Voltammetric detection of lectin using sugar labeled with electroactive substance.

The voltammetric detection of soybean agglutinin (SBA) was investigated on the basis of an interaction between the lectin and a sugar. Because galactose and lactose combined with SBA, the sugars were labeled by a Schiff base with an electroactive daunomycin. After the labeled sugar and SBA were mixed, measurements were carried out by voltammetry. When SBA-sugar binding occurs, a part of daunomycin of the labeled sugar is taken to the binding sites. As a result, SBA is detected by a change in the peak current of daunomycin, and the SBA-sugar interaction is evaluated. The length of the alkyl chain between daunomycin and the sugar was also considered. The electrode response to the concentration of SBA was linear over the range of 0.04-0.8 microg min(-1). The merits of this procedure are the convenient preparation of labeled sugar and a rapid measurement without separation. On the other hand, the detection of sugar at the 10(-9) mol dm(-3) level was achieved by a competitive reaction to limited binding sites of the lectin between the sugar and the labeled sugar.     

Voltammetric behaviors of wheat-germ agglutinin on a chitin-modified carbon-paste electrode.

The voltammetric behavior of wheat-germ agglutinin (WGA) on a chitin-modified carbon-paste electrode (CPE) was investigated using glucose labeled with an electroactive compound. WGA usually consists of two subunits, each with two binding sites for sugars. WGA was immobilized on the electrode surface by selective binding to a N-acetylglucosamine residue of chitin. Because glucose also combines with WGA, the glucose was coupled with electroactive daunomycin to evaluate the binding. When a WGA-labeled glucose complex was formed, the electroactive moiety became electroinactive. The binding caused a decrease in the peak current of the labeled glucose. In a measurement of only daunomycin used as a label, the peak current in a solution with WGA was similar to that in a solution without WGA. Therefore, it is clear that the labeled glucose was held in the remaining binding site of WGA on the electrode surface. Thus, a CPE modified with chitin would be powerful as a reaction field between sugar and lectin.     

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase

Reduced anionic flavin adenine dinucleotide (FADH^?) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV‐damaged DNA. The initial step involves photoinduced electron transfer from *FADH^? to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD‐dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (k_ET) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano‐ or etheno‐bridged Ade between the AN1 and AN6 atoms (e‐FAD and ε‐FAD, respectively) were used to reconstitute apo‐PL, giving e‐PL and ε‐PL respectively. The reconstitution yield of e‐PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε‐PL showed 50% reconstitution yield. The substrate binding constants for ε‐PL and rPL were identical. ε‐PL showed a 15% higher steady‐state repair yield compared to FAD‐reconstituted photolyase (rPL). The acceleration of repair in ε‐PL is discussed in terms of an ε‐Ade radical intermediate vs superexchange mechanism.     

Electrochemical detection of lectin using a galactosamine labeled with daunomycin

To detect a lectin from soybean, an electrochemical procedure was developed by the use of a labeling of galactosamine. Because the lectin has binding sites to galactosamine, galactosamine labeled with daunomycin having electroactivity was prepared. When labeled galactosamine (LG) combines with lectin, the part of daunomycin is taken in the binding sites of the lectin and becomes electroinactive. Therefore, the concentration of the lectin can be estimated by measuring the peak current of the LG. On the other hand, a competitive reaction to the lectin of galactosamine and the LG makes a detection of galactosamine possible. This method has merit that does not require a separation procedure of the free LG from the bound one. An effect of length of spacer between daunomycin and galactosamine was also investigated. It was found that adsorption of reagent on the electrode increased due to introduction of the spacer. Furthermore, the electrode response of the LG was influenced by the type of the spacer.     

Biotin ligands labeled with daunomycin as an electrochemical probe for avidin and biotin interaction

Electroactive biotin ligands were prepared by the reaction of daunomycin with biotinylating reagents with a different spacer. These biotin ligands exhibited similar electrochemical properties to those of daunomycin, but the adsorptivity of the ligands on the electrode increased with increasing length of the spacer. The electrode response of these ligands decreased when specifically bound with avidin. This made it possible to detect electroinactive avidin indirectly. Biotin was detected by observing the competitive reaction between biotin and the ligands for the limited binding sites of avidin. The binding strength of the labeled biotins with avidin was compared with that of unlabeled biotin by using an enzyme assay.     

