Impedimetric immunosensor using avidin-biotin for antibody immobilization

A newly developed electrochemical method--Elimination Voltammetry with Linear Scan (EVLS)--has been applied to the electrochemical study of nucleic acids (NAs) on a silver electrode. Using the linear combination of the currents measured at different scan rates, the EVLS is capable of eliminating one or two selected particular currents. It was shown that the elimination function conserving the reversible diffusion current and eliminating the charging and kinetic currents provides the significant increase of voltammetric signals of DNA. Due to the high sensitivity and resolution power, the EVLS can contribute to study behaviour of nucleic acids on the charged interface and can be applied to nucleic acid analyses and the development of DNA sensors.     

Homogeneous enzyme-linked competitive binding assay for biotin based on the avidin-biotin interaction

A homogeneous enzyme-linked competitive-binding assay for biotin with glucose-6-phosphate dehydrogenase (G6PDH), is described. This assay is based on the interaction between a G6PDH/biotin conjugate with avidin, a natural binder for biotin. In the absence of biotin in the assay mixture, this interaction results in 100% inhibition of the enzyme conjugate. In the presence of biotin, the enzymatic activity of the conjugate is regained in an amount related to the concentration of the vitamin in the sample. Extremely steep, gate-like dose/response curves, attributable to the relative binding affinities of avidin for biotin and the conjugate, are observed. The detection limits of the system vary with the amounts of avidin and enzyme/biotin conjugate used. The method is rapid and sensitive and is evaluated for the direct determination of biotin in vitamin tablets.     

Voltammetric investigation of avidin-biotin complex formation using an electroactive bisbiotinyl compound

Formation of avidin-biotin complex was investigated using bisbiotinyl thionine (BBT) by means of voltammetric techniques. Thionine is an electroactive compound and has two amino groups that are necessary for the reaction with a biotinylation reagent. The biotinylation of thionine produces a new reagent with two biotin moieties at each end of thionine. Three BBTs of different lengths of the spacer that connects the biotin moiety to the thionine moiety were prepared. The avidin-biotin binding assay was achieved by measuring the electrode response of the thionine moiety in BBT. The binding affinity and the conformation of complex, which depended on the length of spacer, are discussed. BBT in which the spacer is shortest (BBT-S, distance between carbonyl group of the two biotin moieties: 11 Å) binds with only one avidin molecule. BBT with medium length of spacer (BBT-M, 28.8 Å) forms the complex with two avidin molecules. BBT with the longest spacer (BBT-L, 46.6 Å) allows binding with two avidin molecules as well as intramolecular binding within one avidin molecule. The affinity constants of BBT-S, BBT-M and BBT-L for avidin were estimated to be 7.0 × 10¹² M⁻¹, 3.2 × 10¹² M⁻¹ and 4.0 × 10¹² M⁻¹, respectively.     

Enzyme immunoassay for the drug of anti-ulcer using avidin-biotin system.

We have developed a competitive enzyme immunoassay for a drug, which was a newly synthesized anti-ulcer agent, using an enzyme immunoassay. The polyclonal anti-drug antibody coupled to biotin, peroxidase labeled drug derivatives as a tracer, and a small column of Sepharose 4B covalently bound to avidin were used in the assay. This assay is simple and rapid, and the sensitivity and the measuring range can be controlled by the flow rate of the substrate solution. The correlation between serum drug concentrations (0.1-10 micrograms/ml) measured by gas chromatography and this assay was good (r = 0.991). This principle for the assay is very practical and applicable to the enzyme immunoassay for small and large molecules.     

