Development of Enzymatic Biosensors to Detect Biocide Disinfectants to Strengthen Self-Monitoring in Industry

: Biocide disinfectants are used in agro-food industries in order to limit the development of pathogens present in environment or on surfaces in contact with food for human or animal consumption. Biocide residues remaining on food surfaces may constitute a toxicological risk for the consumer. Very little feedback from the ﬁeld on the rates of biocide residues in the industry exist due to a lack of simple, fast and responsive self-checking methods. The development of biosensors for the detection of biocide disinfectants represents a promising way to explore this, but most of this research remains limited.


Introduction
The implementation of cleaning and disinfection plans, essential elements in the control of hygiene and food safety for consumers, is a requirement of the "Food Law" (Regulation EC 852/2004) [1].A wide variety of these products are used on a daily basis throughout the food chain to limit the development of undesirable microorganisms present in the environment or on surfaces in contact with food intended for human or animal consumption.Following these treatments, biocidal product residues may persist on the surfaces, in particular if these residues are not completely removed during rinsing operations.It has also been proven that their transfer to food is possible.In 2012, a European Food Safety Agency (EFSA) investigation found excessive amounts of two quaternary ammonium compounds (QACs) [2].The greatest number of positive results (12%) was found in milk and dairy products, with contamination levels ranging from a few tens to a few thousand µg/kg.The presence of residues may be associated with (i) toxicological risks and (ii) emergence of resistance to biocides and cross-resistance with antibiotics.Therefore, cleaning and disinfection procedures must be continuously and effectively monitored to verify their suitability and effectiveness (Biocide Regulation 528/2012) [3].
However, self-checking in food industry is very limited to date and uses insensitive methods.The performances announced by the manufacturers are not subject to any evaluation or official validation.An inventory of tests for the detection of residues of disinfectant biocides has enabled us to show the few commercial methods available for self-monitoring [4].In the context of collaborative projects, we have established that the detection limits were high, in the order of 0.5 to more than 2000 mg/kg, i.e., 1000 times less sensitive than methods based on mass spectrometry [4].In addition, these methods are not very selective (e.g., pH, conductivity) or too specific (e.g., ELISA).Despite the fact that their sensitivity thresholds are high, strip tests are the most used by food industries.This is why it is necessary to develop innovative tools, such as biosensors that are sensitive, fast, inexpensive, portable, and even online, to set up more efficient self-monitoring, and thus better protect consumer health.Many biosensors based on the inhibition of enzymatic activity were built for the detection of organophosphate pesticides (OPP) [5][6][7][8].These works draw inspiration for developing biosensors based on the use of enzymes as a recognition tool for the detection of disinfectant biocides.The results of the work on pesticides make it possible to reach detection limits of the order of µg/kg, i.e., 1000 times more sensitive.

Development of Enzymatic Biosensors for the Detection of Biocide Disinfectants
Due to the structural resemblance of organophosphate pesticides (OPP) to the substrate (i.e., acetylcholine) of acetylcholinesterase (AChE), these pesticides cause a reversible inhibition of this enzyme since they take the place of the substrate at the level of the active site of the enzyme [5].The QAs have a chemical structure similar to acetylcholine, such as OPP.The reversible inhibitory effect of certain QACs (tetraethylammonium ions) on the activity of AChE (e.g., from electric eel) was demonstrated in 1952 by a manome-tric method [9].This project will focus on enzymatic biosensors (i.e., AchE) for the detection of QACs, commonly used in the dairy industry (e.g., benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDAC)) (Figure 1).Three transduction modes (colorimetric, fluorimetric, electrochemical) were selected because they have common advantages: simplicity, speed, low cost, little or no investment in hardware, miniaturization and portability [10].The main challenges will be selectivity, accessibility of the active site of the immobilized enzyme, stability and repeatability.The poster file presented at the 8th International Symposium on Sensor Science (I3S) (17-26 May 2021) will be published as Supplementary Material.
Eng. Proc.2021, 6, 36 2 of 4 fast, inexpensive, portable, and even online, to set up more efficient self-monitoring, and thus better protect consumer health.Many biosensors based on the inhibition of enzymatic activity were built for the detection of organophosphate pesticides (OPP) [5][6][7][8].These works draw inspiration for developing biosensors based on the use of enzymes as a recognition tool for the detection of disinfectant biocides.The results of the work on pesticides make it possible to reach detection limits of the order of µ g/kg, i.e., 1000 times more sensitive.

Development of Enzymatic Biosensors for the Detection of Biocide Disinfectants
Due to the structural resemblance of organophosphate pesticides (OPP) to the substrate (i.e., acetylcholine) of acetylcholinesterase (AChE), these pesticides cause a reversible inhibition of this enzyme since they take the place of the substrate at the level of the active site of the enzyme [5].The QAs have a chemical structure similar to acetylcholine, such as OPP.The reversible inhibitory effect of certain QACs (tetraethylammonium ions) on the activity of AChE (e.g., from electric eel) was demonstrated in 1952 by a manometric method [9].This project will focus on enzymatic biosensors (ie.AchE) for the detection of QACs, commonly used in the dairy industry (e.g., benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDAC)) (Figure 1).Three transduction modes (colorimetric, fluorimetric, electrochemical) were selected because they have common advantages: simplicity, speed, low cost, little or no investment in hardware, miniaturization and portability [10].The main challenges will be selectivity, accessibility of the active site of the immobilized enzyme, stability and repeatability.The poster file presented at the 8th International Symposium on Sensor Science (I3S) (17-26 May 2021) will be published as Supplementary Material.One work focused on developing an electrochemical (conductimetric) biosensor based on AChE for the detection of two QACs (BAC, hexadecylpyridinium bromide) in an aqueous solution [11], while hundreds of articles have been published on the detection of pesticides in this way.Therefore, the strategies to be investigated for the detection of QACs by enzymatic biosensor and the recent development of nanotechnologies (i.e., Nanomaterials) make it possible to predict a sensitivity in line with our expectations [12].One work focused on developing an electrochemical (conductimetric) biosensor based on AChE for the detection of two QACs (BAC, hexadecylpyridinium bromide) in an aqueous solution [11], while hundreds of articles have been published on the detection of pesticides in this way.Therefore, the strategies to be investigated for the detection of QACs by enzymatic biosensor and the recent development of nanotechnologies (i.e., Nanomaterials) make it possible to predict a sensitivity in line with our expectations [12].

Figure 1 .
Figure 1.Description of the principle of biosensors based on the enzymatic inhibition of acetylcholinesterase (AChE) by quaternary ammoniums compounds (QACs).

Figure 1 .
Figure 1.Description of the principle of biosensors based on the enzymatic inhibition of acetylcholinesterase (AChE) by quaternary ammoniums compounds (QACs).