalytic rate constant (k _cat = 6.27 × 10³ M^–1s^–1) have been determined using electrochemical approaches. The amperometric response of the modified electrode is linear against the AA concentration in the range (0.01?0.48 mM). The sensor displays the best activity with a high response signal, a good sensitivity of 74.3 μA/mM, a low detection limit of 2.5 μM (signal/noise = 3) and a fast response time (<3 s). Moreover, the reproducibility, selectivity and applicability of this biosensor are satisfactorily evaluated.     

Kinetic amperometric determination of hydrolase activity

Alkaline phosphatase is determined by using o-hydroxyphenyl or p-aminophenyl phosphate as substrate; for β-glucosidase, p-aminophenol β-D-glucoside is used. The products, p-aminophenol and catechol, are monitored amperometrically. The limit of determination for the hydrolase is about 2.5 × 10^-3 units ml^-1. The apparent Michaelis constants are given.     

An electrochemical sensor for p-aminophenol based on the mesoporous silica modified carbon paste electrode

A mesoporous silica was synthesised and used to modify the surface of carbon paste electrode (CPE). The electrochemical behaviours of p-aminophenol were investigated. Compared to the unmodified CPE, the mesoporous silica-modified CPE obviously lowers the oxidation potential of p-aminophenol, and remarkably increases its oxidation peak current. The effects of pH value, amount of mesoporous silica, accumulation potential and time were examined. As a result, a sensitive, rapid and convenient electroanalytical method was developed for p-aminophenol. The linear range is from 0.025?mg?L^?1 to 3?mg?L^?1, and the limit of detection is 0.01?mg?L^?1 after 2-min accumulation. Finally, the method was successfully used to determine p-aminophenol in water samples.     

New p-aminophenol-based dendritic melamines. Iterative synthesis,structure, and electrochemical characterisation

Avoiding any protective–deprotective step, the synthesis of new (G-0, -1, -2) dendritic melamines is reported. Their construction consisted of chemoselective S_N2–Ar amination of cyanuric chloride with p-aminophenol (peripheral unit) and piperazine or 4,4′-bipiperidine (linkers). This novel class of amino-s-triazines is primarily investigated by DFT calculations (optimal geometry and electronic structure) in tandem with (VT) ¹H NMR spectroscopy providing details of the rotational diastereomerism about the C(s-triazine)-N(exocyclic) partial double bonds, solvation effects and conformation of the linkers. These data are subsequently exploited in electrochemical investigations (cyclic voltammetry on the Pt electrode/DMSO, 0.1 M KCl). Two reversible electron-transfer phenomena have been observed. Thus, depending on the variable π-deficiency strength of the s-triazine ring acting as the EWG on the adjacent NH group and the ability of the latter to undergo redox processes in tandem with the phenolic p-HO group, two electrochemical pathways are proposed, namely the p-benzoquinonimine route and the electropolymerization route.     

IgG anti-gliadin determination with an immunological microfluidic system applied to the automated diagnostic of the celiac disease

In the present article, a novel microfluidic immunosensor coupled with electrochemical detection for anti-gliadin IgG antibody quantification is proposed. This device represents an important tool for a fast, simple, sensitive, and automated diagnostic for celiac disease, which is carried out through detection of anti-gliadin IgG antibodies present in human serum samples. Celiac disease (CD) is an autoimmune disease generated by gluten protein fractions called prolamins. This pathology affects about one in 250 people around the world, produces intestinal inflammation, villous atrophy, and crypt hyperplasia, which causes a range of symptoms including altered bowel habits, malnutrition and weight loss. Our immunosensor consists of a Plexiglas device coupled to a gold electrode, with a central channel containing 3-aminopropyl-modified controlled pore glass (AP-CPG). The quantification of anti-gliadin IgG antibodies was carried out using a heterogeneous, non-competitive enzyme-linked immunosorbent assay (ELISA) in which IgG antibodies bound to gliadin protein, immobilized on AP-CPG, were determined by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP, and the electroactive product was quantified on a gold electrode at 0.250 V. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.52 and 2.72 UR mL⁻¹, respectively, and the within- and between-assay coefficients of variation were below 5.8%. The optimized procedure was applied to the determination of anti-gliadin IgG antibodies in human serum samples.     

