Voltammetric detection of avidin and biotin by biotin labeled with cysteine

An avidin-biotin assay was developed from a voltammetric procedure using biotin labeled with cysteine. Mercury(II) as a marker was used to detect avidin and biotin, because the oxidation wave of mercury decreases when the cysteine part of labeled biotin(LB) complexes with mercury(II).The formation of the mercury(II)-cysteine complex is suppressed when the LB binds to the biotin site of avidin. Accordingly, the concentration of avidin can be estimated from the increasing mercury peak current. Detection of biotin is also carried out by a competitive reaction of biotin and the LB to the binding site on avidin, where the addition of biotin decreases the peak current of mercury. Limits of detection for avidin and biotin were in the 10^–9 mol/L range. The length of the spacer between the cysteine and biotin was investigated. It was observed that the strength of binding increased with increasing length of spacer. Size considerations rules out steric influences, so it is suggested that the binding constant depends on hydrophobic interactions in the binding site.     

Voltammetric homogeneous binding assay of biotin without a separation step using iminobiotin labeled with an electroactive compound.

Avidin, which is one type of glycoprotein, has a strong affinity with biotin (Ka = 10(15) M(-1)). Iminobiotin also forms a complex with avidin (Ka = 10(8) M(-1) at pH 9.5). The avidin-iminobiotin complex changes to the avidin-biotin complex in the presence of biotin because of the difference of the binding constant to avidin. In this study, the interaction between avidin and iminobiotin labeled with an electroactive compound was investigated by voltammetry. After avidin and the labeled iminobiotin (LI) were incubated in 0.1 M phosphate buffer (pH 7.0), the peak currents of LI were measured in various concentrations of biotin. The peak currents increased with increasing the concentration of biotin. Thus, this observation indicates the formation of avidin-biotin complex. On the other hand, the formation of avidin-iminobiotin complex depended on the pH of the solution. LI combines with the avidin at pH 5.6-8.9 and dissociates at pH 4.6.     

Electrochemical evaluation of avidin-biotin interaction using N-iodoacetyl-N-biotinylhexylenediamine

The avidin-biotin assay was investigated by an electrochemical procedure based on the chemical reaction between cysteine containing a thiol group and N-iodoacetyl-N-biotinylhexylenediamine (IB). In the presence of avidin and biotin this reaction, whereby the thiol group combines with IB, is controlled because IB has a biotin part; that is, avidin and biotin are detected indirectly by measurement of iodide ion or cysteine. To achieve a high sensitivity of detection, Hg(II) was introduced as a marker that interacts with cysteine and the oxidation peak from Hg(0) to Hg(II) was measured. The sensitivity of detection of avidin was at the level of 10⁻⁹ M. The relative standard deviation at 1 × 10⁻⁸ M avidin was 4.8% (n = 5). On the other hand, a response curve to detect biotin was obtained by the competitive reaction between IB and biotin for the limited binding sites of avidin. The change in peak current enables the detection of biotin at the level of 10⁻⁹ M. This method has the advantage that it is not necessary to separate free IB from bound IB.     

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.     

A novel,simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry

A simple and sensitive ligand affinity capture method (LAC) was developed to detect biotinylated biomolecules bound to a biotin–avidin base by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI ToF MS). Glass slides covered with a metal film for MALDI MS applications were treated with amino‐silane and derivatized with biotin followed by binding of avidin. Washing buffers with high ionic strength increased the specificity of the subsequent binding of biotinylated biomolecules to the avidin layer. A combined thin layer‐dried droplet method using α‐cyano‐4‐hydroxycinnamic acid (CHCA) in acetone or ethyl acetate resulted in the most intense ions of biotinylated polymyxin B, whereas the matrix conditions did not influence the detection of angiotensin II. Addition of biotinylated biomolecules in the low femtomole to low picomole range resulted in sufficient ion intensity for detection by the LAC method. The LAC concept was extended by binding of biotinylated lipopolysaccharide to the biotin–avidin base followed by preferential capture and specific detection of the binding antagonist polymyxin B. Copyright © 2008 John Wiley & Sons, Ltd.     

