Design of a Vitamin B1‐Selective Electrode Based on an Ion‐Pair and Its Application to Pharmaceutical Analysis

Vitamin B₁‐selective electrodes with PVC membrane were developed that contain ion associates of vitamin B₁ with an inorganic anion, BiI₄^?, and an organic anion, brilliant yellow, as electrode‐active substances. The linearity ranges of the electrode function are 1.0×10^?5–1.0×10^?2 and 1.0×10^?4–1.0×10^?2 M, the electrode function slopes are 33.0±1.0 and 33.1±1.1 mV decade^?1, the detection limits are 5.5×10^?6 and 8.3×10^?5 M for BiI₄^? and brilliant yellow respectively. The working range of pH is 5–12. The efficiency of the use of electrodes for the vitamin B₁ content control in multivitamin pharmaceutical preparations was shown by direct potentiometry and potentiometric titration methods.     

An enzyme electrode forl-lactate with a chemically-amplified response

An enzyme electrode with a chemically-amplified response forl-lactate is constructed from an oxygen electrode and a layer containing immobilized lactate oxidase, to oxidizel-lactate, and lactate dehydrogenase, to regenerate thel-lactate. Regeneration enables oxygen to be consumed beyond the stoichiometric limitation, which results in an electrode response amplified 2–250 times according to the variation in the layer properties such as the V_max and K_m values of the immobilized enzymes and the thickness of the layer. The detection limit is as low as 5 × 10^?9 M. An equation is derived to relate the rate of oxygen consumption in the layer to the experimental parameters; the equation successfully explains the experimental results.     

Ultrasensitive Determination of Vitamin B12 Using Disposable Graphite Screen‐Printed Electrodes and Anodic Adsorptive Voltammetry

A facile, rapid and ultra‐sensitive method for the determination of vitamin B₁₂ (cyanocobalamin) at the sub‐nanomolar concentration range by using low‐cost, disposable graphite screen‐printed electrodes is described. The method is based on the cathodic preconcentration of square planar vitamin B_12s, as occurred due to the electro reduction of Co(III) center in vitamin B_12a to Co(I), at ?1.3 V versus Ag/AgCl/3 M KCl for 40 s. Then, an anodic square wave scan was applied and the height of the peak appeared at ca. ?0.73 V versus Ag/AgCl/3 M KCl, due to the oxidation of Co(I) to Co(II) in the adsorbed molecule, was related to the concentration of the vitamin B₁₂ in the sample. EDTA was found to serve as a key‐component of the electrolyte by eliminating the background signal caused by metal cations impurities contained in the electrolyte (0.1 M phosphate buffer in 0.1 M KCl, pH 3). It also blocks trace metals contained in real samples, thus eliminating their interference effect. The method was optimized to various working parameters and under the selected conditions the calibration curve was linear over the range 1×10^?10–8×10^?9 mol L^?1 vitamin B₁₂ (R²=0.994), while the limit of detection for a signal‐to‐noise ratio of 3 (7×10^?11 mol L^?1 vitamin B₁₂) is the lowest value of any reported in the literature for the electrochemical determination of vitamin B₁₂. The sensors were successfully applied to the determination of vitamin B₁₂ in pharmaceutical products.     

Voltammetric determination of B<Subscript>1</Subscript> and B<Subscript>6</Subscript> vitamins using a pencil graphite electrode

Due to the importance of B₁ and B₆ vitamins for human health it is useful to develop new cheap and rapid methods for their determination. Voltammetric behavior of these vitamins on a pencil graphite electrode was investigated using cyclic voltammetry in different media. Direct quantitative determination of the two vitamins, one in the presence of the other, was done by differential pulse voltammetry. Vitamin B₁ was electroactive only in a NaOH solution generating two irreversible oxidation peaks; the first peak obtained at 250 mV is well-defined and was used in quantitative determinations. In case of vitamin B₆, a well-defined oxidation peak was observed in all investigated supporting electrolytes except for HCl. The linear concentration ranges were 10^?5–10^?3 M for vitamin B₁ in a NaOH solution and 5 × 10^?6–10^?3 M for vitamin B₆ in an acetate buffer solution. The obtained detection limits were 5.34 × 10^?6 M and 2.81 × 10^?6 M for vitamin B₁ and vitamin B₆, respectively. The developed method is simple and rapid and it was successfully applied in the determination of the two vitamins in pharmaceuticals.     

