Enhancing Biocide Safety of Milk Using Biosensors Based on Cholinesterase Inhibition

Abstract

A sensitive and reliable electrochemical biosensor for the detection of benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC), the most commonly used disinfectant biocides in the agri-food industry, is described. Acetylcholinesterase from Drosophila melanogaster (DM AChE) and butyrylcholinesterase from horse serum (BChE) were immobilized by entrapment in a photocrosslinkable polymer on the surface of carbon screen-printed electrodes. Preliminary tests conducted in phosphate buffer showed limits of detection (LODs) of 0.26 µM for BAC using the BChE-based sensor and 0.04 µM for DDAC using the DM AChE sensor. These performances comply with the European regulation for dairy products, which sets a maximum allowable concentration of 0.28 µM for biocides. However, when tested directly in milk samples, a dramatic decrease in the sensitivity of both sensors towards BAC and DDAC biocides was reported. To overcome this problem, a simple liquid-liquid extraction was necessary prior to biosensor measurements, ensuring that the biosensors met European regulatory standards and provided an unbiased response.

Keywords: biocides; biosensor; cholinesterases; quaternary ammoniums.

Figure 1

Principle of electrochemical detection of cholinesterase activity.

Figure 2

Inhibition effect of BAC (a) and DDAC (b) biocides on AChE- and BChE-based biosensors containing a 0.9 mU/electrode. The equations of the calibration curves are the following: (a) DM AChE: y = 19.27ln(x) + 271.23, R² = 0.986; BChE: y = 15.95ln(x) + 251.96, R² = 0.993; (b) DM AChE: y = 9.75ln(x) + 176.67, R² = 0.983; BChE: y = 13.75ln(x) + 221.11, R² = 0.993.

Figure 3

Inhibition effect of BAC biocide on (a) DM AChE- and (b) BChE-based biosensors. Measurements were carried out in whole milk (WM), partially skimmed milk (PSM), and skimmed milk (SM). The equations of the obtained curves are the following: (a) WM: y = 28.04ln(x) + 234.41, R² = 0.989, PSM: y = 18.82ln(x) + 205.61, R² = 0.985, SM: y = 17.3ln(x) + 219.82, R² = 0.999; (b) WM: y = 18.57ln(x) + 194.88, R² = 0.987, PSM: y = 13.43ln(x) + 173.27, R² = 0.939, SM: y = 17.62ln(x) + 248.32, R² = 0.971.

Figure 4

Inhibition effect of DDAC biocide on (a) DM AChE- and (b) BChE-based biosensors. Measurements were carried out in whole milk (WM), partially skimmed milk (PSM), and skimmed milk (SM). The equations of the obtained curves are the following: (a) WM: y = 11.79ln(x) + 123.54, R² = 0.974, PSM: y = 18.81ln(x) + 220.76, R² = 0.968, SM: y = 35.69ln(x) + 443.14, R² = 0.989; (b) WM: y = 27.1ln(x) + 242.06, R² = 0.981, PSM: y = 31.36ln(x) + 320.38, R² = 0.960, SM: y = 41.38ln(x) + 495.08, R² = 0.992.

Figure 5

The recovery rates obtained for BAC after liquid–liquid extraction from whole milk (WM), partially skimmed milk (PSM), and skimmed milk (SM). The samples were spiked with BAC at concentrations corresponding to IC10 (LOD), IC50, and IC80. Measurements were carried out using (a) DM AChE- or (b) BChE-based biosensors.

Figure 6

The recovery rates obtained for DDAC after liquid–liquid extraction from whole milk (WM), partially skimmed milk (PSM), and skimmed milk (SM). The samples were spiked with DDAC at concentrations corresponding to IC10 (LOD), IC50, and IC80. Measurements were carried out using (a) DM AChE- or (b) BChE-based biosensors.