Measurement of conformational changes in the structure of transglutaminase on binding calcium ions using optical evanescent dual polarisation interferometry.

The conformational changes occurring when the protein transglutaminase binds calcium ions have been studied using the optical evanescent technique of dual polarization interferometry (DPI) implemented via a dual slab waveguide structure. Immobilized transglutaminase layers of 4-5 nm in thickness were obtained, which when challenged with calcium ions underwent a contraction of approximately 0.5 nm (depending on the concentration of calcium) and an increase in refractive index of approximately 1 x 10-2. The affinity constant for the calcium binding was found to be in the range of 0.95 +/- 0.2 mM. The results reported are in good agreement with those found in the literature obtained by other techniques. It has also been shown that the structural changes occurring during the binding event are considerably larger than the mass changes that take place; thus, DPI offers a potentially valuable method to study real-time structural changes occurring to proteins when they bind metal ions.

Detection of hormone mimics in water using a miniturised SPR sensor.

The ubiquitous presence of chemicals, both natural and synthetic, in the environment with the potential to mimic hormones that may in turn interfere with the endocrine system in both wildlife and humans has in the last decade become a major international concern. Hormone mimics or endocrine disrupting compounds (EDCs) are especially prevalent in surface and waste-waters and therefore, there is a need for an at-source or at-line analytical device for the monitoring of EDC levels. We have incorporated a miniature integrated surface plasmon resonance (SPR) liquid sensor from Texas Instruments into a field analyser and developed a competition/inhibition assay for a model estrogenic compound in aqueous samples. The analyser has the potential for in situ and semi-continuous analysis of EDCs. A novel regeneration scheme employing the use of a domestic laundry detergent has been used to remove immobilised assay components between each assay cycle. The resultant re-usable sensor has been demonstrated using estrone-3-glucuronide (E3G) as a model EDC and an anti-E3G antibody producing a current detection range of 10 to 150 ng mL(-1).

Photo-modulation of horseradish peroxidase activity via covalent attachment of carboxylated-spiropyran dyes.

The photo-modulation of the enzyme horseradish peroxidase modified with photochromic spiropyran dyes is reported. The degree of photo-modulation, greater than 90% reduction in enzyme activity under visible compared to UV illumination, matches the greatest degree of photo-modulation previously reported in the literature. The observation that only a small fraction of the conjugated photochromic molecules are photo-switched suggests that the dyes influence the enzyme activity via local interaction with the enzyme active site.

Covalent coupling of immunoglobulin G to a poly(vinyl)alcohol-poly(acrylic acid) graft polymer as a method for fabricating the interfacial-recognition layer of a surface plasmon resonance immunosensor.

The synthesis of a terminally thiolated poly(vinyl)alcohol (PVA) grafted with Poly (acrylic acid) (PAA) side chains is described. The PVA-PAA graft polymer (PVAg) was end-tethered to silver surfaces via the terminal thiol functionality and the resultant mobile, hydrophilic polymer matrix exploited for the covalent immobilization of large quantities of polyclonal goat (anti-hIgG) antibody (IgG) with low levels of non-specific adsorption. An SPR immunosensor, fabricated with an IgG-PVA-silver interfacial layer proved capable of performing a sensitive label-free assay of human IgG antigen (hIgG) with minimal non-specific binding interference. A detection limit (DL) for hIgG from serum of 0.8 microgram/ml (5 nM) and an assay sensitivity of 0.66 ng hIgG/mm2/nM are reported.

Covalent coupling of immunoglobulin G to self-assembled monolayers as a method for immobilizing the interfacial-recognition layer of a surface plasmon resonance immunosensor.

Protocols have been developed for the random and site-directed covalent coupling of immunoglobulin G [anti-hIgG] [IgG] to silver surfaces modified with a self-assembled monolayer [SAM] of thioctic acid, mercaptopropionic acid [MPA], L-cysteine or 4-aminothiophenol [PATP]. A surface plasmon resonance [SPR] immunosensor fabricated with a more ordered and hydrophilic IgG-SAM-silver interfacial layer, demonstrates an increased ability for performing sensitive and selective assay of human immunoglobulin G [hIgG] compared with a device fabricated with a physically-adsorbed IgG-silver interfacial-layer due to reduced levels of non-specific binding. Detection limits [DL] for hIgG from serum down to 6 micrograms/ml (40 nM) and assay sensitivities up to 0.24 ng hIgG/mm2/nM are reported.

Physical adsorption of immunoglobulin G on gold studied by scanning tunnelling microscopy.

This paper describes a preliminary scanning tunnelling microscope (STM) study of the physical adsorption of a mouse monoclonal anti-hCG (human chorionic gonadotrophin) immunoglobulin G (IgG) on vacuum-evaporated gold surfaces in air. Ellipsoid-like protrusions of various geometric sizes assembled in different surface patterns were observed on scanning adsorbed IgG molecules. It is believed that both the adsorption process per se and the tip-molecule interactions are key factors which determine the topographical features of adsorbed IgG molecules.

Biosensors.

This review introduces biosensors as analytical devices that respond selectively to analytes in appropriate samples and convert their concentrations into electrical signals via a combination of a biological recognition system and a suitable transducer. The last decade has seen dramatic advances in the design of sensor configurations, the marriage of biological systems with modern monolithic silicon and optical technologies, the development of effective electron-exchange systems and the introduction of direct immunosensors.

Detection of immuno-complex formation via surface plasmon resonance on gold-coated diffraction gratings.

The sensitivity of surface plasmon resonance techniques to changes in local interfacial refractive index has been exploited to detect immuno-complex formation in two model biochemical systems. A gold-coated diffraction grating has been used to excite surface plasmons at the gold/solution interface to which either human immunoglobulin G or the immunoglobulin fraction of sheep antiserum to human serum albumin was physically adsorbed. The complementary proteins, either affinity purified goat anti-human-IgG IgG or human serum albumin was subsequently specifically bound by immuno-complex formation. The binding reactions could be followed with respect to time.

A microelectronic conductimetric biosensor.

The fabrication and operation of a microelectronic conductimetric biosensor is described. The device monitors the change in solution conductance occasioned by the catalytic action of enzymes immobilised over a planar conductance cell comprising serpentined and interdigitated metal conductor tracks. The output of the instrument was linear over a 3 min period on addition of urea to a sample cell overlaid with immobilised urease. The responses to any given urea concentration were reproducible to within approximately +/- 1%. The device responds to urea present in serum samples.