Contact
Sushanta Mitra, Waterloo Institute for Nanotechnology, Department of Mechanical and Mechatronics Engineering
Introduction
The use of pesticides to enhance food production from agricultural is widespread to meet current and future population demands. Carbamates and organophosphates are common in insecticides, one of the largest types of pesticides, and exposure to them can present risks to human and environmental health. Conventionally, the analysis of insecticides in water is carried out using complex analytical methodologies, which are time-consuming and require expensive high-tech instrumentation.
This paper documents a proof-of-concept study for a device that uses a novel configuration of paper to perform a two-step analytical process to detect insecticides in water. It is hoped that this “dip-and-fold” device may represent a simple, rapid and affordable in situ testing platform for the assessment of insecticides in water, in particular in developing countries where access to advanced facilities and instrumentation may be limited.
Methodology
The device was prepared using high-quality blotting and filter papers configured to test water samples for carbamates or organophosphate using a two-step process: acetylcholinesterase enzyme (AChE) inhibition followed by a colourimetric reaction where the interaction between nitrobenzoic acid (DTNB) and acetylthiocholine Iodide (ATChI) produces a yellow chromophore. The device was designed to allow the liquid sample to move vertically through the porous paper from the bottom dipping zone to the top detection zone by capillarity. Chitosan was affixed to the paper in the detection zone to immobilize AChE (Figure 1). Figure 2 shows the dip and fold device testing procedure.
Figure 1: Conceptual diagram of dip-and-fold device (Reprinted with permission from Marques, F. and Mitra, S. K. Dip-and-Fold Device: A Paper-Based Testing Platform for Rapid Assessment of Insecticides in Water Samples. ACS Applied Bio Materials, November 2021. Copyright 2021 American Chemical Society)
Figure 2: Dip-and-fold device testing procedure: (a) immersing the dipping zone perpendicularly into the water sample allowing the liquid to percolate vertically by capillary action to the detection zone, (b) holding the device in a horizontal position for AChE incubation, (c) folding the device to initiate the colourimetric reaction and (d) reading the color result, yellow indicating the detection of insecticides. (Reprinted with permission from Marques, F. and Mitra, S. K. Dip-and-Fold Device: A Paper-Based Testing Platform for Rapid Assessment of Insecticides in Water Samples. ACS Applied Bio Materials, November 2021. Copyright 2021 American Chemical Society)
Samples consisted of stock solutions of the pesticides propoxur, carbofuran (carbamates) and chlorpyriphos (organophosphate). Ellman’s method was used for the colourimetric analysis, while colour intensity results were analyzed using red, green and blue (RGB) measurements from of digital images of resulting paper strips.
Outcomes
The experiment found that the blotting paper was effective in transporting the water samples to the detection zone with a uniform wicking flow, did not tear apart when saturated and facilitated AChE attachment to the paper substrate. The filter paper used for the immobilization of DTNB and ACThI was also found effective, but demonstrated formulation stability limitations over longer periods, particularly for DTNB which is a light-sensitive compound. In contrast to the DTNB and ACThI that have some motility with water flow, AChE needed to be fixed at the detection zone. Results showed that AChE was effectively immobilized by using chitosan as an intermediary agent.
Results indicated that, in general, colour development in the detection zone was quite fast with the standard read-out of the yellow colour taking place between 5 and 10 minutes after unfolding the device. It was observed that the values of the R, G, and B channels were higher as the concentrations of the target analyte increased for the three chemicals studied. The variation of B channel values was higher than R and G values as the visible yellow colour is produced by the absorption of the wavelength energy of the blue light (Figure 3). The device responded satisfactorily to both carbamates, carbofuran and propoxur, showing good contrast of the B values of 0.01, 0.001, and 0.0001 mM, while the chlorpyriphos results showed a nonregular fluctuation on the B values at an intermediary concentration of 0.001 mM. It was also difficult to distinguish between different chlorpyriphos concentrations visually as the presence of methanol in the chlorpyriphos solution resulted in low solubility and influenced the catalytic function of the AChE enzyme.
Figure 3: Mean values of RGB channels versus the concentration of carbofuran, propoxur and chlorpyriphos in standard aqueous samples. Three replicates give the standard deviation of the mean. The images of the yellow-colored devices tested with solutions at 0.1, 0.01, 0.001, and 0.0001 mM and the negative control (no chemicals). (Reprinted with permission from Marques, F. and Mitra, S. K. Dip-and-Fold Device: A Paper-Based Testing Platform for Rapid Assessment of Insecticides in Water Samples. ACS Applied Bio Materials, November 2021. Copyright 2021 American Chemical Society)
Conclusions
The study successfully tested proof of concept for a novel dip-and-fold device found to be a promising alternative for a fast, easy, inexpensive, in situ testing platform for detecting insecticides in water. The fabrication of the testing device, including the selection of blotting and filter papers and its foldable configuration, the use of reagents and active enzyme to immobilize chemicals, and the use of chitosan to fix AChE onto the blotting paper, proved effective. The study presented promising results for detecting carbamates, represented by carbofuran and propoxur, while the results for chlorpyriphos, an organophosphate, were unclear likely due to the presence of methanol in the solution. In addition, the study demonstrated the successful integration of smartphone photography with colourimetry. The semi-quantification of carbamates and organophosphates may be further optimized by developing a scale-grade colour and integrated digital image analysis system.
Further development of the dip-and-fold device should include investigations that use real water samples, test the device’s stability and identify chemical and physical interfering elements from other contaminants. The development of paper-based device platforms for the colourimetric-based detection of other water contaminants that require multistep protocols, such as synthetic organic chemicals, pharmaceuticals or personal care products, could also be investigated.
Marques, F. and Mitra, S. K. (2021). Dip-and-Fold Device: A Paper-Based Testing Platform for Rapid Assessment of Insecticides in Water Samples. ACS Applied Bio Materials, November 2021. https://doi.org/10.1021/acsabm.1c00986
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