Mohamed A. Desai

A study of macromolecular diffusion through native porcine mucus

A diffusion chamber technique based on time-lag analysis for the estimation of effective diffusion coefficients of radiolabelled macromolecules of varying molecular weights through native mucus gel is reported. For all solutes studied, a reduction in effective diffusion coefficients was observed with a retardation of solute flux in both aqueus and mucus layers. Over the molecular weight range of solutes investigated (126–186 000 Daltons), a consistent effect of molecular weight was evident with regard to the retarding effect of mucus. No apparent or absolute molecular weight cut-off for macromolecular transfer was exhibited. However, at high molecular weights (>30 000 Daltons) the retardation was greatly enhanced. The results confirm that mucus can be regarded as a gel with finite pores, but that it does not constitute an absolute barrier to even high molecular weight solutes.

Estimation of effective diffusion coefficients of model solutes through gastric mucus: assessment of a diffusion chamber technique based on spectrophotometric analysis.

A diffusion chamber technique based on spectrophotometric analysis to determine effective diffusion coefficients for solutes with various relative molecular mass (Mr) values and properties, passing through native mucus gel, is reported. For all solutes studied, a reduction in effective diffusion coefficients is observed with a retardation of solute flux by a factor of at least two. For the solutes investigated (with Mr values ranging from 126-14,400 u), no consistent effect of solutes of low Mr was evident with regard to the retarding effect of mucus; however, at high Mr values (greater than 4000 u) the retardation was greatly enhanced. A possible relationship between charged solutes of low Mr and the degree of retardation was observed, which possibly suggests the presence of ionic interactions of the solutes with the largely negatively charged mucus gel. The results provide further evidence for the suggestion that mucus is acting more than simply as a gel support for an unstirred water layer and is perhaps a more potent diffusion barrier to specific solutes.

Poly(vinyl chloride), polysulfone and sulfonated polyether-ether sulfone composite membranes for glucose and hydrogen peroxide perm-selectivity in amperometric biosensors

Membrane materials were evaluated for use in amperometric biosensors. PVC, polysulfone (PS) and sulfonated polyether-ether sulfone–polyether sulfone (SPEES-PES) and SPEES-PES/PVC composites were solvent cast. Varying the plasticizer (isopropyl myristate) content of PS and PVC membranes enabled electrode response to hydrogen peroxide and acetaminophen to be modulated. Omission of plasticizer from PS and PVC resulted in hydrogen peroxide permeable membranes, selective against acetaminophen, ascorbate and urate (ratios 230 : 1 in each case). SPEES–PES copolymer membranes (unplasticized) showed greater hydrogen peroxide permeability (corresponding ratios 1430 : 1). By mixing plasticized PVC and SPEES–PES in suitable proportions, permeability ratios of hydrogen peroxide : acetaminophen were controlled. Glucose sensors using these composite membranes for H2O2 perm-selectivity demonstrated linear responses through the clinical range, and ⩽5% loss of signal on exposure to blood. A novel form of glucose oxidase based biosensor deployed a new membrane as an outer layer selective for the glucose substrate, rejecting interferents. Accordingly, a second selective layer for the enzyme reaction product (hydrogen peroxide) is not required. The new membrane is a plasticized mix of PVC and SPEES–PES polymers incorporating isopropyl myristate. Selectivity is adequate for clinical glucose measurement, and in particular acetaminophen interference is excluded in addition to ascorbate and urate, representing an advance over membranes described hitherto. A linear range of 0–20 mmol l–1 glucose confirms control of substrate diffusion sufficient to extend apparent enzyme KM, and resistance to fouling in blood is also promising for deployment in a clinical assay.

Internal membranes and laminates for adaptation of amperometric enzyme electrodes to direct biofluid analysis

This study describes the use of discrete, highly selective covering inner membranes in amperometric enzyme electrodes for direct biofluid analysis. Such inner membranes when interposed between the enzyme layer and the electrode working surface has been found to be both selective, as well as biocompatible. Cellulose acetate or detergent (Triton X-100 and Tween 80) modified cellulose acetate and polyethersulphone polymeric membranes, were both found to be highly selective against electroactive solutes normally present in biofluids. The inclusion of such inner perm-selective membranes greatly enhanced the biocompatibility of the enzyme electrode and reduced the passivation of the working electrode on exposure to whole blood or serum. The study indicates that electrode drift in biological solutions may also be due to passivation of the working electrode by small diffusible surface active species.

Amperometric enzyme electrode biofouling and passivation in blood: characterisation of working electrode polarisation and inner membrane effects

Signal drift of an H2O2-based glucose enzyme electrode following exposure to whole blood has been studied in relation to the inner membrane barrier used to separate the oxidase enzyme from the working electrode as well as working electrode polarisation.Variation of the inner membrane has been found to have a strong influence on signal drift, confirmed as being due to the passage of some diffusible solutes to the working electrode. The subsequent passivation and loss of current output of up to 70% was observed in 120 min, at a polarisation voltage of +800 mV vs. Ag | AgCl with a 2% w/v cellulose acetate membrane. Signal drift was substantially reduced using less permeable inner membranes, which included microporous polycarbonate of 0.01, 0.03, 0.1 and 1 µm pore radii as well as non-porous cellulose acetate membranes (2% w/v and 5% w/v).Signal drift has also been shown to be critically dependent upon working electrode polarisation; higher over-potentials gave higher losses ranging from 10% at +200 mV to 70% at +800 mV vs. Ag | AgCl after 120 min (for 2% w/v cellulose acetate). There is some indication that, in addition to its permeability, the dielectric properties of the inner membrane may influence the sensor drift.