生物功能电极I: GOD修饰电极的电化学活性

以二环己基碳化二亚胺为活化剂将葡萄糖氧化酶(GOD)共价键接在玻碳电极上, 伏安实验观察到酶与电极基体的直接电子传递, 有观电子传递速度常数约为1s^-^1, 过程归因于全酶中辅基FAD的氧化还原转变。Ag^+离子的存在强烈地阻碍酶辅基的还原, 这与该离子抑制酶活性的机理可能有联系。Ag^+的抑制作用可由EDTA处理或电化学处理而解除, GOD电极对氧和苯醌的电还原有催化作用。测定了苯醌同还原态GOD的化学反应速度常数, 并讨论用苯醌代替氧作为生物电催化中的电子传递体的优点。     

Evaluation of Binding Between Electroactive Biotin Derivative and Streptavidin Immobilized on Chitin Film

Evaluation of the streptavidin‐biotin binding at the surface of chitin film was carried out with voltammetry. Immobilization of streptavidin was attempted to the protonated chitin film, based on an electrostatic interaction that hardly causes any change in the protein structure. The streptavidin‐biotin binding was estimated from changes in the electrode response of biotin labeled with an electroactive compound. Although the response of daunomycin as an electroactive compound did not change at an electrode covered with streptavidin/chitin film, the response of the labeled biotin decreased. This observation shows that streptavidin is immobilized on the chitin film and the biotin binds with immobilized streptavidin. Consequently, it was clear that the chitin film is useful as a reaction field for protein‐ligand binding. Generally, a binding event between protein and its ligand in the living body occurs on the cell surface. The electrochemical evaluation of protein‐ligand binding on a natural polysaccharide like chitin membrane surface is important.     

Voltammetric evaluation for the binding of wheat germ agglutinin to glucosamine-modified magnetic microbead

Binding of wheat germ agglutinin (WGA) on glucosamine-modified magnetic microbeads was investigated with voltammetry. A magnetic bead was considered as a cell, and the beads with amino groups were modified with the sugar by using a cross-linking reagent. To evaluate the binding, glucose labeled with an electroactive daunomycin was prepared as a probe. After WGA and the beads were mixed in 0.1 M phosphate buffer (pH 7.0), the labeled glucose was added to the solution. The binding was monitored from the changes in the electrode response of labeled glucose because the labeled glucose was held to the binding site of WGA for the sugar. In contrast, other lectin not having the binding site to glucosamine or glucose was incubated with the glucosamine-modified beads. As a result, the change of peak current was not observed. Therefore, it is clear that the binding of WGA to glucosamine moiety on the bead surface selectively takes place. This method would be powerful for evaluation of interaction between protein and sugar chain existing at cell surface.     

Electrochemical assay of concanavalin A-ovalbumin binding on magnetic beads

To monitor protein-glycoprotein interactions on magnetic beads, the present study developed an electrochemical assay of the binding between concanavalin A (ConA) and ovalbumin (OVA). The system was a powerful model that could be used to evaluate cell junctions. ConA with an electroactive daunomycin was immobilized on 6 different sizes of magnetic beads (diameter: 1.0-8.9 μm) through a cross-linking agent. Six sizes of OVA-beads (diameter: 1.0-8.9 μm) were also prepared using a similar method. The binding was evaluated using an oxidation peak of ConA with daunomycin because ConA recognized OVA with α-mannose residues. When binding took place on the beads' surface, the peak current was decreased due to the electroactive moieties being covered with OVA. When ConA/daunomycin-OVA binding was evaluated, the change of the peak current obtained by the beads (diameter: 8.9 μm) modified with ConA and daunomycin was the greatest in the presence of OVA-modified beads (diameter: 2.5 μm). In contrast, particle agglomeration was observed for the smallest beads (diameter: 1.0 μm) with ConA/daunomycin and OVA. The results suggested that ConA-OVA binding depended on the size of beads. Thus, this method could be applied to measure protein-glycoprotein interactions on the cell surface.     

