适配体磁纳米探针-激光诱导荧光特异识别微囊藻毒素-LR

doi:10.3964/j.issn.1000-0593(2023)12-3813-07

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摘要：微囊藻毒素-LR（MC-LR）具有强烈的肝毒性、致癌性和发育毒性，亟需严格监测。为实现环境水体中微痕量MC-LR的快速特异灵敏的分析识别，以金属有机框架化合物（MOF）为介导，增强磁纳米粒子比表面积，高效负载纳米金和适配体-cDNA分子杂交荧光探针，研发新型的适配体功能化磁纳米荧光探针（Fe₃O₄@MIL-101-NH₂@Au@aptamer），实现了对MC-LR的适配体特异识别-激光诱导荧光（LIF）超高灵敏分析。论文详细研究了适配体磁纳米探针荧光检测MC-LR的可行性、MC-LR测定的优化条件及其方法性能。结果表明：MOF修饰的磁纳米颗粒Fe₃O₄@MIL-101-NH₂的Brunauer-Emmett-Teller（BET）比表面积达到114.02 m²·g^-1，比单一Fe₃O₄提高了近5倍，适配体-cDNA杂交荧光探针在磁纳米颗粒表面的修饰率达98%以上，适配体磁纳米探针对水体中痕量MC-LR具有很强的荧光响应。在最优条件下（pH 7.5、NaCl浓度500 mmol·L^-1、纳米金尺寸为20 nm，测定时间为30 min），适配体磁纳米探针与MC-LR结合释放出荧光互补链，体系荧光强度与MC-LR含量成正比，线性浓度范围为0.020~3.000 μg·L^-1，检出限（LOD）为0.006 μg·L^-1，灵敏度比文献报道的荧光分析法提高了1.6~22.3倍。所建立的适配体磁纳米探针-LIF法对MC-LR的识别特异性高，在100倍量的干扰物（微囊藻毒素MC-RR、MC-YR、大田软海绵酸OA) 共存情况下，适配体磁纳米探针在混合体系中的荧光响应与在MC-LR单样中的响应强度偏差小于3.3%，交叉反应性小；日内、日间和批间的测定标准偏差（RSD）为1.7%~8.8%，相对误差RE为-4.3%~4.1%，方法稳定性和重现性好。该方法应用于闽江、西湖水和内河水等样品分析，水中MC-LR得到了良好的识别检出，不同加标浓度的MC-LR（0.050，0.100和1.000 μg·L^-1）测定回收率为(90.1%±6.4%)~(104.2%±7.0%) (n=3)，与LC-MS确证方法的测定结果[加标回收率（91.3%±7.0%)~(104.4%±2.0%)，n=3]一致。所建立的适配体磁纳米探针与LIF联用技术对水中痕量MC-LR具有高的特异识别和灵敏检出能力，为环境水中痕量MC-LR的现场特异识别分析提供了一个新的技术。

关键词：适配体；杂交探针；磁纳米颗粒；微囊藻毒素-LR；激光诱导荧光

Abstract：MC-LR possessing strong hepatotoxicity, potential carcinogenicity, and biological toxicity could have an intensive influence on human health and the aquatic ecological environment and need to be monitored critically. For the specific and highly sensitive analysis performance, agold-magnetic composite nanoparticlemodified by aptamer-cDNAfluorescent hybridization probe (Fe₃O₄@MIL-101-NH₂@Au@aptamer) was prepared and applied for sensing detection of MC-LR in water with laser-induced fluorescence (LIF). This work studied the feasibility, optimization of analysis conditions, specificity, stability of the method, and sample analysis application. As a result, a high Brunauer-Emmett-Teller (BET）specific surface area of 114.02 m²·g^-1 was achieved in Fe₃O₄@MIL-101-NH₂@AuNPs, which resulted in a high modification rate of up to 98.8% achieved for aptamer-cDNA fluorescenthybridization probe. With the excitation wavelength of 497 nm, the aptamer-cDNA fluorescent probe has a strong fluorescence response with the emission wavelength of 512 nm to the trace MC-LR. Under the optimal conditions (pH 7.5, NaCl concentration 500 mmol·L^-1, nanogold size 20 nm, analytical time 30 min), a good linear relationship between MC-LR concentration and fluorescence intensity was achieved in a wide linear range (0.020~3.000 μg·L^-1) with the limit of detection (LOD) as low as 0.006 μg·L^-1, which was 1.6~22.3 folds better than that of the most fluorescence methods reported previously. The relative standard deviation (RSD) of intra-day, intra-day, and inter-batch was gained in 1.7%~8.8%, and the relative error (RE) of -4.3%~4.1%. High specific selectivity and low cross-reactivity for MC-LR (1.0 μg·L^-1) were also achieved using theas-prepared fluorescent probe, even if the concentration of interfering toxins (MC-RR, MC-YR, OA) was 100 folds higher than that of MC-LR. The fluorescence responses of MC-LR in these mixtures werewell consistent with that of MC-LR in the standard solution. Applied to the analysis of samples from the Minjiang River, Lake Water, and the inland river, the identification of MC-LR in water samples was satisfactory. The recovery yields of MC-LR for three concentrations (0.050, 0.100 and 1.000 μg·L^-1) rangedin (90.1%±6.4%)~(104.2%±7.0%) (n=3). The results were consistent with those obtained by the LC-MS method [（91.3%±7.0%)~(104.4%±2.0%), n=3]. It might light a new technique for the specific and rapid monitoring technology for tracing MC-LR in the environmental water.

Key words：Aptamer; Hybridization probe; Magnetic nanoparticle; MC-LR; Laser induced fluorescence

收稿日期: 2023-09-11 修订日期: 2023-10-05

中图分类号:

O657.34

基金资助: 国家重点研发计划课题(2018YFC1602902-3), 海洋服务与渔业高质量发展专项(FJHY-YYKJ-2023-1-1)资助

通讯作者: 林旭聪 E-mail: xulin@fzu.edu.cn

作者简介: 齐国敏，1965年生，福州大学化学学院高级工程师 e-mail: 961043190@qq.com

引用本文:

齐国敏，童诗谦，林旭聪. 适配体磁纳米探针-激光诱导荧光特异识别微囊藻毒素-LR[J]. 光谱学与光谱分析, 2023, 43(12): 3813-3819.
QI Guo-min, TONG Shi-qian, LIN Xu-cong. Specific Identification of Microcystin-LR by Aptamer-Functionalized Magnetic Nanoprobe With Laser-Induced Fluorescence. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2023, 43(12): 3813-3819.

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