**5. Acylcholinesterase based sensor for arsenic determination in wine**

Arsenic determination in wine, using the suggested in this work acetylcholinesterase electrochemical sensor, is based on the following reactions:

> acetylthiocholine + H2O *ACh* thiocholine + CH3COOH

#### thiocholine → dithio-bis-choline + 2H+ + 2e-

The acetylcholinesterase Ach (EC 3.1.1.7) catalysed hydrolysis of acetylcholine generates the electroactive product thiocholine. The current of its oxidation is recorded amperometrically at a potential of +0.80 V/SCE. In the presence of As(III), because of the enzyme inhibition that it provokes, the quantity of the produced thiocholine decreases. Thus, the current of its oxidation also decreases as a function of As(III) concentration under similar conditions.

The acetylcholinesterase based electrochemical sensor was prepared as described in our previous works (Stoytcheva et al., 1998a, 1998b), i. e.: acetylcholinesterase was covalently immobilized onto the surface of a rotating disc electrode elaborated from spectrally pure graphite (Ringsdorf Werke, Germany). The analysis was carried out in an electrolysis cell of conventional type, at a temperature of 25oC, with a rotation speed of the working electrode of 1000 rpm. The auxiliary electrode was a glassy carbon electrode. A saturated calomel electrode was used as a reference.

The response of the biosensor was measured for various acetylthiocholine iodide concentrations in the presence of different amounts of As(III) in the form of AsO33- in a Britton-Robinson buffer solution with pH 7. The obtained results are presented in Fig. 2, where I is the difference between the registered steady-state currents of thiocholine oxidation in the absence and in the presence of inhibitor (to note that iodide oxidation to iodine occurs, too).

evaluation using a preliminary constructed calibration curve. The relative error of the

219

The modern food analysis requires sensitive, accurate, and express methods for food safety, food quality, and food technology control. The growing field of the biosensors in food industry represents an answer to this demand. Thus, the method for As(III) determination in wine described in this work is an example demonstrating the viability of the electrochemical

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**6. Conclusion** 

**7. References** 


Fig. 2. Calibration curves for AsO3 3- determination using different substrate concentrations: 1) 0.2 mmol L-1; 2) 0.4 mmol L-1; 3) 0.6 mmol L-1; 4) 1 mmol L-1; 5) 1.2 mmol L-1. pAsO3 is the negative decimal logarithm of the AsO3 3- concentration.

As shown, the linear dynamic range of the calibration curves suitable for AsO3 3 determinations varies from 0.2 nmol L-1 to 0.02 mol L-1. AsO33- concentrations superior to 10 mol L-1 caused an increase of the sensor response, due to the following concurrent reactions:

$$\begin{array}{c} \text{3I} \cdot \text{2e}^{\cdot} = \text{I}\_3\\ \bullet \\ \text{H}\_3\text{AsO}\_3 + \text{I}\_3\text{'} + \text{H}\_2\text{O} = \text{H}\_3\text{AsO}\_4 + \text{3I} + 2\text{H}^+ \end{array}$$

The sensitivity of the determinations, as shown in Table 2, increased with the increase of the acetylthiocholine iodide concentration until the enzyme saturation with 1.0 mmol L-1 acetylthiocholine iodide.


Table 2. Sensitivity of As(III) determination

The method allows As(III) and As(V) differentiation, due to the fact that AsO43- does not inhibit the acetylcholinesterase.

These preliminary results served for the development of a method for As(III) determination in wine. As known, arsenic content in some type of wines exceeds 0.1 mg L-1 (Crecelius, 1997). Arsenic concentration in contaminated illicit whiskey (moonshine) was found to be more than 0.4 mg L-1 (Gerhardt et al., 1980).

For the purposes of the analysis, commercially available wine was artificially contaminated with AsO3 3- 0.0133 mmol L-1 (0.001 mg L-1). The sample, without any pretreatment, was analysed according to the following protocol: (i) registration of the amperometric response of the electrochemical biosensor for a substrate concentration of 1.0 mmol L-1, for which the sensitivity of the AsO33- determination is maximal (25oC, Britton-Robinson buffer 0.1 mol L-1, pH 7, 1000 rpm, +0.80 V/SCE) in the presence of no contaminated wine; (ii) registration of the amperometric response of the electrochemical biosensor in similar conditions, but in the presence of the contaminated wine sample; (iii) I calculation and AsO3 3- concentration evaluation using a preliminary constructed calibration curve. The relative error of the analysis was found to be inferior to 3%.
