Preparation of New Uric Acid Sensors Based on Iodide Selective Electrode

An electrode for uric acid has been prepared by using an iodide selective electrode with the uricase enzyme. The iodide selective electrode used was prepared from 10% TDMAI and PVC according to our previous study. The enzyme was immobilized on the iodide electrode by holding it at pH 7 phosphate buffer for 20 min at room temperature. The H2O2 formed from the reaction of uric acid was determined from the decrease of iodide concentration that was present in the reaction cell. The potential change was linear in the 2 × 10 to 2 × 10 M uric acid concentration (3 34 mg uric acid/100ml blood) range. Uric acid contents of some blood samples were determined with the new electrode and consistency was obtained with a colorimetric method. The effects of pH, iodide concentration, the amount of enzyme immobilized and the operating temperature were studied. No interference of ascorbic acid, glucose and urea was observed.


Introduction
Enzyme electrodes are being used for the measurement of different organic substrates. In most of them potentiometric sensors, e.g. oxygen [1] carbon dioxide [2], or ammonium ion electrodes [3] have been used, but some of them use different voltammetric measuring techniques.
the system resulting from enzymatic reaction of uricase and HRP (horseradish peroxidase), which is involved in oxidizing uric acid to allaintoin and hydrogen peroxide. The linearity of the system toward uric acid was in the concentration range of 125 -1000 µM with detection limit of 125 µM [13].
An electrochemical biosensor based on gold and palladium nano particlesmodified nanoporous stainless steel (Au-Pd/NPSS) electrode has been introduced for the simultaneous determination of levodopa (LD) and uric acid (UA). Differential pulse voltammetry (DPV) was used for the simultaneous determination of LD and UA [14].
In this study, uricase was trapped in plasticized PVC and iodide ion selective electrode was used to monitor iodide. This electrode has an average slope of 63 mV/ten uric acid. When this electrode is used 4 times a day, it has a life of 70 days. The electrode is not sensitive to glucose and ascorbic acid. This study describes the preparation and application of a new potentiometric uric acid sensor.

Apparatus and Reagents
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.

Preparation of Electrode
The iodide electrode in which the enzyme was fixed was prepared according to the procedure developed by us [15].

Result and Discussion
Uric acid is oxidized by air oxygen in the presence of uricase enzyme and hydrogen peroxide is formed, which reacts with iodide ion quantitatively. Thus, this reaction can be used for the determination of uric acid when known concentration of iodide is present. The decrease of iodide concentration will be proportional to uric acid concentration according to the following reactions.  [11]. As can be seen two moles of iodide are used for one mole of uric acid. Thus the change of concentration of iodide after reaction with uric acid can be used for the determination of uric acid. In this work we used iodide electrode for the determination of iodide concentration before and after the reaction with uric acid.

Effect of Iodide Concentration
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

Immobilization of Enzyme
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.

Effect of Temperature
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 O 2 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).

Response and Lifetime
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].

Interference Studies
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.

Measurement of Uric Acid in Blood Serum
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.

Conclusion
A new enzyme based electrode is prepared by using an iodide selective electrode.
Here one electrode works as enzyme holder and at the same time it monitors the iodide concentration. Since the iodide electrode is constructed with an ion ex-