Avidin-biotin Interaction Research Articles

Single and multiple bonds in (strept)avidin–biotin interactions

Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures ( <1 Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.

Kinetics and Thermodynamics of Biotinylated Oligonucleotide Probe Binding to Particle-Immobilized Avidin and Implications for Multiplexing Applications

In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10(-12) M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.

Probing Membrane-Surface Interactions via Brownian Motion of Micro-Sized Beads

Interactions between a free standing bilayer lipid membrane and various surfaces have been investigated by monitoring the Brownian motion of micron-sized silica beads on a GMO membrane. This membrane was suspended across a 200 µm Teflon aperture in either water or buffer solution using the painting method. Silica beads with diameters ranging from 0.7 µm - 5 µm were spread on the membrane and their motions were recorded at a rate of 10 frames/sec. The beads were either left unfunctionalized after cleaning or further functionalized with various chemical groups, including amine, PEG, methyl, octadecyl, avidin as well as the membrane itself. Since the interface interactions between the membrane and the beads leads to increased friction, it would be detected if the bead slowed in comparison with free Brownian motion. Experiments showed that the membrane functions as a near-perfect surface, lacking non-specific interactions. In all cases, only the avidin-biotin and membrane-membrane interactions lead to retarded motion and only in a buffered solution. This study on the Brownian motion of micron-sized beads on a membrane provides a convenient novel probe to detect interface interactions. As the beads used in these experiments have similar sizes to most bacteria and cells, they can be treated as bacteria/cell analogues and used to simulate the adsorption of bacteria /cells on another membraned bioorganism.

Investigating the Avidin-Biotin Interaction on Chiral Soft Structure Platforms

The synthesis of a novel biotin-cholesterol conjugate is described together with its propensity to self-assemble and exhibit interesting optical behaviour, when examined under cross-polarized light. The avidin-biotin interaction performed with this conjugate reveals loss of birefringence suggesting that such constructs could possibly be applied for optical tracking of important biological interactions.

Active Targeting Behaviors of Biotinylated Pluronic/Poly(Lactic Acid) Nanoparticles In Vitro through Three-Step Biotin–Avidin Interaction

In order to prepare targeted drug carriers, previously a biotin group has been attached by our group to the end of Pluronic F87/poly(lactic acid) and Pluronic P85/poly(lactic acid) block co-polymers to obtain B-F87–PLA and B-P85–PLA, respectively. In this paper, the active targeting properties of B-F87–PLA and B-P85–PLA nanoparticles in vitro were investigated through a three-step biotin–avidin interaction by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) tests and fluorescence microscopy (FM). Two kinds of human ovarian cancer cells (OVCAR-3 and SKOV-3) and paclitaxel were chosen for the cytotoxicity tests. CA-125 antigen is over-expressed on OVCAR-3 cells but not on SKOV-3 cells. The loading and release behavior of paclitaxel loaded in B-Pluronic–PLA nanoparticles were also studied. Paclitaxel loaded in both B-F87–PLA and B-P85–PLA nanoparticles shows an initial rapid release followed by a slow release period. Compared with SKOV-3 cells, the cytotoxicity results implied that paclitaxel-loaded B-Pluronic–PLA nanoparticles were delivered more effectively to OVCAR-3 cells due to the specific interaction between the biotin groups on the surface of B-Pluronic–PLA nanoparticles and the avidin/biotinylated MAb X306/CA-125 antigen complexes on the surface of OVCAR-3 cells. The active targeting properties of B-F87–PLA nanoparticles were further confirmed by FM.

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).

Hexon-specific PEGylated adenovirus vectors utilizing avidin-biotin interaction

PEGylation of recombinant adenovirus (Ad) vectors is a promising approach for not only evasion from neutralizing anti-Ad antibodies and uptake by phagocytic cells, but also prolongation of the blood retention time of Ad vectors after systemic administration. However, the conventional PEGylation leads to significant reduction in the transduction activity of Ad vectors, probably because PEG is nonspecifically conjugated to the Ad capsid protein and inhibits the binding of Ad vectors to the primary receptor, coxsackievirus-adenovirus receptor (CAR). In order to PEGylate an Ad vector without significant reduction in the transduction activity, the biotin-binding peptide (BAP) was inserted into the hypervariable region (HVR) 5 of the hexon, which is not involved in the binding to CAR, and PEG was then specifically conjugated to the hexon HVR5 via avidin-biotin interaction. In vitro transduction experiments demonstrated that the hexon-specific PEGylation did not cause an apparent reduction in the transduction efficiency of the Ad vector, although the insertion of the BAP into the HVR5 itself reduced the transduction efficiency by 50-fold, compared with the conventional Ad vector, in the absence of anti-Ad serum. In the presence of anti-Ad serum, the transduction with the Ad vector with the BAP in the hexon HVR5 was significantly blocked; however, anti-Ad serum only slightly inhibited the transduction with the hexon-specifically PEGylated Ad vector (Ad-BAP/Bio/Avi/Bio-PEG-L2). Intravenous administration of Ad-BAP/Bio/Avi/Bio-PEG-L2 resulted in prolonged blood retention, significant reduction in the transduction in the liver, and accumulation in the tumor; however, unexpectedly, the transduction efficiency of Ad-BAP/Bio/Avi/Bio-PEG-L2 in the tumor was almost at the background level.