Electrochemical evaluation of lectin-sugar interaction on gold electrode modified with colloidal gold and polyvinyl butyral

In this work, ConA and CramoLL lectins were immobilized on gold nanoparticles (AuNp) with polyvinyl butyral (PVB), and adsorbed on the surface of gold (Au) electrodes. Electrochemical impedance spectroscopy (EIS), in the frequency range from 100mHz to 100KHz, and cyclic voltammetry (CV), from -0.2 to 0.7V, were performed on these electrodes, in phosphate buffer (PBS) solution containing 10mM K(3)[Fe(CN)(6)]/K(4)[Fe(CN)(6)] (1:1) mixture as a redox probe. EIS and CV measurements showed that redox probe reactions on the modified Au electrodes were partially blocked due to the adsorption of AuNp-ConA-PVB and AuNp-CramoLL-PVB. SEM images showed the presence of aggregates of AuNp-ConA on PVB spherules in a tridimensional structure on the surface of the Au electrode. Bovine serum albumin (BSA) was adsorbed on the AuNp-Lectin-PVB modified electrode in order to block the remaining free gold sites. Both EIS and CV techniques yielded results that confirm positive responses of the lectins to ovalbumin agglutination. These results indicate an improvement of the sensitivity for detection of sugars that can be applicable to construction of a biosensor sensitive to glycoproteins in solution.     

Electrochemical modeling of the silica nanoparticle-biomembrane interaction

The interaction of amorphous colloidal silica (SiO(2)) nanoparticles of well-defined sizes with a dioleoyl phosphatidylcholine (DOPC) monolayer on a mercury (Hg) film electrode has been investigated. It was shown using electrochemical methods and microcalorimetry that particles interact with the monolayer, and the electrochemical data shows that the extent of interaction is inversely proportional to the particle size. Scanning electron microscopy (SEM) images of the electrode-supported monolayers following exposure to the particles shows that the nanoparticles bind to the DOPC monolayer irrespective of their size, forming a particle monolayer on the DOPC surface. A one-parameter model was developed to describe the electrochemical results where the fitted parameter is an interfacial layer thickness (3.2 nm). The model is based on the adsorptive interactions operating within this interfacial layer that are independent of the solution pH and solution ionic strength. The evidence implies that the most significant forces determining the interactions are van der Waals in character.     

Electrochemical oxidation of ochratoxin A at a glassy carbon electrode and in situ evaluation of the interaction with deoxyribonucleic acid using an electrochemical deoxyribonucleic acid-biosensor

Ochratoxin A (OTA) is a fungal metabolite that occurs in foods, beverages, animal tissues, human blood and presents carcinogenic, teratogenic and nephrotoxic properties. This study concerns the redox properties of OTA using electrochemical techniques which have the potential for providing insights into the biological redox reactions of this molecule. The in situ evaluation of the OTA interaction with DNA using a DNA-electrochemical biosensor is also reported.The oxidation of OTA is an irreversible process proceeds with the transfer of one electron and one proton in a diffusion-controlled mechanism. The diffusion coefficient of OTA was calculated in pH 7 phosphate buffer to be D_O = 3.65 × 10⁻⁶ cm² s⁻¹. The oxidation of OTA is also pH dependent for electrolytes with pH < 7 and involves the formation of a main oxidation product which adsorbs strongly at the GCE surface undergoing reversible oxidation. In alkaline electrolytes OTA undergoes chemical deprotonation, the oxidation involving only the transfer of one electron.The electrochemical dsDNA-biosensor was also used to evaluate the possible interaction between OTA and DNA. The experiments have clearly proven that OTA interacts and binds to dsDNA strands immobilized onto a GCE surface, but no evidence of DNA-damage caused by OTA was obtained.     

Antibody coating using various avidin-biotin complexes employed to an enzyme immunoassay for estradiol

Conclusion We conclude that the antibody LC-biotin avidin complex on polystyrene plates is a suitable component for a sensitive enzyme immunoassay system.

