基于CRISPR/Cas12a偶联金核球形核酸的磺胺二甲嘧啶电化学检测方法  

Electrochemical Method for Sulfamethazine Detection Based on CRISPR/Cas12a Coupled with Spherical Nucleic Acid with the Core of Gold Nanoparticle

作　　者：黄悦 王乾 张岩 祁贤菊 王耀松 HUANG Yue;WANG Qian;ZHANG Yan;QI Xianju;WANG Yaosong(College of Light Industry and Food Engineering,Nanjing Forestry University,Nanjing 210037,China)

机构地区：[1]南京林业大学轻工与食品学院,江苏南京210037

出　　处：《食品工业科技》2025年第8期302-310,共9页Science and Technology of Food Industry

基　　金：国家自然科学基金(31901771)。

摘　　要：磺胺二甲嘧啶在畜禽养殖、水产养殖等农业生产中被广泛用于预防或治疗细菌性疾病。然而,不合理用药会导致其在动物性食品中残留量超标,进而危害人体健康。因此,开展磺胺二甲嘧啶的快速、高灵敏检测对于保障食品安全和人体健康具有重要意义。本工作基于CRISPR/Cas12a偶联金核球形核酸构建了一种新型磺胺二甲嘧啶电化学检测方法,以实现对磺胺二甲嘧啶的快速、高灵敏和高特异性检测。以目标物磺胺二甲嘧啶的适体序列为CRISPR/Cas12a的激活链,磺胺二甲嘧啶与其适体序列的特异性结合会抑制CRISPR/Cas12a的反式切割活性,从而使得预修饰于金电极表面的底物探针得以保留,进而引入金核球形核酸以触发杂交链式反应,最终产生显著的电化学信号放大。为了获得最佳检测性能,对靶标磺胺二甲嘧啶与适体的孵育时间、CRISPR/Cas12a的浓度和作用时间、发夹探针的浓度和反应时间进行了优化,结果分别为30 min、120 nmol/L、30 min、1µmol/L、100 min。所构建的电化学检测方法对磺胺二甲嘧啶的线性检测范围为0.006~600 ng/mL,检测限低至6 pg/mL,同时对目标抗生素与其他干扰抗生素表现出较好的区分能力。此外,本方法成功应用于对实际食品样本中磺胺二甲嘧啶的检测,测得回收率在98.07%~106.90%之间,展现出潜在的食品安全检测应用价值。Sulfamethazine was widely used to prevent or treat bacterial diseases in agricultural production such as livestock breeding and aquaculture.However,irrational use of sulfamethazine would lead to excessive residual amount in animal food,which would harm human health.Therefore,rapid and sensitive detection of sulfamethazine was of great significance for food safety and human health.In this work,a novel electrochemical method for the detection of sulfamethazine was constructed based on CRISPR/Cas12a coupled with spherical nucleic acid of gold nanoparticle core to realize rapid,highly sensitive and specific detection of sulfamethazine.The aptamer sequence of the target sulfamethazine was designed as the activator of CRISPR/Cas12a,and the specific binding of sulfamethazine to its aptamer sequence would inhibit the transcleavage activity of CRISPR/Cas12a,so that the substrate probe pre-modified on the surface of the gold electrode could be retained,and then the spherical nucleic acid could be introduced to trigger the hybridization chain reaction,producing significantly amplified electrochemical signal.To obtain the best analytical performance,several key conditions including the incubation time of sulfamethazine with aptamer,the concentration and cleavage time of CRISPR/Cas12a,the concentration and reaction time of hairpin probes were optimized,and the results were 30 min,120 nmol/L,30 min,1µmol/L,100 min,respectively.Under the optimal conditions,the proposed electrochemical method exhibited prominent quantitative detection performance with the linear detection range of 0.006~600 ng/mL and the detection limit as low as 6 pg/mL.Meanwhile,the method showed good distinguishing ability between target antibiotic and other interfering antibiotics.In addition,the method was successfully applied to the detection of sulfamethazine in actual food samples with the recoveries ranging from 98.07%~106.90%,showing potential application value for food safety detection.

关 键 词：CRISPR/Cas12a 金纳米颗粒 球形核酸 磺胺二甲嘧啶 电化学检测 

分 类 号：TS201.6[轻工技术与工程—食品科学]