Enzyme Inhibition-Mediated Distance-Based Paper Biosensor for Organophosphate Pesticide Detection in Food Samples

Analytical improvements shown over four interlaboratory studies of perfluoroalkyl substances in environmental and food samples

An integrated lateral flow test strip with an electrochemical sensor (LFTSES) device with rapid, selective, and sensitive response for quantification of exposure to organophosphorus (OP) pesticides and nerve agents has been developed. The principle of this approach is based on parallel measurements of postexposure and baseline acetylcholinesterase (AChE) enzyme activity, where reactivation of the phosphorylated AChE is exploited to enable measurement of the total amount of AChE (including inhibited and active) which is used as a baseline for calculation of AChE inhibition. Quantitative measurement of phosphorylated adduct (OP-AChE) was realized by subtracting the active AChE from the total amount of AChE. The proposed LFTSES device integrates immunochromatographic test strip technology with electrochemical measurement using a disposable screen printed electrode which is located under the test zone. It shows a linear response between AChE enzyme activity and enzyme concentration from 0.05 to 10 nM, with a detection limit of 0.02 nM. On the basis of this reactivation approach, the LFTSES device has been successfully applied for in vitro red blood cells inhibition studies using chlorpyrifos oxon as a model OP agent. This approach not only eliminates the difficulty in screening of low-dose OP exposure because of individual variation of normal AChE values but also avoids the problem in overlapping substrate specificity with cholinesterases and avoids potential interference from other electroactive species in biological samples. It is baseline free and thus provides a rapid, sensitive, selective, and inexpensive tool for in-field and point-of-care assessment of exposures to OP pesticides and nerve agents.

An analytical method was developed for determining organophosphate pesticides (OPP) and pyrethroid pesticides (PYR) in duplicate-diet solid food. The method consisted of pressurized liquid extraction (PLE) with dichloromethane followed by cleanup with gel permeation and solid phase extraction columns and gas chromatography/mass spectrometry (GC/MS) analysis. Quantitative recoveries (73–117 %) of the target pesticides were obtained for spiked duplicate-diet food samples. The percent standard deviation (% RSD) of replicate food samples was within ± 20 %. Another method was developed for determining a common OPP metabolite, 3, 5, 6-trichloro-2-pyridinol (TCP) in duplicate-diet food. The method consisted of a PLE with methanol followed by liquid-liquid partitioning, derivatization, and GC/MS analysis. Recoveries of TCP ranged from 83 to 101 % for spiked duplicate-diet food samples. The % RSD of replicate food samples was within ± 15 %. The results confirmed that these methods are reliable and robust, and that they can be used in routine analysis. In addition, a storage stability study for a common OPP, chlorpyrifos (CPF), in solid food samples was performed. The fortified 15N-13C-labeled CPF was stable over 16 mo storage at −20° C in the dark. The developed analytical methods were successfully applied to 278 duplicate-diet food samples from preschool children, demonstrating that these methods are robust and suitable for routine analysis in future exposure monitoring studies.

Review of recent developments (2018–2020) on acetylcholinesterase inhibition based biosensors for organophosphorus pesticides detection

Many efforts have been made to evaluate organophosphorus (OP) toxicity by sensitive biomarkers. Therefore, the aim of this work is to evaluate cholinesterase and paraoxonase (PON1) enzymes activities as diagnostic tools in acute & chronic organophosphorus toxicity with poisoning severity assessment.The present study was conducted on 90 adult men after taking their informed consent, they were divided into three groups; group (I) included thirty patients who were acutely exposed to organophosphorus insecticides (OPI). Group (II) included thirty farm workers chronically exposed to OP. Group (III) included thirty healthy matched volunteers served as control group. The severity of symptoms and signs of acute OP poisoning was graded into mild, moderate and severe grade. Long term pesticide exposure intensity was estimated depending on mixing, application methods, repair activities and use of personal protective equipment (PPE). Exposure intensity score = (Mix + Apply + Repair) х PPE. Butyrylcholinesterase (BuChE), acetylcholinesterase (AChE) and paraoxonase (PON1) enzymes activities were determined. The result of the current study revealed significant decrease in BuChE, AChE and PON1 in groups I and II when compared to group III. Moreover, BuChE and AChE enzymes were significantly decreased in group I when compared to group II. Significant positive correlation was detected between AChE enzyme activity and both BuChE and PON1 enzymes activities in group I. However, group IIshowed significant positive correlations between BuChE enzyme activity and the activity of each of AChE and PON1. Moreover, there was significant decrease in AChE enzyme activity in severe cases compared to mild and moderate cases in group I. Nevertheless, group II registered significant decrease in BuChE, AChE and PON-1 when Pesticide Exposure Intensity Score is more than 10.

