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An amplified fluorescence biosensor for intracellular telomerase determination and in situ imaging based on thioflavin T and conjugated polymer nanoparticles
Sensors and Actuators B: Chemical ( IF 8.4 ) Pub Date : 2022-08-05 , DOI: 10.1016/j.snb.2022.132485
Xingfen Liu , Jixiang Song , Chao Wang , Ruining Yang , Pengfei Sun , Yanqin Huang , Lei Zhang , Quli Fan

Telomerase, as a significant tumor biomarker, is highly associated with cancers. Highly sensitive determination of telomerase activity is beneficial to accurate diagnosis and treatment of malignant tumors. A new simple and elegant fluorescence sensing strategy based on thioflavin T (ThT) and conjugated polymer nanoparticles was established to determine telomerase activity in living cells. In the presence of telomerase, the primer is prolonged with repeated sequence of (TTAGGG)n. This long G-riched sequence can form G-quadruplex in the mediation of ThT. The combination of ThT and G-quadruplex allows ThT to emit robust fluorescence. The fluorescence can be further amplified by positively charged conjugated polymer nanoparticles (PPE-PLL). The remarkable amplification is attributed to the disaggregation of PPE-PLL nanoparticles and a good superposition of fluorescence emission from ThT and PPE-PLL. The results demonstrated a new amplification assay for the determination of telomerase activity in cell extracts and living cells with high specificity and sensitivity. The limit of detection is estimated to be 3 HeLa cells in 400 μL. It is not only more sensitive than most reported biosensors for telomerase detection. Still, it is extremely simple, rapid, and low-cost since it is PCR-free, label-free, and enzyme-free.



中文翻译:

基于硫代黄素 T 和共轭聚合物纳米粒子的用于细胞内端粒酶测定和原位成像的放大荧光生物传感器

端粒酶作为一种重要的肿瘤生物标志物,与癌症高度相关。端粒酶活性的高灵敏度测定有利于恶性肿瘤的准确诊断和治疗。建立了一种基于硫黄素 T (ThT) 和共轭聚合物纳米粒子的简单而优雅的新型荧光传感策略,用于测定活细胞中的端粒酶活性。在端粒酶存在的情况下,引物以 (TTAGGG) n的重复序列延长. 这种富含 G 的长序列可以在 ThT 的介导中形成 G-四链体。ThT 和 G-四链体的结合使 ThT 能够发出强烈的荧光。荧光可以通过带正电荷的共轭聚合物纳米粒子 (PPE-PLL) 进一步放大。显着的放大归因于 PPE-PLL 纳米粒子的解聚以及来自 ThT 和 PPE-PLL 的荧光发射的良好叠加。结果证明了一种新的扩增测定法,用于测定细胞提取物和活细胞中的端粒酶活性,具有高特异性和灵敏度。检测限估计为 400 μL 中的 3 个 HeLa 细胞。它不仅比大多数报道的用于端粒酶检测的生物传感器更敏感。尽管如此,它还是非常简单、快速和低成本,因为它是无 PCR、无标记和无酶的。

更新日期:2022-08-05
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