In a recent article published in the journal Biosensors, researchers highlight the growing concerns about food safety due to the increased use of biocides in agriculture, particularly in dairy products.
Study: Enhancing Biocide Safety of Milk Using Biosensors Based on Cholinesterase Inhibition. Image Credit: Roman Melnyk/Shutterstock.com
Benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC), two commonly used biocides, can pose health risks if residues persist in food. This study focuses on the development and validation of electrochemical enzymatic biosensors for detecting these biocides in cow milk, offering a reliable and sensitive method for monitoring residue levels to enhance food safety and ensure compliance with regulatory standards.
Background
Biocides are widely employed in agriculture to control microbial growth, but their residues in food can have adverse effects on consumer health. Traditional detection methods often rely on complex, time-consuming procedures that are unsuitable for routine analysis. To address this limitation, the authors of this study have proposed an innovative approach using biosensors based on cholinesterase (ChE) inhibition.
Cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are enzymes that hydrolyze acetylcholine. Certain biocides inhibit these enzymes, and this inhibition can be quantitatively measured to provide a reliable detection mechanism. The study’s goal was to design a biosensor leveraging this principle, combining rapid detection with sensitivity for real-world applications.
Study Details
The researchers employed a systematic method to develop the biosensors, starting with the preparation of cholinesterase solutions at a concentration of 0.3 U/mL, stored at 4 °C before use. The biosensors were fabricated using a semi-automatic screen-printing machine, creating a three-electrode system: a carbon working electrode, a carbon counter electrode, and a silver/silver chloride reference electrode. To enhance electron transfer, the working electrode was modified with cobalt phthalocyanine.
Enzyme immobilization, a critical step, was achieved by trapping the enzymes in a polyvinyl alcohol gel, ensuring stability and protection from environmental factors. The biosensors’ performance was assessed by measuring enzyme activity before and after exposure to BAC and DDAC, calculating inhibition rates based on the differences in current responses.
For sample preparation, the study employed liquid-liquid extraction. Milk samples were spiked with BAC and DDAC stock solutions to create the desired concentrations and then treated with formic acid and an extraction solvent. After vortexing, the samples were centrifuged, and the supernatant was evaporated to produce a dry extract suitable for electrochemical analysis.
Results and Findings
The study revealed that the biosensors showed high sensitivity and specificity for detecting BAC and DDAC in milk. Calibration curves demonstrated a strong linear relationship between biocide concentrations and enzyme inhibition rates, with correlation coefficients (R2) indicating a robust fit for both AChE and BChE.
The biosensors were tested on various milk matrices, including whole, partially skimmed, and skimmed milk, confirming their applicability to real-world scenarios. Stability tests showed consistent responses across multiple measurements, and the sensors reliably detected low biocide concentrations, highlighting their suitability for routine monitoring.
The authors also noted significant advantages of electrochemical biosensors over traditional methods. These include faster response times, simpler procedures, and minimal sample preparation, making the technology particularly appealing for use in the dairy industry. Importantly, the findings emphasized the need for ongoing monitoring of biocide levels in food products to protect public health and comply with stringent safety standards.
Conclusion
This study successfully developed and validated electrochemical enzymatic biosensors capable of detecting BAC and DDAC residues in cow milk. The sensors demonstrated impressive sensitivity, specificity, and stability, offering a practical tool for improving food safety. By utilizing cholinesterase inhibition, this innovative method provides a rapid and effective way to monitor biocides in dairy products.
The authors underscore the importance of routine testing for biocide residues to ensure consumer safety and regulatory compliance. Future research could focus on optimizing the biosensor design further and broadening its application to other food matrices, contributing to enhanced safety measures across the agri-food industry.
Journal Reference
Mouawad L., Istamboulie G., et al. (2025). Enhancing Biocide Safety of Milk Using Biosensors Based on Cholinesterase Inhibition. Biosensors 15(1), 26. DOI: 10.3390/bios15010026, https://www.mdpi.com/2079-6374/15/1/26