Avidin-biotin Interaction Research Articles

Electric field and Charged Molecules Mediated Self-Assembly for Electronic Devices

ABSTRACTIn this paper we present techniques, utilizing dielectrophoresis and electrohydrodynamics, which can possibly be used for assembling devices suspended in a solution onto a binding site on a substrate. We explored the concepts using micro-scale negatively charged polystyrene beads and rectangular silicon blocks. Dielectrophoretic forces on devices in buffer solutions were examined as a function of frequency of the applied AC signal. The observed results can be explained by taking in account electro-thermal and AC electroosmotic effects. The study described in the paper can be used for placing and assembling micro and nano-electronic devices and objects at specific sites on various substrates, in combination with bio-inspired biological binding techniques such as DNA hybridization, antigen-antibody interactions, and ligand-receptor (avidin-biotin) interactions.

Electric field and Charged Molecules Mediated Self-Assembly for Electronic Devices

AbstractIn this paper we present techniques, utilizing dielectrophoresis and electrohydrodynamics, which can possibly be used for assembling devices suspended in a solution onto a binding site on a substrate. We explored the concepts using micro-scale negatively charged polystyrene beads and rectangular silicon blocks. Dielectrophoretic forces on devices in buffer solutions were examined as a function of frequency of the applied AC signal. The observed results can be explained by taking in account electro-thermal and AC electroosmotic effects. The study described in the paper can be used for placing and assembling micro and nano-electronic devices and objects at specific sites on various substrates, in combination with bio-inspired biological binding techniques such as DNA hybridization, antigen-antibody interactions, and ligand-receptor (avidin-biotin) interactions.

A Visualization Method of Filamentous Phage Infection and Phage-Derived Proteins in Escherichia coli Using Biotinylated Phages

Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin–biotin–peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin–avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses.

Fabrication of organic phase biosensors based on multilayered polyphenol oxidase protected by an alginate coating

We describe herein, the creation of an organic phase enzyme electrode (OPEE) via avidin–biotin interactions built over an electrogenerated polymer. Multilayered polyphenol oxidase (PPO) assemblies were transferred into an organic solvent (chloroform) for the catechol detection at −0.2 V. In conjunction with an alginate gel, as a hydrophilic additive, the biosensor performance was widely enhanced. The effects of biotinylated polypyrrole film and alginate gel on the diffusion process through the biosensor coating are studied by rotating disk electrode experiments carried out in chloroform with hydroquinone as electroactive permeant.

Temperature dependence of ultrasonic enhancement with a site-targeted contrast agent.

Molecular imaging contrast agents specifically detect the biochemical "signatures" of disease before anatomical manifestations are apparent. Sensitive and specific localization of fibrin both in vivo and in vitro has been demonstrated with the use of a ligand-directed liquid perfluorocarbon nanoparticle. Since the acoustic properties of perfluorocarbons are known to vary with temperature, it was hypothesized that temperature could be used to augment the magnitude of enhancement imparted by targeted nanoparticles. Accordingly, the acoustic backscatter of two different substrates, nitrocellulose membrane and human plasma clot, targeted by the nanoparticles was measured at temperatures ranging from 27 degrees to 47 degrees C in 5 degrees C increments. Classic avidin-biotin interactions were utilized to couple biotinylated nanoparticles to avidin-conjugated nitrocellulose membranes. Ultrasonic contrast enhancement of the nitrocellulose membrane at 25 MHz, measured by acoustic microscopy, increased from 2.0+/-0.3 dB at 27 degrees C to 3.7+/-0.4 at 47 degrees C. In a similar experiment, antifibrin nanoparticles bound to human plasma clots also exhibited temperature-dependent ultrasonic signal enhancement ranging from 13.9+/-1.5 dB at 27 degrees C to 18.1+/-1.5 dB at 47 degrees C. The increase in ultrasonic contrast enhancement measured was well described by a simple, acoustic transmission line model with temperature-dependent impedance. These results suggest that temperature-dependent changes in acoustic backscatter may be used to further differentiate tissues targeted with site-specific nanoparticles from surrounding normal soft tissues.

A Collection of Long, Flexible PEG-Crosslinkers for Molecular Recognition Force Microscopy on Single Molecules

Attachment of a single probe molecule (e.g. an antibody) to the measuring tip of an atomic force microscope yields a powerfull sensor for specific detection of single target molecules. As evidenced by many studies, target recognition by the probe is much facilitated when inserting a poly(ethylene glycol) (PEG) chain (∼8 nm) between probe and tip [1]. This presentation points out future directions of coupling via long, flexible spacers. Starting from highly pure NH2 -PEG-COOH, a variety of heterobifunctional PEG derivatives with amino-, carbonyl-, and different thiol-reactive end groups, as well as with biotin or nitrilotriacetic acid (NTA) termini, were synthesized and applied in recognition force microscopy (Fig. 1, next page) [2]. Tip-bound PEG with an NTA terminus proved useful to “snatch” a His6 -tagged probe (the ectodomain of a virus receptor) to study the interaction with the ligand sites on immobilized viruses. The avidin-biotin interaction was utilized for methodical purposes (Fig. 2, next page). Attachment of biotin-PEG-COOH to aminofunctionalized tips and adsorption of avidin to mica provided for easy model experiments with clear-cut controls that allowed developing new modes of recognition imaging in dynamic force microscopy [3].

