Ammonia-sensitive photonic structures fabricated in Nafion membranes by laser ablation.

Here we report the fabrication and characterization of photonic structures in Nafion membranes sensitive to ammonia in the 0.19%-12.5% concentration range. The photonic structures were recorded by laser ablation of silver nanoparticles synthesized in situ by diffusion. The particles showed an average diameter of 17 nm with a narrow size distribution. After ablation, the nanoparticles generated a diffracting structure giving colorful reflections at defined peak wavelengths. The reflectivity at these wavelengths was directly proportional to concentration after ammonia exposure. The concentration range that can be measured with these membranes encompasses the fatal limit of exposure and the lower flammable limit of gaseous ammonia. Interrogation by reflection spectroscopy makes them suitable for remote sensing and real-time monitoring of gases.

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.

Synthetic affinity ligands as a novel tool to improve protein stability.

Cutinase from Fusarium solani pisi is the model-system for a new approach to assess and enhance protein stability based on the use of synthetic triazine-scaffolded affinity ligands as a novel protein-stabilizing tool. The active site of cutinase is excluded from the main surface regions postulated to be involved in early protein's thermal unfolding events. Hence, these regions are suitable targets for binding complementary affinity ligands with a potential stabilizing effect. A random solid-phase combinatorial library of triazine-bisubstituted molecules was screened for binding cutinase by a rapid fluorescence-based method and affinity chromatography. The best binding substituents were combined with those previously selected by screening a rationally designed library. A second-generation solid-phase biased library was designed and synthesized, following a semi-rational methodology. A dual screening of this library enabled the selection of ligands binding cutinase with higher affinity while retaining its functionality. These compounds were utilized for thermostability assessment with adsorbed cutinase at 60 degrees C and pH 8.0. When bound to different types of ligands, the enzyme showed markedly distinct activity retention profiles, with some synthetic affinity ligands displaying a stabilizing effect on cutinase and others a clearly destabilizing effect, when compared with the free enzyme.

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.

De novo design, synthesis and screening of a combinatorial library of complementary ligands directed towards the surface of cutinase from Fusarium solani pisi.

The protein surface is the interface through which a protein molecule senses the external world. The composition of this interface, in charged, polar and/or hydrophobic residues is crucial for both the activity and stability of the protein. Protein immobilization on surfaces has been extensively explored as one of the most effective approaches for stabilization. The mechanism of stabilization, however, is still poorly understood, and usually the success of any method is more a matter of trial and error rather than the result of rational concepts. The importance of local unfolding processes in a number of biologically significant processes has been recognized and attracted increasing attention. Unfolding regions have been localized in different proteins including the recombinant cutinase from Fusarium solani pisi. The study of three structural surface regions associated with early cutinase unfolding events was the basis for the approach followed in this work. A 64-member solid-phase combinatorial library of ligands was synthesized on a triazine-substituted agarose matrix using a modified 'mix and split' procedure. The combinatorial library was assessed for binding to cutinase from Fusarium solani pisi in a biologically active form. Four lead ligands (3/5, 3/7, 4/5, 4/7) have been selected in which immobilized cutinase presented a relative activity of 30-60% as compared to the free enzyme.

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.

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.

Glucose-sensitive holographic sensors.

Holographic sensors for monitoring glucose were fabricated from hydrogel films containing chemical ligands based on phenylboronic acid. The films were transformed into reflection holograms using a diffusion method coupled with exposure to laser light. The diffraction wavelength of the holograms was used to monitor the swelling of the hydrogel film in the presence of glucose. Fully reversible changes in diffraction wavelength were demonstrated, highlighting the potential for using these holograms as glucose sensors.

The application of the acoustic spectrophonometer to biomolecular spectrometry: a step towards acoustic "fingerprinting".

A tunable acoustic biosensor for investigating the properties of biomolecules at the solid-liquid interfaces is described. In its current, format the device can be tuned to frequencies between 6.5 MHz and 1.1 GHz in order to provide a unique detection feature: a variable evanescent wave thickness at the sensor surface. The key to its successful implementation required the careful selection of antennae designs that could induce shear acoustic waves at the solid-liquid interface. This non-contact format makes it possible to recover resonant shear acoustic waves over 100 different harmonic frequencies as a result of the electrical characteristics of the spiral coil. For testing this multifrequency sensing concept the surface of a quartz disc was exposed to solutions of immunoglobulin G (IgG) to form an adsorbed monolayer, whence protein A and IgG were added again in order to form multilayers. Spectra at frequencies between 6 and 600 MHz were generated for each successive layer and revealed two characteristic phases: an initial phase at the low megahertz frequencies consistent with the conventional Sauerbrey relation, and a possible additional phase towards the high megahertz to gigahertz frequencies, that we believe relates to the structure of the biomolecular film. This two-phase behaviour evident from differences between high and low frequencies, rather than from any distinct frequency transition, was anticipated from the reduction in evanescent wave thickness down to nanometre dimensions, and thin film resonance phenomena that are known to occur for film and fluid systems. These measurements suggested that the single element acoustic biosensor we present here may form the basis from which to generate acoustic molecular spectra, or "acoustic fingerprints", in a manner akin to optical spectroscopy.

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.

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.

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.

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.

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.

Design, synthesis and evaluation of biomimetic affinity ligands for elastases.

A low-molecular-weight biomimetic affinity ligand selective for binding elastase has been designed and synthesized. The ligand was based on mimicking part of the interaction between a natural inhibitor, turkey ovomucoid inhibitor and elastase, and modelled from the X-ray crystallographic structure of the enzyme-inhibitor complex. Limited solid-phase combinatorial chemistry was used to synthesize 12 variants of the lead ligand using the triazine moiety as the scaffold for assembly. The ligand library was screened for its ability to bind elastase and trypsin, and two ligands were studied further. Ligand C4/6 [2-alanyl-alanyl-4-tryptamino-6-(alpha-lysyl)-s-triazine] was found to bind porcine pancreatic elastase, but not trypsin, with a dissociation constant of 6 x 10(-5) M and a binding capacity of 21 mg elastase per ml gel. The adsorbent was used to purify elastase from a crude extract of porcine pancreas. Immobilized ligand C4/5 6 [2-alanyl-alanyl-4-tyramino-6-(alpha-lysyl)-s-triazine] was similarly chosen for optimal binding of elastase from cod and used to purify the enzyme from a crude extract of cod pyloric caeca. Ligand C4/6 was subsequently synthesized in solution and its structure verified by 1H-NMR.

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.

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.

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.

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.

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.