Investigations on Bioanalytical Chemistry Part I. On Differential Pulse Voltammetry of Bilirubin

Abstract

The electrochemical behaviour of the bilirubin in many kinds of supporting electrolytes on a glassy carbon electrode (GCE) and a hanging mercury drop electrode (HMDE) was investigated by means of anodic or cathodic differential pulse voltammetry. The influences of different methods of pre-treatment of the glassy carbon electrode was also discussed. In Na₂B₄.O₇-KH₂PO₄ buffer solution, the linear range was 2×10^?9-1×10^?9 mol/l and the detection limit was 3.3×10^?9 mol/l by anodic differential pulse voltammetry at GCE. A linear relationship holds between the peak current and the concentration of bilirubin in a concentration range of 1×10^?9-4×10^?7 mol/l with good precision and accuracy, and the limit of detection was 2×10^?10 mol/l, when cathodic differential pulse adsorption voltammetry at HMDE was used.     

Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes

Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognize Escherichia coli and Salmonella typhimurium flagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equation K _D?=?k _d/k _a, showed values of 13.8?×?10^?9 and 7.73?×?10^?9?M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, against S. typhimurium. The anti-flagellin affinity results for E. coli showed K _D of 84.1?×?10^?8?M for SPR assays and K _D of 3.5?×?10^?8?M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.     

Electrocatalysis of Horseradish Peroxidase Immobilized on Cobalt Nanoparticles Modified ITO Electrode

Abstract

It is the first time that Horseradish peroxidase (HRP) was successively immobilized on the magnetic cobalt nanoparticles modified ITO (indium tin oxide) electrode. Morphologies of electrode surface were featured by the field emission‐scanning electron microscope (FSEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the modified process of electrode. Direct electrochemistry and electrocatalysis of HRP immobilized on nano‐Co/ITO were investigated. The biosensor exhibited high sensitivity, good stability, and excellent electrocatalytic activity to the reduction of H₂O₂. Under the optimized experimental conditions, a calibration curve over 2.0×10^?9～2.0×10^?8 mol l^?1 and 2.0×10^?7～2.0×10^?6 mol l^?1, with a limit of detection of 1.9×10^?9 mol l^?1 was obtained. The apparent Michaelis‐Menten constant (K _M ^app ) for HRP/nano‐Co/ITO electrode was calculated to be 0.79 mmol l^?1, indicating a higher affinity of HRP attached on the modified electrode.     

Sensitive electrochemical determination of Sudan II in food samples using a poly(aminosulfonic acid) modified glassy carbon electrode

In this paper, a novel poly(aminosulfonic acid) modified glassy carbon electrode (PASA/GCE) for the determination of Sudan II was fabricated through electrochemical polymerizat ion. The electrochemical behavior of Sudan II at the modified electrode was studied by cyclic voltammetry. Results show that the modified electrode exhibits excellent electrocatalytic activity toward the electrochemical redox reaction of Sudan II. Under optimal experimental conditions, the oxidation peak current is linearly proportional to the concentration of Sudan II in the ranges of 4.0 × 10^?8 to 1.0 × 10^?6 mol L^?1 and 1.0 × 10^?6 to 1.2 × 10^?5 mol L^?1. The linear regression equations are i _pa(A) = 2.87c + 3.74 × 10^?6, r = 0.9977 and i _pa(A) = 0.78c + 6.11 × 10^?6, r = 0.9982, respectively, and the detection limit is 4.0 × 10^?9 mol L^?1. The novel method shows good recovery, reproducibility and sensitivity for the voltammetric determination of Sudan II in food samples.     

Immobilized enzyme electrode for the determination of salicylate in blood serum

This determination of salicylate in blood serum is based on application of an immobilized enzyme electrode. Salicylate hydroxylase (E.C.1.14.13.1) is chemically immobilized onto a pig intestine mounted on an oxygen electrode. The signals are monitored amperometrically and the resulting output voltage is read using a simple adapter. The experimental parameters and possible interferences are discussed. Samples containing 1.0 × 10^?5?1.87 × 10^?3 M (1.6–300 μg ml^?1) salicylate were assayed with relative standard deviations between 1.3% and 6% and recoveries between 98.7 and 103%. Results obtained by the proposed method and by the established clinical method for randomly spiked pooled serum samples correlated well (r = 0.99).     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

Efficient Synthesis of a New Podand and Application as a Suitable Carrier for Silver Ion‐Selective Electrode

A new podand of 1,1′‐thia‐bis‐[1‐(chloroethan‐2‐acetamid‐α‐oxy)] naphtol was synthesized and used as a suitable carrier for Ag⁺ PVC membrane electrode. The electrode exhibited linear response with a Nernstian slope of (59.5±0.8 mV/decade) within a wide concentration range of 1.0×10^?7 to 1.5×10^?2 mol L^?1 silver ions. The electrode had a fast response time of <10 s and detection limit of 8.6×10^?8 mol L^?1 with a working pH range from 3.7 to 9.0. The electrode was highly selective for Ag(I) ions over a large number of cations such as alkali, alkaline earth, and heavy metal ions. The proposed sensor has been applied as an indicator electrode for indirect determination of vitamin B₁ in tablets by determination of Cl^? ions in this compound with a standard solution of Ag(NO₃).     