Use of thiazolidine-mediated ligation for site specific biotinylation of mouse EGF for biosensor immobilisation

Biosensors provide a sophisticated and discriminating means of probing biomolecular interactions. Specific ligands such as peptides and proteins can be immobilized onto sensor surfaces by a number of means including covalent attachment via amine, thiol or aldehyde chemistry, capture via biotin-avidin interaction or the use of specific tags. We have devised a simple chemoselective ligation method to selectively conjugate an anchoring functionality onto N-terminal serine or threonine residues of peptides and proteins allowing them to be immobilised onto the sensor surface in a defined orientation. It is based on the specific reaction of the 1,2-aminothiol of cysteine with an aldehyde under acidic conditions to form a stable thiazolidine product. The carbonyl precursors are derived from the 1,2-aminoalcohols of Ser or Thr that can be selectively and rapidly converted to the aldehyde form by periodate oxidation. Biotinylation of the aldehyde is then achieved via simple conjugation with a novel water-soluble dipeptide that contains a lysine residue bearing an Ne-cysteine-derived 1,2-aminothiol and an Nα-biotin moiety. Use of this method allowed selective biotinylation of a native form of murine EGF (mEGF2-53) that has an N-terminal serine residue. This derivative was then immobilised onto a streptavidin biosensor surface, and the resultant surface activity compared with those obtained by immobilising recombinant human EGF or the soluble extracellular domain of the EGF receptor (sEGFR1-621) using amine coupling (NHS/EDC) chemistry. The surface recognised the recombinant sEGFR with a similar K D to that of human EGF immobilised using NHS/EDC chemistry, or if the receptor was immobilised and murine EGF injected.

Bilayer lipid membrane glucose biosensors with improved stability and sensitivity

Bilayer lipid membranes (BLMs) can be successfully formed on polymers electrodeposited on a solid metallic support. Avidin–biotin interactions were employed for immobilisation of the enzyme on the surface of the BLM. Improved long-term stability and better selectivity towards certain interfering electroactive species were observed for a BLM glucose biosensor based on glucose oxidase immobilised on a platinum support modified with any of several polymers. However, the best results were obtained for the mediated system in which the BLM was formed on a Pt support covered with a layer of evaporated Nafion with incorporated ferrocene. The stable and sensitive response with significant elimination of the influence of electroactive interferences on the signal magnitude should allow a practical application of such miniaturised biosensors.

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic β-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin–biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.

Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain.

A fusion protein, consisting of biotin carboxyl carrier protein (BCCP) domain from Escherichia coli and firefly luciferase (FL) from Photinus pyralis, was immobilized through the biotin-avidin interaction on 6% cross-linked agarose beads. Several properties of the immobilized BCCP-FL were studied. Immobilized and free enzymes showed no significant difference in thermal stability; both retained at least 91% activity after incubation at 4 degrees C and 25 degrees C for 22 h. Incubation at 37 degrees C for 22 h caused significant activity loss. K(M) and k(cat) values were determined for both free and immobilized enzymes. K(M) values were similar between free and immobilized enzymes; however, k(cat) of immobilized BCCP-FL was one-third of the k(cat) of the free enzyme. 294 micromol/L Co-enzyme A (CoA) and 44 mmol/L dithiothreitol (DTT) enhanced the total bioluminescence output. Triton X-100, Tween 20, PEG 8,000, PVP 40,000 and PVP 360,000 did not enhance the bioluminescence reaction of immobilized BCCP-FL.

Bioadhesive Microdevices for Drug Delivery: A Feasibility Study

A variety of delivery systems have been devised to improve the oral bioavailability of drugs including enterically coated tablets, capsules, particles, liposomes, and others. Microfabrication technology, however, may offer some potential advantages over conventional drug delivery technologies. This technology, combined with appropriate surface chemistry, may permit the highly localized and unidirectional release of drugs, permeation enhancers, and/or promoters. In this study, we demonstrate the fabrication of prototype reservoir containing microparticles and a surface chemistry protocol that can be used to bind lectin via avidin-biotin interactions to silicon microparticles. In vitro studies show enhanced bioadhesion of these lectin conjugated microparticles. Such an approach can be used to improve the absorption of pharmacologically active biopolymers such as peptides, proteins and oligonucleotides into circulation at targeted sights in the GI system. Moreover, the use of microfabrication allows one to tailor the size, shape, reservoir volume, and surface characteristics of the drug delivery vehicle.

