Development of Microfluidic Paper Based Analytical Devices (Μ-Pad) for the Determination of Organophosphate and Carbamate Pesticides from Environmental Samples

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Date

2023-06

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Addis Ababa University

Abstract

This thesis focuses on the development of microfluidic paper-based analytical devices (μ-PADs) and sample preparation techniques, namely dispersive liquid-liquid microextraction (DLLME) and the combination of QuEChERS (quick, easy, cheap, effective, rugged, and safe) with DLLME for colorimetric determination of organophosphate (OP) and carbamate (CM) pesticides from water and lettuce samples. In the colorimetric quantification, the yellow color is generated by the interaction between acetylcholinesterase (AChE), acetylthiocholine iodide (ATChI), and 5, 5- dithiobis- (2-nitrobenzoic acid) (DTNB). The digital image of the yellow color was captured and analyzed using a Canon scanner and ImageJ software, respectively. Key experimental conditions (concentrations and volumes of AChE, ATChI, and DTNB, incubation time, storage stability, and image capturing time) were systematically optimized using a univariate approach. Under optimized conditions, the method showed a wide linear range (1-16 mg L-1), repeatability (3-5% RSD), and intermediate precision (7-10% RSD), and robustness (≤ 1.85% RSD), with R2 ≥ 0.9945. Limits of detection (LOD) were in the range of 0.24-0.33 mg L-1. An acceptable mean recovery (87-94%) was observed at a 1 mg L-1 fortification level. Following that, the μ-PAD operational parameters (reagent concentrations and volumes) were optimized using multivariate techniques based on the Box-Behnken design (BBD). The analysis of variance revealed that high regression and fitting values were obtained between the experimental and predicted responses at a 95% confidence level. Satisfactory linearity (R2 ≥ 0.9990) in the range of 0.5-16 mg L-1, LOD (0.12-0.17 mg L-1), repeatability (3.75-7.98% RSD), intermediate precision (7.15-10.98% RSD), and accuracy (78-97%) were achieved. The results of the univariate and multivariate optimization were compared and found that the multivariate optimization requires a much lower concentration of the reagents and improved LOD values of the univariates by 29-64% for the studied pesticides. Therefore, the multivariate approach has been demonstrated to be more efficient and effective than the univariate approach. For further improving sensitivity, the μ-PAD was coupled with an enrichment method called DLLM. The experimental parameters (volume of hexane and acetone, extraction time, and salt amount) were optimized using response surface methodology (RSM). The enrichment factors (EF) and LOD values were found in the range of 317-1471 and 0.17-0.41 μg L-1, respectively, which is below the maximum residue limits of EU for drinking water. The matrixmatched calibration curve showed a wide linear range (0.625-40 μg L-1) with R2 ≥ 0.9968. The obtained extraction recovery varied from 79-97% with repeatability (2.21-6.01% RSD) and intermediate precision (5.60-10.41% RSD). Thus, the proposed method is low cost, easy to operate, efficient, and sensitive, showing great potential for the determination of pesticides in water samples. Finally, the combination of QuEChERS, DLLME, and a μ-PAD was developed for the extraction, preconcentration and quantification of target analytes from lettuce samples, respectively. RSM using Plackett-Burman and central composite design were used for the screening and optimization of significant factors, respectively. The matrix-matched calibration curves held excellent linearity (R2 ≥ 0.9988) in the range of 0.01-0.64 mg kg-1. The EFs and LODs were 21-31 fold and 0.006-0.009 mg kg-1, respectively. The recoveries were in the range of 79-97% and good precision was obtained with RSD below 11%. Thus, the experimental results revealed that the QuEChERS-DLLME method is suitable for the routine pesticides analysis from lettuce vegetables in terms of high accuracy, precision, and efficiency.

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Keywords

Acetylcholinesterase, Pesticides, Mean Color Intensity, Microfluidic Paper-Based Anal Ytical Devices, Dispersive Liquid- Liquid Microextraction, Box-Behnken Design, Inhibition Percent, Response Surface Methodology, Quechers

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