Voltammetric sensing of sugar by an electrode covered with wheat germ agglutinin/chitin film

The binding between wheat germ agglutinin (WGA) and N-acetylglucosamine at the electrode covered with chitin film was investigated with voltammetry. Chitin, β-1,4-poly-N-acetylglucosamine, is one of the biolpolymers which have a high biocompatibility. WGA is immobilized to the surface of chitin film by the affinity of WGA to N-acetylglucosamine residue of chitin. To investigate the binding event of WGA on the chitin modified electrode, N-acetylglucosamine labeled with an electroactive compound was prepared. The binding causes the changes in the electrode response of labeled sugar. The peak current of labeled sugar decreased due to the specific binding with WGA on the chitin film modified at the electrode. N-Acetylglucosamine was successfully determined by using the competitive reaction with labeled sugar to WGA on the chitin film electrode.     

Efficient wheat germ agglutinin purification with a chitosan‐based affinity chromatographic matrix

An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini‐spheres cross‐linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA – calculated from the corresponding isotherms – was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini‐spheres cross‐linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP‐HPLC was developed.     

Recognition between a divalent sialyl molecule and wheat germ agglutinin

Structural divalency between a designed N-acetyl-neuraminic acid (NeuAc)-containing molecule and lectin wheat germ agglutinin (WGA) is investigated. The sialyl molecule was designed based on the NeuAc-WGA complex in the Protein Data Bank and featured polyethylene glycol linkers connecting to an aromatic scaffold. Our results elucidate the divalent recognition association constant between WGA and the multivalent-NeuAc molecules to be 10⁷ by surface plasmon resonance.     

Voltammetric evaluation of the binding between wheat germ agglutinin and thionine/glucose-modified magnetic microbeads.

The binding between glucose residues and wheat germ agglutinin (WGA) on thionine/glucose-modified magnetic microbeads was evaluated using voltammetry. Thionine is an electroactive compound and has two amino groups. Thionine was immobilized to magnetic beads via cross-linking of the amino groups on the beads with an amino group on thionine. Glucose was bound to the other amino group of thionine via the formation of a Schiff base. The beads were only several micrometers in size the same size, as cells. WGA-binding to glucose on the bead surface blankets the thionine moiety. Thus, WGA-binding could be detected as a decrease in peak current of the thionine moiety.     

Generation of a dynamic combinatorial library using sialic acid aldolase and in situ screening against wheat germ agglutinin

This paper describes the generation of a dynamic combinatorial library of sialic acid analogues using sialic acid aldolase. Addition of wheat germ agglutinin to the equilibrating libraries results in selective amplification of one or more members.     

麦胚凝集素亲和色谱富集糖肽过程中的肽键裂解

蛋白质的糖基化是最重要的翻译后修饰之一,与蛋白质结构和功能的关系密切。凝集素亲和色谱是蛋白质糖基化研究中很常用的工具,不同的凝集素可以对不同的单糖或寡糖有特异的富集作用。麦胚凝集素（WGA）由于其特异作用的糖型广泛存在而成为使用最多的凝集素之一。在本研究中,发现将WGA用于糖肽亲和富集会导致部分肽段的降解,从而导致后续的肽段序列分析的失败。本文用4种标准蛋白质对这种现象进行了验证,结果表明肽段的降解可以发生在多个位点,其中较多地发生在酪氨酸、苯丙氨酸及亮氨酸的羧基端。这一结果提示：在糖蛋白质组研究中,如果应用WGA富集糖肽并采用质谱进行鉴定,则采用半酶切或非特异性酶切的检索策略更为合适。     

Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin-I for capturing sub-glycoproteomics from breast cancer and disease-free human sera

In this study, a liquid-phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub-glycoproteomics that are affected in cancers, e.g. breast cancer. The platform is comprised of three monolithic columns with surface immobilised lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con A have specificities directed towards the core portion of N-glycans on the glycoprotein surface, RCA-I specifically interacts with the non-reducing terminal moieties of the outer chain structures of N-glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA → Con A → RCA-I (denoted as WCR) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease-free and breast cancer sera, respectively, corresponding to 75 and 65 non-redundant proteins, respectively. Using mass spectral count ratios and Q-Q plots yielded a panel of 23 non-redundant differentially expressed proteins (i.e. a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate.     

Cyclic Voltammetric Response of Tetrathiafulvalene-Glucose Oxidase-Modified Electrode and Results for Digital Simulation

Abstract

An amperometric enzyme electrode for the determination of glucose (<16mmol^?1) is constructed by incorporating electron mediators tetrathiafulvalene in Nafion and subsequently coating with immobilized glucose oxidase. The results obtained from measurements of glucose in fermentation samples containing biomass or molasses indicate the utility of the electrode for glucose assay in such media over at least 12 weeks. Digital simulation is employed to study the cyclic voltammetry (CV) of Tetrathiafulvalene-Glucose Oxidase-Modified Glassy Carbon Electrode (TTF-GOD-GCE); the digital model is built and the effects of kinetic parameters on CV-curves are discussed. The response process of the Tetrathiafulvalene-Glucose Oxidase-Modified Electrode is partly explained by the simulation results and further research is expected to guide the design of biosensors and improve the properties of the enzyme-mediator modified electrode.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells.

