Biomedical subjects

Elizabeth A H Hall

Publications and source records attributed to Elizabeth A H Hall.

10 recordsLinked to original sources

PubMed2006

K+-selective nanospheres: maximising response range and minimising response time.

Cross-linked K(+) ion-selective copolymer nanospheres have been prepared by free-radical photo-initiated polymerization of n-butyl acrylate (nBA) with hexanedioldiacrylate (HDDA). Nanospheres (<200 nm) containing H(+)-chromoionophore (ETH 5294) and lipophilic salt (KTClPB) for H(+)-sensors, or ETH 5294, a K(+)-selective ionophore (valinomycin) and anionic sites for K(+)-sensors were compared, and the effect of varying the normalised concentrations for beta (R(T)(-)/L(T)) and gamma (C(m)(T)/L(T)) was studied. Experimental data were fitted to theoretical curves for the dynamic response range, based on the effect of changes in the concentration of these lipophilic sensing components incorporated into the spheres, and conditions identified for maximising the response range. A complex valinomycin-K(+) formation constant, log K(IL) = 13.13 +/- 2.22, was obtained in the nBA matrix, and from the calibration curves the apparent acid-dissociation equilibrium constant (pK(a) = 12.92 +/- 0.03) was extracted for the H(+)-sensing system, and the equilibrium exchange constant (pK(exch) = 6.16 +/- 0.03, at pH 7) calculated for the K(+)-sensing nanospheres. A basis for establishing optimum performance was identified, whereby response range and response time were balanced with maximum fluorescence yield. Parameters for achieving nanospheres with a response time <5 minutes, covering 2-3 orders of magnitude change in activity were identified, demanding nanospheres with radius <300 nm and beta(crit) approximately 0.6. An RSD(%) approximately 3% was obtained in a study of the reproducibility of the response of the proposed nanospheres, and selectivity was also evaluated for a K(+)-selective nanosensor using several cations as interfering agents. In most cases, the fluorescent emission spectra showed no response to the cations tested, confirming the selectivity of nanospheres to potassium ion. The nanosensors were satisfactorily applied to the determination of K(+) in samples mimicking physiological conditions.

Anions↗

PubMed2006

pH response of carboxy-terminated colorimetric polydiacetylene vesicles.

Carboxy-terminated polydiacetylene vesicles are known to undergo dramatic color transitions in response to exposure to external stimuli such as pH, temperature, and receptor-ligand binding. FTIR spectroscopy was used to identify the breakdown in the interfacial hydrogen-bonding interactions of the carboxylic acid headgroups of polymerized 10,12-tricosadiynoic acid (TRCDA) vesicles in aqueous solution during pH chromic transition. The headgroup structure was monitored as the chromic transition takes place and the dissociation dependence of the pKa was determined. Due to the attenuated acidity of the interfacially confined carboxy groups, which exhibit pKa values in the range 9.5-9.9, it was found that the deprotonation-triggered blue-red chromic transition occurred in the pH range 9.0-10.1 and that the mechanism of the transition required interaction with the surface carboxyl group, which is of importance in the design of a biochromic mechanism using PDA assemblies. Transmission electron microscopy and FTIR spectroscopy revealed that the surface ionization and the pH-induced chromogenic transition was also accompanied by a dramatic vesicle-planar morphological transition alongside subtle changes to the alkyl chain conformation and packing. A two-step mechanism was implicated as causing the chromic transition that first involves surface deprotonation and then specific cation binding, which can aid the design of sensitive surface-ligand chemistry for new PDA structures.

Binding Sites↗

PubMed2006

Manometric transduction in enzyme biosensors.

The combination of enzymatic recognition and manometric transduction is explored, using enzymes that consume or evolve a gas with low solubility in aqueous media. A design is discussed whereby change in partial pressure of a gas in the headspace is related to the turnover of analyte by the enzyme. Headspace and sample volume dimensions are considered, demonstrating the influence of flux at the air-water interface. The relative importance of diffusion and reaction for the enzyme solution is shown. When enzyme kinetics dominate, the concentration gradient is low and the overall kinetics are determined by the total amount of active enzyme, reducing either enzyme concentration or enzyme layer thickness will reduce the diffusion limitation. A Teflon-enzyme composite is presented to allow a reuseable immobilised enzyme preparation and a disc with stirring magnet identified as an efficient configuration. A glucose oxidase system was tested in the monitoring of glucose consumption during fermentation. Application to other enzyme systems is discussed.

Biosensing Techniques↗

PubMed2004

Surface plasmon resonance: theoretical evolutionary design optimization for a model analyte sensitive absorbing-layer system.

