Xia Jiaoyun, Xu Tong, Qing Jing, Wang Lihua, Tang Junlong
School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, China.
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
Front Chem. 2021 Mar 31;9:631959. doi: 10.3389/fchem.2021.631959. eCollection 2021.
The detection of single nucleotide polymorphisms (SNPs) is of great significance in the early diagnosis of diseases and the rational use of drugs. Thus, a novel biosensor based on the quenching effect of fluorescence-embedded SYBR Green I (SG) dye and graphene oxide (GO) was introduced in this study. The probe DNA forms a double helix structure with perfectly complementary DNA (pcDNA) and 15 single-base mismatch DNA (smDNA) respectively. SG is highly intercalated with perfectly complementary dsDNA (pc-dsDNA) and exhibits strong fluorescence emission. Single-base mismatch dsDNA (SNPs) has a loose double-stranded structure and exhibits poor SG intercalation and low fluorescence sensing. At this time, the sensor still showed poor SNP discrimination. GO has a strong effect on single-stranded DNA (ssDNA), which can reduce the fluorescence response of probe DNA and eliminate background interference. And competitively combined with ssDNA in SNPs, quenching the fluorescence of SG/SNP, while the fluorescence value of pc-dsDNA was retained, increasing the signal-to-noise ratio. At this time, the sensor has obtained excellent SNP resolution. Different SNPs detect different intensities of fluorescence in the near-infrared region to evaluate the sensor's identification of SNPs. The experimental parameters such as incubation time, incubation temperature and salt concentration were optimized. Under optimal conditions, 1 nM DNA with 0-10 nM linear range and differentiate 5% SNP were achieved. The detection method does not require labeling, is low cost, simple in operation, exhibits high SNP discrimination and can be distinguished by SNP at room temperature.
单核苷酸多态性（SNP）的检测在疾病的早期诊断和药物的合理使用中具有重要意义。因此，本研究引入了一种基于荧光嵌入的SYBR Green I（SG）染料与氧化石墨烯（GO）猝灭效应的新型生物传感器。探针DNA分别与完全互补DNA（pcDNA）和15种单碱基错配DNA（smDNA）形成双螺旋结构。SG与完全互补双链DNA（pc-dsDNA）高度嵌入并表现出强烈的荧光发射。单碱基错配双链DNA（SNP）具有松散的双链结构，SG嵌入性差，荧光传感能力低。此时，该传感器对SNP的区分能力仍较差。GO对单链DNA（ssDNA）有很强的作用，可降低探针DNA的荧光响应并消除背景干扰。并与SNP中的ssDNA竞争性结合，猝灭SG/SNP的荧光，同时保留pc-dsDNA的荧光值，提高信噪比。此时，该传感器获得了优异的SNP分辨率。通过检测不同SNP在近红外区域的荧光强度差异来评估传感器对SNP的识别能力。对孵育时间、孵育温度和盐浓度等实验参数进行了优化。在最佳条件下，实现了对0 - 10 nM线性范围内1 nM DNA的检测，能够区分5%的SNP。该检测方法无需标记，成本低，操作简单，对SNP具有高区分能力，且可在室温下通过SNP进行区分。
