Holographic detection of hydrocarbon gases and other volatile organic compounds.

There is a need to develop sensors for real-time monitoring of volatile organic compounds (VOCs) and hydrocarbon gases in both external and indoor environments, since these compounds are of growing concern in human health and welfare. Current measurement technology for VOCs requires sophisticated equipment and lacks the prospect for rapid real-time monitoring. Holographic sensors can give a direct reading of the analyte concentration as a color change. We report a technique for recording holographic sensors by laser ablation of silver particles formed in situ by diffusion. This technique allows a readily available hydrophobic silicone elastomer to be transformed into an effective sensor for hydrocarbon gases and other volatile compounds. The intermolecular interactions present between the polymer and molecules are used to predict the sensor performance. The hydrophobicity of this material allows the sensor to operate without interference from water and other atmospheric gases and thus makes the sensor suitable for biomedical, industrial, or environmental analysis.

Holographic sensors for the detection of bacterial spores.

Holographic sensors for the detection of Bacillus species spore germination and vegetative growth are described. Reflection holograms were fabricated using a diffusion method for the distribution of ultra-fine silver bromide grains into pre-formed polymer films, followed by holographic recording using a frequency doubled Nd:YAG (532 nm) laser. Changes in holographic replay wavelength or diffraction intensity were used to characterise the swelling behaviour or structural integrity of a range of holographic matrices in response to various extracellular products of bacterial spore germination and vegetative metabolism. Divalent metal ion-sensitive holograms containing a methacrylated analogue of nitrilotriacetic acid (NTA) as the chelating monomer were successfully used to monitor Ca2+ ions released during B. subtilis spore germination in real-time, which was within minutes of sample addition; the holographic response manifested as a 16 nm blue-shift in diffraction wavelength over the progress of germination. Similarly, pH-sensitive holograms comprising methacrylic acid (MAA) as the ionisable monomer were responsive to changes in pH associated with early vegetative metabolism following germination of B. megaterium spores; a visually perceptible blue-shift in holographic replay wavelength of 75 nm was observed. Casein and starch-based holographic matrices, prepared by co-polymerisation of the appropriate substrate with acrylamide, were used to detect exo-enzymes released during later stages of B. megaterium and B. subtilis vegetative cell growth; holographic responses of both matrices were visible as a reduction in diffraction intensity due to progressive fringe disruption caused by enzymatic cleavage. The combined monitoring of various germination and growth events using the range of aforementioned holographic sensors provides a novel, comprehensive means for the detection of viable bacterial spores.

Nanobiotechnology: the fabrication and applications of chemical and biological nanostructures.

Biology can teach the physical world of electronics, computing, materials science and manufacturing how to assemble complex functional devices and systems that operate at the molecular level. Our present capability to fabricate simple molecular tools, devices, materials and machines is primitive compared with the sophistication of nature. Nevertheless, the nanomanufacturing of 'biomimetic' devices is moving ahead strongly. Recent developments have been made in the use of biological systems in molecular self-assembly, spatial positioning, microconstruction, biocomposite fabrication, nanomachines and biocomputing.

Design, synthesis, and application of a protein A mimetic.

Low-molecular-weight synthetic molecules that mimic the activity of native biological macromolecules have therapeutic potential, utility in large-scale production of biopharmaceuticals, and the capacity to act as probes to study molecular recognition events. We have developed a nonpeptidyl mimic for Staphylococcus aureus Protein A (SpA). The specific recognition and complexation elements between the B domain (Fb) of SpA and the Fc fragment of IgG were identified from the x-ray crystallographic structure. Computer-aided molecular modeling was used to design a series of biomimetic molecules around the Phe132-Tyr133 dipeptide involved in its binding to IgG. One of the ligands binds IgG competitively with SpA in solution and when immobilized on agarose beads, with an affinity constant of 10(5)-10(6) M-1. The immobilized artificial Protein A was used to purify IgG from human plasma and murine IgG from ascites fluid, and to remove bovine IgG from fetal calf serum.

Maternal age and birth rank of women with breast cancer.

Data from a large international case-control study of breast cancer suggested that women born to young mothers had a 25% lower risk of breast cancer. The association was not secondary to a tendency for these women themselves to have had children at early ages. The data provided no indication of a meaningful association between breast cancer risk and birth rank. Confounding was controlled by stratification according to a summary confounder score.

Affinity chromatography on immobilised triazine dyes. Studies on the interaction with multinucleotide-dependent enzymes.