Surface plasmon resonance (SPR) has been widely used in a Kretschmann configuration to study optical thickness changes of layers on a Au surface in response to an analyte. The method has been popularized and optimized for protein layers, but has also been used in the same format for other layers without further optimization including those absorbing at the incident wavelength. In this paper, we examine whether SPR remains the "best"attenuated reflectivity format for absorbing overlayers. Experimental data from the SPR response of a copper phthalocyanine film to nitrogen dioxide are used as an input example for a design process using an evolutionary algorithm. The data showed a trend toward thinner gold layer systems ( approximately 25 nm gave an contrast-enhancement of 42.9% compared with approximately 50-nm Au) or Au-free solutions including a layer with low refractive index. From the evolutionary design predictions, further modification could be tested based on available materials and "redundant layers" could be eliminated from the final selection. By inclusion of the external optics, a design could be selected to accommodate poor precision (+/-0.5 degrees ) in the incident angle and a possible multilayer solution was shown using Teflon AF 1600, with refractive index approximately 1.3. The predicted NO(2) response showed an improvement compared with the classical SPR configuration, and the incident angle chosen by the SGA for the interrogation of these layers was close to a stationary point in the absolute response curve, thus offering very good tolerance to automatic position referencing to the reflectivity minimum.

Equipment Design↗

PubMed2004

A strand exchange FRET assay for DNA.

A new displacement hybridisation method is reported using a single strand DNA probe, labelled with an acceptor fluorophore (oregon green 488). Detection of double stranded sample target is shown, with discrimination between the probe, duplexed during the assay, and free single stranded probe DNA achieved through the FRET from a donor grove fluorophore (Hoechst 33258). A model for the kinetics of the displacement assay is presented and the course of the assay predicted according to probe/target ratios and sequence. The modelled predictions are consistent with the experimental data showing single base pair mismatch discrimination. The pattern of response according to the mismatch/perfect complement ratio in a mixed sample is also considered with an allele-discrimination ratio lying between the homozygous gene and total mismatch case, according to ratio. The assay is shown to be tolerant of different probe concentrations and ratios and through the dual wavelength recorded signals from donor and FRET acceptor, internal baseline correction is achieved with excellent noise reduction through ratiometric measurement.

Base Pair Mismatch↗

PubMed2004

Examination of bilayer lipid membranes for 'pin-hole' character.

BLM prepared on electrode substrates by supporting or tethering were tested for 'pin-hole' character, comparing data from cyclic voltammetry (CV), surface plasmon resonance (SPR) and rotating disc electrodes (RDE). 1-hexadecylamine tethered BLMs on SAM modified gold electrodes were compared with BLMs assembled on modified polyHEMA or sol-gel layers. BLM formation followed by SPR showed that the initial phase of the assembly was complete in 5-20 minutes and produced layers of thickness >5 nm, compared with the expected final BLM thickness of approximately 3 nm. The CVs of the K(3)[Fe(CN)(6)] couple were significantly suppressed irrespective of the method of BLM assembly, without major differences emerging for the different methods. However, data from the RDE distinguished the 'pin-hole' character of the different preparations. The data were consistent with incomplete initial (<1 h, SPR estimated BLM thickness >5 nm) vesicle fusion leaving 'pin-holes' of approximately 2 microm (HDA-11-mercaptoundecanoic acid (MUA) tethered BLM) to approximately 3 microm (tetraethylorthosilicate sol-gel supported BLM) followed by a slow maturation (>15 h; impedance spectroscopy estimated thickness approximately 3 nm) and lateral spreading and fusion, resulting in loss of 'pin-hole' character (<1 microm). The BLM could be used in conjunction with potentiometric measurement to observe the incorporation of nystatin into the BLM and the rate of incorporation adjusted according to original permeability of the BLM. The 'pin-hole-free' BLM construction with lowest permeability (TEOS supported, 4 x 10(-10) cm s(-1) compared with HDA-MUA, 3 x 10(-9) cm s(-1)) gave a potentiometric signal independent of bulk ion-concentration across 5 decades change in concentration. Formed on an ion-selective electrode, nystatin incorporation could be followed as a change in potential, over >2 h, whereas the TEOS supported BLM with permeability 1 x 10(-9) cm s(-1) shows nystatin incorporation within 1 h. In this instance, addition of ConA reduced the potential to the same value as prior to nystatin incorporation, consistent with nystatin channel closure.

Biosensing Techniques↗

PubMed2004

An experimental study of membrane materials and inner contacting layers for ion-selective K+ electrodes with a stable response and good dynamic range.