Flow screen-printed amperometric detection of p-nitrophenol in alkaline phosphatase-based assays

p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system the detection limit achieved for p-nitrophenol was 2×10⁻⁸ mol L⁻¹, almost two orders of magnitude lower than that obtained by measuring the absorbance of the compound. By use of this electrochemical detection method, measurement of 7×10⁻¹⁴ mol L⁻¹ alkaline phosphatase was achieved after incubation for 20 min. The feasibility of coupling immunoassay to screen-printed carbon electrode amperometric detection has been demonstrated by performing an ELISA for detection of pneumolysin, a toxin produced by Streptococcus pneumoniae, which causes respiratory infections. The method is simple, reproducible, and much more sensitive than traditional spectrophotometry.     

The determination of p-cresol in chloroform with an enzyme electrode used in the organic phase

An enzyme electrode that operates in chloroform is described. Polyphenol oxidase (tyrosinase; EC.1.14.18.1) is used to detect p-cresol via electrochemical reduction of the product, 4-methyl- 1,2-benzoquinone, at a graphite foil electode. The response is linear for p-cresol concentrations of 0–0.10 mM, with a limit of detection of 1 μM. After an initial rise from 1.9 μA to 4.0 μA in the first three assays, the response of the electrode to 0.10 mM p-cresol remained stable for twelve consecutive assays ($\text{x}$=4.6, SD=0.49). After intermittent usage for 204 days with appropriate storage, the enzyme electrode remained active. The electrode is sensitive to a broad range of phenols. The feasibility of detecting p-cresol contamination of water is demonstrated.     

Plastified poly(ethylene terephthalate) (PET)-toner microfluidic chip by direct-printing integrated with electrochemical detection for pharmaceutical analysis

Rapid separation and determination of acetaminophen and its hydrolysate with end-channel electrochemical (EC) detection integrated on a plastified poly(ethylene terephthalate) (PET)-toner microchip capillary electrophoresis (CE) system was investigated. In this separation and detection system, a Pt ultramicroelectrode integrated on a three-dimensional adjustor was used as working electrode. Factors influencing the separation and detection were investigated and optimized. Results show that acetaminophen and p-aminophenol can be well separated within 84 s with R.S.D. < 1% for migration time and R.S.D. < 3.6% for detection current for both analytes. Detection limits for both analytes are determined to be 5.0 μM (S/N = 3). This method has been successfully applied to the detection of trace p-aminophenol in paracetamol tablets. The results demonstrate that the PET-toner microchips can obtain better performance than PDMS microfluidic devices but at much lower cost.     

Single step modification of copper electrode for the highly sensitive and selective non-enzymatic determination of glucose

A non-enzymatic sensor was developed for the determination of glucose in alkaline medium by anodisation of copper in sodium potassium tartrate solution. The morphology of the modified copper electrode was studied by scanning electron microscopy, and its electrochemical behavior by cyclic voltammetry and electrochemical impedance spectroscopy. The electrode enables direct electrocatalytic oxidation of glucose on a CuO/Cu electrode at 0.7 V in 0.1 M sodium hydroxide. At this potential, the sensor is highly selective to glucose even in the presence of ascorbic acid, uric acid, or dopamine which are common interfering species. The sensor displays a sensitivity of 761.9 μA mM^?1 cm^?2, a linear detection range from 2 μM to 20 mM, a response time of <1 s, and a detection limit of 1 μM (S/N = 3). It was tested for determination of glucose level in blood serum.     