Unadulterated Glucose Biosensor Based on Direct Electron Transfer of Glucose Oxidase Encapsulated Chitosan Modified Glassy Carbon Electrode

Glucose oxidase (GOD) was encapsulated in chitosan matrix and immobilized on a glassy carbon electrode, achieving direct electron transfer (DET) reaction between GOD and electrode without any nano‐material. On basis of such DET, a novel glucose biosensor was fabricated for direct bioelectrochemical sensing without any electron‐mediator. GOD incorporated in chitosan films gave a pair of stable, well‐defined, and quasireversible cyclic voltammetric peaks at about ?0.284 (E_pa) and ?0.338 V (E_pc) vs. Ag/AgCl electrode in phosphate buffers. And the peak is located at the potentials characteristic of FAD redox couples of the proteins. The electrochemical parameters, such as midpoint potential (E_1/2) and apparent heterogeneous electron‐transfer rate constants (k_s) were estimated to ?0.311 V and 1.79 s^?1 by voltammetry, respectively. Experimental results indicate that the encapsulated GOD retains its catalytic activity for the oxidation of glucose. Such a GOD encapsulated chitosan based biosensor revealed a relatively rapid response time of less than 2 min, and a sufficient linear detection range for glucose concentration, from 0.60 to 2.80 mmol L^?1 with a detection limit of 0.10 mmol L^?1 and electrode sensitivity of 0.233 μA mmol^?1. The relative standard deviation (RSD) is under 3.2% (n=7) for the determination of practical serum samples. The biologic compounds probably existed in the sample, such as ascorbic acid, uric acid, dopamine, and epinephrine, do not affect the determination of glucose. The proposed method is satisfactory to the determination of human serum samples compared with the routine hexokinase method. Both the unique electrical property and biocompatibility of chitosan enable the construction of a good bio‐sensing platform for achieved DET of GOD and developed the third‐generation glucose biosensors.     

Factor analysis of spectroelectrochemical reduction of FAD reveals the pKa of the reduced state and the reduction pathway

The free flavin adenine dinucleotide (FAD) cofactor is known to exhibit a pH‐dependent midpoint potential involving a simultaneous two‐electron transfer step (n = 2). Uv‐vis spectroelectrochemical reductions of FAD at constant pH, ranging from 5 to 9, were recorded and analyzed by factor analysis. Principal factor analysis was used to determine the number of species present at each pH. The results indicate that only two composite forms of FAD are present: the oxidized and the reduced forms. Window factor analysis was used to extract the concentration profiles of the controlling species. The oxidized form was found to be a single pH‐independent species, whereas the reduced form consists of two species. The pH‐dependent spectroscopic changes of reduced FAD were best modeled by a single proton transfer step involving two different ionization states with an apparent pK_a = 6.3. This value compares favorably with those obtained from NMR and from midpoint potential measurements. At pH 6, the reduction of FAD was found to be first order, whereas at pH 9 the reduction is zero order; these observations are explained in terms of the reaction pathway involving xanthine oxidase, its substrate, and the pH. Copyright © 2007 John Wiley & Sons, Ltd.     

Molecularly Imprinted Electrochemical Luminescence Sensor Based on Enzymatic Amplification for Ultratrace Isoproturon Determination

A new molecularly imprinted electrochemical luminescence sensor (MIP‐ECL sensor) was developed for isoproturon (IPU) determination based on the competition reaction between IPU and glucose oxidase labeled IPU (GOD‐IPU). After competition, hydrogen peroxide produced by residual GOD‐IPU on the MIP reacted with luminol to emit electrochemiluminescence (ECL) signal. The ECL intensity decreased when the GOD‐IPU molecules were replaced by IPU molecules in the samples. IPU could be determined in the concentration range from 9×10^?11 mol/L to 5.1×10^?9 mol/L with a detection limit of 3.78×10^?12 mol/L. Water samples were assayed and recoveries ranging from 98.5 % to 102.1 % were obtained.     

