基于适配体胶体金侧向层析试纸快速检测卡那霉素的方法研究

Rapid Detection of Kanamycin Based on Aptamer Colloid Gold Lateral Flow Test Strip

该文基于适配体以及非巯基化核酸修饰的胶体金纳米探针(AuNPs@polyA-DNA),建立了一种新型的卡那霉素胶体金侧向层析试纸条。分析了试纸条各组装元件,包括适配体浓度、链霉亲和素(SA)与Biotin-DNAT的比例、SA-生物素(Biotin)-DNAT偶联物浓度以及孵育时间与温度等,对显色反应的影响。最佳实验条件为:缓冲液为4xSSC(0.5%Tween 20),SA与Biotin-DNAT的最佳摩尔比为1∶6,检测区喷涂偶联物SA-Biotin-DNAT浓度为4μmol/L,适配体浓度为10 nmol/L,室温孵育时间为20 min。在优化条件下,该试纸条对卡那霉素的肉眼分辨浓度为25 ng/mL,线性范围为5.0~125 ng/mL,检出限为1.5 ng/mL。用于蜂蜜中卡那霉素的检测,其回收率为95.1%~105%,相对标准偏差(RSD)为3.4%~8.5%。该试纸条具有灵敏度高、特异性好、架构简单、重复性高等优点,可用于实际样品中卡那霉素的检测。

In this paper,a new type of colloidal gold lateral flow test strip(AuNPs@polyA-DNA)was fabricated based on aptamer recognition of kanamycin molecule and non-thiolated DNA modified colloidal gold nanoprobe.Effects of the strip components on color development were investigated,including concentration of aptamer,proportion of streptavidin(SA)to Biotin-DNAT,concentration of SA-Biotin-DNAT,conjugate reaction time and temperature.The optimized experimental conditions were as follows:Running buffer:4xSSC(0.5%Tween 20),molar ratio of SA to Biotin-DNAT:1∶6,concentartion of SA-Biotin-DNAT conjugate sprayed at T line:4μmol/L,concentartion of aptamer:10 nmol/L,incubation of aptamer:ambient temperature for 20 min.The results demonstrated that 25 ng/mL kanamycin could be discriminated with the naked eyes.The quantitative determination of kanamycin in the range of 5.0-125 ng/mL could be carried out by a colloidal gold reader,with a detection limit of 1.5 ng/mL.The test strip was applied to the determination of kanamycin in honey,with recoveries of 95.1%-105%and relative standards deviation(RSD)of 3.4%-8.5%.The strip could be used for the detection of kanamycin in real samples with the advantages of high sensitivity,high specificity,simple structure and high repeatability.