Affinity 2009

Affinity 2009

Electrochemical strategy for detection of phosphorylation based on enzyme-linked electrocatalysis

In this paper, we report an electrochemical strategy for the detection of phosphorylation based on enzyme-linked electrocatalysis. In this strategy, the substrate peptides modified on a gold electrode surface are firstly phosphorylated by protein kinase, and then biotinylated via the recognition and combination with the specific biotin-labeled antibody. After that, gold nanoparticles (Au-NPs) which are modified with horseradish peroxidase (HRP) loaded streptavidin are immobilized on the surface of the electrode through biotin–avidin interaction. As a result, HRP on the electrode surface may take catalytic reaction towards the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), and the generated electrochemical signals can be used to monitor phosphorylation. This proposed electrochemical strategy can be employed to detect kinase activity with a satisfactory detection limit of 1 × 10 −3 unit/mL and linear range from 1 × 10 −3 to 1 × 10 −2 unit/mL. Furthermore, the inhibition of protein kinase has been also studied by using this strategy.

Ultrasensitive quantum dots-based DNA detection and hybridization kinetics analysis with evanescent wave biosensing platform

Ultrasensitive DNA detection was achieved using a new biosensing platform based on quantum dots (QDs) and total internal reflection fluorescence, which featured an exceptional detection limit of 3.2 amol of bound target DNA. The reusable sensor surface was produced by covalently immobilizing streptavidin onto a self-assembled alkanethiol monolayer of fiber optic probe through a heterobifunctional reagent. Streptavidin served as a versatile binding element for biotinylated single-strand DNA (ssDNA). The ssDNA-coated fiber probe was evaluated as a nucleic acid biosensor through a DNA–DNA hybridization assay for a 30-mer ssDNA, which were the segments of the uidA gene of Escherichia coli and labeled by QDs using avidin–biotin interaction. Several negative control tests revealed the absence of significant non-specific binding. It also showed that bound target DNA could easily be eluted from the sensor surface using SDS solution (pH 1.9) without any significant loss of performance after more than 30 assay cycles. A quantitative measurement of DNA binding kinetics was achieved with high accuracy, indicating an association rate of 1.38 × 10 6 M −1 s −1 and a dissociation rate of 4.67 × 10 −3 s −1. The proposed biosensing platform provides a simple, cheap, fast, and robust solution for many potential applications including clinical diagnosis, pathology, and genetics.

Surface modification of permalloy (Ni80Fe20) nanoparticles for biomedical applications

We report a simple and novel method for surface biofunctionalization onto recently reported Ni80Fe20 permalloy nanoparticles (~71 nm) and the immobilization of a model protein, IgG from human serum. The strategy of protein immobilization involved attachment of histidine-tagged streptavidin to the Ni80Fe20 nanoparticles via a non-covalent ligand binding followed by biotinylated human IgG binding on the nanoparticle surface using the specific high affinity avidin–biotin interaction. The biofunctionalization of Ni80Fe20 permalloy nanoparticles was confirmed by Fourier Transform InfraRed (FTIR) spectroscopy and protein denaturing gel electrophoresis (lithium dodecyl sulfate-polyacrylamide gel electrophoresis, LDS-PAGE). This protocol for surface functionalization of the novel nanometer-sized Ni80Fe20 permalloy particles with biological molecules could open diverse applications in disease diagnostics and drug delivery.

Theoretical Optimization Method of Buffer Ionic Concentration for Protein Detection Using Field Effect Transistors

Field effect transistors (FETs) may respond to charge variations occurring when proteins are adsorbed to the gate surface. The electrolyte electrostatic screening allows a practical detection of charges that are only within a distance of approximately the double layer thickness, which is related to the Debye length in the electrolyte. Hence, operating the FET biomolecular sensors at the highest possible ionic concentration improves reproducibility; however, it may reduce the signal if the concentration is too high. Here, we propose an optimization method of the buffer ionic concentration using a figure of merit “charge number,” , which is defined as the ratio between the effective number of the surface charges and the number of immobilized molecules. The theoretical was calculated using a three-dimensional conformation data taken from the Protein Data Bank. The was obtained from the total number of four charged amino acids of avidin, arginine (positive), lysine (positive), aspartic acid (negative), and glutamic acid (negative), which are within the double layer length from the surface. To verify this model, we chose avidin–biotin interaction. The experimental , which was obtained for three buffer concentrations, matched the theoretical . This method shortens the solution calibration time and reduces the analyte amount.

