Biomolecular Interactions In Real Time Research Articles

Nucleic acid testing using surface plasmon resonance fluorescence detection.

The completion of the Human Genome Project (1) is likely to accelerate the demand for nucleic acid diagnostics, one of the fastest growing segments of in vitro diagnostics. To meet this demand, fully automated, sensitive, and specific technologies for nucleic acid detection are needed. Surface plasmon resonance fluorescence (SPRF) is an optical detection technology that offers kinetic analysis of biomolecular interactions in real time (2). SPRF technology uses a sensor composed of a dielectric material coated with a thin layer of a noble metal. An evanescent field is generated at the metal-dielectric interface through interaction of p -polarized light with surface plasmons (oscillating electrons) under a resonance condition. The evanescent field excites a fluorophore localized near the metal surface, which in turn emits evanescent waves, generating surface plasmons at the fluorescence emission frequency. These propagating waves (plasmon-mediated emission) radiate through the metal film and dielectric material. This emission emerges as a cone of radiation in the dielectric material. Application of SPRF for nucleic acid testing involves immobilization of synthetic oligonucleotide capture probes on the sensor surface (Fig. 1⇓ ). Biotin-tagged nucleic acid amplicons are hybridized to target-specific capture probes and detected with a streptavidin-fluorophore conjugate by monitoring of the plasmon-mediated emission over time. Because the evanescent field penetrates a short distance into the medium above the metal surface, the hybridized amplicons are selectively excited and detected. This feature of SPRF allows sensitive detection without a wash step to separate the free from the hybridized nucleic acids. The fluorescent emission is collected by a patented optical system, providing higher sensitivity than other similar techniques (2). The rate of …

A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real time on a surface.

This paper presents a new label-free optical method to study biomolecular interactions in real time at the surface of an optically transparent substrate. The method relies on the change in the absorbance spectrum of a self-assembled monolayer of colloidal gold on glass, as a function of biomolecular binding to the surface of the immobilized colloids. Using this approach, we demonstrate proof of principle of a label-free optical biosensor to quantify biomolecular interactions in real time on a surface in a commercially available UV-visible spectrophotometer and of a colorimetric end-point assay using an optical scanner. The spectrophotometric sensor shows concentration-dependent binding and a detection limit of 16 nM for streptavidin. The sensor is easy to fabricate, is reproducible in its performance, has minimal technological requirements, namely, the availability of an UV-visible spectrophotometer or an optical scanner, and will enable high-throughput screening of biomolecular interactions in real time in an array-based format.

The use of biosensor technology for the engineering of antibodies and enzymes.

Recently developed scientific instrumentation featuring surface plasmon resonance detection allows the detection of biomolecular interactions in real time and without chemical modification of the binding partners. These biosensors are proving invaluable tools in protein engineering, particularly in research aimed at the isolation and improvement of protein binders and catalysts from macromolecular repertoires containing billions of individual members. This article reviews the use of biosensor technology for the isolation and characterization of engineered antibodies and enzymes.

Instrumental biosensors: new perspectives for the analysis of biomolecular interactions.

The use of instrumental biosensors in basic research to measure biomolecular interactions in real time is increasing exponentially. Applications include protein-protein, protein-peptide, DNA-protein, DNA-DNA, and lipid-protein interactions. Such techniques have been applied to, for example, antibody-antigen, receptor-ligand, signal transduction, and nuclear receptor studies. This review outlines the principles of two of the most commonly used instruments and highlights specific operating parameters that will assist in optimising experimental design, data generation, and analysis.

Surface plasmon resonance: a study of the effect of biotinylation on the selection of antibodies for use in immunoassays

Surface plasmon resonance is a valuable optical phenomenon for monitoring biomolecular interactions in real time. In this project anti-mouse-Fc was coupled to the carboxymethyl dextran coating on the surface of a CM5 sensor chip (BIAcore) using amine coupling. Monoclonal antibodies (MAbs) to Luteinizing Hormone (LH) were then captured on this surface in the correct orientation for binding. LH (500 IU/l) was injected over the surface and the subsequent binding and dissociation events were monitored. The resulting optical response curves allowed fast analysis of the binding interactions of eight selected MAbs. It was possible to develop a two-site immunometric assay for LH using a pair of these MAbs. The effect of biotinylating the MAbs, using various biotin:antibody coupling ratios, on their subsequent binding to both LH and avidin conjugated alkaline phosphatase was also investigated. This approach has allowed rapid evaluation of the effect of changes in both reagent and reaction conditions on immunoassay performance and appears to be a valuable adjunct to immunosensor and immunoassay development.

Biomolecular interaction analysis: affinity biosensor technologies for functional analysis of proteins

The introduction of affinity-based biosensors has permitted label-free functional analysis of biomolecular interactions in real time. A variety of methods are now based on BIACORE ® and IAsys technology and have mainly been used to determine kinetics and affinity constants.