A systematic investigation into the interaction of several triazinyl dyes with two enzymes from purine metabolism, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14( and adenylosuccinate synthetase (IMP: L-aspartate ligase (GDP-forming), EC 6.3.4.4) has been conducted. Evidence from kinetic inhibition studies, enzyme inactivation with specific affinity labels and specific elution techniques from agarose-immobilised dyes indicate that triazine dyes such as Procion Blue H-B (Cibacron Blue F3G-A), Red HE-3B and Red H-3B are able to differentiate between the nucleotide-binding sites of these enzymes. This information has been exploited to design specific elution techniques for the purification of these enzymes by affinity chromatography.

Metal ion-promoted binding of proteins to immobilized triazine dye affinity adsorbents.

Low concentrations of metal ions, particularly those of the first row transition series such as Zn2+, Co2+, Mn2+, Ni2+, Cu2+, and, to a lesser extent, the group IIA ions, Ca2+ and Mg2+, promotes binding of carboxypeptidase G2, alkaline phosphatase and yeast hexokinase to immobilized Procion Red H-8BN, Procion Yellow H-A and Cibacron Blue F3G-A respectively. The binding of ovalbumin to immobilized Cibacron Blue F3G-A and Procion Orange MX-G is selectively enhanced in the presence of AI3+. With ovalbumin and alkaline phosphatase, the effect is almost totally specific for both the metal ion and dye, whereas with carboxypeptidase G2 and hexokinase, metal ions such as Co2+, Ni2+, Mn2+, Cu2+, Ca2+ and Mg2+ also promote binding to varying degrees. Almost all other monovalent and trivalent metal ions appear to be ineffective. Metal ion-bound enzymes can subsequently be eluted with appropriate chelating agents of the amine, aminocarboxylate or substituted pyridine classes.

Combinatorial approaches to affinity chromatography.

A new armoury of protein purification tools is required to support rapid advances in high-throughput genomics and proteomics, which are predicted to lead to the discovery, isolation, characterisation and manufacture of a number of new biopharmaceutical proteins. Computer-aided molecular design, combinatorial (bio)chemistry and high-throughput screening techniques are now being exploited to identify highly selective ligands for use in the purification of these proteins by affinity chromatography.

Chemoselective biosensors.

New opportunities for biosensors are now appearing in clinical and genetic diagnostics, genomics, environmental protection, food processing and safety, drug discovery and bioprocess monitoring. Concerns about the cost, stability and selectivity of previous sensor technologies are being addressed by developing new recognition systems and their integration into transducers, micro- and nanofabricated devices, array technologies and novel magnetic, acoustic and optical transduction systems.

Planar coil excitation of multifrequency shear wave transducers.

A transducer format that replaces the electrode of an acoustic resonator with a planar spiral coil is used to extract multifrequency spectral information from adsorbed protein films. Both amorphous silica and crystalline piezoelectric resonators are driven to resonance by forces induced across an air gap by magnetic direct generation and piezoelectric excitation induced by the electromagnetic field of the coil. Inspection of the harmonic frequencies between 6 MHz and 0.6 GHz indicates that the response of these two resonator types is described by different families of shear acoustic standing waves, with similar acoustic features to the quartz crystal microbalance. Exposure of the devices to protein solutions results in reproducible shifts of their harmonic frequencies, up to a maximum of 15 kHz and increasing linearly with frequency and operating mode. The gradient, determined from the ratio of the frequency change to the operating frequency was determined as 21.5 x 10(-6) for the quartz device and 60.9 x 10(-6) for the silica device. Consistency with the Sauerbrey equation for the piezoelectric linear shear mode was comparable at a predicted value of 22.5 x 10(-6), but not for the radial shear mode of the silica device at 12.7 x 10(-6). Opportunities resulting from the wide bandwidth of the planar coil excitation and choice of acoustic mode are discussed with respect to acoustic fingerprinting of adsorbed proteins.

Immunosensors.

Immunosensors are important analytical tools for monitoring antibody-antigen reactions in real time. Recent developments in immunosensors have produced systems that allow rapid and continuous analysis of the binding event without the requirement for added reagents or separation/washing steps. As a result, great interest has focused on commercializing immunosensors for applications in areas such as clinical, environmental and food analysis.

Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography.

Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.

Mortality from ischaemic heart disease--inter-town variation and its association with climate in England and Wales.

