Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging.
IF: 18.027


The hybrid chain reaction (HCR), an isothermal and enzyme-free amplification strategy, has found extensive use in fluorescent in situ hybridization (FISH) assays. However, the existing HCRs are limited, being time-consuming processes and low-efficiency imaging due to weak signal, significantly restricting their application in transcriptomic assays. To address the limitations, we developed nine orthogonal HCR hairpin-pair (hp) probes in this study to enable efficient signal amplification for multiplex assays. To enhance the efficiency and imaging quality of multiplex assays using these HCR probes, we employed two strategies. First, we coupled fluorescent molecules to HCR hairpins via disulfide bonds, facilitating easy removal through chemical cleavage. As a result, the workflow was greatly simplified. Second, we combined HCR with in situ rolling circle amplification (ISRCA), creating ISRCA-HCR, which achieved a 17-fold signal amplification. ISRCA-HCR demonstrated a high-level imaging capability for spatial cell type assays. This study shows the application for cell typing based on the developed HCR probes, enabling accurate and high-level signal amplification for multiplex FISH imaging. This provides an effective research tool for transcriptome and spatial cell type analysis.


cell types
fluorescent in situ hybridization (FISH)
hybrid chain reaction (HCR)
rolling circle amplification (RCA)
spatial transcriptome

MeSH terms

In Situ Hybridization, Fluorescence
Diagnostic Imaging
Nucleic Acid Hybridization
Biosensing Techniques


Cao, Dongjian
Qin, Xinxin
Wang, Wenjing
Zhang, Ying
Peng, Sunxiang
Gong, Hui
Luo, Qingming
Yang, Jie