Improved Vitamin B12 Fermentation Process by Adding Rotenone to Regulate the Metabolism of Pseudomonas denitrificans

Our previous research had revealed that the dissolved oxygen limitation was more favorable for vitamin B₁₂ fermentation, due to its inducement to the increased glycolytic flux in Pseudomonas denitrificans. In this paper, a novel strategy was implemented to further investigate the metabolic characteristics of P. denitrificans under different oxygen supply levels, by exogenously adding rotenone (a respiratory chain inhibitor interfering with the oxygen consumption) to the fermentation broths. Compared to the fermentation process without rotenone treatment, it was observed that 5 mg/L rotenone treatment could significantly strengthen the glycolytic flux of P. denitrificans via activating the key glycolytic enzymes (phosphofructokinase and pyruvate kinase), resulting in the accelerated generations of anterior precursors (glutamate and 5-aminolevulinic acid) for vitamin B₁₂ biosynthesis. Although 5 mg/L rotenone treatment had a negative effect on cell growth of P. denitrificans, the vitamin B₁₂ yield was increased from 48.28?±?0.62 mg/L to 54.70?±?0.45 mg/L, which further proved that an increased glycolytic flux in P. denitrificans was a consequence of higher vitamin B₁₂ production.     

Adsorptive stripping voltammetry of chlordiazepoxide at the hanging mercury drop electrode

Controlled adsorptive accumulation at the hanging mercury drop electrode enables 0.8–11 × 10^?5 M chlordiazepoxide to be quantified by differential-pulse stripping voltammetry with accumulation times of 1–3 min. With 3-min accumulation, the peak current is enhanced 12-fold for 1.0 × 10^?7 M chlordiazepoxide compared to the current from differential pulse polarography. The detection limit is 0.9 × 10^?9 M for 4-min accumulation. The procedure is applied to spiked human serum after preseparation of the drug on a Sep-Pak C₁₈ cartridge.     

Direct electrochemistry and electrocatalysis of hemoglobin on a gold ion implantation-modified indium tin oxide electrode

In this paper, a gold nanoparticle-modified indium tin oxide electrode (Au/ITO) was prepared without the use of any cross-linker or stabilizer reagent. The prepared Au/ITO was used as a new platform to achieve the direct electron transfer between Hb and the modified electrode. The proposed electrode exhibited a pair of well-defined redox peaks with a formal potential of ?0.073 V (vs. Ag/AgCl). The immobilized Hb showed excellent electrocatalytic activity toward H₂O₂ and the electrocatalytic current values were linear with the increasing concentration of H₂O₂ ranging from 1.0?×?10^?6?M to 7.0?×?10^?4?M. The detection limit was 2.0?×?10^?7?M (S/N?=?3) and the Michaelis–Menten constant was calculated to be 0.2 mM. The proposed electrode also showed high selectivity, long-term stability, and good reproducibility.     

A Novel NH2/ITO Ion Implantation Electrode: Preparation,Characterization, and Application in Bioelectrochemistry

A novel NH₂⁺ ion implantation‐modified indium tin oxide (NH₂/ITO) electrode was prepared. Acid‐pretreated, negatively charged MWNTs were firstly modified on the surface of NH₂⁺ ion implantation electrode, then, positively charged Mb was adsorbed onto MWNTs films by electrostatic interaction. The assembly of MWNTs and Mb was characterized with electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized Mb showed a couple of quasireversible cyclic voltammetry peaks in pH 7.0 phosphate buffer solution (PBS). The apparent surface concentration of Mb at the electrode surface was 1.06×10^?9 mol cm^?2. The Mb/MWNTs/NH₂/ITO electrode also gave an improved electrocatalytic activity towards the reduction of hydrogen peroxide. The catalysis currents increased linearly to the H₂O₂ concentration in a wide range from 9×10^?7 to 9.2×10^?5 M with a correlation coefficient of 0.999. The detection limit was 9.0×10^?7 M. The experiment results demonstrated that the modified electrode provided a biocompatible microenvironment for protein and supplied a necessary pathway for its direct electron transfer.     

Voltammetric determination of trace doxorubicin at a nano-titania/nafion composite film modified electrode in the presence of cetyltrimethylammonium bromide

