Biomedical subjects

C R Lowe

Publications and source records attributed to C R Lowe.

At least 19 recordsLinked to original sources

PubMed2006

Wireless excitation of quartz crystals immersed in an aqueous fluid.

A novel toroidal coil geometry able to induce remote acoustic waves in quartz crystals has been evaluated for the development of (bio)sensors. Remote acoustic generation in air was obtained for two alternative toroidal coils, with corresponding electrical impedance changes of 40 Omega for a PDMS- and 140 Omega for a ferrite-supported toroid respectively. It was found that the range of remote acoustic generation relative to the spiral coil standard was much improved, increasing the axial separation of their resonant sensing element from 0.1 mm to 20 mm, thereby allowing electromagnetic wave penetration across glass walls and fluid media to be utilised. Consideration of the transduction mechanism, along with measured cyclic changes in acoustic signal as a function of rotation, indicated that the large PDMS toroidal coil produced an asymmetric electric field. It was shown for the first time that a quartz crystal blank fully immersed in an aqueous fluid could support chemically sensitive shear acoustic standing waves that were excited and detected remotely. A signal to noise ratio of 30 ratio 1 at 20.13 MHz was achieved by placing a ferrite supported toroidal coil on the lower side of a glass beaker containing a 12 x 0.25 mm AT crystal blank and 1 mL of water. This discovery allows wireless shear acoustic wave measurements to be performed with total separation between the electronic detection system and assays undertaken in fluidic systems.

Acoustics↗

PubMed2005

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.

Electronics↗

PubMed2003

The acoustic spectrophonometer: a novel bioanalytical technique based on multifrequency acoustic devices.

A measurement technique similar to optical absorption spectroscopy but based on evanescent acoustic waves is described in this paper. This format employs a planar spiral coil to vibrate a single crystal of quartz from 6 to 400 MHz, in order to measure multifrequency acoustic spectra. Consistency with the defined Sauerbrey and Kanazawa terms K1 and K2 when applied to multiple frequencies was found for these specific operating conditions in terms of a significant fit between the measured and calculated values: For an IgG surface density of 13.5 ng mm(-2) the measured value of K1 is 22.5 x 10(-6) and the calculated value is 20.4 x 10(-6), whilst for glycerol viscous loadings of 5.131 cP the measured value of K2 is 0.47 and the calculated value is 0.54. Thus for these specific surface loadings the multifrequency data fits to the predictions of the Sauerbrey model to within 10% and to Kanazawa model within 13%. However collective frequency shifts for 5.131 cP solutions of sucrose, dextran and glucose were found to exhibit an unanticipated additional variability (R2 < 0.4) with frequency, but retained a square root of frequency dependency within a factor 2 of the interpolated K2 values. The response to the 5.131 cP dextran solution was found to be significantly below the other isoviscous solutions, with a substantially reduced frequency shift and K2 value than would be expected from its bulk viscosity. In comparison with these viscous solutions, IgG protein films consistently produced linear frequency shifts with little scatter (R2 > 0.96) that were proportional to the operating frequency, and fully consistent with the Sauerbrey model under these specific conditions. A t-test value of 14.52 was calculated from the variance and mean of the two groups, and demonstrates that the acoustic spectrophonometer can be used to distinguish between the acoustic impedance characteristics of two chemical systems that are not clearly differentiable at a single operating frequency.

Acoustics↗

PubMed2003

Hypersonic evanescent waves generated with a planar spiral coil.

A planar spiral coil has been used to induce hypersonic evanescent waves in a quartz substrate with the unique ability to focus the acoustic wave down onto the chemical recognition layer. These special sensing conditions were achieved by investigating the application of a radio frequency current to a coaxial waveguide and spiral coil, so that wideband repeating electrical resonance conditions could be established over the MHz to GHz frequency range. At a selected operating frequency of 1.09 GHz, the evanescent wave depth of a quartz crystal hypersonic resonance is reduced to 17 nm, minimising unwanted coupling to the bulk fluid. Verification of the validity of the hypersonic resonance was carried out by characterising the system electrically and acoustically: Impedance calculations of the combined coil and coaxial waveguide demonstrated an excellent fit to the measured data, although above 400 MHz a transition zone was identified where unwanted impedance is parasitic of the coil influence efficiency, so the signal-to-noise ratio is reduced from 3000 to 300. Acoustic quartz crystal resonances at intervals of precisely 13.2138 MHz spacing, from the 6.6 MHz ultrasonic range and onto the desired hypersonic range above 1 GHz, were incrementally detected. Q factor measurements demonstrated that reductions in energy lost from the resonator to the fluid interface were consistent with the anticipated shrinkage of the evanescent wave with increasing operating frequency. Amplitude and frequency reduction in contact with a glucose solution was demonstrated at 1.09 GHz. The complex physical conditions arising at the solid-liquid interface under hypersonic entrainment are discussed with respect to acceleration induced slippage, rupture, longitudinal and shear radiation and multiphase relaxation affects.

Biophysical Phenomena↗

PubMed2001

New developments in affinity chromatography with potential application in the production of biopharmaceuticals.

Affinity chromatography is likely to play an increasingly important role in the purification of pharmaceutical proteins. This review describes new approaches to the design and synthesis of affinity ligands based on the ability to combine knowledge of X-ray crystallographic or NMR structures with defined or combinatorial chemical synthesis. The de novo design process is based on peptidal templates, complementarity to surface exposed residues and mimicking natural biological recognition. Examples of ligands designed to bind specifically to kallikrein, elastase, immunoglobulin G, insulin, alpha(1)-antitrypsin, clotting factor VII and glyco-proteins are given. The exceptional selectivity and stability of these synthetic ligands allows their use in harsh manufacturing environments.