Binding assay for cholera toxin based on sequestration electrochemistry using lactose labeled with an electroactive compound

A simple electrochemical binding assay for cholera toxin (CT) was developed using lactose labeled with daunomycin as an electroactive compound. The labeled lactose (LL) was determined with high sensitivity by adsorptive stripping voltammetry (AdSV). The electrochemical behaviors of LL at glassy carbon (GC), plastic formed carbon (PFC) and carbon nanotubes paste (CNTP) electrode were investigated. The CNTP electrode showed the greatest accumulation capacity for LL. The assay for CT based on the sequestration electrochemistry was demonstrated. The binding event of the LL to CT was detected by the decrease in the electrochemical response of daunomycin as an electroactive label without a separation process to remove the free LL from the one bound with CT before any measurements can be made. The detection limit of the CT assay using the CNTP electrode was 0.5 nM (42 ng mL(-1)).     

Reconstituted Enzymes on Electropolymerizable FAD‐Modified Metallic Nanoparticles: Functional Units for the Assembly of Effectively “Wired” Enzyme Electrodes

Au nanoparticles are functionalized with thioaniline electropolymerizable units and mercaptophenyl boronic acid ligands. Flavin adenine dinucleotide (FAD) is linked to the boronic acid ligands and apo‐glucose oxidase, apo‐GOx, is reconstituted on the FAD cofactor units to yield enzymes in a structurally‐aligned configuration in respect to the Au NPs. Electropolymerization of the enzyme‐functionalized Au NPs on a thioaniline‐modified Au electrode yields a three‐dimensional bis‐aniline‐crosslinked Au NPs/reconstituted glucose oxidase matrix on the electrode that reveals effective electrical contacting with the electrode.     

Low‐Potential Detection of Glucose with a Biosensor Based on the Immobilization of Glucose Oxidase on Polymer/Manganese Oxide Layered Nanocomposite

A nanocomposite with poly(diallyldimethylammonium), PDDA, intercalated between manganese oxide layers is constructed on a graphite electrode surface through one‐step electrodeposition and used to adsorb glucose oxidase (GOD). The immobilized GOD displays a pair of stable and quasireversible redox peaks with a formal potential of ?468 mV in pH 7.0 buffer solutions and exhibits excellent electrocatalysis to the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, indicating that the immobilized GOD kept its bioactivity. Thus a reagentless biosensor for glucose at a low detection potential was established. The linear concentration range is from 0.02 to 2.78 mM with a detection limit of 9.8 μM. The proposed glucose biosensor was insensitive to common interferences such as ascorbic and uric acids etc.     

NIR Mega‐Stokes Fluorophores for Bioorthogonal Labeling and Energy Transfer Systems–An Efficient Quencher for Daunomycin

A set of new azide‐ and alkyne‐bearing lepidinium‐based fluorophores were synthesized for bioorthogonal labeling schemes. These fluorescent dyes all show large Stokes‐shifts with emission maxima in the near‐infrared (NIR) region of the electromagnetic spectrum. The applicability of these dyes in the construction of energy‐transfer systems was tested using one of these new fluorescent tags and daunomycin (Dau), an anticancer drug with fluorescent features. These daunomycin conjugates are the very first examples of fluorescently modulated constructs of this anticancer agent. The dually labeled architectures proved that the applied fluorescent dye can be utilized as an efficient quencher for daunomycin. Enzymatic cleavage of a dually labeled enzyme substrate resulted in full recovery of the fluorescence of daunomycin. Such fluorescently modulated Dau conjugates can provide useful information for the mechanism of action of Dau‐regulated cell death processes.     

