Bao Yunxiang, Zhu Dengzhao, Zhao Yang, Li Xinzhu, Gu Chunmei, Yu Hansong
College of Food Science and Engineering Jilin Agricultural University Changchun China.
Division of Soybean Processing Soybean Research & Development Center Chinese Agricultural Research System Changchun China.
Food Sci Nutr. 2022 Jan 19;10(3):953-963. doi: 10.1002/fsn3.2729. eCollection 2022 Mar.
Kunitz trypsin inhibitor (KTI), a harmful protein, seriously affects food hygiene and safety. Therefore, a sensitive, efficient, and rapid method for KTI detection is urgently needed. Aptamers are short and single-stranded (ss) DNA that recognize target molecules with high affinity. This work used graphene oxide-SELEX (GO-SELEX) to screen KTI aptamers. The positive and reverse screening was designed to ensure the high specificity and affinity of the selected aptamers. After 10 rounds of screening, multiple nucleic acid chains were obtained, and the chains were sequenced. Three aptamers with better affinity were obtained, and the values of the dissociation constant ( ) were calculated to be 52.6 nM, 22.7 nM, and 67.9 nM, respectively. Finally, a colorimetric aptamer biosensor based on gold nanoparticles (AuNPs) was constructed. The biosensor exhibited a broader linear range of 30-750 ng/ml, with a lower detection limit of 18 ng/ml, and the spiked recovery rate was between 98.2% and 103.3%. This experiment preliminary demonstrated the potential of the application of KTI aptamer in the real sample tests.
Kunitz胰蛋白酶抑制剂（KTI）是一种有害蛋白质，严重影响食品卫生与安全。因此，迫切需要一种灵敏、高效、快速的KTI检测方法。适体是能以高亲和力识别靶分子的短单链（ss）DNA。本研究采用氧化石墨烯-指数富集的配体系统进化技术（GO-SELEX）筛选KTI适体。通过正向和反向筛选确保所选适体具有高特异性和亲和力。经过10轮筛选，获得多条核酸链，并对这些链进行测序。得到了3条亲和力较好的适体，其解离常数（ ）值分别计算为52.6 nM、22.7 nM和67.9 nM。最后，构建了基于金纳米颗粒（AuNPs）的比色适体生物传感器。该生物传感器线性范围更宽，为30 - 750 ng/ml，检测下限为18 ng/ml，加标回收率在98.2%至103.3%之间。本实验初步证明了KTI适体在实际样品检测中的应用潜力。
