Séamus P.J. Higson

Poly(o-phenylenediamine) ultra-thin polymer-film composite membranes for enzyme electrodes

Abstract An ultra-thin poly( o -phenylenediamine) film composite membrane has been used as the outer covering barrier in an amperometric glucose oxidase enzyme electrode. The composite membrane was formed via the electropolymerisation of o -phenylenediamine dihydrochloride at a gold sputter-coated porous polycarbonate membrane. Homogeneous poly( o -phenylenediamine) films of approximately 30xa0nm thickness acted as an effective diffusional barrier and were supported by the underlying host gold-coated polycarbonate membrane. Oxygen and glucose permeability coefficients were determined using diffusion chamber apparatus. Composite membranes of this type were also found to offer some permselective screening of anionic interferent solutes such as ascorbate. On exposure to whole blood, a 30% signal drift with time was observed over a period of 1xa0h; after this time responses remained almost stable. Whole blood glucose determinations compared with standard hospital laboratory analyses showed a close correlation ( r 2 =0.92).

Ultra-thin-polysiloxane-film-composite membranes for the optimisation of amperometric oxidase enzyme electrodes.

An outer ultra-thin-polydimethyldichlorosiloxane film composite membrane has been used as the outer covering barrier in an amperometric glucose oxidase enzyme electrode biosensor. The composite membrane was formed via the condensation polymerisation of dimethyldichlorosilane at the surface of a host porous alumina membrane. Homogeneous polydimethyldichlorosiloxane films of <100 nm thickness acted as effective substrate diffusional barriers and were supported by the underlying porous alumina surface. Glucose and oxygen permeability coefficients were determined using diffusion chamber apparatus. Polysiloxane composite membranes were found to offer some screening functionality towards anionic biological interferents such as ascorbate. On exposure to blood an approximate 25% signal drift was observed during the first 2 h exposure to blood; after this time responses remained almost stable. Whole blood glucose determinations showed a close correlation (r(2)=0.98) to analyses performed via standard hospital analyses.

Diamond-like carbon coated microporous polycarbonate as a composite barrier for a glucose enzyme electrode

Diamond-like carbon (DLC) coated microporous polycarbonate membranes were used as a outer covering membranes in clusoe enzyme electrodes. With optimised DLC deposition and the use of 0.01 μm microporous polycarbonate, biocompatibility has been seen to improve with only a loss of 6% response after 30 min exposure to whole blood, and a correlation to within 1 mM concentration with a standard laboratory method. In addition, control fo the coating process allowed substrate diffusion-limiting properties to the bulk enzyme to be finely tuned permitting extensions in linearity ranges from 5 to > 80 mM glucose. Furthermore the higher biocompatibility coupled with the degree of permselectivity exhibited by DLC has enabled operation within whole blood without a second hydrogen peroxide selective barrier membrane. Permeability coefficients of glucose and O2 determined for corresponding membranes by a classical diffusion chamber technique suggest that both the glucose/O2 permeability coefficient ratios and the absolute glucose permeability influenced the linearity range.

Amperometric enzyme electrode for the determination of urine oxalate

Abstract An enzyme electrode has been developed for the simplified determination of oxalate in human urine. Oxalate oxidase has been co-immobilised with bovine serum albumin (BSA) using glutaraldehyde between polycarbonate or haemodialysis external membranes and an internal cellulose acetate membrane to form a classical oxidase enzyme laminate construction. The underlying cellulose acetate permselective barrier confers the required selectivity for H 2 O 2 over the interferent constituents of urine. Prior dilution and acidification of urine samples has enabled optimisation of enzyme activity and therefore electrode response characteristics for low level oxalate assay. Sequestering of singly and doubly charged cations in urine by ethylenediaminetetraacetic acid (EDTA) has ensured release of bound oxalate, so permitting near total (> 98%) determination of urinary oxalate. An outer substrate diffusion limiting membrane has proved critical to an electrode linear response within the clinical concentration range. Determination of human urinary oxalate in the concentration range 2–200 μM in diluted urine (corresponding to 80–800 μM oxalate in undiluted urine) has been possible. Results correlate well ( r 2 = 0.971; y = 1.11 x + 4.20; n = 20) with standard spectrophotometric determinations ( x ) performed within a hospital laboratory.