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10(7) binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37 degrees C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Cathodized Gold Nanoparticle‐Modified Graphite Pencil Electrode for Non‐Enzymatic Sensitive Voltammetric Detection of Glucose

A highly sensitive enzymeless electrochemical glucose sensor has been developed based on the simply prepared cathodized gold nanoparticle‐modified graphite pencil electrode (AuNP‐GPE). Cyclic voltammetry (CV) experiments show that AuNP‐GPE is able to oxidize glucose partially at low potential (around −0.27) whereas the bare GPE cannot oxidize glucose in the entire tested potential windows. Besides, fructose and sucrose cannot be oxidized at potential lower than +0.1 V at AuNP‐GPE. As a result, the glucose oxidation peak at around −0.27 V is suitable enough for selective detection of glucose in the presence of fructose and sucrose. Cathodization of AuNP‐GPE under optimum condition (‐1.0 V for 30 s) in the same glucose solution before voltammetric measurement enhanced glucose oxidation peak current around −0.27 V to achieve an efficient electrochemical sensor for glucose with a detection limit of 12 μM and dynamic range between 0.05 to 5.0 mM with a good linearity (R²= 0.999). Almost no interference effect was observed for sensing of glucose in the presence of ascorbic acid, alanine, phenylalanine, fructose, sucrose, and NaCl.     

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.     

Access to Phylogeny from Voltammetric Fingerprints of Seeds: the Asparagus Case

A methodology for characterizing vegetal taxonomic groups from the voltammetric fingerprints of polyphenolic components of seeds is described. It is based on recording the voltammetric response of microparticulate films deposited on glassy carbon electrodes from seed extracts using different organic solvents. The obtained responses in contact with aqueous electrolytes provided characteristic voltammetric profiles at the level of genera/subgenera and/or families using bivariant and multivariant chemometric methods. The voltammograms of 14 species from 5 different families provided family‐characteristic patterns. Analysis of voltammetric responses for a set of 20 species of the Asparagus genus from four continents permits to discriminate the genetic lines represented by the Asparagus , Protasparagus and Myrsiphyllum subgenera.     

Fabrication of tunable microreactor with enzyme modified magnetic nanoparticles for microfluidic electrochemical detection of glucose

A microfluidic device was designed for amperometric determination of glucose by packing enzyme modified magnetic nanoparticles (MNPs) in its microchannel as an enzyme microreactor. Glucose oxidase was covalently attached to the surface of MNPs and localized in the microchannel by the help of an external magnetic field, leading to a tunable packing length. By changing the length of microreactor from 3 to 10 mm, the performance for glucose detection was optimized. The optimal linear range to glucose was from 25 μM to 15 mM with a detection limit of 11 μM at a length of 6 mm. The inter- and intra-day precisions for determination of 1.0 mM glucose were 0.8% and 1.7%, respectively, and the device-to-device reproducibility was 95.6%. The enzyme reactor remained its 81% activity after three-week storage. Due to the advantages of the device and fracture sampling technique, serum samples could be directly sampled through the fracture to achieve baseline separation from ascorbic acid, and proteins in the samples did not interfere with the detection. This work provided a promising way for pretreatment-free determination of glucose with low cost and excellent performance.     

Frontal analysis microchip capillary electrophoresis to study the binding of ligands to receptors derivatized on magnetic beads

The model binding of the glycopeptide antibiotic teicoplanin (Teic) from Actinoplanes teichomyceticus, immobilized on magnetic microspheres, to d-Ala-d-Ala terminus peptides was assessed using microchip capillary electrophoresis (MCE) with continuous frontal analysis (FA). Teic is closely related to vancomycin (Van), historically, the drug of last resort used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to the d-Ala-d-Ala terminus on the cell wall of Gram-positive bacteria via hydrogen bonds, thereby preventing the enzyme-mediated cross-linking of peptidoglycan and eventual cell death. In this work direct and competitive bead-based assays in a microfluidic chip are demonstrated. The binding constants obtained using the technique are comparable with values reported in the literature.     

酸性铬蓝K-H2O2-HRP伏安酶联免疫分析法测定HRP及其标记物

本文提出了一种新的辣根过氧化物酶(HRP)的底物--酸性铬蓝K(ACBK),它本身具有电化学活性,能够在汞电极上发生还原反应.以H2O2为氧化剂,HRP的加入能加快氧化反应的进行,使酸性铬蓝K被氧化分解,其平衡浓度降低,对应的还原峰电流降低,峰电流的降低值同HRP的加入量在8.0×10-8～1.0×10-6 g/mL之间呈线性关系.用于IgG-HRP的测定,最高稀释比为1∶5 000.     