In September 2014, a small gastroenteritis outbreak occurred in a Greek restaurant. Primary investigations by official food surveillance revealed significant hygienic problems in the premises. Food samples and environmental samples were analyzed for the presence of bacterial and viral food pathogens. Norovirus genogroup I (GI) was detected in 2 environmental samples and in mixed salad. At the same time, stool samples from patients were analyzed and norovirus GI was detected. Further investigations revealed the presence of norovirus GI on some of the restaurant employees. Comparison of nucleic acid sequences revealed full sequence homology between norovirus RNA genotype 1.2 in food, environmental and stool samples, suggesting a common source of contamination and infection. Sequence analysis of food and environmental samples was facilitated by application of a system for total RNA amplification. Despite the fact that original source of contamination could be determined doubtlessly, observed weaknesses in the food production that caused this outbreak were discussed. The mixed salad could have been contaminated either by the lettuce contaminated at primary production or by one of the food-handlers. The investigation of the path of infection is necessary for the kind of legal consequences to be directed by authorities and may contribute to measures to eliminate possible sources of food contamination.

Facile ultrasonication-assisted synthesis of Purpald-functionalized silver nanoparticles for the rapid spectrophotometric detection of acetamiprid pesticide in food and environmental samples

Foodborne illness associated with food service establishments is an important food safety issue in Korea. In this study, foodborne pathogens (Bacillus cereus, Clostridium perfringens, Escherichia coli, pathogenic Escherichia coli, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and Vibrio parahaemolyticus) and hygiene indicator organisms [total viable cell counts (TVC), coliforms] were analyzed for food and environmental samples from foodservice establishments at schools in Gyeonggi province. Virulence factors and antimicrobial resistance of detected foodborne pathogens were also characterized. A total of 179 samples, including food (n=66), utensil (n=68), and environmental samples (n=45), were collected from eight food service establishments at schools in Gyeonggi province. Average contamination levels of TVC for foods (including raw materials) and environmental samples were 4.7 and 4.0 log CFU/g, respectively. Average contamination levels of coliforms were 2.7 and 4.0 log CFU/g for foods and environmental swab samples, respectively. B. cereus contamination was detected in food samples with an average of 2.1 log CFU/g. E. coli was detected only in raw materials, and S. aureus was positive in raw materials as well as environmental swab samples. Other foodborne pathogens were not detected in all samples. The entire B. cereus isolates possessed at least one of the diarrheal toxin genes (hblACD, nheABC, entFM, and cytK enterotoxin gene). However, ces gene encoding emetic toxin was not detected in B. cereus isolates. S. aureus isolates (n=16) contained at least one or more of the tested enterotoxin genes, except for tst gene. For E. coli and S. aureus, 92.7% and 37.5% of the isolates were susceptible against 16 and 19 antimicrobials, respectively. The analyzed microbial hazards could provide useful information for quantitative microbial risk assessment and food safety management system to control foodborne illness outbreaks in food service establishments.

Traditional peroxidase-like nanozyme-based sensors suffer from self-decomposition and high toxicity of H2O2, as well as the interference of color from nanozymes themselves and testing samples. In this work, we adopt nanozymes (two-dimension (2D) MnO2 sheets, manganese dioxide nanosheets (MnNS)) with oxidase-like and peroxidase-like properties as advanced catalysts to develop a novel homogeneous electrochemical sensor for organophosphate pesticides (OPs) using dissolved O2 as a coreactant without the interference of H2O2 and color. Owing to the large surface area and unique catalytic activity of MnNS, a large amount of tetramethylbenzidine (TMB) is catalyzed oxidation, leading to a significantly declined differential pulse voltammetry (DPV) current. Obviously, MnNS display an excellent response to thiocholine, deriving from the catalyzing hydrolysis of acetylthiocholine (ATCh) by acetylcholinesterase (AChE), which switches a homogeneous electrochemical OP detection process based on the depressing AChE activity with a limit of detection (LOD) of 0.025 ng mL-1. The as-proposed strategy on using nanozymes with oxidase-like and peroxidase-like properties to develop a homogeneous electrochemical sensor will provide a new pathway for improving the performance of nanozyme-based sensors, and the established MnNS-based homogeneous electrochemical sensor will find more applications for OP residue determination in food samples.

