David C. Cowell

Voltammetric studies of lead at a 1-(2-pyridylazo)-2-naphthol modified screen-printed carbon electrode and its trace determination in water by stripping voltammetry

A method for the trace determination of Pb2+, using a disposable 1-(2-pyridylazo)-2-naphthol (PAN) drop-coated modified screen-printed carbon electrode (PAN-SPCE), has been developed. Lead ions (Pb2+) are accumulated from ammonia buffer, on the PAN-SPCE surface by the formation of a chemical complex at open circuit. The resulting SPCE with complexed Pb2+ is then transferred to 0.1 M HCl and subjected to differential pulse anodic stripping voltammetry. Conditions were optimised with respect to the pH and concentration of the accumulation medium, preconcentration time and the mass of drop-coated PAN. A 5 min preconcentration time resulted in a limit of detection of 15 ng ml(-1). The calibration plot was found bimodal with linear ranges between 0 and 700 ng ml(-1) and 700-5000 ng ml(-1) Pb2+. The principal metal interference was copper, but this problem was successfully overcome by the addition of iodide to the ammonia buffer prior to the accumulation step. The method was evaluated by carrying out lead determinations on spiked potable water samples; the recovery of Pb2+ was 92.5% and the coefficient of variation was found to be 4.4% (n = 5) using a separate PAN-SPCE for each determination. Therefore, the performance data suggests that the method is reliable at the concentrations examined in this study; in addition, the data compared favorably with that obtained by a published method.

Voltammetric behaviour and trace determination of copper at a mercury-free screen-printed carbon electrode.

Screen-printed carbon electrodes (SPCEs), without chemical modification, have been investigated as disposable sensors for the measurement of trace levels of Cu(2+). Cyclic voltammetry was employed to elucidate the electrochemical behaviour of Cu(2+) at these electrodes in a variety of supporting electrolytes. For all of the electrolytes studied the anodic peaks, obtained on the reverse scans, showed that the Cu(2+) had been deposited as a thin layer on the surface of the SPCE. The anodic peak of greatest magnitude was obtained in 0.1 M malonic acid. The possibility of determining Cu(2+) at trace levels using this medium was examined by differential pulse anodic stripping voltammetry (DPASV). The effect of Bi(3+), Cd(2+), Fe(3+), Hg(2)(2+), Pb(2+), Sb(3+) and Zn(2+) on the Cu stripping peak was examined and under the conditions employed, only Hg(2)(2+) was found to significantly effect the response gained. The sensors were evaluated by carrying out Cu(2+) determinations on spiked and unspiked serum and water samples. The mean recovery was found in all cases to be >90% and the performance characteristics indicated the method holds promise for trace Cu(2+) levels by employment of Hg-free SPCEs using DPASV.

Voltammetric Behavior and Trace Determination of Lead at a Mercury‐Free Screen‐Printed Carbon Electrode

Abstract Screen-printed carbon electrodes (SPCEs), without chemical modification, have been investigated as disposable sensors for the measurement of trace levels of Pb. Cyclic voltammetry was employed to elucidate the electrochemical behavior of Pb at these electrodes in a variety of supporting electrolytes. For all of the electrolytes studied the anodic peaks obtained on the reverse scans, showed that the Pb had been deposited as a thin layer on the surface of the SPCE. The anodic peak of greatest magnitude was obtained in 0.1 M HCl. The possibility of determining Pb at trace levels using this medium was examined by anodic stripping voltammetry using the differential pulse waveform in the measurement step. Models are suggested for the deposition of Pb onto the SPCE surface under stated conditions. Calibration plots were found to be linear in the range 6.3 ngmL-1 to 24 ngmL-1 and 24 to 50 ngmL-1; the detection limit was 2.5 ngmL -1 and the coefficient of variation, determined on one single electrode, at a concentration of 12.6 ngmL-1 was calculated to be 2.4 % (n = 5). The effect of copper, cadmium and zinc on the Pb stripping peak was examined and, under the conditions employed, no significant change in current was found. The sensors were evaluated by carrying out Pb determinations on spiked and unspiked pond water. The recovery was calculated to be 97.4 % and the coefficient of variation was 3.2% at a concentration of 12.6 ngmL-1. These performance characteristics indicate that reliable data may be obtained for Pb measurements in natural waters.

