基于适配体-MOF模拟酶比色检测bPAG9  

A Colorimetric Detection of bPAG9 Based on Aptamer and Peroxidase-like Mimics of Metal Organic Framework

作　　者：卢春霞 刘长彬[2] 张振良[2] 卢守亮 吴世嘉[1,3] LU Chun-xia;LIU Chang-bin;ZHANG Zhen-liang;LU Shou-liang;WU Shi-jia(School of Advanced Agriculture and Bioengineering,Yangtze Normal University,Chongqing 408100,China;State Key Laboratories of Sheep Genetic Improvement and Healthy Production,Xinjiang Academy of Agriculture and Reclamation Science,Shihezi 832000,China;School of Food Science and Technology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]长江师范学院现代农业与生物工程学院,重庆408100 [2]新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子832000 [3]江南大学食品学院,江苏无锡214122

出　　处：《分析测试学报》2024年第5期714-721,共8页Journal of Instrumental Analysis

基　　金：兵团重点领域科技计划(2023AB009-01)。

摘　　要：该研究基于金属有机框架Fe-MIL-101和双适配体夹心原理建立了一种比色检测牛妊娠相关糖蛋白9(bPAG9)的新方法。将生物素化适配体1固定在微孔板上作为捕获探针,Fe-MIL-101功能化的适配体2作为信号探针,当测试样品中含有bPAG9时,适配体1和适配体2与bPAG9结合,在固相载体表面形成双适配体夹心结构,随后适配体2偶联的Fe-MIL-101催化无色过氧化物酶底物产生比色输出信号,通过比色信号的变化即可实现对bPAG9的定性和定量分析。结果显示:在优化条件下,bPAG9在缓冲液中的线性检测范围为0.5~50 ng/mL(r=0.996),检出限为0.13 ng/mL。bPAG9在空白血清中的加标回收率为89.4%~104%,相对标准偏差小于10%。将该方法应用于人工受精28 d的奶牛血清检测,妊娠诊断结果显示,该法的诊断敏感性、特异性和准确率分别为92.5%、90.0%和91.5%。所建方法具有无创、简便、经济等特点,在奶牛早孕检测领域具有良好的应用前景。The aim of this study is to establish a new colorimetric method for the detection of bovine pregnancy associated glycoprotein 9(bPAG9)based on the metal-organic framework Fe-MIL-101 and double aptamer sandwich principle.In a typical experimental procedure,biotinylated aptamer1 was immobilized onto microplate to act as capture probes.Fe-MIL-101 functionalized aptamer 2 act as a signal probe.In the presence of bPAG9,aptamer 1 and aptamer 2 bind with bPAG9 and forma double aptamer sandwich structure on solid phase carrier.Fe-MIL-101 coupled to aptamer 2 enable to catalyze the colorless peroxidase substrate to yield a colorimetric output signal.Thus,the qualitative and quantitative analysis of bPAG9 could be realized by the change of colorimetric signal.The results showed that under the optimal conditions,the linear detection range were 0.5-50 ng/mL for bPAG9 in buffered solution,with a limit of detection(LOD)of 0.13 ng/mL.The average recoveries of bPAG9 in blank serum samples were in the range of 89.4%-104%,and the relative standard deviations were less than 10%.Subsequently,the serum samples from Holstein cows at 28 days after insemination were analyzed by the developed assay.The test results were confirmed with ultrasonography and compared with PAG-ELISA test kit.The diagnostic sensitivity,specificity,and accuracy of the developed assay were 92.5%,90.0%and 91.5%,respectively.The developed assay has such advantages as simple,noninvasive,and economical.It has a good application prospect in the field of cow early pregnancy detection.

关 键 词：妊娠相关糖蛋白 适配体 金属有机框架 妊娠诊断 奶牛 

分 类 号：O629.7[理学—有机化学] S826[理学—化学]