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Single-molecule imaging technologies, especially those based on fluorescence, have been developed to probe both the equilibrium and dynamic properties of biomolecules at the single-molecular and quantitative levels. In this review, we provide an overview of the state-of-the-art advancements in single-molecule fluorescence imaging techniques. We systematically explore the advanced implementations of in vitro single-molecule imaging techniques using total internal reflection fluorescence (TIRF) microscopy, which is widely accessible. This includes discussions on sample preparation, passivation techniques, data collection and analysis, and biological applications. Furthermore, we delve into the compatibility of microfluidic technology for single-molecule fluorescence imaging, highlighting its potential benefits and challenges. Finally, we summarize the current challenges and prospects of fluorescence-based single-molecule imaging techniques, paving the way for further advancements in this rapidly evolving field. [ABSTRACT FROM AUTHOR]

Nucleocapsid protein (N protein) is an appropriate target for early determination of viral antigen-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have found that β-cyclodextrin polymer (β-CDP) has shown a significant fluorescence enhancement effect for fluorophore pyrene via host–guest interaction. Herein, we developed a sensitive and selective N protein-sensing method that combined the host–guest interaction fluorescence enhancement strategy with high recognition of aptamer. The DNA aptamer of N protein modified with pyrene at its 3′ terminal was designed as the sensing probe. The added exonuclease I (Exo I) could digest the probe, and the obtained free pyrene as a guest could easily enter into the hydrophobic cavity of host β-CDP, thus inducing outstanding luminescent enhancement. While in the presence of N protein, the probe could combine with it to form a complex owing to the high affinity between the aptamer and the target, which prevented the digestion of Exo I. The steric hindrance of the complex prevented pyrene from entering the cavity of β-CDP, resulting in a tiny fluorescence change. N protein has been selectively analyzed with a low detection limit (11.27 nM) through the detection of the fluorescence intensity. Moreover, the sensing of spiked N protein from human serum and throat swabs samples of three volunteers has been achieved. These results indicated that our proposed method has broad application prospects for early diagnosis of coronavirus disease 2019. [ABSTRACT FROM AUTHOR]

DNA-templated fluorescent nanoclusters (NCs) have attracted increasing research interest on account of their prominent features, such as DNA sequence-dependent fluorescence, easy functionalization, wide availability, water solubility, and excellent biocompatibility. Coupling DNA templates with complementary DNA, aptamers, G-quadruplex, and so on has generated a large number of sensors. Additionally, the preparation and applications of DNA-templated fluorescent NCs in these sensing have been widely studied. This review firstly focuses on the properties of DNA-templated fluorescent NCs, and the synthesis of DNA-templated fluorescent NCs with different metals is then discussed. In the third part, we mainly introduce the applications of DNA-templated fluorescent NCs for sensing metal ions. At last, we further discuss the future perspectives of DNA-templated fluorescent NCs in the synthesis and sensing metal ions in the environmental and biological fields. [ABSTRACT FROM AUTHOR]