Potential measurements were made with JENWAY 3030 Ion Analyser. “A double junction Ag/AgCl electrode 9240368” was used as the outer reference electrode. The enzyme was immobilized on our previously prepared iodide electrode. For the pH measurements, the ion analyzer with 924005 combined pH electrode is used. All measurements were made with a 30 ml glass cell prepared for this purpose. A magnetic stirrer was used throughout the experiments. All reagents used were analytical reagent grade (Merck). Triply distilled water was used for the preparation of solutions.

The iodide electrode in which the enzyme was fixed was prepared according to the procedure developed by us [15]. For this purpose, approximately 180 mg of PVC and 60 mg of ion exchanger (tridodecylmethylammonium iodide) are dissolved in 5 ml of tetrahydrofuran (THF). Then 0.2 ml of plasticizer (dibutyl phthalate) is added and mixed. After evaporation of the solvent the film membrane is cemented to a PVC tube with inner diameter of 10 mm, the tube is filled with 0.1 M KI and 0.1 M NaCl solution. A home-made Ag/AgCl electrode is immersed as the inner reference.

For the immobilization procedure, first 10 mg of enzyme is dissolved in 5 ml phosphate buffer (pH = 7). The iodide electrode prepared as the above given procedure is kept in it for 2 h at room temperature. This electrode was stored in pH 7 buffer at +2˚C when not in use. The measurements are made in 19 ml pH 7 phosphate buffer, 1 ml 1 × 10⁻³ M Mo(VI) and in the presence of 0.01 M iodide solution.

Since one mole of uric acid uses two moles of iodide its concentration has to be higher than uric acid concentration. The optimum concentration of iodide has to be determined for a uric acid concentration that is in the range of glucose present in blood. For this purpose, solutions with 19 ml pH 7 phosphate buffer and 1ml 1 × 10⁻³ M (Mo(VI)) have been prepared containing various iodide concentrations of 1 × 10⁻⁴, 1 × 10⁻³ and 1 × 10⁻² M. Their potentials were measured and then after each uric acid addition once more the potentials were measured. The uric acid concentrations in the cell were in the range of blood serum, changing from 2 × 10⁻⁵ M (3 mg/100ml) to 2 × 10^-4 M (34 mg/100ml blood). As can be seen from Figure 1 the slope was the highest for 10⁻² M iodide concentration.

Adjustment of pH is important both for the immobilization of enzyme and for the reaction between hydrogen peroxide and iodide. At different pH values changing from 9 to 5.5 the response of electrode has been measured against uric acid concentration when 0.01 M iodide was present. Whereas there was nearly no response at pH values of 5.5, 6, 6.5, 8 and 9 the slope was the largest at pH 7. At this pH the ion exchanger (mentioned in Section 3.3) is positive and enzyme

is negative, thus it is the most convenient pH for immobilization and for the reaction.

Phosphate buffer has been chosen because of its pH working area. Its response for uric acid has been investigated at buffer concentrations changing from 1.0 to 10⁻³ M. The change of potential against uric acid concentration is given in Figure 2. As can be seen 0.1 M buffer concentration had the largest slope.

The ion exchanger (TDMAI) on the iodide electrode becomes a positive charge at pH = 7, at this pH the enzyme becomes minus charge and thus the enzyme will be immobilized on the electrode surface. The quantity of enzyme will be important since only one part of it can be immobilized on the electrode surface. For this purpose, iodide electrode was dipped into pH 7 phosphate buffer solutions each containing 5, 10 and 20 mg/ml enzyme for 2 h. It was washed with distilled water and its response has been measured for uric acid concentrations in the presence of 0.01 M iodide. As can be observed from Figure 3, the slope was the largest for 10 mg/ml enzyme.

Optimum temperature is very important since the enzyme activity will increase with temperature, but on the other hand at high temperatures there may be thermal deactivation of the enzyme and also decrease of O₂ concentration. In a solution containing 0.01 M iodide, the potential of a solution of 1 × 10⁻⁴ M uric acid has been followed between temperatures of 30˚C - 60˚C within 5˚C intervals. The maximum activity of the enzyme was obtained at 50˚C (Figure 4).

This electrode did not lose its activity for 45 days when used 4 times a day. The response time was measured at different uric acid concentrations. As can be seen from Figure 5 the response was almost immediate. The lifetime of the electrode is also very good compared with other electrodes [6] [8] [10] [11] [15].

The product of enzymatic reaction is hydrogen peroxide, thus reducing agents such as glucose and ascorbic acid, two compounds commonly found in biological fluids, may interfere [8] [9]. The strong interference of glucose and ascorbic acidin a former study [9] was eliminated after pretreatment with hydrogen peroxide. With our new electrode in the presence of 0.01 M iodide and 2 × 10⁻⁵ M uric acid there was no interference from the above mentioned substances in the concentration ranges that are commonly encountered in biological fluids (0 - 2.5 mM). The selectivity constants determined by using the mixed solution method [16] are given in Table 1.

It was found that, with this new electrode the uric acid could be determined with high accuracy and precision (Table 2). For a solution containing 5 mg/100ml the result obtained for 4 measurements was 4 ± 0.1 mg/100ml.

The blood samples that were analyzed for their uric acid quantity were obtained from the University Health Center. They were first centrifuged with a speed of 9000 round/min and these were used for uric acid determination. First the potential of a solution containing 19 ml buffer (pH = 7), 1 ml 1 × 10⁻³ M Mo (VI) and 0.01 M iodide was measured. A 0.1 ml of serum sample was added and the potential was once more measured. Then two standard additions of 0.1 M uric acid (each 0.1 ml) were made and potentials were measured. From the change of potentials, the amount of uric acid in blood was determined. Blood samples shall not wait long time; otherwise uric acid will be lost because of destruction. If it has to wait additions of fluoride or ascorbic acid is needed. Glucose quantities for three different blood samples are given in Table 3 with the results of University Health Center (colorimetric) for comparison.

^aA: Uric acid; B: Interfering ion.

CI: 95%, N = 4.

95% CI, N = 4.