The characterization of liposomal glucose oxidase electrodes for the measurement of glucose.

Many biosensors have been described for the measurement of glucose in order to monitor diabetic patients. Glucose oxidase has been used commonly in the construction of glucose sensors but the performance of this enzyme is limited by enzyme saturation kinetics, which restrict the measurement of clinically relevant glucose concentrations (0 to 25 mM). Diffusion limiting membranes have been described that result in the exposure of the enzyme to lower concentrations of glucose than are present in the bulk test solution. Recently a liposomal enzyme electrode was reported whereby glucose oxidase was encapsulated within liposomes so that the lipid bilayer was the diffusion limiting membrane. It was shown that the electrode response was defined by the lipid constituents of the liposome, and that a linear response to glucose could be achieved up to 40 mM. This paper describes research undertaken to improve the methods of production of a liposomal enzyme electrode. Improved immobilization of liposomes is demonstrated with the use of poly-L-lysine solution. The variation in electrode response with respect to the amount of glucose oxidase liposomally encapsulated is reported. The new method allows a greater number of sensors to be produced from a single batch of liposomes. Studies also show the biofouling effects of the lipid constituents of ruptured liposomes on the response of the electrode to glucose over time.

Diamond like carbon coated films for enzyme electrodes; characterization of biocompatibility and substrate diffusion limiting properties

The biocompatibility and substrate diffusion limiting properties for a range of diamond like carbon (DLC) coated microporous polycarbonate and DLC coated dialysis (haemodialysis) membranes have been studied. This characterisation builds upon previous findings where DLC coated membranes imparted enhanced enzyme electrode performance. In this study electrode linear ranges have been extended from 10 mM glucose for a 0.01 μm pore size membrane to 160 mM. These findings correlated with the duration of DLC deposition and associated reductions in permeability for glucose. Permeability coefficient ratios for both microporous and dialysis membranes were also found to be important with low glucose/O2 permeability ratios imparting extensions in glucose linear response range. DLC coated membranes employed within enzyme electrodes have also been shown to exhibit enhanced haemocompatibility as determined by both sensitivity change and surface deposition of blood components examined by scanning electron microscopy. Correlations are made between the reduced losses in sensor response to biofouling/ working electrode passivation processes, and extended linear ranges that DLC coated membranes may impart to enzyme electrode performance. Particular reference is made to the determination of glucose levels within whole blood.

Diamond like carbon films for enzyme electrodes ; characterisation of novel overlying permselective barriers

Abstract Diamond like carbon (DLC) coated microporous polycarbonate membranes have been studied for use as novel composite permselective barriers membranes for a glucose enzyme electrode. Permeability coefficients, P, for key electrochemically active interferents across uncoated and DLC coated polycarbonate membranes has been compared. Interferent responses have then been assessed for sensors incorporating such membranes, and their relationship to differing DLC depositions assessed. Membranes with smaller pore sizes (0.03 and 0.01 μm) and extended DLC depositions (up to 7 min coating), while imparting some enhanced selectivity towards glucose, failed to show major discrimination for glucose over interferents as shown by P values: maximum glucose-to-interferent P ratios being 1.36 and 1.25 for ascorbate and urate, respectively. The implications of these findings are discussed.