基于室温离子液体的有序多孔金膜葡萄糖传感器

将1-丁基-3-甲基咪唑四氟硼酸盐（[BMIm][BF4]）、N,N-二甲基甲酰胺（DMF）与葡萄糖氧化酶（GOD）的混合物修饰于三维有序大孔（3DOM）金膜电极上,构建了一种新型的葡萄糖传感器．固定的GOD在pH7．0的磷酸缓冲液（PBS）中展现出一对可逆性好的氧化还原峰,这归因于GOD的活性中心黄素腺嘌呤二核苷酸（FAD）的直接电化学行为．研究表明,离子液体（IL）、DMF以及3DOM金膜对GOD的直接电化学都起到了重要的作用．3DOM金膜修饰电极作为基底提高了酶的负载量,加速了GOD与电极表面的电子传递;IL的应用增加了固定GOD的电化学活性;DMF与IL、GOD的协同作用更好地保持了GOD的生物活性．固定在电极表面的GOD对葡萄糖显示出良好的催化性能,其检测线性范围为10～125nmol／L,检测限为3．3nmol／L（S／N=3）,酶催化反应的表观米氏常数Km为0．018mmol／L．     

Preparation of the glucose sensor based on three-dimensional ordered macroporous gold film and room temperature ionic liquid

A novel type of glucose sensor was fabricated based on a glucose oxidase (GOD)-N,N-dimethtylformamide (DMF)-[BMIm][BF₄] composites modified three-dimensional ordered macroporous (3DOM) gold film electrode. The immobilized GOD exhibits a pair of well-defined reversible peaks in 50 mM pH 7.0 phosphate buffer solutions (PBS), which could be attributed to the redox of flavin adenine dinucleotide (FAD) in GOD. The research results show that ionic liquid ([BMIm][BF₄]), DMF and 3DOM gold film are crucial for GOD to exhibit a pair of stable and reversible peaks. It is believed that the large active area of 3DOM gold film can increase the amount of immobilized GOD. Simultaneously, the application of IL enhances the stability of GOD and facilitates the electron transfer between GOD and the electrode. The synergetic effect of DMF can help the GOD to maintain its bioactivity better. GOD immobilized on the electrode exhibits the favorable electrocatalytic property to glucose, and the prepared sensor has a linear range from 10 to 125 nM with a detection limit of 3.3 nM at a signal-to-noise ratio of 3σ. The apparent K _m (Michaelis- Menten constant) for the enzymatic reaction is 0.018 mM.     

孔蛋白-磷脂仿生膜的葡萄糖氧化酶电化学

采用孔蛋白（MspA）和双肉豆蔻磷脂酰胆碱（DMPC）在玻碳（GC）基底表面成功构建有仿生特性的纳米通道膜,同时将葡萄糖氧化酶（GOD）修饰于膜上. 使用循环伏安法研究GOD/MspA-DMPC/GC电极的GOD直接电化学过程以及其对氧气和葡萄糖的响应. 研究发现,MspA与DMPC形成的仿生纳米通道膜内,GOD在接近生物体系FAD/FADH标准电位处实现了自身两质子、两电子表面控制的电化学反应. MspA与DMPC的仿生纳米通道膜体系为GOD提供了理想活性环境.     

Electrochemically monitoring the binding of concanavalin A and ovalbumin

To evaluate protein-protein interactions, a new voltammetric method was developed using a protein labeled with an electroactive compound. Concanavalin A (ConA), which is a lectin, recognizes α-mannose residues. Because the ConA was to be bound to ovalbumin (OVA), which has a high-mannose sugar chain, ConA labeled with daunomycin was prepared as the probe to monitor the binding. The binding to OVA was caused by the label modification of the ConA. As a result, the electrode response of the labeled ConA decreased as the OVA concentration increased. The electrode response of the labeled ConA was linearly over the range of 1.5 × 10⁻¹⁰ and 1.5 × 10⁻⁹ M OVA. The relative standard deviation of 1.5 × 10⁻⁸ M labeled ConA and 1.5 × 10⁻¹⁰ M OVA was 6.9% (n = 5). The labeled ConA-OVA binding could then be conveniently monitored based on the change in response. In contrast, interactions between the labeled ConA and a protein with no specific sugar chain also were investigated. Incubation scarcely influenced the peak current of the labeled ConA. When several concentrations of OVA were added to a serum, good recovery determined it. Consequently, this method could be applied to the measurement of protein-protein interactions.