Non-isotopic receptor assay for benzodiazepines using a biotin-labeled ligand and biotin-immobilized microtiter plate.

A non-isotopic receptor assay for benzodiazepine drugs was developed using a biotin-labeled ligand, biotin-1012S. Biotinylated bovine serum albumin (biotin-BSA) was immobilized onto the wall of microtiter plate wells by simple adsorption. Avidin peroxidase conjugate could be extracted from solution owing to its strong interaction with biotin. The amount of avidin peroxidase taken up on the wall was then determined by measuring the enzyme activity. The competition between immobilized biotin on the wall and free biotin for avidin provided the basis for a solid-phase avidin-biotin binding assay. By this binding assay, not only biotin but also biotin-1012S could be measured sensitively. Because 1012S is a ligand with high affinity to benzodiazepine receptors, biotin-1012S could be utilized as a probe ligand for a non-isotopic receptor assay. Based upon the competition between biotin-1012S and various benzodiazepine drugs for the receptor binding sites, a non-isotopic receptor assay was demonstrated.     

Determination of biotin on a protein by quantitative sodium dodecyl sulfate-capillary gel electrophoresis of monomeric avidin

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS-CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17,000. In the presence of a biotin-protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin and the initial amount is the portion of monomeric avidin bound to the conjugate. Accordingly, the number of biotin on the protein can be calculated. The assay is linearly responsive to increasing biotin loading in a biotinylation reaction of a protein. Accuracy of the assay is also demonstrated by good sample dilution recovery. Excellent quantitative reproducibility < 2% (relative standard deviation) is obtained for both intra- and inter-day measurements. Main advantages of the method include the use of monomeric avidin that minimizes steric hindrance to capture biotin on a protein and assay automation on a capillary electrophoresis apparatus.     

Visual observation of avidin-biotin affinity by fluorescent G4.5 poly(amidoamine) dendrimer

An air-treated G4.5 poly(amidoamine) (PAMAM) dendrimer displayed the enhanced fluorescence enough to be utilized as a fluorescence marker to visualize avidin-biotin affinity: On a fluorescence microscopic image, the avidin labeled by a fluorescent G4.5 PAMAM dendrimer was observed to be selectively bound on the biotin pattern that was prepared by amide-bonding of biotin on a carboxylic acid-terminated self-assembled monolayer and in turn by UV-irradiation with a photomask on the monolayer.     

Directed self-assembly of gold-tipped CdSe nanorods

Gold-tipped CdSe rods (nanodumbbells) were solubilized in an aqueous phase and self-assembled in a head-to-tail manner using biotin disulfide and avidin. The disulfide end of the biotin molecule attaches to the gold tip of the nanodumbbell, and the biotin end of the molecule is able to conjugate to an avidin protein. The avidin can strongly conjugate up to four biotin molecules. Changing the ratios of biotin to nanodumbbells leads to the formation of dimers, trimers, and flowerlike structures. To further improve the distribution of chain lengths, a separation method based upon weight was applied using a concentration gradient. The gold tips provide effective anchor points for constructing complex nanorod structures by self-assembly.     

Electrochemical Evaluation of the Interaction between Avidin and Biotin at Biotinylated Polypyrrole Electrode Using a Redox Marker

The interaction between avidin and biotin was evaluated electrochemically by monitoring the change in the electrode response of redox markers. Biotin was immobilized on the electrode surface by means of the electrochemical polymerization of biotinylated pyrrole and pyrrole. When avidin was introduced onto the biotinylated polypyrrole electrode surface, the large change in the electrode response of the redox marker was detected. The fact that the change in the electrode response of a marker ion could be attributed to the electrostatic interaction between avidin on the electrode surface and the redox marker ion present in a solution was verified by replacing avidin with NutrAvidin. At a pH lower than the isoelectric point of avidin, the electrode response of ferrocyanide as an anionic marker ion increased linearly within the range of 5.0×10^?9 ?3.0×10^?8 M avidin. The relative standard deviation at 1.5×10^?8 M avidin was about 5.4% (n=5). The detection of biotin was also performed using a competitive reaction between biotin in solution and biotin that had been immobilized on the electrode surface in the form of the biotinylated polypyrrole.     