Biological evaluation of avidin-based tumor pretargeting with DOTA-Triazole-Biotin constructed via versatile Cu(I) catalyzed click chemistry

Background: The biotin-avidin interaction remains a gold standard for the two-step pretargeting approach to image tumor sites. We aim to develop two-step pretargeting systems utilizing 99mTc labeled biotin functionalized macrocyclic chelating agents synthesized using the highly efficient Cu(I) catalyzed azide–alkyne cycloaddition for potential radioimaging applications.Methods: A facile synthesis of DOTA-Triazole-Biotin, radiocomplexation with 99mTc and the pretargeting protocol is described. The synthesis features Cu(I) catalyzed click conjugation between biotinylated azide and propynyl functionalized DO3A. 99mTc radiolabeling was performed to detect the accumulation of avidin as the pretargeting agent. Cytotoxicity was determined using the trypan blue exclusion assay, macrocolony, and MTT assay. Cell uptake studies were performed using radiolabeled DOTA-Triazole-Biotin and compared with avidin treated cells for 2 h. Tumor imaging was performed in U-87MG cell line implanted tumor bearing nude mice and uptake of the radiotracer was estimated.Results: All compounds have been successfully characterized by NMR and MS spectroscopy. More than 96% radiolabeling efficiency was obtained and the radioconjugate exhibited sufficient stability under physiological conditions.Conclusion: To summarize, a new candidate for avidin based two-step pretargeting of tumors has been synthesized and evaluated for potential imaging and diagnostic applications. The chelate possesses high stability under physiological conditions, exhibits effective interaction with its avidin target, and low nonspecific retention in vivo.

Development of transferrin functionalized poly(ethylene glycol)/poly(lactic acid) amphiphilic block copolymeric micelles as a potential delivery system targeting brain glioma

The aim of present study is to conceive a biodegradable poly(ethylene glycol)-polylactide (PEG-PLA) copolymer nanoparticle which can be surface biofunctionalized with ligands via biotin-avidin interactions and used as a potential drug delivery carrier targeting to brain glioma in vivo. For this aim, a new method was employed to synthesize biotinylated PEG-PLA copolymers, i.e., esterification of PEG with biotinyl chloride followed by copolymerization of hetero-biotinylated PEG with lactide. PEG-PLA nanoparticles bearing biotin groups on surface were prepared by nanoprecipitation technique and the functional protein transferrin (Tf) were coupled to the nanoparticles by taking advantage of the strong biotin-avidin complex formation. The flow cytometer measurement demonstrated the targeting ability of the nanoparticles to tumor cells in vitro, and the fluorescence microscopy observation of brain sections from C6 glioma tumor-bearing rat model gave the intuitive proof that Tf functionalized PEG-PLA nanoparticles could penetrate into tumor in vivo.

QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases damaging both fresh and marine fish species. VHS caused by VHSV and diagnosis of VHSV has been dependent on the conventional methods, such as cell culture and RT-PCR, which takes a few days or several hours. This study demonstrates a rapid and sensitive QCM biosensor for diagnosis of VHSV infection in fish. The QCM biosensor was developed to detect a main viral RNA encoding G protein in VHSV using the specific DNA probe. To maximize the sensitivity of the biosensor, we prepared three different DNA probes which modified 3′ end of DNA by thiol, amine, or biotin and compared three different immobilisation methods on quartz surface coated with gold: immobilisation of thiol labelled probe DNA on naked gold surface, immobilisation of amino labelled probe DNA on gold surface prepared as carboxyl chip using MPA followed by EDC/NHS activation, and immobilisation of biotin labelled probe DNA on gold surface after immobilising avidin on carboxyl chip prior to biotin. As a result, immobilisation method using avidin–biotin interaction was most efficient to immobilise probe DNA and to detect target DNA. The QCM biosensor system using biotinylated probe DNA was stable enough to withstand 32 times of repeated regenerations and the detection limit was 0.0016 μM. Diagnosis using the QCM biosensor system was more sensitive and much faster than a conventional RT-PCR analysis in detecting the viral RNA.

Small Noncytotoxic Carbon Nano‐Onions: First Covalent Functionalization with Biomolecules

Small carbon nano-onions (CNOs, 6-8 shells) were prepared in high yield and functionalized with carboxylic groups by chemical oxidation. After functionalization these nanostructures were soluble in aqueous solutions. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 tetrazolium (MTS) tests showed excellent cytocompatibility of all CNOs analyzed at 30 and 300 microg mL(-1), so these carbon nanostructures can be safely used for biological applications. The first covalent functionalization of oxidized CNOs (ox-CNOs) with biomolecules, by using biotin-avidin interactions is reported here. Multilayers were prepared on a gold surface by layer-by-layer assembly and the process was monitored by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Covalent binding of molecules to the short amine-terminated organosulfur monolayers was assessed by Fourier transform infrared spectroscopy using total attenuated reflactance mode (FT-IR/HATR).