Development of a Carbon Nanotube Modified Ionic Liquid Electrode for the Voltammetric Determination of Methyldopa Levels in Urine

A glassy carbon electrode was modified with carbon nanotubes and the ionic liquid N‐butyl pyridinium trifluoromethyl methanesulfonate for the determination of methyldopa in urine samples. Methyldopa exhibited a well‐defined anodic signal over a broad pH range of 2–10 and the peak current increased approximately 100 fold over that of the unmodified electrode. Accordingly, a novel method for the determination of methyldopa was proposed using differential pulse voltammetry. The peak current was linear over a methyldopa concentration range from 21 to 2111 ng mL^?1 with a LOD of 6.9 ng mL^?1 and a LOQ of 7.4 ng mL^?1. The method was applied to determine the excretion profile of methyldopa in urine without sample pretreatment.     

Voltammetric Determination of Favipiravir Used as an Antiviral Drug for the Treatment of Covid-19 at Pencil Graphite Electrode

This work describes the sensitive voltammetric determination of favipiravir (FAV) based on its reduction for the first time with a low-cost and disposable pencil graphite electrode (PGE). In addition, the determination of FAV was also performed based on its oxidation. Differential pulse (DP) voltammograms recorded in 0.5 M H₂SO₄ for the reduction of FAV show that peak currents increase linearly in the range of 1.0 to 600.0 μM with a limit of detection of 0.35 μM. The acceptable recovery values (98.9–106.0 %) obtained from a pharmaceutical tablet, real human urine, and artificial blood serum samples spiked with FAV confirm the high accuracy of the proposed method.     

A Novel Voltammetric Method for the Determination of Maleic Acid Using Silver Amalgam Paste Electrode

An efficient voltammetric method was developed for the determination of maleic acid at a silver amalgam paste electrode (AgA‐PE) in Britton–Robinson buffer pH 2.0. The experimental parameters, such as pH of Britton–Robinson buffer, type of the supporting electrolyte and activation of the electrode surface were optimized. Under the optimal conditions, a linear response was observed over the 2×10^?6–1×10^?4 mol L^?1 maleic acid concentration range, determination limit being 5×10^?7 mol L^?1. A highly stable response, with a relative standard deviation (RSD) of 1.6% for 45 repetitive measurements of 1×10^?4 mol L^?1 maleic acid showed that there was no apparent surface passivation indicating the suitability of the method. The method was successfully applied for direct determination of maleic acid in drinking and river water.     

Modification of the Electrode Surface by Ag Nanoparticles Decorated Nano Diamond‐graphite for Voltammetric Determination of Ceftizoxime

A modified glassy carbon electrode with a film of nano diamond? graphite nano mixture decorated with Ag nanoparticles (AgNPs? NDG/GCE) was constructed and used for sensitive voltammetric determination of ceftizoxime (CFX). Morphology of AgNPs? NDG/GCE has been examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Experimental variables such as deposited amount of the modifier suspension, pH of the supporting electrolyte and accumulation potential and time were optimized by monitoring of CV and LSV responses of CFX. The results illustrate that AgNPs? NDG/GCE exhibits an excellent electrocatalytic effect in the electro‐oxidation of CFX that leads to a considerable improvement in the corresponding anodic peak current. This also allows the development of a highly sensitive voltammetric sensor for the determination of CFX in pharmaceutical and clinical samples. Under the optimum conditions, the modified electrode showed a linear response to the concentration of CFX in the range of 0.02–7 µM with detection limit of 6 nM. The prepared modified electrode has some remarkable electrochemical properties such as simple preparation, high sensitivity, excellent repeatability and reproducibility and long‐term stability.     

Different methods for the fractionation of magnetic fluids

 Magnetic fluids are used in many fields of application, such as material separation and biomedicine. Magnetic fluids consist of magnetic nanoparticles, which commonly display a broad distribution of magnetic and nonmagnetic parameters. Therefore, upon application only a small number of particles contribute to the desired magnetic effect. In order to optimize magnetic fluids for applications preference is given to methods that separate magnetic nanoparticles according to their magnetic properties. Hence, a magnetic method was developed for the fractionation of magnetic fluids. Familiar size-exclusion chromatography of two different magnetic fluids was carried out for comparison. The fractions obtained and the original samples were also magnetically characterized by magnetic resonance and magnetorelaxometry, two biomedical applications. The size-exclusion fractions are similar to those of magnetic fractionation, despite the different separation mechanisms. In this respect, magnetic fractionation has several advantages in practical use over size-exclusion chromatography: the magnetic method is faster and has a higher capacity. The fractions obtained by both methods show distinctly different magnetic properties compared to the original samples and are therefore especially suited for applications such as magnetorelaxometry. Received: 12 July 1999/Accepted in revised form: 9 November 1999