Rapid colormetric detection of pesticide residues based on enzyme inhibition method

Abstract: In recent years, due to the frequently reported food safety events, people generally care about the food safety. According to former research, the application amount of organophosphorus pesticides and carbamate pesticides possesses about 70% of the total application amount of pesticides. So, it is necessary to develop fast, sensitive and accurate analysis of pesticide residues to assure the quality of agricultural products. However, the existing food quality supervision system is generally based on the instrument analysis technology, which is time-consuming and high-costing, and will delay the actual release of information for the food safety determination. So it cannot provide the rapid response to the consumer queries. Therefore, there is an urgent need to develop the rapid detection techniques for food safety.A novel disposable enzyme tablet was developed for rapid colorimetric detection of pesticide residues in agricultural products, which based on the inhibition of acetylcholinesterase (AChE) activity by pesticides and the AChE-catalyzed hydrolysis of chromogenic substrate, indoxyl acetate. As known, indoxyl acetate can be catalyzed to indole with the existence of AChE. Indole was oxidized in air quickly, turning to be blue-green, which could be easily distinguished by the naked eye.Firstly, the AChE was immobilized onto the Hybond N+ nylon membrane. It was a crucial work to choose a suitable immobilization method. In our study, the physical absorption method seemed more easy-operative and suitable than the cross-linking since it rarely has no inference on the results. Besides, immobilization temperature, time and methods of drying were optimized in the immobilization process. The enzymatic tablets were immobilized under 4℃ for 30 min, then freeze-dried in a vacuum chamber, the recovery rate of enzyme activity was as high as 27.3%, which considered to be optimal. The other parameters such as substrate concentration, inhibition time and coloring time were optimized. The results were as follows: the best amount of acetate solution was 10 mmol/L, and the inhibition time and the color develop time were 15 and 10 min, respectively.In the measurement process, the color of the enzyme tablet was blue-green. Through analyzing the changes of color intensity, the limit of detection (LOD) of this assay system were 1 μg/mL for omethoate, 0.05 μg/mL for chlorpyrifos, 1.5 μg/mL for carbaryl, and 0.8 μg/mL for pirimicarb, all of them have attained the requirements of the maximum residue limits in food. Detection results of pesticide residues in real samples of grape and cabbage showed that the enzyme tablet had high sensitivity, high degree of accuracy, and good reproducibility, suggesting its great potential for practical application in rapid qualitative determination of organophosphorus (OP) and carbamate (CM) pesticide residues in agricultural products. Furthermore, this assay has long storage time at 60 days. In sum, this work provides a feasible way to develop fast, sensitive detection for pesticide residue.