Tests of the Prepared Biosensor

3.2.1. Electrochemical Measurements

Acetylthiocholine chloride (ATChCl) will be used as the substrate. Carry out the electrochemical measurements as follows:

1. Make chronoamperometric measurements at room temperature in PBS by using a potentiostat, imposing a constant potential of 100 mV between screen-printed working and reference (Ag/AgCl) electrodes (see Note 6).

2. To perform the measurements, immerse the biosensor in a cell containing 5 mL PBS under magnetic stirring and measure the stabilized electrical current (nA) (see Notes 6 and 7).

3. Immerse the biosensor in the same cell containing 4950 |L of PBS under magnetic stirring, and add 50 |L of 0.1 M ATChCl solution (substrate final concentration: 10-3 M). Register the stabilized electrical current (nA) after substrate addition.

3.2.2. Optimization of Substrate Concentration

1. Prepare solutions of varied concentrations (0.1-10 mM) in PBS and test the biosensor response (current, in nA) according to the previously described procedure. Construct a calibration curve of substrate concentration (mM) vs current (nA), and select the minimum substrate concentration to furnish a current intensity of approx 100 nA (see Note 8).

3.2.3. Stabilization of the Biosensor

After selecting the best ATCh concentration, repeat the electrochemical measurement during specific enzyme-substrate reaction at least 10 times in order to verify the biosensor stability (see Note 9).

3.2.4. Inhibition Assays

Paraoxon ethyl and dichlorvos of different concentrations will be tested. Carry out the inhibition assays as follows:

1. Record the stabilized current intensity after electrochemical measurement with the optimized substrate solution (So).

2. Carefully wash the biosensor with PBS and immerse it in the pesticide solution. Let it incubate in the pesticide solution for 10 min.

3. Carefully wash the biosensor with PBS and carry out another electrochemical measurement with the same substrate solution. Record the stabilized current intensity (nA) (5j).

4. Calculate the percentage of relative inhibition (RI [%]), according the following equation:

5. Repeat the inhibition assay with the two pesticides at different concentrations and prepare two inhibition curves (pesticide concentration vs relative inhibition). Observe and compare the curves shapes (see Note 10).

3.2.5 Determination of the Detection Limits

Calculate and compare the limits of detection (LOD) for both OP pesticides. This LOD value is calculated with basis on 10% inhibition of the acetylcholinesterase, according to the inhibition assay previously described. Express the results in millimolarity (mM and parts per billion (ppb).

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