pH dependence of hydrochloric acid diffusion through gastric mucus: correlation with diffusion through a water layer using a membrane-mounted glass pH electrode

Solute diffusion coefficients (D) can indicate a dependence upon actual solute concentrations. Here a single compartment has been utilized, in which effective HCl diffusion to a membrane-mounted glass pH electrode can be measured across the pH spectrum. The study has investigated HCl diffusion through both mucus and water layers as a function of HCl concentration. The observed dynamic responses of a liquid-film and mucus-coated electrodes over a range of HCl concentrations suggest that the speed at which equilibrium is attained is pH dependent; equilibrium was reached rapidly under more acidic and alkaline conditions. Estimated values of DHCl also indicate a strong pH dependence for both liquid film and mucus. In both instances, a greater than 10-fold reduction in DHCl at pH 7.5 as compared with that at pH 3.5 has been demonstrated. Furthermore, estimated values of DHCl are approximately 4-fold smaller through the mucus gel, as compared with a water layer. The findings indicate that the most powerful influence on diffusional resistance is pH itself, whereby a marked drop in H+ diffusion is likely to occur towards neutral pH irrespective of the composition of the gel barrier. Possible implications of the findings are discussed in relation to mucosal protection from acid.

Electrochemical determination of the permeability of porcine mucus to model solute compounds.

Abstract— An electrochemical approach to the determination of permeability through native mucus gel of simple electrochemically active solutes is reported. For all the the solutes studied, a reduction in effective diffusion coefficients was observed, with retardation of solute flux by a factor of at least two. However, NADH and the dicarboxylic acid derivative of ferrocene demonstrated a substantial, almost ten‐fold, reduction in permeability through mucus. Results for the controls were in reasonable agreement with literature values where available. No consistent effect of molecular weight was evident with regard to the barrier properties of mucus over the molecular weight range of solutes investigated (34–660 daltons). The results suggest that mucus is acting more than as a gel support for an unstirred water layer.

An in Vitro Study of Enhanced H+ Diffusion by Urease Action on Urea: Implications for Helicobacter pylori-Associated Peptic Ulceration

The in vitro effect of urea and hydrolysis of urea by urease on mucus H+ permeability is reported here. The effective DHCl values indicate a strong pH dependence for H+ diffusion in both water and mucus layers, with no apparent trend at concentrations between 1 and 50 mM urea. However, the estimated DHCl at near-neutral and alkaline pH are 4- to 10-fold lower through mucus than through aqueous films. Moreover, the pKa values of HCO3- and NH3 (generated by urease action on urea) had a profound effect on measured DHCl. These in vitro studies suggest that a high local concentration of NH3 and HCO3- within the mucus layer, generated by the action of Helicobacter pylori urease on endogenous intragastric urea, could greatly accelerate proton flux to the surface epithelium by operation of a buffer shuttle. This results in enhanced H+ permeability, particularly at pKa values of HCO3- and NH3, and in extreme circumstances it may result in gastric ulcer formation.

Glucose oxidase enzyme electrode: relation between inner membrane permeability and substrate response

Abstract Permeability coefficients, P (cm 2 s −1 ), for H 2 O 2 across microporous polycarbonate membranes of varying pore radii together with similar membranes coated with an organosilane have been determined, using a classical diffusion chamber. A correlation has been attempted between the permeability of such membranes with their performance when used as inner membranes of glucose oxidase electrodes. Organosilane coated polycarbonate membranes were found to impart extended linearity ranges ( > 40 mM) to glucose enzyme electrodes. The O 2 /glucose permeability coefficient ( P ) ratio of such inner membranes was found to be critical in determining sensor performance. It is proposed that for the oxidase based electrodes used, extensions in linearity were due to the cumulative effect of two phenomena. Firstly the relative augmentation of O 2 returning into the enzyme layer following the oxidation of H 2 O 2 helps free the sensor from the effects of low ambient pO 2 levels. Secondly inner membrane barriers offering increased diffusional resistances towards H 2 O 2 maintain linear diffusion of H 2 O 2 to the working electrode at higher substrate concentrations.

Chemical sensors and biosensors: nearer the patient

Rapid biochemical testing can expedite therapy in key patient categories, particularly intensive care. Dry reagent chemistries have gone some way to satisfying some of these requirements. However, chemical and biosensors offer the prospects for more robust and operationally simpler near-patient analysers which, furthermore, are well suited to the assay of optically opaque biofluids, as well as the development of cheaper instrumentation, The wide range of transduction principles on offer with sensing technology not only enhances prospects for success in t his area, but permits more precise tailoring of sensor performance to clinical requirements. Background considerations for extra-laboratory testing are reviewed here, together with discussion of key sensor properties and fabrication needs that are of relevance to measurement in these less controlled environments. An outline will then be given of the main transducer t ypes, including ion-selective and amperometric electrodes, thermistors, piezoelectric crystals and optical wave guides. Use of biomolecules will centre on a description of enzyme and antibody incorporation into practical systems.