Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jan 6;15(1):26.
doi: 10.3390/bios15010026.

Enhancing Biocide Safety of Milk Using Biosensors Based on Cholinesterase Inhibition

Lynn Mouawad  1   2 , Georges Istamboulie  1   2 , Gaëlle Catanante  1   2 , Thierry Noguer  1   2
Affiliations

Enhancing Biocide Safety of Milk Using Biosensors Based on Cholinesterase Inhibition

Lynn Mouawad et al. Biosensors (Basel). .

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Principle of electrochemical detection of cholinesterase activity.
Figure 2
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
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
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
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
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.
See this image and copyright information in PMC

References

    1. Vereshchagin A.N., Frolov N.A., Egorova K.S., Seitkalieva M.M., Ananikov V.P. Quaternary ammonium compounds (QACs) and ionic liquids (ILs) as biocides: From simple antiseptics to tunable antimicrobials. Int. J. Mol. Sci. 2021;22:6793. doi: 10.3390/ijms22136793. - DOI - PMC - PubMed
    1. Gaudin V., Soumet C. Development of enzymatic biosensors to detect biocide disinfectants to strengthen self-monitoring in industry. Eng. Proc. 2021;6:36. doi: 10.3390/I3S2021Dresden-10077. - DOI
    1. Choi Y., Kang M., Huh D., Chae W., Moon K.W. Priority setting for management of hazardous biocides in Korea using chemical ranking and scoring method. Int. J. Environ. Res. Public Health. 2020;17:1970. doi: 10.3390/ijerph17061970. - DOI - PMC - PubMed
    1. Bertuzzi T., Pietri A. Determination of benzalkonium homologues and didecyldimethylammonium in powdered and liquid milk for infants by hydrophilic interaction liquid Chromatography–Mass spectrometry. Food Anal. Methods. 2014;7:1278–1284. doi: 10.1007/s12161-013-9745-y. - DOI
    1. Journal of the European Union, EU Law n° 1119/2014. [(accessed on 12 September 2024)]. Available online: www.eur-lex.europa.eu.

LinkOut - more resources