激活式复合功能核酸化学发光传感器检测葡萄酒中的赭曲霉毒素A  

Chemiluminescence Sensor Based on Composite FunctionalNucleic Acid for Detection of Ochratoxin A in Wine

作　　者：杨成[1] 张亚旗 林黛琴 刘洋[3] 孙冰冰[1] YANG Cheng;ZHANG Ya-Qi;LIN Dai-Qin;LIU Yang;SUN Bing-Bing(State Key Laboratory of Fine Chemicals,School of Chemistry,College of Chemical Engineering,Dalian University of Technology,Dalian 116024,China;Jiangxi Product Quality Supervision Testing Institute,Nanchang 330052,China;Key Laboratory of Life Organophosphorus Chemistry and Chemical Biology of Ministry of Education,Tsinghua University,Beijing 100084,China)

机构地区：[1]大连理工大学化工学院化学系,精细化工国家重点实验室,大连116024 [2]江西省产品质量监督检测院,南昌330052 [3]清华大学生命有机磷化学及化学生物学教育部重点实验室,北京100084

出　　处：《分析化学》2021年第4期496-503,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.201874080);中央高校基本科研业务费专项资金项目(Nos.DUT17LAB18,DUT15RC(3)064);辽宁省博士科研启动基金项目(No.201501167);电分析化学国家重点实验室开放课题(No.SKLEAC202105);农业部生物毒素检测重点实验室开放课题资助。

摘　　要：构建了以赭曲霉毒素A(OTA)核酸适配体为“裂开型”核酶激活“开关”的化学发光传感器,用于葡萄酒中OTA的高灵敏度检测。K+诱导富含G碱基的寡聚核苷酸折叠成平行G-四链体结构,平行G-四链体与血红素复合生成具有类过氧化物酶活性的核酶,可催化过氧化氢氧化鲁米诺产生化学发光。将OTA核酸适配体序列插入可形成核酶的“裂开型”核酸链中,使核酸适配体成为激活核酶催化活性的“开关”。当体系中不存在OTA时,OTA的核酸适配体呈自由链状,“裂开型”核酶的两段核酸序列距离较远,无法有效形成具有酶活性的复合物,催化活性较低;当OTA存在时,OTA诱导核酸适配体形成反平行G-四链体结构,适配体的两端序列相互杂交,从而拉近“裂开型”核酶的两段核酸序列,使其可形成平行G-四链体结构,并与血红素复合,生成具有明显的类过氧化物酶活性的核酶。以鲁米诺为底物,采用化学发光法实现了OTA的高灵敏检测。本方法的线性范围为0.10~2.00 nmol/L,检出限为0.10 nmol/L。A chemiluminescence sensor,using ochratoxin A(OTA)aptamer as the activation switch of the split DNAzyme,is established to achieve high-sensitivity detection of OTA in wine.Guanine-rich oligonucleotide can fold into a parallel G-quadruplex in the presence of K+.Hemin as a ligand is able to specifically bind to parallel G-quadruplexes.Hemin-G-quadruplex complexes display peroxidase-like activity,named as DNAzyme.The activity of the DNAzyme can be measured by chemiluminescence via the H 2O 2 mediated-oxidation with luminol.Guanine-rich oligonucleotide splits into two fragments,and when they are near to each other,they can reform DNAzyme.OTA aptamer as a switch is inserted into the nucleic acid sequence of DNAzyme.In the absence of OTA,the unfolded OTA aptamer inhibits the split DNAzyme effectively self-assemble to form a structure with enzymatic activity.In the presence of OTA,OTA induces the aptamer to form an antiparallel G-quadruplex and draws the tail of the aptamer closer,so that the two fragments of split DNAzyme refold and complexes with hemin.Folding degree of the aptamer will directly affect the activity of DNAzyme.The high-sensitive detection of OTA can be obtained through monitoring the enhancing ratio of chemiluminescence intensity.The linear range of this method is 0.10-2.00 nmol/L,and the limit of detection is 0.10 nmol/L.

关 键 词：功能核酸 核酸适配体 裂开型核酶 化学发光 赭曲霉毒素A 

分 类 号：TS262.6[轻工技术与工程—发酵工程] O657.3[轻工技术与工程—食品科学与工程]