Acetylcholinesterase- and Butyrylcholinesterase-Based Biosensors for the Detection of Quaternary Ammonium Biocides in Food Industry

Abstract

A sensitive and robust electrochemical cholinesterase-based sensor was developed to detect the quaternary ammonium (QAs) biocides most frequently found in agri-food industry wash waters: benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC). To reach the maximum residue limit of 28 nM imposed by the European Union (EU), two types of cholinesterases were tested, acetylcholinesterase (AChE, from Drosophila melanogaster) and butyrylcholinesterase (BChE, from horse serum). The sensors were designed by entrapping AChE or BChE on cobalt phthalocyanine-modified screen-printed carbon electrodes. The limits of detection (LOD) of the resulting biosensors were 38 nM for DDAC and 320 nM for BAC, using, respectively, AChE and BChE. A simple solid-phase extraction step was used to concentrate the samples before biosensor analysis, allowing for the accurate determination of DDAC and BAC in tap water with limits of quantification (LOQ) as low as 2.7 nM and 5.3 nM, respectively. Additional assays demonstrated that the use of a phosphotriesterase enzyme allows for the total removal of interferences due to the possible presence of organophosphate insecticides in the sample. The developed biosensors were shown to be stable during 3 months storage at 4 °C.

Keywords: biocides; biosensor; cholinesterases; phosphotriesterase; quaternary ammoniums; screen-printed electrodes.

Figure 1

Chemical structures of BAC (a) and DDAC (b) biocides.

Figure 2

Schematic representation of the biosensor immersed in the thermostated cell.

Figure 3

Response of the biosensor as a function of substrate (AtCh) concentration at different temperatures: (a) BChE-based biosensor (b) AChE-based biosensor.

Figure 4

Inhibition effect of BAC (a) and DDAC (b) biocides on AChE- and BChE-based biosensors. Biocides were either prepared in distilled water (DW) or tap water (TW). Equations of the obtained curves are the following: (a) AChE-DW: y = 19.273ln(x) + 271.23 (R² = 0.9864); AChE-TW: y = 19.625ln(x) + 277.62 (R² = 0.9863); BChE-DW: y = 16.775ln(x) + 261.12 (R² = 0.9883); BChE-TW: y = 16.952ln(x) + 265.6 (R² = 0.9913). (b) AChE-DW: y = 9.7538ln(x) + 176.67, R² = 0.9839); AChE-TW: y = 9.8656ln(x) + 181.03 (R² = 0.9736); BChE-DW: y = 13.757ln(x) + 221.11 (R² = 0.9935); BChE-TW: y = 12.983ln(x) + 213.91 (R² = 0.9920).

Figure 5

Relative response of AChE (dark bars) and BChE (white bars) biosensors during storage at 4 °C. The biosensor response was tested in presence of acetylthiocholine at 1 mM, as described in inhibition experiments.

Figure 6

Comparison of AChE (a) and BChE (b) sensor inhibition by BAC and DDAC at different concentrations, in absence (dark bars) or presence (white bars) of PTE at 0.8 U/mL and PO insecticide at 10 µM.

Figure 6

Comparison of AChE (a) and BChE (b) sensor inhibition by BAC and DDAC at different concentrations, in absence (dark bars) or presence (white bars) of PTE at 0.8 U/mL and PO insecticide at 10 µM.