Comparison of Two Automated Adrenocorticotropic Hormone Assays

The measurement of adrenocorticotropic hormone (ACTH 1-39; M r 4500) plays a key role in the evaluation of hyper- and hypocortisolism (1)(2)(3), but low ACTH concentrations make accurate measurement challenging. Two automated, nonisotopic ACTH assays have become available in recent years. Because it is usual for rather small series to be assayed for ACTH in a clinical setting and because nonisotopic methods with stable calibration are of great practical interest, we compared these assays. Both assays investigated were solid-phase, sandwich immunoassays that use chemiluminescence for signal generation and are implemented on benchtop, multichannel, random-access analyzers with ready-to-use reagents. The Nichols Advantage ACTH assay (Nichols Institute Diagnostics) uses one acridinium ester-labeled mouse monoclonal antibody that specifically binds to the C-terminal region of ACTH and one biotin-labeled goat polyclonal antibody that binds to the N-terminal region. The sample is incubated for 21 min at 37 °C with both antibodies simultaneously, leading to the formation of a soluble sandwich complex in the presence of ACTH molecules. Streptavidin-coated magnetic particles are then added, and the reaction mixture is incubated for 10 min, during which the sandwich complex binds to the particles by biotin-avidin interaction. The particles are fixed to the wall of the single-use reaction cell magnetically, and unbound, labeled antibody is separated by aspiration and subsequent washing. Finally, two trigger solutions are injected into the reaction cells to initiate the chemiluminescent reaction, and the light emission is quantified over 2 s by a luminometer. A lot-specific master calibration curve is loaded by barcode and adjusted weekly with two calibrators by the user. The reported measuring range extends to 1500 ng/L. Barcode-labeled sample tubes are processed directly. The sample throughput is ∼90/h, and the time to first result is 37 min with a start-up time of ∼15 min. In the …

Affinity Biosensor for Avidin Using a Double Functionalized Dendrimer Monolayer on a Gold Electrode

We have developed an affinity biosensor system based on avidin–biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 × 10−12 mol/cm2 on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.

Experimental determination of phase velocity of perfluorocarbons: Applications to targeted contrast agents

Targeted acoustic contrast agents are designed to enhance the sensitivity and specificity of ultrasonic diagnoses. We have previously developed a ligand targeted ultrasonic contrast system that is a lipid-encapsulated, liquid-perfluorocarbon emulsion. The emulsion particles are small (250 nm) and have inherently low echogenicity unless bound to a surface by a pretargeted ligand through avidin-biotin interactions. We have recently proposed a simple acoustic transmission line model that treats the emulsion particles as a thin layer over the targeted surface. In this model, the acoustic reflectivity of the sample increases for perfluorocarbons with smaller velocities of longitudinal sound or lower densities. In this study, we measure and report the velocity of longitudinal sound for 20 perfluorocarbons using a broadband phase spectroscopic approach for estimating phase velocities. Experimentally determined velocities ranged from 520+/-2 m/sec (perfluorohexane) to 705+/-5 m/s (perfluorodecalin). No measurable dispersion was observed over the useful bandwidth of 2 to 22 MHz. Increasing carbon backbone chain length and fluorine substitution with halogens of greater atomic weight increased the measured speed of sound. Our experimental data were consistent (R=0.87) with a published empirical model that predicts velocity as a function of molecular structure. These data provide a rational basis for optimizing targeted perfluorocarbon-based contrast agents and offer further insight into the physical mechanisms responsible for the observed enhancement of surface acoustic reflectivity.

Piezoelectric Quartz Crystal Biosensors: Recent Immobilisation Schemes

ABSTRACT A Mini Review on recent immobilisation techniques for the realisation of piezoelectric biosensors is presented. The reactions considered are affinity reactions such as DNA hybridisation and antigen/antibody systems. The immobilisation procedures considered are based on avidin-biotin interaction, adsorption and self-assembled monolayers.

