| 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/LMgCl2、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 100 μL,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、Mg2+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=3 639.8X+2 341.5[R2=0.972 5, 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。 |
| 关键词: 非标记型 噻唑橙 石房蛤毒素 适配体传感器 |
| DOI：10.16781/j.CN31-2187/R.20230750 |
| 投稿时间：2023-12-19修订日期：2024-01-05 |
| 基金项目:国家自然科学基金(82273894),上海市军民融合发展专项(2020-jmrh1-kj11). |
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| Highly sensitive detection of saxitoxin by a label-free aptasensor |
| XI Xiaoyu1,LIU Ying2,LIAN Zhilan2,LU Feng1,2* |
(1. Department of Pharmaceutical Analysis, School of Pharmacy, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China
*Corresponding author) |
| Abstract: |
| 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 MgCl2, 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 100 μL. 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 Mg2+ 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=3 639.8X+2 341.5 (R2=0.972 5; 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. |
| Key words: label-free thiazole orange saxitoxin aptasensor |
