基于适配体-金属有机框架模拟酶比色检测牛妊娠相关糖蛋白9  

Colorimetric detection of bovine glycoprotein pregnancy associated glycoprotein 9 based on competition analysis and aptamer-metal organic framework mimetic enzyme

作　　者：卢春霞 刘长彬[2] 张振良[2] 卢守亮 倪建宏[2] 王超丽[3] LU Chun-Xia;LIU Chang-Bin;ZHANG Zhen-Liang;LU Shou-Liang;NI Jian-Hong;WANG Chao-Li(School of Advanced Agriculture and Bioengineering,Yangtze Normal University,Chongqing 408100,China;Key Laboratories of Sheep Breeding and Reproduce,Xinjiang Academy of Agriculture and Reclamation Science,Shihezi 832000,China;Xinjiang Western Animal Husbandry Co.,Ltd.,Shihezi 832000,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]新疆西部牧业股份有限公司,新疆石河子832000

出　　处：《化学研究与应用》2025年第2期335-343,共9页Chemical Research and Application

基　　金：兵团重点领域科技计划项目(2023AB009-01)资助。

摘　　要：本研究以金属有机框架Fe-MIL-101作为过氧化物模拟酶,bPAG9适配体为识别探针,基于间接竞争原理建立了一种比色检测bPAG9方法。在方法建立过程中,将互补于适配体的生物素-cDNA固定在微孔板上,加入适配体功能化Fe-MIL-101,适配体与互补链cDNA杂交,当测试样品中含有bPAG9时,适配体与bPAG9结合后从互补链上脱落,然后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实现对bPAG9的定性和定量分析。在优化条件下,bPAG9在缓冲液中线性检测范围为1~100 ng·mL^(-1)(R>0.996),检出限为0.25 ng·mL^(-1)。bPAG9在空白血清中加标回收率为85.2%~102.4%,相对标准偏差小于10%。将本方法应用人工受精28d的奶牛血清检测,妊娠诊断结果显示,本方法诊断敏感性、特异性和准确率分别为91.6%、92.1%和91.8%。本方法具有无创、简便、准确性高等特点,具有良好的应用前景。In this work,a new colorimetric method was developed for the detection of Pregnancy associated bovine glycoprotein pregnancy associated glycoprotein 9(bPAG9)based on indirect competition principle was established,using metal-organic framework FeMIL-101 as peroxide mimicase and bPAG9 aptamer as an recognition probe.In a typical experimental procedure,the biotinylated cDNA complementary to the aptamer was immobilized onto microplate as a capture probes.Fe-MIL-101 functionalized aptamer was added,which was hybridized with complementary cDNA.In the presence of bPAG9,aptamer bind with bPAG9 and then falls off the complementary chain.The Fe-MIL-101enable 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.Under the optimal conditions,the linear range were 1~100 ng·mL^(-1)for bPAG9 in buffered solution,with a limit of detection(LOD)of 0.25 ng·mL^(-1).The average recoveries of bPAG9 in blank serum samples were in the range of 85.2%~102.4%,and the relative standard deviations were less than 10.0%.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 91.6%,92.1%and 91.8%,respectively.The developed assay has such advantages as simple,non trauma,and high accuracy.It has a good application prospect.

关 键 词：妊娠相关糖蛋白 适配体 金属有机框架 早期妊娠诊断 奶牛 

分 类 号：O657.3[理学—分析化学]