With the continuous development of biosensors, researchers have focused increasing attention on various signal amplification strategies to pursue superior performance for more applications. In comparison with other signal amplification strategies, hybridization chain reaction (HCR) as a powerful signal amplification technique shows its certain charm owing to nonenzymatic and isothermal features. Recently, on the basis of conventional HCR, this technique has been developed and improved rapidly, and a variety of HCR-based biosensors with excellent performance have been reported. Herein, we present a systematic and critical review on the research progress of HCR in biosensors in the last five years, including the newly developed HCR strategies such as multibranched HCR, migration HCR, localized HCR, in situ HCR, netlike HCR, and so on, as well as the combination strategies of HCR with isothermal signal amplification techniques, nanomaterials, and functional DNA molecules. By illustrating some representative works, we also summarize the advantage and challenge of HCR in biosensors, and offer a deep discussion of the latest progress and future development trends of HCR in biosensors.

The Recent Development of Hybridization Chain Reaction Strategies in Biosensors.

With the continuous development of DNA nanotechnology, various spatial DNA structures and assembly techniques emerge. Hybridization chain reaction (HCR) is a typical example with exciting features and bright prospects in biosensing, which has been intensively investigated in the past decade. In this Spotlight on Applications, we summarize the assembly principles of conventional HCR and some novel forms of linear/nonlinear HCR. With advantages like great assembly kinetics, facile operation, and an enzyme-free and isothermal reaction, these strategies can be integrated with most mainstream reporters (e.g., fluorescence, electrochemistry, and colorimetry) for the ultrasensitive detection of abundant targets. Particularly, we select several representative studies to better illustrate the novel ideas and performances of HCR strategies. Theoretical and practical utilities are confirmed for a range of biosensing applications. In the end, a deep discussion is provided about the challenges and future tasks of this field.

Recent Progress in DNA Hybridization Chain Reaction Strategies for Amplified Biosensing.

Nonlinear hybridization chain reaction-based functional DNA nanostructure assembly for biosensing, bioimaging applications

A novel cascade signal amplification strategy integrating CRISPR/Cas13a and branched hybridization chain reaction for ultra-sensitive and specific SERS detection of disease-related nucleic acids.

As an amplification tool, DNA walker nanomachine exhibits promising potential in the fields of biosensing. To further improve amplification efficiency of DNA walker, a number of cascade amplification systems have been developed, but they still suffer from separated steps and long reaction time. Herein, by combining entropy-driven catalytic (EDC) amplification with DNAzyme reaction, a highly integrated cascade three-dimensional (3D) DNA walker is constructed, which allows the autonomous DNA circuit to be completed in one step. Further combined with element tag and inductively coupled plasma mass spectrometry (ICP-MS) detection, the cascade walker can be used for quantification of miRNA-21. Specifically, the reactant of EDC (WLT complex) and the substrate of DNAzyme (Substrate-Tb) are both fixed on magnetic beads (MBs) to assemble the track of walker. Once cascade DNA walker is triggered by target miRNA, EDC amplification runs first and keeps circulating to liberate many DNAzyme strands. DNAzyme can repeatedly cleave Substrate-Tb with autonomously walking along the substrate on MBs. As a result, a great number of fragments of Substrate-Tb, as output signal, are generated within 1 h, resulting in elemental signal response in ICP-MS and reflecting the amount of miRNA-21. A good linear relationship was obtained with miRNA-21 concentration in the range of 0.02−1 nmol L−1, and the detection limit of 5 pmol L−1 was achieved. The recoveries of miRNA-21 in human serum were 92.0–107 %. The developed method provided a novel idea for constructing highly integrated and efficient cascade DNA walker with ICP-MS strategy for bioanalysis.

Highly integrated and one-step triggered cascade DNA walker based on entropy-driven catalytic and DNAzyme amplification

Hybridization chain reaction (HCR) was a significant discovery for the development of nanoscale materials and devices. One key challenge for HCR is the vulnerability to background leakage in the absence of the initiator. Here, we systematically analyze the sources of leakage and refine leak-resistant rule by using molecular thermodynamics and dynamics, biochemical and biophysical methods. Transient melting of DNA hairpin is revealed to be the underlying cause of leakage and that this can be mitigated through careful consideration of the sequence thermodynamics. The transition threshold of the energy barrier is proposed as a testing benchmark of leak-resistance DNA hairpins. The universal design of DNA hairpins is illustrated by the analysis of hsa-miR-21-5p as biomarker when used in conjunction with surface-enhanced Raman spectroscopy. We further extend the strategy for specific signal amplification of miRNA homologs. Significantly, it possibly provides a practical route to improve the accuracy of DNA self-assembly for signal amplification, and that could facilitate the development of sensors for the sensitive detection of interest molecules in biotechnology and clinical medicine.

Elucidation of leak-resistance DNA hybridization chain reaction with universality and extensibility.

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Elucidation of leak-resistance DNA hybridization chain reaction with universality and extensibility.