Simple test system for single molecule recognition force microscopy

We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG₈₀₀ diamine was glutarylated, the mono-adduct NH₂-PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy.     

Effect of Functionalization of Colloidal Gold on Controlled Flocculation by the Ligand-Receptor Mechanism

Modification of colloidal gold particles with octadecanthiol and alkyl biotin, with the subsequent flocculation of these particles via introduction of a tetramer protein avidin, was studied.     

New fluorometric enzyme immunoassay for 17beta-estradiol by homogeneous reaction using biotinylated estradiol

A new fluorometric enzyme immunoassay for 17β-estradiol (E2) using biotinylated estradiol (BE) as a probe ligand, is described. In this method, E2 is detected indirectly by a solid-phase avidin-biotin binding assay, in which the biotin is immobilized on a microtiter plate (biotin-plate). After the competitive reaction between E2 and BE for the anti-E2 antibody in solution, the free E2 and BE are separated from the bound forms by means of ultrafiltration. The concentration of BE in the solution is determined from the reaction between the biotin immobilized on the plate and the free BE for the limited biotin binding sites of avidin conjugated with horseradish peroxidase (avidin-HRP), which is added to the solution. The enzymatic reaction of HRP was measured by a fluorometric analysis with the QuantaBlu™ Fluorogenic Peroxidase Substrate (QFPS) in order to detect of the avidin-biotin binding with a high degree of sensitivity. The detection limit and linear range for the determination of E2 were 0.12 nM and from 0.12 to 25 nM, respectively. The relative standard deviations (R.S.D.) for the E2 assay were between 2.2 and 9.1% (n = 3). The cross-reactivity for several other estrogens was also evaluated.     

Separation of polyethylene glycols and maleimide‐terminated polyethylene glycols by reversed‐phase liquid chromatography under critical conditions

The separation of polyethylene glycols and maleimide‐substituted polyethylene glycol derivatives based on the number of maleimide end‐groups under critical liquid chromatography conditions has been investigated on a reversed‐phase column. The critical solvent compositions for nonfunctional polyethylene glycols and bifunctional maleimide‐substituted polyethylene glycols were determined to be identical at about 40% acetonitrile in water on a reversed‐phase octadecyl carbon chain‐bonded silica column using mixtures of acetonitrile and water of varying composition as the mobile phase at 25°C. The maleimide‐functionalized polyethylene glycols were successfully separated according to maleimide functionality (with zero, one, two, or three maleimide end‐groups, respectively) under the critical isocratic elution conditions without obvious effect of molar mass. The separation was mainly due to the hydrophobic interaction between the maleimide end‐groups and the column packing. Off‐line matrix‐assisted laser desorption/ionization time of flight mass spectrometry was used to identify the repeating units and, especially, the end‐groups of the maleimide‐substituted polyethylene glycols. Liquid chromatography analysis at critical conditions could provide useful information to optimize the synthesis of functional polyethylene glycols. To our knowledge, this is the first report of the baseline separation of maleimide‐functionalized polyethylene glycols based on the functionality independent of the molar mass without derivatization by isocratic elution.     

Strategies for the reversible immobilization of enzymes by use of biotin-bound anti-enzyme antibodies

Glucose oxidase (E.C. 1.1.3.4) is reversibly immobilized in a reactor coupled to a flow-injection analysis system using an immunological reaction. The antibody used is irreversibly immobilized on the reactor support by an avidin-biotin linkage. The bond between avidin and biotin is nearly irreversible under normal elution conditions for antibody-antigen reactions. The reactor is packed with a support on which avidin is covalently attached and a biotin-bound second antibody is passed over the reactor packing, which immobilizes this antibody. An immune complex of the enzyme, or first anti-enzyme antibody followed separately by enzyme, is introduced into the flow system, resulting in enzyme immobilization. The reactor produced can be used in the determination of 1 x 10(-11) -1 x 10(-6) mole of glucose with a sample size of 20 mul and a sample throughput of 20-30/hr. These results are comparable to or better than those obtained with glucose oxidase directly immobilized on the same support. The enzyme can be removed by elution with low-pH buffers, and the reactor regenerated by injection of the anti-enzyme antibody and the enzyme.     