The diffusion limited oxidase-based glucose enzyme electrode: relation between covering membrane permeability and substrate response

Permeability coefficients, P (cm2-sec−1, for glucose, oxygen, acetaminophen (paracetamol), p-aminophenol (PAP), 5-trihydroxybenzene (phloroglucinol) and ascorbate through polycarbonate (PC) neutron beam track-etch membranes of pore radii 0.015–0.4 μm have been determined at (22 ± 1°C) using a diffusion chamber and the principle of steady state analysis. P values were found to increase with increasing membrane pore radius. The ratio of glucose/O2P values for membranes of pore radii 0.4, 0.05, 0.03 and 0.015 μm were determined to be 0.64, 0.36, 0.22 and 0.045 respectively and therefore decreased with decreasing pore radius. The P value ratios for glucose with respect to other organic species were in the range 0.7–1.2, but showed little dependence on membrane pore radius. It has been found that lower glucose/O2P ratios characteristic of smaller pore radii membranes, were associated with extended linearity ranges for glucose analysis when utilised as upper membranes in a conventional glucose oxidase enzyme laminate electrode construction.

A lactate dehydrogenase amperometric pyruvate electrode exploiting direct detection of NAD+ at a poly(3-methylthiophene):poly(phenol red) modified platinum surface

Abstract An amperometric L(+) lactic dehydrogenase, pyruvate sensing electrode has been developed in which NADH is regenerated at a poly (3-methylthiophene)/poly (phenol red) electrode. A key part of the sensor is the use of a dual poly (3-methylthiophene)/poly (phenol red) film which permits the reduction of enzymically active NAD + at an over potential of only −0.2 V (vs. Ag/AgCl) in comparison to − 1 V at a bare electrode. NAD + reduction is proposed to take place via the quinone group of poly (phenol red) to poly (3-methylthiophene) facilitating electron transfer from the base Pt electrode. A pyruvate sensor was constructed using gel entrapped L(+) lactic dehydrogenase. The pH, cofactor concentration, buffer type and concentration all had effects on the pyruvate response. In phosphate saline buffer, pyruvate gave a response independent of L(+) lactic dehydrogenase activity, and were attributed to the acid doping of the poly(3-methylthiophene) film. With 2-[ N -morpholino(ethanesulfonic acid)] (MES) saline buffer the pyruvate electrode gave a decrease in cathodic currents. The nonspecific doping effect by pyruvate was negligible and this behaviour was therefore attributed to an increase in poly(3-methylthiophene) conductivity during interaction with enzymically produced NAD + . Under optimised assay conditions of (40 mM MES pH 6 containing 50 mM KCl, 1 mM NADH) the drop in cathodic response could be related to solution pyruvate concentration. The K M for pyruvate reduction by L(+) lactic dehydrogenase was determined to be 3.7 mM. The different properties of poly(3-methylthiophene)/poly(phenol red) modified electrodes in phosphate and MES buffers were studied using impedance spectroscopy. It is proposed that phosphate entry contributes significantly to the charge transfer resistance of the dual polymer film despite the presence of mobile K + and Cl − ions. However, in MES buffer the K + and Cl − ions appeared to be the dominant charge carriers. The reasons for the different sensitivities of poly (3-methylthiophene)/poly(phenol red) modified electrodes to pyruvate in phosphate and MES buffers are discussed in terms of the ion exchange properties of the dual film.

Identification of novel mammalian squalene synthase inhibitors using a three-dimensional pharmacophore.

Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerisation of farnesyl diphosphate in a [1-4] head to head fashion to form squalene, and is the first committed step in cholesterol biosynthesis. Specific inhibitors of squalene synthase would inhibit cholesterol formation and allow production of other important compounds derived from the cholesterol biosynthetic pathway, namely the ubiquinones (co-enzyme Q(10)), dolichol, and would also allow the isoprenylation process of ras by farnesyl-protein transferase. The construction of a hypothetical squalene synthase three-dimensional pharmacophore is presented. It serves as a template for the identification of several new potential classes of inhibitors. The synthesis, anti-microbial and mammalian pig liver squalene synthase activities of analogues based on the bicyclo[3.2.0]heptane and bicyclo[3.3.0]octane ring systems are reported. Analogues of the latter system are pro-drug type inhibitors and exhibit promising biological activity.