Institute of Agrochemistry and Food Technology, Spanish Council for Scientific Research (IATA-CSIC), Av. Agustí Escardino 7, Paterna, 46980, Valencia, Spain.
Department of Analytical Chemistry, University of Valencia, Dr. Moliner 50, Burjassot, 46100, Valencia, Spain.
Anal Bioanal Chem. 2024 Dec;416(30):7395-7404. doi: 10.1007/s00216-024-05428-0. Epub 2024 Jul 9.
Proquinazid is a new-generation fungicide authorized in the EU for combating powdery mildew infections in high-value crops. Due to the perishable nature of fruits, alternative analytical methods are necessary to protect consumer's health from pesticide residues. Currently, immunoassays are a well-established approach for rapidly monitoring chemical contaminants. However, the production of high-quality immunoreagents, such as antibodies and bioconjugates, is essential. This study presents a newly designed hapten that maintains the characteristic moieties of proquinazid unmodified. The linear aliphatic substituents of this molecule were used to introduce the spacer arm. A three-step synthesis strategy was optimized to prepare a hapten that displays the entire 6-iodoquinazolin-4(3H)-one moiety with excellent yields. The N-hydroxysuccimidyl ester of the hapten was activated and purified to prepare a protein conjugate with high hapten density, which was used as an immunogen. Antibodies were raised and competitive enzyme-linked immunosorbent assays were developed. To enhance the assay's sensitivity, two additional heterologous haptens were prepared by modifying the halogenated substituent at C-6. The optimized assays demonstrated low limits of detection in buffer, approximately 0.05 μg/L. When applied to the analysis of proquinazid in QuEChERS extracts of strawberry samples, the immunoassays produced precise and accurate results, particularly in the 10-1000 μg/kg range.
丙嗪菌胺是一种新一代杀菌剂，在欧盟获准用于防治高价值作物的白粉病感染。由于水果易腐，需要替代的分析方法来保护消费者的健康免受农药残留的影响。目前，免疫分析是快速监测化学污染物的一种成熟方法。然而，生产高质量的免疫试剂，如抗体和生物缀合物，是必不可少的。本研究提出了一种新设计的半抗原，它保持了丙嗪菌胺未修饰的特征部分。该分子的线性脂肪取代基被用来引入间隔臂。优化了三步合成策略，以制备以高收率展示整个 6-碘喹唑啉-4(3H)-酮部分的半抗原。半抗原的 N-羟基琥珀酰亚胺酯被激活并纯化，以制备具有高半抗原密度的蛋白质缀合物，用作免疫原。制备了抗体，并开发了竞争性酶联免疫吸附测定法。为了提高测定的灵敏度，通过修饰 C-6 上的卤代取代基，制备了另外两个异源半抗原。优化后的测定法在缓冲液中的检测限约为 0.05μg/L。当应用于草莓样品的 QuEChERS 提取物中丙嗪菌胺的分析时，免疫分析产生了精确和准确的结果，特别是在 10-1000μg/kg 范围内。