Biopharmaceutics↗

PubMed2001

A strategy for chemical sensing based on frequency tunable acoustic devices.

A new acoustic sensor geometry, the magnetic acoustic resonant sensor (MARS), is described. The device comprises a circular 0.5-mm-thick resonant plate fabricated from a wide variety of nonpiezoelectric materials and coated on the underside with a 2.5-microm-thick aluminum film. Harmonic radial shear waves over at least a 2 orders of magnitude frequency range can be induced in the resonant plate by enhanced magnetic direct generation using a noncontacting rf coil and NdFeB magnet. Mass loading with adherent aluminum films produced frequency changes of 106 Hz/nm (40.8 Hz/ng-mm(-2)), while contact with viscous fluids resulted in maximum changes of 15 446 Hz/cP. At an operating frequency of 50 MHz, the device detected viscosity changes as low as 0.0006 cP. The adsorption of proteins such as human IgG and the binding of a complementary antigen, goat anti-human IgG, on the upper nonmetallized surface of the device has been monitored with a detection limit of approximately 75 ng/mL. The binding of substrates and allosteric effectors to glycogen phosphorylase b has provided evidence that the device is very sensitive to viscoelastic changes in adsorbed proteins. The MARS device generates radial shear acoustic waves over a broad bandwidth that are unaffected by the conductivity of the solution. These results suggest that simple metal, glass, crystalline, or polycrystalline plates can be used as a new type of tunable acoustic immunosensor.

Acoustics↗

PubMed2001

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.

Chromatography, Affinity↗

PubMed2000

C-Terminal labeling of immunoglobulin G with a cysteine derivative by carboxypeptidase Y catalyzed transpeptidation.

We describe a new method for the site-specific incorporation of an extrinsic cysteine to the C-termini of immunoglobulin G (IgG) using carboxypeptidase Y (CPase Y) catalyzed transpeptidation. The transpeptidase activity of CPase Y was employed to attach cysteine esters to the C-termini of the IgG molecule (cysteinylation) at alkaline pH. No CPase Y catalyzed transpeptidation products were found when native IgG was used as the substrate or when cysteine was used as the nucleophile. However, C-terminal labeling occurred when cysteine ethyl ester (CysOEt) or cysteine isobutyl ester (CysOiBu) was used as the nucleophile and IgG methyl ester as the substrate. When CysOiBu was used as the nucleophile, the maximal labeling yield obtained with IgG methyl ester as substrate was 25%, assuming all four C-termini in the IgG molecule were labeled equally. The C-terminal labeling pattern of cysteinylated IgG was determined by autoradiography followed by the integration of radiodensity. It revealed that both the C-termini of the heavy and light chains of IgG methyl ester were labeled with CysOiBu.

Carboxypeptidases↗

PubMed2000

Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin.

A combination of rational design based on mimicking natural protein-carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, 1H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 micromol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M alpha-D-methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for alpha-D-mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of alpha-L-fucose. Interestingly, alpha-L-fucose and alpha-D-mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of D-mannose, D-glucose, L-fucose and D-galactose with immobilised 18/18 establish that the affinity constants (K(AX)) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3x10(5) M(-1), 1.9X10(4) M(-1) and 1.2x10(4) M(-1) respectively. 1H-NMR studies on the interaction of alpha-D-methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of alpha-D-methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.

Adsorption↗

PubMed2000

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.

Alcohol Dehydrogenase↗

PubMed2000

New strategy for the design of ligands for the purification of pharmaceutical proteins by affinity chromatography.

A new approach for the identification of ligands for the purification of pharmaceutical proteins by affinity chromatography is described. The technique involves four steps. Selection of an appropriate site on the target protein, design of a complementary ligand compatible with the three-dimensional structure of the site, construction of a limited solid-phase combinatorial library of near-neighbour ligands and solution synthesis of the hit ligand, immobilisation, optimisation and application of the adsorbent for the purification of the target protein. This strategy is exemplified by the purification of a recombinant human insulin precursor (MI3) from a crude fermentation broth of Saccharomyces cerevisiae.

Amino Acid Sequence↗

PubMed2000

Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.

A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.

Animals↗

PubMed2000

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.

Animals↗

PubMed1999

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.

Biotechnology↗

PubMed1999

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.

Biosensing Techniques↗

PubMed1998

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.

Biosensing Techniques↗

PubMed1998

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.

Acrylic Resins↗

PubMed1998

A holographic sensor based on a rationally designed synthetic polymer.

A new silver halide-containing holographic recording material has been designed and developed specifically for holographic chemical sensors. The hologram enables very small volume changes to be measured in a polymer layer throughout which the hologram is located. The holographic film is based on a fine-grain silver bromide emulsion suspended in a poly(vinyl alcohol) matrix crosslinked with Cr(III) ions. Cross-linking gives the material sufficient spatial integrity to allow a holographic image to be recorded, while maintaining adequate porosity and elasticity of the polymer matrix for sensing applications. The new material has been characterized with respect to its response to pH and compared with a traditional gelatin holographic film. The response to some ions and small molecules typically found in analytical samples has also been measured. Functional groups introduced covalently into the poly(vinyl alcohol) matrix transform the base matrix into a pH-responsive polymer with predictable swelling properties and which can be further derivatized to incorporate specific ligands. A rationally designed holographic sensor for trypsin has been developed from chemically synthesized artificial polymers. A trypsin substrate, the poly(amino acid) poly(L-lysine), was incorporated into poly(vinyl alcohol) holograms to create a 'designed' holographic material which was degraded in a concentration-dependent manner by trypsin. Extensions of this approach to other hydrolytic enzymes are briefly discussed.

Animals↗