LOD of paper strip for pesticide in spiked food sample
In the present study, different groups of pesticide concentrations are prepared in organic solvent and detected using the paper strip assay (Figure 1A). In the presence of pesticide residues like organophosphate, carbamate, organochlorine, and fungicide and herbicide group, the bacillus spores present in the tubes were germinated into vegetative cells followed by the release of marker enzyme, and activity of the marker enzyme get inhibited due to the presence of pesticides residues present in the food system. Therefore, there was no color change due to no hydrolysis of a specific chromogenic substrate, and the strip remains colorless. In the absence of pesticide residues in food, marker enzyme released during spore germination will be intact and hydrolyze the specific chromogenic substrate with the production of blue color (Figure 1B).
The developed paper strip assay was evacuated with a different group of pesticides including organophosphate, carbamate, organochlorine, and fungicide and herbicide group spiked in a pure system as well as a different food system in the range from 100 ppm to 1ppb in an organic solvent for the detection of Limit of Detection (LOD) of the developed assay. The limit of detection in a pure solvent system ranges from 1 to 10 ppb, 1-50ppb, 250-500ppb, 1-50ppb, and 1ppb for organophosphate, carbamate, organochlorine, and fungicide, and herbicide, respectively (Table 1). Further, different concentrations of pesticides spiked inhomogeneous food samples like milk, cereal-based food, and fruit juice sample. After extraction of pesticide, the pesticide residues analyzed by paper strip assay protocol and the LOD of an organophosphate pesticide, carbamate pesticide, organochlorine pesticide, fungicide, and herbicide group in milk ranges from 1-10 ppb, 1-50ppb, 250-500ppb, 1-50ppb, and 1ppb, respectively by strip-based sensor same as obtained in pure system. The LOD for cereal-based food was found to be in the range of 1-100ppb, 10ppb, 1ppb, and 1-100ppb for organophosphate, organochlorine, fungicide, pyrethroid ester, and herbicide, respectively. The LOD for fruit juices are in the range from 1ppb, 1-10ppb, and 1ppb, respectively for organophosphate, fungicide, and herbicide groups. There was no difference in a detection limit of pesticide in the pure system and food sample was observed using paper strips.
Screening of food sample using paper strip assay
Cereal based foods
Cereal-based foods, fruit juices, and milk were screened for pesticide detection. Cereal-based food comprises a total of 19 samples of rice flour, 23 samples of wheat flour, 12 samples of maize flour, 3 samples of cornflakes, and 13 samples of cookies were screened. Among these, 3 each sample of wheat flour, rice flour, and maize flour samples have shown the presence of pesticide residues by not changing the color from colorless to blue color (Figure 2A). There were no positive samples found for corn flakes and cookies. While the using modified QuEChERS and GC-MS were used for the detection of pesticide in whole wheat flour samples collected from the south Brazil Region . The detection of fenitrotion in 20% of maize flour samples found less than permissible limits . The results indicate that different flour and products need continuous checking to warrant food safety.
In the case of fruit juices, a Total of 57 samples of fruit juice collected from the market comprises 12 grapes juice samples, 15 apple juice samples, 15 mixed fruit samples, and 14 pomegranate juice samples. All sample screens for pesticide detection using the paper strip-based sensors. Among the 57 samples, 2 raw un-washed pomegranates and 2 raw un-washed apple juice found positive for pesticide at their MRL level (Figure 2B). However, these pesticides are harmful and not deteriorate naturally and they will present on plant tissue and appear in the pulp and juice. However, the pesticide cannot completely remove from the pulp and juice. Since wherein natural fruit juice has less concentration of pesticide than the processed juice . Children consume more juice than adults and thus the children are more susceptible than adults .
Milk is considered a complete food with a source of protein and major minerals. A total of 125 samples of milk collected which comprises raw milk, pasteurized milk, and UHT milk were screened for pesticide using paper strip-based senor. Among 125 samples of milk comprising 89 raws, 12 pasteurized and 24 UHT milk among that 31 samples of raw milk and 2 samples of pasteurized milk found positive for pesticide (Figure 2C). The assessment and detection of organochlorine pesticide (OC) residues in bovine milk of different places in the Bundelkhand region of India were studied by Nag and Raikwar . They revealed that the concentration of pesticide levels in milk was decreased over the previous studies of particular in India but still contamination present in low concentration. Overall, 33 milk samples, 6 cereal flour samples, and 4 fruit juice samples were found positive for pesticide residues (Figure 2D) by developed paper strip sensors for detection of pesticide residues.
Among 43 positive samples out of 159 samples including milk, cereal products, and fruit juice samples by paper strip sensor for detection of pesticide residues were evaluated for pesticide residues quantitatively by GC-MS/MS. The condition required for multiple reactions monitoring (MRM) method using GC-MS/MS was optimized for the analysis of pesticide residues in food samples (Table 2). In GC-MS/MS analysis about 11 pesticides were targeted from two groups that include organochlorine (Aldrin, dieldrin, endosulfan, and DDT) and organophosphate (Fenithrothion, Chlorpyrifos- methyl, Monochrotofos, Diazinon, Malathion, Phorate, and Chloropyrifos). Among the 43 samples, 7 samples (Raw milk, pasteurized milk, rice flour, wheat flour, maize flour, apple juice, and pomegranate juice) were found positive for different groups of pesticides at trace levels or above the MRL using GC-MS/MS analysis (Figure 3). Three samples of pasteurized milk, rice flour, and wheat flour were positive for the presence of Chlorpyrifos and chlorpyrifos-methyl pesticide residues, respectively at above MRL level prescribed by the Codex Alimentarius Commission (Table 3). In case of other pesticide residues found at trace level that includes DDT, DDD, α- Endosulfan, β – Endosulfan that are found at the level below MRL using GC-MS/MS and these samples were showing positive by our developed paper strip assay. Based on the above comparative study it can be concluded that the developed paper strip assay can be a potential tool in the screening of pesticide residues in a large number of milk samples, cereal flour samples, and fruit juice samples. Similar results were reported by Kowalska et al.  in the products of plant origin and the pesticides identified by HPLC-MS/MS includes azoxystrobin (22.5%), linuron (20.6%), chlorpyrifos, and carbendazim (8.1%), metalaxyl and metalaxyl M (6.9%), and acetamiprid (4.4%). In India, Dwivedi et al.  have reported the presence of Chlorpyrifos in orange juice (1.08 mg/kg) & Deltamethrin (1.28 mg/kg) in ginger garlic at or above the permissible limit of FSSAI. Garcı´a-Reyes et al.  were also reported the presence of pesticide residues in fruit-based soft drinks extracts based on the application of liquid chromatography-electrospray time of-flight mass spectrometry (LC-TOF MS). The contamination of milk with hexachlorocyclohexane (HCH), dichloro-diphenyl trichloroethane (DDT), endosulfan, cypermethrin, cyhalothrin, permethrin, chlorpyrifos, ethion, and profenophos pesticides was also reported in peri-urban bovine milk at or above respective maximum residue limits (MRLs) for pesticide using GC-MS .