A reagentless bioactive paper-based solid-phase biosensor was developed for detection of acetylcholinesterase (AChE) inhibitors, including organophosphate pesticides. The assay strip is composed of a paper support (1 x 10 cm), onto which AChE and a chromogenic substrate, indophenyl acetate (IPA), were entrapped using biocompatible sol-gel derived silica inks in two different zones (e.g., sensing and substrate zones). The assay protocol involves first introducing the sample to the sensing zone via lateral flow of a pesticide-containing solution. Following an incubation period, the opposite end of the paper support is placed into distilled deionized water (ddH(2)O) to allow lateral flow in the opposite direction to move paper-bound IPA to the sensing area to initiate enzyme catalyzed hydrolysis of the substrate, causing a yellow-to-blue color change. The modified sensor is able to detect pesticides without the use of any external reagents with excellent detection limits (bendiocarb approximately 1 nM; carbaryl approximately 10 nM; paraoxon approximately 1 nM; malathion approximately 10 nM) and rapid response times (approximately 5 min). The sensor strip showed negligible matrix effects in detection of pesticides in spiked milk and apple juice samples. Bioactive paper-based assays on pesticide residues collected from food samples showed good agreement with a conventional mass spectrometric assay method. The bioactive paper assay should, therefore, be suitable for rapid screening of trace levels of organophosphate and carbamate pesticides in environmental and food samples.

High-resolution mass spectrometry-based screening and dietary intake assessment of organophosphate esters in foodstuffs from South China

Human noroviruses (HuNoVs) are the major cause of non-bacterial acute gastroenteritis worldwide. RT-qPCR is a widely used method to detect HuNoVs. However, the method is unable to extract a virus from environmental samples and to discriminate between infectious and non-infectious viruses. In this study, we explored a new in situ capture RT-qPCR (ISC-RT-qPCR) methodology to estimate the infectivity of HuNoV in environmental and food samples. This assay was based on capturing encapsidated HuNoV by viral receptors, followed by in situ amplification of the captured viral genomes by RT-qPCR. We demonstrated that ISC-RT-qPCR did not capture and enable signal amplification of the heat-denatured Tulane virus (TV) and HuNoVs. Therefore, ISC-RT-qPCR provides better estimates for infectivity of HuNoV than RT-qPCT. We then utilized the ISC-RT-qPCR to detect HuNoV in environmental water samples and food samples, as compared to a conventional RT-qPCR procedure. The presence of HuNoV was examined in 36 oyster samples from retail markets using by both assays for detection. The detection rates of HuNoV in gill, digestive glands, and other tissues were 33.3%, 25%, and 19.4%, respectively, by ISC-RT-qPCR; and were 5.6%, 11.1%, and 11.1%, respectively, by RT-qPCR. ISC-RTqPCR is more sensitive than RT-qPCR for the detection of HuNoV in oysters. By contrast, the HuNoV detection rate by ISC-RTqPCR is lower for environmental samples. Of the 72 water samples that tested positive for HuNoV by RT-qPCR, only 20 (27.8%) of these tested positive by ISC-RT-qPCR, suggesting that 72.2% of RT-qPCR-positive samples were unlikely to be infectious. A better detection rate by ISC-RT-qPCR in oyster samples indicates the likelihood of infectious HuNoV that accumulated in oysters, and a lower detection rate of HuNoV in environmental water by ISC-RT-qPCR, indicating that the majority of RT-qPCR-positive samples were from non-infectious viral RNA.

Chapter 8 Media for Aeromonas spp., Plesiomonas shigelloides and Pseudomonas spp. from food and environment

Media for Aeromonas spp., Plesiomonas shigelloides and Pseudomonas spp. from food and environment

Ultrasensitive electrochemiluminescence biosensor for organophosphate pesticides detection based on carboxylated graphitic carbon nitride-poly(ethylenimine) and acetylcholinesterase