Printing patterns of biospecifically-adsorbed protein

The advancement of elastomeric patterning techniques in recent years has significantly enhanced our ability to spatially control biomaterial surface chemistry at the micrometre level. The application of this technology to the patterning of biomolecules onto solid surfaces has created many potential applications including the development of advanced biosensors, combinatorial library screening and the formation of tissue engineering templates. In this paper, we describe the direct patterning of protein by microcontact printing. An important consideration for the fabrication of protein micropatterns intended for these applications is the nature of the protein immobilization to a substrate. To date, the patterning of proteins by direct microcontact printing (μCP) has relied on the non-covalent adsorption to a substrate. Ideally, the proteins need to be firmly anchored onto a surface without adversely effecting their activity. Here, the high affinity avidin-biotin receptor-ligand interaction has been exploited to form arrays of avidin molecules onto a polymeric substrate expressing biotin moieties. This has created a generic technique by which any biotinylated species can be subsequently immobilized into defined patterns. Utilizing atomic force microscopy (AFM), the patterned surfaces have been characterized to molecular resolution. The micropatterned sample supported cell adhesion when biotin-(G)11-GRGDS was bound to the avidin bearing arrays.

Nanocomposites of colloidal gold particles and fatty acids formed by the high-affinity biotin–avidin interaction

Download options Please wait... Article information DOI https://doi.org/10.1039/B000182L Article type Paper Submitted 06 Jan 2000 Accepted 28 Mar 2000 First published 03 May 2000 Download Citation Phys. Chem. Chem. Phys., 2000,2, 2461-2466 BibTex EndNote MEDLINE ProCite ReferenceManager RefWorks RIS Permissions Request permissions Nanocomposites of colloidal gold particles and fatty acids formed by the high-affinity biotin–avidin interaction N. Lala and M. Sastry, Phys. Chem. Chem. Phys., 2000, 2, 2461 DOI: 10.1039/B000182L To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page. Read more about how to correctly acknowledge RSC content. Social activity Tweet Share Search articles by author Neeta Lala Murali Sastry

A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons.

We have prepared a novel optical fiber evanescent wave DNA biosensor using a newly developed molecular beacon DNA probe. The molecular beacons (MB) are oligonucleotide probes that become fluorescent upon hybridization with target DNA/RNA molecules. Biotinylated MBs have been designed and immobilized on an optical fiber core surface via biotin-avidin or biotin-streptavidin interactions. The DNA sensor based on a MB does not need labeled analyte or intercalation reagents. It can be used to directly detect, in real-time, target DNA/RNA molecules without using competitive assays. The sensor is rapid, stable, highly selective, and reproducible. We have studied the hybridization kinetics of the immobilized MB by changing the ionic strength of the hybridization solution and target DNA concentration. Our result shows divalent cations play a more important role than monovalent cations in stabilizing the MB stem hybrids and in accelerating the hybridization reaction with target DNA/RNA molecules. The concentration detection limit of the MB evanescent wave biosensor is 1.1 nM. The MB DNA biosensor has been applied to the analysis of specific gamma-actin mRNA sequences amplified by polymerase chain reaction.

Electrochemical Detection of Biotin Using an Interaction between Avidin and Biotin Labeled with Ferrocene at a Perfluorosulfonated Ionomer Modified Electrode

The electrochemical behavior of biotin labeled with ferrocene was investigated at a perfluorosulfonated ionomer (Nafion®) film-coated glassy carbon electrode (GCE). The electrode response of the labeled biotin (LB) was improved by about fifty-fold in sensitivity by using a Nafion®-coated GCE, compared with a plain GCE. It was found by a solid-phase avidin–biotin binding assay that the labeling with ferrocene hardly affected the affinity of a biotin moiety in the labeled biotin to avidin. The avidin-biotin interaction was estimated from the change of the electrode response caused by the formation of a complex with avidin and the labeled biotin. Furthermore, the detection of biotin was performed by a competitive reaction between the labeled biotin and unlabeled biotin for the limited binding sites of avidin.

Rapid Detection of Escherichia coli O157:H7 in Ground Beef Using a Fiber-Optic Biosensor

A portable fiber-optic biosensor was used to detect Escherichia coli O157:H7 in seeded ground beef samples. The principle of the system is a sandwich immunoassay using cyanine 5 dye-labeled polyclonal anti-E. coli O157:H7 antibodies for generation of a specific fluorescent signal. Signal acquisition is effected by launching light from a 635-nm diode laser into a dual tapered 600-μm silica fiber. Fluorescent molecules within approximately 100nm of the fiber surface are excited by the evanescent field, and a portion of the emission recouples into the fiber. A photodiode allows for quantitation of the collected emission light at wavelengths of 670 to 710 nm. Biotin–avidin interactions are used to attach polyclonal antibodies specific for E. coli O157:H7 to the final 7.5 cm of the fiber probe. The biosensor was able to detect E. coli O157:H7 to 3 to 30 CFU/ml in seeded ground beef samples. The reaction was highly specific. Signals with Listeria monocytogenes, Salmonella Typhimurium, or E. coli nonO157:H7 were 2 to 3% of those observed with a similar concentration of E. coli O157:H7. Assays were conducted at or near real-time with results obtained within 20 min of sampling.