非标记型适配体传感器对石房蛤毒素的高灵敏度检测  

Highly sensitive detection of saxitoxin by a label-free aptasensor

作　　者：奚潇雨 刘英 连志兰 陆峰[1,2] XI Xiaoyu;LIU Ying;LIAN Zhilan;LU Feng(Department of Pharmaceutical Analysis,School of Pharmacy,Naval Medical University(Second Military Medical University),Shanghai 200433,China;School of Health Science and Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China)

机构地区：[1]海军军医大学(第二军医大学)药学系药物分析学教研室,上海200433 [2]上海理工大学健康科学与工程学院,上海200093

出　　处：《海军军医大学学报》2024年第4期441-447,共7页Academic Journal of Naval Medical University

基　　金：国家自然科学基金(82273894);上海市军民融合发展专项(2020-jmrh1-kj11).

摘　　要：目的建立基于荧光染料噻唑橙和核酸适配体45e-1的非标记型适配体传感器,以快速、灵敏地检测石房蛤毒素(STX)。方法将50 nmol/L 45e-1溶解在缓冲液(20 mmol/L Tris-HCl、100 mmol/L NaCl、2.5 mmol/L MgCl_(2)、5 mmol/L KCl,pH=7.5)中,在95℃水浴中加热10 min,冰水浴中冷却5 min。随后加入STX标准溶液,充分混匀,室温孵育10 min后。再加入噻唑橙溶液,添加缓冲液使反应体系体积为100μL,充分混合后室温孵育2 min。最后在496 nm激发波长下测定体系的荧光强度。结果当噻唑橙与45e-1的摩尔浓度比为5∶1、噻唑橙与45e-1的孵育时间为2 min、STX与45e-1的孵育时间为10 min、Mg^(2+)的浓度为2.5 mmol/L、K^(+)的浓度为5 mmol/L时,反应体系的荧光强度与STX浓度呈良好的线性关系,拟合线性方程为Y=3639.8X+2341.5[R^(2)=0.9725,X为STX浓度(nmol/L)的常用对数],检测限为0.67 nmol/L,对其他常见海洋毒素的交叉反应可以忽略。在海水中,该方法的回收率为90.7%~108.7%,RSD为7.1%~10.9%。结论所建立的非标记型适配体传感器可以定量检测STX,具有良好的特异性和重现性。Objective To establish a label-free aptasensor based on the fluorescent dye thiazole orange(TO)and aptamer 45e-1 for rapid and sensitive detection of saxitoxin(STX).Methods The aptamer 45e-1(50 nmol/L)was dissolved in buffer(20 mmol/L trihydroxymethyl aminomethane-HCl,100 mmol/L NaCl,2.5 mmol/L MgCl_(2),5 mmol/L KCl,pH=7.5).It was then heated in a water bath at 95℃for 10 min,followed by cooling in an ice-water bath for 5 min.The STX standard solution was added,thoroughly mixed,and then incubated at room temperature for 10 min.After that,TO solution was added,and buffer was also added to make the reaction system volume 100μL.The mixture was thoroughly mixed and incubated for 2 min at room temperature.Finally,the fluorescence intensity of the system was measured at excitation wavelength of 496 nm.Results When the molar concentration ratio of TO to 45e-1 was 5∶1,the incubation time of TO and 45e-1 was 2 min,the incubation time of STX and 45e-1 was 10 min,the concentration of Mg^(2+)was 2.5 mmol/L,and the concentration of K^(+) was 5 mmol/L,the changes in fluorescence intensity showed a good linear relationship with STX concentration.The fitting linear equation was Y=3639.8X+2341.5(R^(2)=0.9725;X was the common logarithm of STX concentration[nmol/L]),and the detection limit was 0.67 nmol/L.The cross-reaction with other common marine toxins was negligible.In seawater,the recovery rate was 90.7%-108.7%,and the relative standard deviation was 7.1%-10.9%.Conculsion The label-free aptasensor established in this study can achieve the quantitative detection of STX with good specificity and reproducibility.

关 键 词：非标记型 噻唑橙 石房蛤毒素 适配体传感器 

分 类 号：R284.1[医药卫生—中药学] TP212[医药卫生—中医学]