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Antikörper-Beschichtung mittels verschiedener Avidin-Biotin-Komplexe verwendet in einem Enzymimmunoassay für Estradiol

     

Electrochemical investigation of the interaction of 2,4-D and double stranded DNA using pencil graphite electrodes

2,4-dichlorophenoxyacetic acid (2,4-D) is an auxinic herbicide used to control broadleaf weeds. It is also a threatening factor for not only aquatic life but also human health due to its genotoxicity and endocrine disruptive property. Herein, the interaction between 2,4-D and double stranded DNA was investigated by using single-use pencil graphite electrodes (PGE) in combination with electrochemical techniques. The detection mechanism was based on the monitoring of the changes at the guanine oxidation signal obtained before/after surface-confined interaction of 2,4-D and DNA at the surface of PGE. The electrochemical characterization of the interaction was studied by using microscopic and electrochemical techniques. The response obtained by interaction in the presence of another herbicide, glyphosate, which is widely used with 2,4-D for weed control, was compared to the one occurred in the presence of 2,4-D. Electrochemical monitoring of the interaction between the herbicide whose active molecule was 2,4-D and DNA was also investigated. The detection (LOD) and quantification limits (LOQ) for 2,4-D and the herbicide could be obtained in the linear concentration ranges of 30–70 µg/mL and 10–30 µg/mL, respectively and LOD and LOQ values were found to be 2.85 and 9.50 µg/mL for both 2,4-D and the herbicide. The sensitivity of the biosensor was calculated as 0.087 µA.mL / µg.cm² .This is the first study in literature by means of not only voltammetric detection of 2,4-D and DNA interaction but also the herbicide-DNA interaction at the surface of PGE based on the changes at the guanine signal.     

Electrochemical study on interaction of vincristine with tubulin

In Tris (0.005 mol/L)‐NaCl (0.05 mol/L) buffer solution (pH = 7.10), keeping temperature at 37°C, a highly sensitive reduction peak of the antitumor agent was obtained by linear sweep voltammetry. The peak potential is‐1.56 V (vs. SCE). The peak current is proportional to the concentration of vincristine over the range of 2.1 ± 10^?7‐4.2 ± 10^?6 mol/L with the detection limit of 1.0 ± 10^?7 mol/L. The behavior of the binding of vincristine to tubulin was studied. The results showed that the reaction of tubulin dimer with vincristine formed an electrochemically active complex to be 1:2. Its stability constant is 2.5 ± 10¹⁴. The reduction process of the complex is irreversible with adsorptive characteristics.     

Electrochemical studies of NiTMpyP and interaction with DNA

In this paper, cyclic voltammetry, linear sweep voltammetry and chronocoulometry in connection with the hang mercury drop electrode were used to study NiTMpyP and its mixture with DNA. The reduction of NiTMpyP in our experimental conditions involves in 4e reduction of TMpyP. NiTMpyP interacting with DNA forms electrochemically non-active complex DNA-2NiTMpyP, which can not be reduced on the Hg electrode. The peak potential of NiTMpyP does not shift and its electrochemical kinetic parameters indicate no significant change in the presence of DNA. However, the reduction current of NiTMpyP decreases obviously due to the formation of DNA-2NiTMpyP, which implies its equilibrium concentration decreases when DNA was mixed. The decrease of peak current is proportional to DNA concentration, which can be applied to estimate DNA concentration.     

Electrochemical oxidation of morin and interaction with DNA

A poly (tetrafluroethylene)-deoxyribonucleate acid (PTFE-DNA) film-modified glassy carbon electrode (GCE) has been fabricated. The electrochemical oxidation behaviors of morin as well as its interaction with DNA have been studied at PTFE-DNA film-modified GCE and bare GCE by electrochemical methods. This modified electrode shows an enhanced effectiveness towards the oxidation of morin. Importantly, as to the interaction between morin and DNA in solution, characteristic parameters such as the binding stoichiometry and association equilibrium constant according to the Hill model for cooperative binding have been determined on the basis of linear sweep voltammetry and chronocoulometry.     

Protein assemblies by site-specific avidin-biotin interactions

Exploiting self-assembly systems with biological building blocks is of significant interest in the fabrication of advanced biomaterials. We assessed the potential use of site-specific ligand labeling of protein building blocks in designing functional protein self-assemblies by combining site-specifically biotinylated bacterial alkaline phosphatase (as a bidentate or tetradentate ligand unit) and streptavidin (as a tetrameric receptor).     

