Nanogap antennas are plasmonic nanostructures with a strong electromagnetic field generated at the gap region of two neighboring particles owing to the coupling of the collective surface plasmon resonance. They have great potential for improving the optical properties of fluorophores. Herein, nanogap antennas are constructed using an aqueous solution‐based method to overcome the defects of weak fluorescence and photobleaching associated with traditional organic dyes, and a highly sensitive nanogap antenna‐based sensing strategy is presented for the detection of low‐abundance nucleic acid biomarkers via a target‐triggered strand displacement amplification (SDA) reaction between two DNA hairpins that are tagged to the tips of gold nanorods (Au NRs). In the presence of targets, end‐to‐end Au NR dimers gradually form, and the fluorophores quenched by the Au NRs exhibit a dramatic fluorescence enhancement due to the plasmon‐enhanced fluorescence effect of nanogap antennas. Meanwhile, the SDA reaction results in secondary amplification of fluorescence signals. Combined with single‐molecule counting, this method applied in miRNA‐21 detection can achieve a low detection limit of 97.2 × 10−18 m. Moreover, accurate discrimination between different cells through miRNA‐21 imaging demonstrates the potential of this method in monitoring the expression level of low‐abundance nucleic acid biomarkers.
Tyrosinase (TYR) plays a vital role in melanin biosynthesis and is widely regarded as a relatively specific marker for melanocytic lesions which involve vitiligo, malignant cutaneous melanoma, Parkinson's disease (PD), etc. However, the detection of TYR in living cells with fluorescent probes is usually interfered by diverse endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS). Herein, we synthesized a melanosome-targeting near-infrared (NIR) fluorescent probe (HB-NP) with a large Stokes shift (195 nm), achieving a highly sensitive and selective in situ detection for intracellular TYR, by incorporating a m-hydroxybenzyl moiety that recognizes TYR specifically and the morpholine unit which facilitates the probe accumulating in the melanosome into a salicyladazine skeleton. When treated with TYR, the probe itself with weak fluorescence is lit up via an inhibited photoinduced electron-transfer (PET) effect and HB-NP shows a strong fluorescence signal (nearly 48-fold enhancement) with a low detection limit of 0.5 U mL. HB-NP has been successfully applied in visualizing and in situ quantification of the intracellular TYR activity. Moreover, owing to the different expression levels of TYR, two human uveal melanoma cells with different invasive behaviors are distinguished by means of bioimaging and the effects of the inhibitor, kojic acid, and the up-regulating treatment, psoralen/ultraviolet A, on TYR activity of the two melanoma cells are evaluated. HB-NP is expected to be a useful tool to monitor diseases associated with the abnormal level of melanin and screen medicines for TYR disorder more effectively.
DNA‐templated silver nanoclusters (DNA‐AgNCs) have been extensively studied in recent years. The enhancement of fluorescence emission from DNA‐AgNCs is still being explored. Herein, a study on the fluorescence enhancement of DNA‐AgNCs induced by metal ions is reported. The enhancement is greatly dependent on the primary sequence and secondary structure of the DNA strands. Thus, a label‐free AgNCs‐based molecular beacon (MB) was explored for the detection of telomerase activity. Nonfluorescent MB‐AgNCs in phosphate buffer emit dramatic red fluorescence when Mg2+ is introduced, whereas Mg2+ has a limited effect on the weak fluorescence of DNA‐AgNCs when the hairpin structure of MB is opened. Telomerase primer can be elongated by telomerase, which results in unfolding of MB in a strand‐displacement reaction. On the basis of the different brightnesses of AgNCs produced by the two DNA templates, telomerase activity can be detected. The MB‐AgNCs sensing platform provides a simple and low‐cost method to detect telomerase activity and shows great potential in the construction of cost‐effective probes for biomolecular detection.
A comparative study of plasmonic-enhanced single-molecule fluorescence (PESMF) induced by four gold nanoantennas is reported. The gold triangular nanoplate (Au TNP) is the optimal PESMF substrate for Cy5.5 owing to its sharpest point and strongest electric fields. The Au TNP is chosen for the preparation of a telomerase sensor.
Sequencing
by synthesis is a significant method for high-throughput
DNA sequencing. Herein, we synthesized terminal aggregated-induced-emission
luminogen (AIEgen) labeled nucleotides (dNTPs-HCAP) that could serve
as substrates for some polymerases and applied them into the sequencing
of small DNA fragments. In the process of DNA amplification, ratiometric
AIEgens are released from dNTPs-HCAP and aggregate through the effects
of phosphatase, which results in changes in the ratiometric fluorescent
signals. With the AIEgen-labeled nucleotides, we accomplished the
sequencing of small DNA fragments through double changes in fluorescence.
In addition, we achieved the differentiation of single nucleotide
polymorphisms through rolling circle amplification reactions without
the addition of signal probes, which is fast and cost-effective. The
introduction of ratiometric AIEgens into DNA synthesis makes the detection
of DNA sequences more efficient and accurate. Therefore, the development
of AIEgen-labeled nucleotides is meaningful for the study of DNA sequencing
methods.
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