Three-Dimensional CHA-HCR System Using DNA Nanospheres for Sensitive and Rapid Imaging of miRNA in Live Cells and Tissues.

Isothermal, enzyme-free amplification techniques, such as the hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), have gained increasing attention for miRNA analysis. However, current methodological challenges, including slow kinetics, low amplification efficiency, difficulties in efficient cellular internalization of DNA probes, and concerns regarding the intracellular stability of nucleic acids, need to be addressed. To this end, we propose a novel strategy for sensitive miRNA detection based on a three-dimensional (3D) CHA-HCR system. This system comprises two DNA nanospheres, named DS-13 and DS-24, which are functionalized with CHA and HCR hairpins. Target miR-21 initiates CHA between the two nanospheres, thereby activating downstream HCR and bringing cyanine 3 (Cy3) and cyanine 5 (Cy5) into proximity. The 3D CHA-HCR process leads to the formation of large DNA aggregates and the generation of fluorescence resonance energy transfer signals. In this strategy, the employment of a cascaded reaction and spatial confinement effect improve sensitivity and kinetics, while the use of DNA nanocarriers facilitates cellular delivery and protects nucleic acid probes. The experimental results in vitro, in living cells, and in clinical tissue samples demonstrated the desirable sensing performance. Collectively, this approach holds promise as a valuable tool for cancer diagnosis and biomedical research.

Simultaneous detection and imaging of two specific miRNAs using DNA tetrahedron-based catalytic hairpin assembly.

Improving the accuracy, sensitivity and speed of intracellular miRNA imaging is essential for early diagnosis of cancer. To achieve this goal, we herein present a strategy for imaging two distinct miRNAs by DNA tetrahedron-based catalytic hairpin assembly (DCHA). Two nanoprobes, DTH-13 and DTH-24, were prepared by one-pot synthesis. The resultant structures were DNA tetrahedrons functionalized with two sets of CHA hairpins, which respectively responded to miR-21 and miR-155. Using these structured DNA nanoparticles as the carriers, the probes could easily enter living cells. The presence of miR-21 or miR-155 could trigger CHA between DTH-13 and DTH-24, leading to independent fluorescence signals of FAM and Cy3. In this system, the sensitivity and kinetics were significantly enhanced owing to the strategy of DCHA. The sensing performance of our method was thoroughly investigated in buffers, fetal bovine serum (FBS) solutions, living cells, and clinical tissue samples. The results validated the potential of DTH nanoprobes as a diagnostic tool for early stages of cancer.

Recent progress in the development of DNA-based biosensors integrated with hybridization chain reaction or catalytic hairpin assembly.

As isothermal, enzyme-free signal amplification strategies, hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) possess the advantages such as high amplification efficiency, excellent biocompatibility, mild reactions, and easy operation. Therefore, they have been widely applied in DNA-based biosensors for detecting small molecules, nucleic acids, and proteins. In this review, we summarize the recent progress of DNA-based sensors employing typical and advanced HCR and CHA strategies, including branched HCR or CHA, localized HCR or CHA, and cascaded reactions. In addition, the bottlenecks of implementing HCR and CHA in biosensing applications are discussed, such as high background signals, lower amplification efficiency than enzyme-assisted techniques, slow kinetics, poor stability, and internalization of DNA probes in cellular applications.

A novel 1,8-naphthalimide-based fluorescent chemosensor for the detection of HSA in living cells.

Human serum albumin (HSA) is an essential protein for maintaining human health. Accurate detection and quantification of HSA are of great significance for disease diagnosis and biochemical research. Here, a new HSA fluorescent probe BNPE based on the 1,8-naphthalimide fluorophore was designed and synthesized. The probe could recognize HSA through a twisted intramolecular charge transfer mechanism, effectively avoid the interference of most substances, and realize HSA fluorescence imaging in living cells.

Ratiometric and amplified fluorescence nanosensor based on a DNA tetrahedron for miRNA imaging in living cells.

Enzyme-free signal amplification approaches have attracted considerable attention in the field of intracellular miRNA analysis. However, the application of nucleic acid amplification has been limited by intracellular delivery of multiple oligonucleotide components with precise stoichiometry. In this work, we propose a new DNA tetrahedron (DTN)-based sensing platform addressing the delivery and stoichiometric control of nucleic components for enzyme-free amplification. The nanosensor is composed of two DTN probes; DTN-F served as the target recognition and signal output unit, and DTN-H served as the signal amplification unit. DTNs could facilitate the cell internalization of the nucleic acid probes and protect them from nuclease degradation. In the absence of target miRNA, the fluorescent strands (F) hybridize with the hanging sequences of DTN, and FAM and TAMRA labeled on F will be separated, blocking fluorescence resonance energy transfer (FRET). In the presence of the target miRNA, F will be displaced by the target and the hairpin structure will be restored, bringing the FRET pair into close proximity and inducing a FRET signal. Moreover, the helper strands (H) on DTN-H could liberate target miRNA through strand displacement, which will initiate a new round of reaction, generating an amplified FRET signal. The DTN nanosensor realized sensitive and selective detection of let-7a in buffer solution and 10% FBS solution. In addition, imaging of miRNA in the different cell lines and monitoring of intracellular miRNA fluctuations were carried out The developed method offers a new tool for bioanalytical and biomedical research.