Specimen Preparations

7.1.1 Electrodes

Dual band microelectrodes (prepared by Agmet-ESL, Reading, UK, to a design of Prof. D.E. Williams) were used (35). These were made by screen-printing patterns of conductors and insulators on the surface of an alumina tile, then snapping carefidly a pre­ scribed line of weakness to expose a clean electrode surface. The resulting patterns had a thickness o f about 10-15 pm. Figure 7.1 shows a diagram of the screen-printed microbands. Two types o f dual bands were employed in this study: gold-silver and gold- gold bands. Although past studies have revealed impurities in these gold printed electrodes, they are electro active only at potentials below -500 mV with respect to saturated calomel reference electrode (see) (36). Therefore, in terms o f analysis, screen printed electrodes can be used effectively to determine concentrations of redox active species that can be either reduced or oxidized at potential more anodic than -500 mV with respect to see.

The preparation o f these microbands before use is discussed in greater detail in the next chapter under the relevant sections. However, in the case when Au-Au dual bands were employed, it was not necessary to clean them prior to taking electrochemical measurements. Nevertheless, the behavior o f these bands was checked by looking at their cyclic voltammograms. If cleaning was required, then they were cycled in nitric acid (IM ) continuously (-0.1 V to +0.5 V with respect to Ag/AgCl electrode with KCl internal electrolyte) until a reproducible voltammogram was obtained. When a reproducible CV could not be obtained , a new snapped microband was enq)loyed.

7.1.2 Membranes

A number of different membranes were used in this study. Commercially supplied membranes were cut into 1 cm ^ pieces and mounted on glass slides by ‘wetting’ with 10 pi o f the phosphate buffer. After mounting, the membrane preparation comprised the following steps. First, the GOx enzyme solution and the mediator solution o f the desired concentration and volume was dispersed onto the membrane and the water allowed to evaporate. For measurement, glucose solution was introduced into the membrane in the same manner (Figure 7.2). It was important both to prevent the membrane from drying out and to avoid flooding with the solutions. If a membrane did dry out, then it needed to be carefully re-hydrated, by adding 2 pi ahquots o f phosphate buffer. Any excess electrolyte was carefully wiped off using a tissue. For every electrochemical measurement made, a new membrane piece was employed.

Membranes containing skin fluid (which we will refer to as the ‘German membranes’) were supphed by collaborators at Potsdam, Germany (37). When not in use, these membranes were stored in a refrigerator at 4 °C. The membranes used for skin fluid extraction were cuprophane', prepared using DMSO (60%) solution mixture. The presence o f this organic solvent was necessary to trap and preserve skin fluid. For reasons o f commercial confidentiahty, no further details o f the membranes or the skin fluid extraction procedure were revealed by these collaborators.

7.1.3 Reagents and apparatus

Materials and reagents used in the investigation were as follows:

a) The supporting electrolyte was phosphate buffered sodium chloride: 50 mM K2H P O4,

50 mM K H2P O4, 0.1 NaCl (pH = 6.8). Solutions were prepared from triply distilled

deionised water and were degassed with Ar for 20 minutes to remove any dissolved O2

prior to use.

^ Cuprophane is a type o f cellulose membrane, consisting of sequential cellobiose (4-0-beta-D-gluco- pyranosy-l-D-glycopyranose) units. It is prepared by the dispersion of cellulose fibers in solutions of cuoxam (cuprammonium hydroxide) and the regeneration of the insoluble cellulose structure upon membrane formation. Because o f their great degree o f swelling, these membranes are classified as hydrogels (with pore dimensions of 1.72 nm). One disadvantage is that in contrast to hydrogels formed from acrylic polymers, they do not readily absorb globular proteins (Lloyd D.R. Materials Science of Synthetic Membranes ACS Symposium Series 269 (1985) American Chemical Society, 92-103.).

b) D-glucose and potassium ferricyanide (K3Fe(CN)6) were obtained from commercial

sources. GOx (hom Asperigillus niger), type 7-S was purchased from Bioenzyme and use without further purifrcation.

Stock solutions o f D-glucose, potassium ferricyanide and GOx enzyme were prepared in the phosphate buffer. To prevent oxidation with dissolved O2, the GOx and

ferricyanide solutions were prepared immediately prior to use. Standard solutions of glucose of the desired concentration were prepared and left to equilibrate overnight at room temperature. Glucose solutions were used within 2 days of preparation.

In the case o f HPLC studies^ aU chemicals used were o f the highest commercial purity HPLC grade: water, acetonitrile, ethanol and methanol (used to clean the syringe between measurements).

In the HPLC analysis, sample preparation involved the extraction of glucose from the ‘German membranes’. Sample preparation can play an important part in deterrnining the accuracy o f quantification. The most important requirement in any isolation procedure is, o f course, that the compound o f clinical interest is not denatured or destroyed. Solvent mixtures such as alcohol and water have been frequently used to extract sugars from food (as the mixture is an excellent sugar extraction solvent) (38, 39). The extraction procedure carried out in this study will be discussed in more detail in the relevant chapter.

The electrochemical cell incorporating the dual microband electrode was set up as

illustrated in Figure 7.3. The cell set up was inside a Faraday cage and measurements were carried out at room temperature. Unless stated otherwise, a two-electrode configuration was enqjloyed with one o f the bands acting as the working electrode and the other band as the counter-reference electrode. In between measurements, the bands were cleaned by carefully rinsing the surface of the electrode with water and afterwards wiping it with a damp cloth.

7.2. Electrochemical Techniques

The electrochemical techniques used for the detection o f glucose were performed using a commercial computer-controlled electrochemistry system (Ecochimie: Autolab^ ). :

^ The Autolab is a computer-controlled electrochemical measurement system designed for low current applications, in which low noise levels are required. It consists of a data-acquisition system and a potentiostat. The maximum voltage output equals about 10 V. The maximum current capability is 30 mA in which there are six current ranges, between 100 nA and 10 mA. The current is recorded with a good

a) Cyclic voltammetry

This technique was described in detail in Chapter 2.1.2.

In the study, cyclic voltammetry provided a simple means o f monitoring the transition from a frilly reversible^ to irreversible behavior.

b) Chronoamperometry

The measurements of an individual transient for a step to a given potential is a valuable experiment called chronoamperometry. The potential is the variable to be perturbed and the response to the potential perturbation will be the resulting current. The idea behind this technique is that an excited system will have the tendency to reach an equihbrium. The set up for chronoanq)erometric measurements involved stepping the potential for a specified length o f time (in seconds).