Fig. 2. Two-channel screen-printed electrodes used for AChE-biosensor construction.

and enzyme activity may be determined with these solutions by employing the Ellman method (9).

3. Methods

3.1. Preparation of the Screen-Printed Electrodes

A single-channel two-electrode sensing strip (7 mm x 44 mm x 0.5 mm) will be used in this work. Figure 2 presents a two-channel sensor strip, prepared as follows (10,11):

1. Use PVC supports to prepare the strips with a DEK 248 screen-printer (DEK).

2. Print the conducting silver track (Electrodag PF410, Acheson) that forms the electrical connections between reference and working electrodes.

3. Print the graphite layer (Electrodag 423SS, Acheson) over the silver tracking to prevent contact between the tracking and applied analyte solution.

4. Print the silver/silver chloride ink (Electrodag 603SS, Acheson) reference electrode over the graphite layer on alternative silver tracks.

5. Print the working electrode prepared by mixing graphite T15 (Lonza) with TCNQ-mediator and hydroxyethyl-cellulose (HEC) over the remaining graphite-coated silver tracks. Prepare the graphite-TCNQ-HEC mixture by diluting 1 g graphite in 75 mL acetone followed by vacuum-evaporation for 30 min; then mix 0.9 g of the resulting powder with 5.8 mL of a 3% (w/v) HEC aqueous solution.

6. After homogenization, deposit the resulting paste by printing it on the working electrode. After applying the mediator layer, dry the electrodes at 60°C before enzyme immobilization for a minimum of 2 d.

7. Finally, deposit 2 ||L of the sensitive paste, consisting of a mixture of determined volumes of PVA-SbQ and AChE solution (final amounts: 30% [v/v] for PVA-SbQ and 1 U/mL for AChE) (see Note 5).

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