Development of an amperometric sulfite biosensor based on sulfite oxidase with cytochrome c, as electron acceptor, and a screen-printed transducer

An amperometric biosensor for sulfite has been developed. The enzyme sulfite oxidase (SOD) and electron acceptor cytochrome c are mixed into the carbon ink that is deposited onto the working electrode of a screen-printed strip. A silver–silver chloride electrode is printed alongside the working electrode and serves as reference/counter electrode. The electrochemical behaviour of the biosensor surface in plain buffer has been investigated by cyclic voltammetry. In the voltage range −0.5 to +0.5 V, a well-defined anodic peak appeared at −0.15 V and a less well-defined anodic peak at about +0.2 V. In the presence of SO32−, the cyclic voltammogram obtained with the biosensor exhibited an increase in magnitude of the more positive peak; this was considered to result from the electrocatalytic oxidation of SO32− involving SOD and the heme (Fe2+/Fe3+) centre of cytochrome c. Amperometry in stirred solution was used to construct a hydrodynamic voltammogram for SO32− using the biosensor; this exhibited a single wave with a plateau beginning at +0.3 V. This wave corresponds to the electrocatalytic response observed by cyclic voltammetry. The pH and concentration of buffer components have been optimised for the determination of SO32− by amperometry in stirred solution. Using these conditions, a detection limit of 4 ppm was obtained. The stability of the biosensors was examined after storage in 0.05 M phosphate buffer pH 7.4 at 4°C; it was found that the initial response was retained for at least 45 days. The proposed biosensors were evaluated on samples of unspiked and spiked estuarine, river and tap waters. The recovery and precision data indicated that the devices could be expected to give reliable data in these waters.

Development of disposable amperometric sulfur dioxide biosensors based on screen printed electrodes.

The possibility of developing amperometric biosensors for the measurement of SO(2) in flowing gas streams has been examined. Screen-printed carbon electrodes (SPCEs) were tailored with the enzyme sulfite oxidase and cytochrome c and the response is generated through the resulting enzymatic and electrocatalytic reactions involving SO(3)(2-), formed when SO(2) gas is dissolved in the supporting electrolyte. Two methods of integrating the enzyme and cytochrome c with the SPCE were investigated. In one design (b-type biosensor), the components were mixed thoroughly with the same ink used to produce the SPCEs, then the modified ink was spread over the working electrode. In the second approach the bio-components were dissolved in the supporting electrolyte and simply deposited on top of the transducer (s-type biosensor). Both devices gave linear responses over the range 4--50 ppm but the sensitivity of the s-type was approximately twice that of the b-type biosensor. In addition, the time taken to reach 90% of the maximum response (t(90%)) was 110 s for the s-type biosensor compared with 200 s for the b-type biosensor. These studies illustrate the successful use of biosensors for the detection of sulfur dioxide at the relatively low potential of +0.3 V versus Ag.AgCl and should provide useful alternatives for decentralised environmental studies.

The detection and identification of metal and organic pollutants in potable water using enzyme assays suitable for sensor development

A panel of five pollutants have been identified as a model system to study: the herbicide atrazine, phenol, pentachlorophenol, cadmium and chromium. The non-specific inhibition of the activity of 23 enzymes was studied for these five pollutants at 1000 times the statutory limits. An array of six enzymes was chosen to develop four computer models from which the resultant pattern of inhibition could be interpreted using artificial neural nets. The models clearly demonstrated that the identification of pollutants using this novel approach is a feasible proposition. No false negatives were demonstrated at the concentrations tested by any of the models studied and the false positive rate was acceptably minimal. The models further demonstrated their ability to be semi-quantitative with regard to the level of pollutant present.

Development of an anodic stripping voltammetric assay, using a disposable mercury-free screen-printed carbon electrode, for the determination of zinc in human sweat

This paper reports on the development of a novel electrochemical assay for Zn(2+) in human sweat, which involves the use of disposable screen-printed carbon electrodes (SPCEs). Initially, SPCEs were used in conjunction with cyclic voltammetry to study the redox characteristics of Zn(2+) in a selection of supporting electrolytes. The best defined cathodic and anodic peaks were obtained with 0.1 M NaCl/0.1 M acetate buffer pH 6.0. The anodic peak was sharp and symmetrical which is typical for the oxidation of a thin metal film on the electrode surface. This behaviour was exploited in the development of a differential pulse anodic stripping voltammetric (DPASV) assay for zinc. It was shown that a deposition potential of -1.6 V versus Ag/AgCl and deposition time of 60 s with stirring (10 s equilibration) produced a well-defined stripping peak with E(pa) = -1.2 V versus Ag/AgCl. Using these conditions, the calibration plot was linear over the range 1x10(-8) to 5x10(-6) M Zn(2+). The precision was examined by carrying out six replicate measurements at a concentration of 2x10(-6) M; the coefficient of variation was calculated to be 5.6%. The method was applied to the determination of the analyte in sweat from 10 human volunteers. The concentrations were between 0.39 and 1.56 microg/mL, which agrees well with previously reported values. This simple, low-cost sensitive assay should have application in biomedical studies and for stress and fatigue in sports studies.

