基于NaYF4:Yb,Er上转换荧光纳米颗粒精准检测DNA

Accurate Detection of DNA Based on the NaYF_4∶Yb,Er Upconversion Nanoparticles

建立了一种利用碱基堆积原理并以上转换纳米粒子荧光作为内参的精准检测DNA的方法。该方法首先利用热分解法制备NaYF_4∶Yb,Er上转换荧光纳米颗粒(upconversion nanoparticles,UCNPs),再通过表面羧基化变性牛血清蛋白修饰后与氨基化探针核酸单链共价偶联,形成上转换荧光标记显示探针。最后再基于碱基堆积原理进行杂交检测。研究结果表明以NaYF_4∶Yb,Er荧光强度为内参,根据FAM/UCNP的强度比来定量检测目标DNA浓度比单一的以报告DNA中FAM荧光强度定量检测目标DNA浓度要更为精准,有效地避免了实验中出现的人为操作和仪器误差。本方法不需要进行扩增,检测底限可达到5 nmol·L^(-1),且在较大的浓度范围内有较好的线性关系,同时该方法也有着良好的特异性,能有效区分单碱基错配序列。

A new method for improving the accuracy of DNA detection has been developed, which was based on the base stacking principle and the fluorescence of NaYF4：Yb, Er UCNPs. Firstly, the NaYF4：Yb, Er UCNPs were synthesized by thermal decomposition method and functionalized with denatured bovine serum albumin. Then, the denatured bovine serum albumin-functionalized OA/NaYF4：Yb, Er UCNPs were conjugated with amino group- modified DNA probes to form upconversion fluorescence labeled probes and detect DNA. The results showed that the method of using the fluorescence of NaYF4：Yb, Er UCNPs as a reference standard to quantitatively detect target DNA concentration had higher accuracy than that of only using a single FAM fluorescence intensity, and the operation and equipment errors was effectively avoid during the experiment. Moreover, this method can reach the detection limit as low as 5 nmol .L-1 without amplification, displays a good linear relationship in wide concentration range and a high specificity, and also can effectively differenciate single-base mismatch sequences.