Electrochemical evaluation and determination of antiretroviral drug fosamprenavir using boron-doped diamond and glassy carbon electrodes

Fosamprenavir is a pro-drug of the antiretroviral protease inhibitor amprenavir and is oxidizable at solid electrodes. The anodic oxidation behavior of fosamprenavir was investigated using cyclic and linear sweep voltammetry at boron-doped diamond and glassy carbon electrodes. In cyclic voltammetry, depending on pH values, fosamprenavir showed one sharp irreversible oxidation peak or wave depending on the working electrode. The mechanism of the oxidation process was discussed. The voltammetric study of some model compounds allowed elucidation of the possible oxidation mechanism of fosamprenavir. The aim of this study was to determine fosamprenavir levels in pharmaceutical formulations and biological samples by means of electrochemical methods. Using the sharp oxidation response, two voltammetric methods were described for the determination of fosamprenavir by differential pulse and square-wave voltammetry at the boron-doped diamond and glassy carbon electrodes. These two voltammetric techniques are 0.1 M H₂SO₄ and phosphate buffer at pH 2.0 which allow quantitation over a 4 × 10⁻⁶ to 8 × 10⁻⁵ M range using boron-doped diamond and a 1 × 10⁻⁵ to 1 × 10⁻⁴ M range using glassy carbon electrodes, respectively, in supporting electrolyte. All necessary validation parameters were investigated and calculated. These methods were successfully applied for the analysis of fosamprenavir pharmaceutical dosage forms, human serum and urine samples. The standard addition method was used in biological media using boron-doped diamond electrode. No electroactive interferences from the tablet excipients or endogenous substances from biological material were found. The results were statistically compared with those obtained through an established HPLC-UV technique; no significant differences were found between the voltammetric and HPLC methods.     

Electrochemical evaluation of the viability of platelets

The difference between the open-circuit redox potentials of a platinum electrode measured in blood plasma before and after the cryolysis of platelets depends linearly on the content of granule-rich platelets in the plasma. This phenomenon can be used to determine the viability of platelets.     

Electrochemical behaviors of nicotine and its interaction with DNA

The studies of the interaction between nicotine and DNA on the solid electrode were proposed for the first time. The electrochemical behaviors of nicotine and its interaction with DNA were explored by differential pulse voltammetry (DPV) at the DNA modified glassy carbon electrode (DNA/GCE). In 0.05 M Na₂C₂O₄ (pH 4.24), nicotine at DNA/GCE showed an irreversible reductive behavior in the range of ?1.6 to ?1.1 V. The presence of DNA led to the decrease in the peak current of nicotine and the negative shift in the potential, indicating that nicotine could interact with DNA by electrostatic mode. The binding ratio between nicotine and DNA was calculated to be 2:1 and the binding constant was 8.31(±0.36) × 10². The interaction between nicotine and DNA could be further verified by UV–vis spectroscopy.     

泛昔洛韦与牛血清白蛋白相互作用的伏安行为研究

在pH 7的B-R缓冲底液中,应用循环伏安法和微分脉冲法在玻碳电极上对泛昔洛韦与牛血清白蛋白相互作用进行了研究,与相同浓度单独存在于溶液中泛昔洛韦相比,峰电位基本不变,峰电流有所降低,讨论了pH、反应时间、扫速、富集时间、静止时间对二者作用的影响,建立了对牛血清白蛋白浓度的检测方法.计算了泛昔洛韦加入BSA前后转移的质子数和电子数不变,扩散系数变小,推测泛昔洛韦进入BSA的疏水空腔内,使游离药物浓度变小.     

Electrochemical detection of interaction between Thioflavin T and acetylcholinesterase

The electrochemical oxidation of the benzothiazole dye Thioflavin T (ThT) was found to be modulated by its interaction with electric eel acetylcholinesterase (AChE). Modifications of AChE by trace amounts of small molecule inhibitors such as carbachol and paraoxon were detectable electrochemically using minimal reagents and with greater sensitivity than attainable through conventional fluorescence approaches. This property appears to be unique to ThT, since its closely related neutral derivative BTA-1 only interacts with AChE, but is not significantly affected by the presence of small molecule inhibitors.