基于核酸适配体的微囊藻毒素LR纳米金生物传感检测方法的建立及其识别机制研究

Establishment of gold nanoparticles biosensor method for detection of microcystin LR based on aptamer and research on its recognition mechanism

中文关键词:  微囊藻毒素-LR  纳米金生物传感器  核酸适配体  识别机制

英文关键词:microcystin-LR  gold nanoparticles biosensor  aptamer  recognition mechanism

中文摘要:

      目的 建立微囊藻毒素-LR(microcystin-LR, MC-LR)的纳米金生物传感比色检测法, 并探究核酸适配体截短后对此方法检测效果的影响。方法 采用柠檬酸钠还原法制备纳米金, 通过优化盐浓度、适配体浓度、适配体与MC-LR的反应时间, 建立了MC-LR的纳米金生物传感比色检测法。然后, 采用组合型核酸适配体截短策略, 探究了截短后的核酸适配体对比色法检测MC-LR的影响。结果 NaCl浓度为0.9 mol/L, 适配体浓度为0.4 μmol/L, MC-LR与核酸适配体的反应时间为10 min时为其最适检测条件。在最适条件下, 该方法的目测最低检测限为0.4 μg/mL, 仪器读数最低检测限为(1.05±0.002) ng/mL, 线性检测范围为0~1.1 μg/mL。探究得出核酸适配体截短后会影响其对MC-LR的识别活性。结论 该方法简单、易行, 线性检测范围宽, 为小分子危害物核酸适配体的快速鉴定奠定了理论基础, 同时, 对截短核酸适配体与小分子危害物的识别活性研究提供了技术支撑。

英文摘要:

      Objective To establish a nano-gold biosensing colorimetric assay for microcystin-LR (MC-LR), and investigate the effect of nucleic acid aptamer truncation on the detection effect. Methods The gold nanoparticles were prepared by sodium citrate reduction method. After optimizing the concentration of salt, concentration of aptamer, reaction time, gold nanoparticles biosensor colorimetric method for the detection of MC-LR was developed. Then, the effect of nucleic acid aptamer contrast method on MC-LR detection was investigated by using combinatorial aptamer truncation strategy. Results The concentration of NaCl was 0.9 mol/L, the concentration of adaptor was 0.4mumol /L, and the reaction time for MC-LR conjugation with aptamer was 10 min. Under the optimal conditions, the limit of detection (LOD) for MC-LR with naked eye was 0.4 μg/mL, the LOD for instrument reading was (1.05±0.002) ng/mL, and the linear response range extended from 0 to 1.1 μg/mL. It was concluded that nucleic acid aptamer truncation would affect its recognition activity to MC-LR. Conclusion This method is simple and easy to implement, and the linear detection range is wide. In addition, it lays a theoretical foundation for the rapid identification of aptamers specific for small molecules, and provides technical support for the recognition activity of truncate nucleic acid aptamers and small molecule hazards.