Biotin-β-Cyclodextrin: A New Host-Guest System for the Immobilization of Biomolecules

The formation of stable supramolecular interactions between biotin and β-cyclodextrin was studied. An association constant of 3 × 10(2) M(-1) could be determined by NMR measurements by mapping the high field shift differences of the β-cyclodextrin protons (H-3) at different biotin concentrations. With the aim to demonstrate a new alternative for the immobilization of bioreceptors, biotin and β-cyclodextrin tagged biomolecules were immobilized on transducer surfaces, which were functionalized with the correspondent host-guest partner. The reliability of this new affinity system was investigated using two enzymes (glucose oxidase and polyphenol oxidase) as biomolecule models. This supramolecular inclusion complex shows clear advantages to the classic biotin-(strept)avidin-biotin system due to a detrimental effect of the additional avidin layer reducing the transduction efficiency. A 7-fold increase in the maximum current density and an almost 20 times higher sensitivity were exhibited by the immobilized biological layer obtained using this new host-guest system.     

Water cluster calibration reduces mass error in electrospray ionization mass spectrometry of proteins

Herein we report a novel calibration routine for use in positive ion mode electrospray ionization mass spectrometry (ESI-MS). Monoisotopic masses were calculated for a series of water clusters and used as calibration reference files (available at http://www.hbcg.utmb.edu/xray). The water cluster series contains singly charged peaks every 18 Da, which allows calibration curves to be precisely defined over a broad mass-to-charge ratio range. Water clusters, induced by a combination of high flow rate and high cone voltage, were used to accurately calibrate a quadrupole mass spectrometer from 100 to 1900 m/z. Calibration curves thus generated have many more data points and greatly reduced standard deviations compared to those obtained from myoglobin, sodium iodide, cesium iodide, or poly(ethylene glycol) based calibration standards. This calibration routine reduces the error in protein mass measurements by a factor of 3, from ±0.01% to ±0.0035% at the 95% confidence limit. The implications of this increased mass accuracy and wider calibrated mass-to-charge ratio scale for the study of protein sequence, structure, and folding by ESI-MS are discussed.     

A spectrophotometric assay for biotinbinding sites of immobilized avidin

A rapid and sensitive spectrophotometric assay was developed for the measurement of biotin-binding sites of immobilized avidin. The method is based on the reaction of avidin with excess biotin followed by assay of the unbound biotin using the HABA (2-[4′-hydroxyazobenzene] benzoic acid) method. Three solids possessing variable amounts of monomeric avidin were examined; viz., succinamidopropyl-controlled-pore glass (CPG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B**), and crosslinked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB₁. Results indicate that the total biotin-binding sites of monomeric avidin immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0.093, and 0.218 μmol biotin per mL beads, respectively. Assays for exchangeable biotinbinding sites gave values greater than 90% of the total sites. The spectrophotometric HABA method described is an alternative to assays based on tracers, thus the handling of radioactive material is avoided. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Chip-Based CE for Avidin Determination in Transgenic Tobacco and Its Comparison with Square-Wave Voltammetry and Standard Gel Electrophoresis

Avidin transgenic plants are a potential tool for providing resistance against various species of insect pests due to the sequestration of vitamin H (biotin) in the plant from the insect pests. In this project we compared three techniques for avidin determination in transgenic tobacco plants, a novel chip-based capillary electrophoretic method (Experion), classical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) and a square wave voltammetric method using a carbon paste electrode. We determined that the automated chip-based capillary electrophoretic method is rapid, sensitive and the results obtained are well repeatable. The avidin content measured in transgenic tobacco leaves using chip-based capillary electrophoresis varied from 15 to 854 ng per mg of fresh mass depending on the individual plant.     

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.