Development of Electrogenerated Chemiluminescence-Based Enzyme Linked Immunosorbent Assay for Sub-pM Detection

This paper reports the development and characterization of a highly sensitive enzyme linked immunosorbent assay realized by the electrogenerated chemiluminescence (ECL) detection of a thiol monolayer formed by an enzyme labeled antibody. We used two monoclonal anti tumor necrosis factor-alpha (TNF-alpha) antibodies for a sandwich immunoassay. One was a capture antibody, and the other was a detection antibody labeled with an enzyme via an avidin-biotin interaction. Acetylcholinesterase was used as the labeling enzyme to convert acetylthiocholine to thiocholine. Then the thiocholine was collected on a gold electrode surface by gold-thiol binding. A bright and distinctive emission was observed at 1150 mV (vs Ag-AgCl) on the gold electrode with a thiocholine monolayer as a coreactant in the presence of tris(2,2'-bipyridyl)ruthenium complex. This method can greatly enhance the immunoassay signal since a large number of coreactant molecules can be generated by the enzymatic reaction, which is advantageous compared with a previously reported ECL based immunoassay that directly labels the detection antibody with a coreactant or luminophore. In addition, a surface accumulated coreactant is superior to the previously reported coreactant system in a bulk solution, because ECL emission occurs only very close to an electrode surface. As a result, high sensitivity and a low detection limit of 0.2 pM (3.4 pg/mL) TNF-alpha were achieved with excellent reproducibility by optimizing the conditions for the immuno-reaction, thiocholine accumulation, and ECL generation.

Polymer–lipid microbubbles for biosensing and the formation of porous structures

Polymer–lipid microbubbles (PLBs) are generated by microfluidic flow-focusing devices to form a new class of long-lasting hybrid particles. The specific PLB construct developed is an elastic gas-filled microsphere with a polydimethylsiloxane (PDMS) shell containing phospholipids conjugated to functionalized polyethyleneglycol (PEG). Digital “droplet-based” microfluidics technology enables control of particle composition, size, and polydispersity ( σ < 10%). Use of PDMS as a shell component improves the functionality and stability (lifetime > 6 months) of the hybrid particles due to the thermally maneuverable solidification process. With a gas core, they serve as a template material for creating three-dimensional porous structures and surfaces, requiring no cumbersome post-processing removal steps. By adding biotinylated PEG–lipid derivatives that offer targeting capabilities, we demonstrate the immobilization of fluorescent IgG antibodies on stationary PDMS–lipid microbubbles through biotin–avidin interactions and on-chip trapping for immunoassays. A PDMS–lipid composition offers several advantages such as biocompatibility and biodegradability for future in vivo use as porous engineered scaffolds, packing materials, or delivery (e.g. therapeutic) agents with cell targeting capability.

Biodegradable PEG Hydrogels Cross-linkedUsing Biotin-Avidin Interactions

Poly(ethylene glycol) (PEG) hydrogels are water-swellable, non-toxic, non-immunogenic, and biocompatible. In this paper, we describe the generation of biodegradable PEG hydrogels by cross-linking biotinylated PEG oligomers containing intrinsic disulfide bonds via biotin-avidin interactions. The biotinylated PEG oligomers were synthesized by the condensation reaction between PEG and 3,3′-dithiodipropionic acid, followed by the reaction with biotin. This methodology obviates the need for potentially toxic chemical cross-linking agents that are usually used in the common preparation of hydrogels. Therefore it may be particularly useful in biomedical or pharmaceutical applications.

New oligoethylene glycol linkers for the surface modification of an ultra-high frequency acoustic wave biosensor

This work describes the application of acoustic wave technology for the real-time and label-free detection of biotin–avidin interactions. Biosensing surfaces are constructed onto unelectroded piezoelectric quartz discs as functionalizable mixed self-assembled monolayers (SAM) produced from previously unreported linker and diluent molecules. Biotinthiol can subsequently be immobilized for detection purposes in a straightforward and coupling-free manner. Specific and non-specific adsorptions of avidin are measured at ultra-high frequencies (1.06 and 0.82 GHz) with an electromagnetic piezoelectric acoustic sensor (EMPAS) using micromolar avidin-spiked buffer solutions. These biosensing surfaces, especially the oligoethylene glycol SAM-based variety, display high specificity for avidin, with moderate to excellent reproducibility. This preliminary work constitutes the first application of SAM chemistry and EMPAS technology in the bioanalytical field.