The goal was to identify formulations for use in valinomycin K(+) ion-selective electrodes that could routinely achieve a detection limit of <10(-6) M, even after repeated use and exposure at higher K+ activity (0.1 M) and without the requirement for special pretreatment or conditioning in low K+ activity (10(-3) M). Electrodes that would be characterized by high potential stability were sought in this work. Valinomycin-containing membranes with diffusion coefficient of approximately 10(-11) cm(2) s(-1), formulated from methacrylic/acrylic polymers with or without plasticizer, were compared with plasticized PVC membranes (diffusion coefficient 10(-8) cm(2) s(-1)). The methacrylic/acrylic membranes without plasticizer were shown to give an order of magnitude lower detection limit, when compared with PVC-dioctyl sebacate and o-nitrophenyl octyl ether plasticized methacrylic/acrylic polymers under the same conditions, highlighting the influence of plasticizer on the detection limit. As predicted from current theoretical derivation, the inner contacting layer in the ion-selective electrode construction was shown to be highly influential in maintaining the detection limit below 10(-6) M with use and with poly(pyrrole) providing the inner contact ion-to-electron transduction function, a further order of magnitude improvement in the lower detection limit could be maintained for both chloride and hexacyanoferrate doped poly(pyrrole), when 2% ionophore was employed in the ion-selective membrane. This formulation showed extraordinary stability and reproducibility in terms of measurement range and drift over extended measurement testing, with close to Nernstian slopes. At higher ionophore concentrations (4%), the apparent selectivity of the electrode was improved at the expense of detection limit and the nature of the poly(pyrrole) dopant ion became important in determining the dominant exchange processes at the poly(pyrrole)/ion-selective membrane interface.

Journal Article↗

PubMed2003

Short peptide receptor mimics for atherosclerosis risk assessment of LDL.

Short peptides sequences were selected that showed binding selectivity towards healthy or oxidised (unhealthy) low density lipoprotein (LDL), respectively. These were investigated for application in atherosclerosis risk monitoring. Comparison was also made with the LDL receptor ligand repeat peptide (LR5). The peptides were immobilised on a gold surface plasmon resonance surface and LDL binding detected as a shift in the resonance. 3.7x10(7) (+/-5.6x10(6)) LDL/mm(2)/microg/ml solution LDL were bound on GlySerAspGlu-OH and 6.8x10(7) (+/-9.2x10(6)) LDL/mm(2)/microg/ml on GlyCystineSerAspGlu, compared with approximately 10(8) LDL/mm(2)/microg/ml on LR5. In this first group, binding of LDL decreased with oxidation level and a good correlation was found between LDL binding and residual amino groups on the apoprotein of the LDL following oxidation, or the change in relative electrophoretic mobility (REM) of LDL. The decrease in binding was 1.1x10(7) LDL particles/mm(2) per% oxidation for GlySerAspGlu-OH, 1.8x10(7) LDL particles/mm(2) per% oxidation for GlyCystineSerAspGlu and 2.4x10(7) LDL particles/mm(2) per% oxidation for LR5. A second group of three peptides were also selected showing increased binding with LDL oxidation: GlyCystineCysCys (1.5x10(7) LDL/mm(2) per microg/ml), GlyLysLysCys-SH (10(7) LDL/mm(2) per microg/ml) and GlyLysLys-OH (5.6x10(7) LDL/mm(2) per microg/ml). The latter gave a linear increase in LDL binding with oxidation level (1.2x10(7) LDL particles/mm(2) per% oxidation). LDL concentration is around 2-3 mg/ml in plasma compared with the low detection levels with this method (1-10 microg/ml), allowing a strategy to be developed requiring the minimum sample volume and diluting with physiological buffer prior to assay. By using a comparative reading between LDL adsorption on surfaces from the first and second group of peptides (e.g. GlyCystineSerAspGlu and GlyLysLys-OH, respectively), LDL oxidation could be determined without knowledge of LDL concentration. Higher binding was seen on GlyCystineSerAspGlu than GlyLysLys-OH below 30% LDL oxidation, whereas above 30% oxidation the binding on the latter surface was greater. Simple correlation of this form could provide good tests for atherosclerosis risk.

Adsorption↗

PubMed2003

Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode. Part 1. Wild-type enzyme redox mediation.

An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe-4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe-4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (k(s) = 0.93 x 10(5) M(-1) s(-1)) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0-1.0. Plots of kcat/KM revealed two ionisations with pKa values of 7.5 and 10. The pKa of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pKa of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA-N per lOOg wet fish muscle). The obtained results compared well with a reference method based on picric acid.

Animals↗

PubMed2002

Direct toxicity assessment of wastewater: Baroxymeter, a portable rapid toxicity device and the industry perspective.

Direct toxicity assessment of wastewater is becoming necessary, and new legislation may render it compulsory for the water industry. At present such assessment is performed at a laboratory away from a site, at considerable cost, and results often come too late, after a toxic event has occurred and the toxin has been released into the environment. Some of the rapid toxicity tests available today require certain conditions to function properly, or their results do not always correlate with other methods. The objective of this study was to assess a portable device, the Baroxymeter, for its suitability as an instrument to test wastewater toxicity. The way the device works is based on monitoring respiration of a bacterial culture by pressure measurements and using respiration inhibition as a toxicity alert. It has been shown that it is possible to detect toxic substances such as 3,5-dichlorophenol and bronopol within 5 min from a 1-mL sample. The benefits and future applications of the Baroxymeter as a high-throughput, cost-effective alternative for toxicity screening are discussed in this article.

Bacterial Physiological Phenomena↗