A sensitive electrochemical method was developed for the determination of doxorubicin at a glassy carbon electrode (GCE) modified with a nano-titania (nano-TiO₂)/nafion composite film. Nano-TiO₂ was dispersed into nafion to give a homogeneous suspension. After solvent evaporation, a uniform film of nano-TiO₂/nafion composite was obtained on the GCE surface. The nano-TiO₂/nafion composite film modified GCE exhibited excellent electrochemical behavior toward the reduction of doxorubicin. Compared to the reduction of doxorubicin at the bare GCE, the reduction current of doxorubicin at the nano-TiO₂/nafion composite film modified GCE was greatly enhanced. Based on this, a novel voltammetric method was applied to the determination of doxorubicin. The experimental parameters that influence the reduction current of doxorubicin, were optimized. Under optimal conditions, a linear response of doxorubicin was obtained in the range from 5.0?×?10^?9 to 2.0?×?10^?6 mol L^?1 (R?=?0.998) and with a limit of detection (LOD) of 1.0?×?10^?9 mol L^?1(S/N?=?3). The RSD of the measurement is 4.7%, and the RSD of the inter-electrode is of 5.1% which indicate the reproducibility of this method. The current response decreased only by around 3.8% of its initial response after 2 weeks exposing the electrode in air. The procedure was applied to assay doxorubicin in human plasma samples with the recoveries of 94.9–104.4%.     

Hemoglobin co-immobilized with silver–silver oxide nanoparticles on a bare silver electrode for hydrogen peroxide electroanalysis

Hemoglobin (Hb) and silver–silver oxide (Ag–Ag₂O) nanoparticles were co-immobilized on a bare silver electrode surface by cyclic voltammetry, and were characterized by UV–vis reflection spectroscopy, scanning electron microscopy, and electrochemical impedance spectroscopy. The immobilized Hb was shown to maintain its biological activity well. Direct electron transfer between Hb and the resulting electrode was achieved without the aid of any electron mediator. The reduction currents to hydrogen peroxide (H₂O₂) at co-immobilized electrodes showed a linear relationship with H₂O₂ concentration over a concentration range from 6.0?×?10^?6 to 5.0?×?10^?2 mol L^?1, and a detection limit of 2.0?×?10^?6 mol L^?1 (S/N?=?3).     

Electrochemical DNA impedance biosensor for the detection of DNA hybridization with polymeric film, single walled carbon nanotubes modified glassy carbon electrode

In this work, we report the fabrication of a sensitive electrochemical DNA impedance biosensor for the detection of sequence-specific target DNA. p-Aminobenzoic acid was first immobilized on the surface of the electrode modified with single walled carbon nanotubes with carboxylic acid groups (SWCNTs) by cyclic voltammetry (CV). A single-stranded DNA probe with a NH₂ group at the end (H₂N-ssDNA) was then covalently immobilized on the surface of polymeric film at room temperature. The impedance measurement was performed in a solution containing 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]. The change of interfacial charge transfer resistance (R _CT) was confirmed the hybrid formation. The difference of R _CT was linear with the logarithm of complementary oligonucleotides concentrations in the range of 1.0 × 10^?12 to 1.0 × 10^?7 M, with a detection limit of 3.5 × 10^?13 M (S/N = 3).     

Carbon Composite Electrode Modified with Copper(II) Phosphate Immobilized in a Polyester Resin for Voltammetric Determination of Catechin in Teas

A carbon composite electrode modified with copper (II) phosphate immobilized in a polyester resin (Cu₃(PO₄)₂-Poly) was proposed for the voltammetric determination of catechin in teas. The modified electrode allows the determination of catechin (CAT) at lower potential than that observed at an unmodified electrode. Several parameters that can influence the voltammetric response of the proposed electrode such as carbon composite composition, pH of electrolyte, and others were investigated. The peak current was proportional to the concentration of catechin in the range from 9.9 × 10^?8 to 1.2 × 10^?6 mol L^?1, with a detection limit of 5.8 × 10^?8 mol L^?1. The stability and repeatability of the electrode for the determination of catechin were discussed, and the modified electrode was applied with success in the determination of catechin in teas.     

Study on the Interaction of Vitamin B12 with DNA by Spectroscopy and Electrochemical Methods

The Interaction between vitamin B₁₂ (VB₁₂) and fish sperm DNA was investigated in physiological buffer (pH 7.4) using the methylene blue (MB) dye as a spectral probe by spetcrophotometery, viscosity measurements and cyclic voltammetry. The apparent binding constant of vitamin B₁₂ with DNA was found to be 3.2×10⁵ mol⁻¹·L. The voltammetric behavior of vitamin B₁₂ has been investigated at glassy carbon electrode using cyclic voltammetry. Thermodynamic parameters including ΔH⁰, ΔS⁰ and ΔG⁰ for the interaction between VB₁₂ and DNA have determined as −2.3×10⁴, 27.54 and −3.1×10⁴J·mol⁻¹·K⁻¹ respectively. One indication of DNA binding mode with VB₁₂ was the change in viscosity when a small molecule associates with DNA. The diffusion coefficients of VB₁₂ in the absence (D₀)_f and presence of DNA (D₀)_b was calculated as 5.04×10⁻⁶ and 1.13×10⁻⁶ cm²·s⁻¹ respectively. The results indicated that vitamin B₁₂ can bind to DNA and the major binding mode was intercalative binding.