Bead-based immunoassays with microelectrode detection

The suitability of a microelectrode as the detector for a small-volume, bead-based enzyme-labeled immunoassay for later use in a microfluidic device was investigated. The microelectrode helps to overcome consumption of the electroactive species by the electrode (depletion) that is encountered with macroelectrodes such as the rotating disk electrode (RDE) and allows the volume of the detection cell to be reduced. Microelectrodes also allow the chemical reactions to be monitored in real time due to the electrodes close proximity to the assay site. A bead-based sandwich immunoassay for mouse IgG was developed with alkaline phosphatase (AP) as the enzyme label, p-aminophenyl phosphate (PAPP) as the enzyme substrate, and microelectrode detection. The diffusion coefficient of the product of enzymatic hydrolysis, p-aminophenol (PAP), was determined to be 7.2±0.9×10^–6 cm² s^–1. The detection limits were determined for free (0.52 ng mL^–1) and bead-bound AP (10 ng mL^–1). The number of binding sites for AP per bead was calculated to be 9.6×10⁴ molecules/bead, and under saturation conditions the minimum detectable number of beads was 2500. Lower detection limits could be achieved with the microelectrode than the RDE while maintaining similar reproducibility. The microelectrode also made it possible to work with lower sample volumes (down to 10 L) than with the RDE (minimum volume of 40 L). Depletion of PAP was not observed with the microelectrode. The results obtained here with a microelectrode showed great promise for later use of microelectrodes in microfluidic devices with limited sample volumes. RDE detection cannot be used in a microfluidic system due to its complex set-up that includes a motor for rotation.Abbreviations: Ab Antibody - Ag Antigen - AP Alkaline phosphatase - NSA Nonspecific adsorption - PAP p-aminophenol - PAPP p-aminophenyl phosphate - RDE Rotating disk electrode - NSA Nonspecific adsorption - PBS Phosphate-buffered saline     

A new voltammetric sensor for the determination of sulfite in water and wastewater using modified-multiwall carbon nanotubes paste electrode

The electrochemical response of a modified-carbon nanotubes paste electrode with p-aminophenol was investigated as an electrochemical sensor for sulfite determination. The electrochemical behaviour of sulfite was studied at the surface of the modified electrode in aqueous media using cyclic voltammetry and square wave voltammetry. It has been found that under the optimum condition (pH 7.0) in cyclic voltammetry, the oxidation of sulfite occurs at a potential about 680?mV less positive than that of an unmodified-carbon nanotubes paste electrode. Under the optimized conditions, the electrocatalytic peak current showed linear relationship with sulfite concentration in the range of 2.0?×?10^?7–2.8?×?10^?4?mol?L^?1 with a detection limit of 9.0?×?10^?8?mol?L^?1 sulfite. The relative standard deviations for ten successive assays of 1.0 and 50.0?µmol?L^?1 sulfite were 2.5% and 2.1%, respectively. Finally, the modified electrode was examined as a selective, simple and precise new electrochemical sensor for the determination of sulfite in water and wastewater samples.     

Poly(aquachlorobis(1,10–phenanthroline)copper(II)iodidemonohydrate)/GCE for simultaneous determination of caffeine and theophylline in human serum,tea, and tablet samples

This paper presents metal complex based polymer film modified electrode for simultaneous determination of caffeine, and theophylline. Potentiodynamic fabrication of poly(aquachlorobis(1,10– phenanthroline)copper(II)iodidemonohydrate) modified glassy carbon electrode (poly(ACP₂CuIH)/GCE) was verified using cyclic voltammetric and electrochemical impedance spectroscopic techniques. In contrast to the unmodified glassy carbon electrode, the poly(ACP₂CuIH)/GCE in equi-molar mixture of theophylline and caffeine revealed sufficiently separated oxidative peaks with much enhanced peak current showing electrocatalytic property of the polymer film towards the oxidation of theophylline and caffeine. Under optimized solution pH and square wave voltammetric parameters, oxidative peak current response of poly(ACP₂CuIH)/GCE showed linear dependence on the concentration of caffeine and theophylline in the concentration range 1.0–200.0 µM with limit of detection 8.92 × 10^-3 µM for theophylline, and 1.02 × 10^{-2 µ}M for caffeine. Spike recovery in the range 97.0-102.4% for theophylline, and 95.4-100.0% for caffeine, interference recovery in the range 96.0–101.0% for theophylline, and 95.7–104.3% for caffeine, agreement of the detected amounts of theophylline and caffeine in tablet samples with the nominal values, and stability of the modified electrode all validated the developed method for simultaneous determination of theophylline and caffeine in wide range of real samples. The method was applied for simultaneous determination of both theophylline and caffeine in three tea brands (Black lion, Addis, and Wush wush), pharmaceutical tablet brands (Panadol extra, and Theodrine), and human blood serum samples making the method an excellent candidate.