Rapid electrochemical detection and identification of catalase positive micro-organisms

The rapid detection and identification of bacteria has application in a number of fields, e.g. the food industry, environmental monitoring and biomedicine. While in biomedicine the number of organisms present during infection is multiples of millions in the other fields it is the detection of low numbers of organisms that is important, e.g. an infective dose of Escherichia coli O157:H7 from contaminated food is less than 100 organisms. A rapid and sensitive technique has been developed to detect low numbers of the model organism E. coli O55, combining Lateral Flow Immunoassay (LFI) for capture and amperometry for sensitive detection. Nitrocellulose membranes were used as the solid phase for selective capture of the bacteria using antibodies to E. coli O55. Different concentrations of E. coli O55 in Ringers solution were applied to LFI strips and allowed to flow through the membrane to an absorbent pad. The capture region of the LFI strip was placed in close contact with the electrodes of a Clarke cell poised at +0.7 V for the detection of hydrogen peroxide. Earlier research identified that the consumption of hydrogen peroxide by bacterial catalase provided a sensitive indicator of aerobic and facultative anaerobic microorganisms numbers. Modification and application of this technique to the LFI strips demonstrated that the consumption of 8 mM hydrogen peroxide was correlated with the number of microorganisms presented to the LFI strips in the range of 2 x 10(1)-2 x 10(7) colony forming units (cfu). Capture efficiency was dependent on the number of organisms applied and varied from 71% at 2 x 10(2) cfu to 25% at 2 x 10(7) cfu. The procedure was completed in less than 10 min and could detect less than 10 cfu captured from a 200 microl sample applied to the LFI strip. The approached adopted provides proof of principle for the basis of a new technological approach to the rapid, quantitative and sensitive detection of bacteria that express catalase activity.

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays.

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 microM (270 microg l(-1)). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml(-1) LDH and 0.8 U ml(-1) LOD in reactions containing 246 microM pyruvate and 7.5 microM NADPH. PCP detection limits were an EC(10) of 800 nM (213 microg l(-1)) and a minimum inhibition detectable (MID) limit of 650 nM (173 microg l(-1)). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 microM. PCP detection limits were obtained for an assay containing 5 U ml(-1) LDH, 0.8 U ml(-1) LOD and 0.1 U ml(-1) GDH with 246 microM pyruvate, 400 mM glucose and 2 microM NADPH. The EC(10) limit was 150 nM (39.9 microg l(-1)) and the MID was 100 nM (26.6 microg l(-1)). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.

Characterization and purification of the vitamin K1 2,3 epoxide reductase system from rat liver

The enzyme vitamin K1 2,3 epoxide reductase is responsible for converting vitamin K1 2,3 epoxide to vitamin K1 quinone thus completing the vitamin K cycle. The enzyme is also the target of inhibition by the oral anticoagulant, R,S‐warfarin. Purification of this protein would enable the interaction of the inhibitor with its target to be elucidated. To date a single protein possessing vitamin K1 2,3 epoxide reductase activity and binding R,S‐warfarin has yet to be purified to homogeneity, but recent studies have indicated that the enzyme is in fact at least two interacting proteins. We report on the attempted purification of the vitamin K1 2,3 epoxide reductase complex from rat liver microsomes by ion exchange and size exclusion chromatography techniques. The intact system consisted of a warfarin‐binding factor, which possessed no vitamin K1 2,3 epoxide reductase activity and a catalytic protein. This catalytic protein was purified 327‐fold and was insensitive to R,S‐warfarin inhibition at concentrations up to 5 mm. The addition of the S‐200 size exclusion chromatography fraction containing the inhibitor‐binding factor resulted in the return of R,S‐warfarin inhibition. Thus, to function normally, the rat liver endoplasmic reticulum vitamin K1 2,3 epoxide reductase system requires the association of two components, one with catalytic activity for the conversion of the epoxide to the quinone and the second, the inhibitor binding factor. This latter enzyme forms the thiol‐disulphide redox centre that in the oxidized form binds R,S‐warfarin.