In both seasonal and inter-town variation, the mortality from ischaemic heart disease in England and Wales in 1969-71 is very highly correlated with temperature. Also in the inter-town variation, IHD mortality is highly correlated with rainfall and with socio-economic index. It is postulated that the same mechanism of body cooling underlies seasonal, inter-town and socio-economic variations in IHD mortality. The first law of thermodynamics states the heat and energy are equivalent. Therefore to maintain body temperature against an increased thermal gradient (caused by a lower environmental temperature as in seasonal variation, or a temperature-rainfall interaction as in inter-town variation) more energy must be expended by the body or, alternatively, the body must reduce heat loss by increasing thermal insulation. It is to be expected that there will be many accompanying changes in physiological parameters and several of these are discussed.

Purification and characterisation of an NAD(+)-dependent secondary alcohol dehydrogenase from Pseudomonas maltophilia MB11L.

A constitutive NAD(+)-linked alcohol dehydrogenase was purified 338-fold from cells of Pseudomonas maltophilia MB11L grown on glucose. Maximum activity was observed with cyclic and linear secondary alcohols, with little activity seen against primary or aromatic alcohols. Substrate oxidation activity was maximal at pH 10.0, while substrate reduction was optimal at pH 4.5. The Km values for propan-2-ol, NAD+ and acetone were 87, 413 and 143 microM respectively. The enzyme is a tetramer with subunit Mr of approximately 44,000. It has an isoelectric point of 4.75, and was inhibited by chelating agents, thiol reagents and certain metal ions.

Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200.

The structural gene, adhA, for a thermostable primary alcohol dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. Constitutive expression from its own promoter was observed in Escherichia coli. The nucleotide sequence of adhA corresponded to an open reading frame of 1197 bp, encoding a polypeptide of 399 amino acids with a calculated Mr of 43 192. Amino acid sequence analysis showed 67-69% identity with alcohol dehydrogenases from two archaeal species and 29-37% identity with bacterial type III alcohol dehydrogenases. This represents the first reported cloning of an alcohol dehydrogenase from a bacterial species that is both thermostable and active against primary long-chain alcohols.

A single mode fibre-optic evanescent wave biosensor.

This paper reports experimental developments in the construction and operation of a single-mode fibre-optic evanescent wave biosensor using an exposed core silica single-mode fibre embedded in a silica block. The device was able to monitor the concentration of a blue dye, Procion Blue MX-G, in overlayers of various refractive indices. The practicality of such a biosensor has been demonstrated with a colorimetric enzyme assay system. Penicillin G in the 0-0.4 mM concentration range was monitored at 633 nm by the decoloration of the starch-iodine reagent when Bacillus cereus penicillinase was immobilized over the exposed core of the monomode fibre.

An optical biosensor for monitoring recombinant proteins in process media.

This paper describes the construction of a sensor for the direct monitoring of a recombinant protein, the human insulin analogue (MI3). The surface plasmon resonance (SPR) sensor incorporates an immobilised, sterilisable affinity-ligand that has been designed to bind to MI3. In practice, gold SPR devices were fabricated with; a 2D assembly of ethanethiol-modified ligand, a 2D mixed-assembly of ethanethiol-modified ligand and mercaptoethanol, a 3D coating of ligand-modified terminal-thiolated poly(vinyl)alcohol (PVA) or a 3D hydrogel of dextran coupled to a self-assembled monolayer (SAM) of mercaptohexaneundecanl-ol. Routine measurement of the concentration MI3 in the concentration range 1-100 mg/l in pilot-scale samples of crude fermentation broth have been achieved with high sensitivity levels and a high signal-to-noise ratio. Analysis can be achieved within < 10 min with the active surface being regenerable for at least 60 cycles over a 6 month period. The coupling of a robust, sterilisable and highly-selective sensor-coating with suitable transducer technologies promises to deliver sensors that are capable of direct in situ monitoring of biopharmaceuticals in industrial bioprocesses.

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.

An amperometric opiate assay.

Current techniques for the detection and measurement of diacetylmorphine (heroin), morphine and their principal metabolite morphine-3-glucuronide (M3G) are based mainly on chromatography or immunoassay. No enzymatic method for the detection of these compounds has yet been reported. Two novel microbial enzymes have been isolated and characterized in this laboratory: an acetylmorphine carboxyesterase (heroin esterase) and a morphine dehydrogenase (MDH). These highly specific enzymes have been incorporated in an amperometric assay for heroin and morphine using phenazine methosulphate as a mediator. The assay gives a rapid and sensitive response to heroin and morphine, with a detection limit for morphine of 6.8 micrograms ml-1 (23.7 microM).

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.