Construction of Dual-Color Probes with Target-Triggered Signal Amplification for In Situ Single-Molecule Imaging of MicroRNA

Abstract

The in vitro detection of low abundance biomolecules via nonenzymatic signal amplification is an attractive strategy. However, it remains a challenge to monitor targets of interest in situ in living cells by low-background interference and visualized enzyme-free signal amplification strategies. Taking advantage of the single-molecule imaging and dynamic DNA nanotechnologies, we have achieved the target-triggered self-assembly of nanostructure-based dual-color fluorescent probes (NDFPs) by an enzyme-free toehold-mediated strand displacement cascade. NDFPs will facilitate the simple and visualized monitoring of microRNA (miRNA) at the femtomolar level. The recycled miRNA can be considered as the catalyst for the assembly of multiple H1/H2 duplexes. This generated the fluorescence signal of the enhanced target expression, indicating both in vitro and in vivo signal-amplified imaging. Moreover, the NDFPs improved the measurement accuracy by dual-color colocalization imaging to greatly avoid false-positive signals and enabled the successful in situ imaging of miRNA in living cells in real time. This work provides a strategy to visually monitor and study the integration of signal amplification detection and single-molecule imaging. NDFPs may be an important step toward the enzyme-free amplified monitoring and imaging of various biomolecules in living cells at the single-molecule level.

DNA‐Based Architectures for in situ Target Biomolecule Analysis in Confined Nano‐space†

“…In addition, single molecule detection (SMD), which has been rapidly developed nowadays, is considered an ideal tool for ultrasensitive biosensors. [ 62‐63 ] At the single‐molecule scale, this technology has shown brilliant potential in detecting biomolecules with low abundance and achieving in situ imaging in complex biological environments, broadening the application fields in bioimaging and biomedicine. Taken together, in situ target biomolecule analysis in confined nano‐space based on DNA architectures is a promising field, which might herald a new era in biological applications.…”

Advances in single‐molecule fluorescent nanosensors

“…In single‐molecule fluorescent nanosensor, the AuNP and MnO 2 nanosheet can quench the fluorescent labels. When target biomolecule is present, it causes the separation of fluorescent labels from the quencher, leading to the recovery of fluorescent signal, which can be quantified by single‐molecule detection (Y. Han, Chen, et al, 2019; Y. Han, Ye, et al, 2019; B. Li et al, 2020; X. Li et al, 2018).…”

“…(c) AuNP‐based single‐molecule fluorescent nanosensor for microRNA detection using toehold‐mediated strand displacement cascade. Reprinted with permission from B. Li et al (2020). Copyright 2020 American Chemical Society.…”

“…Due to their different compositions, dimensions, sizes, and shapes, the nanoscaled materials exhibit unique chemical, optical, mechanical, and electronic properties, and they can be easily functionalized with different biomolecules through electrostatic binding, physical adsorption, ligand exchange, biorecognition, and covalent bonding for the construction of diverse nanosensors (Bellan et al, 2011; Ma, Zhang, & Zhang, 2020; Rosi & Mirkin, 2005; Sheehan & Whitman, 2005). Recently, a variety of nanomaterials have been employed for the development of novel single‐molecule fluorescent nanosensors (Y. Han, Chen, et al, 2019; Y. Han, Ye, et al, 2019; B. Li et al, 2020; W. Li et al, 2016; X. Li et al, 2018; Ma, Ren, & Zhang, 2017; Ma, Wei, & Zhang, 2019; Pei et al, 2018; Z. Wu et al, 2019; J. Zhou et al, 2013). The use of functional nanomaterials including quantum dots, gold nanoparticles, upconversion nanoparticles, fluorescent conjugated polymer nanoparticles, nanosheets, and magnetic nanoparticles as fluorescent labels, quenchers, separators, and carriers has greatly improve the sensitivity, accuracy, and applications of single‐molecule detection.…”

Entropy-driven strand displacement reaction for ultrasensitive detection of circulating tumor DNA based on upconversion and Fe3O4 nanocrystals

“…They can measure the biochemical signals of entire cells by physical devices such as fluorescent biosensors, optical transducers, surface-enhanced Raman scattering (SERS), localized surface plasmon resonance (LSPR), and electrochemical biosensors [15][16][17][18][19][20][21][22]. Thus, the biosensors offer a more conducive approach to detect ctDNA because of their convenience and precision [23][24][25][26][27][28][29].…”