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Chemosensors
Volume 13
Issue 9
10.3390/chemosensors13090343
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

High-Intensity In Situ Fluorescence Imaging of MicroRNA in Cells Based on Y-Shaped Cascade Assembly

by
Yan Liu
1,2,
Xueqing Fan
1,2,
Xinying Zhou
1,2,
Zhiqi Zhang
1,2,
Qi Yang
2,
Rongjie Yang
2,
Yingxue Li
2,
Anran Zheng
2,
Lianqun Zhou
1,2,*,
Wei Zhang
1,2 and
Jinze Li
1,2,*
1
School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
2
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
*
Authors to whom correspondence should be addressed.
Chemosensors 2025, 13(9), 343; https://doi.org/10.3390/chemosensors13090343 (registering DOI)
Submission received: 2 August 2025 / Revised: 27 August 2025 / Accepted: 5 September 2025 / Published: 6 September 2025
(This article belongs to the Special Issue Advancements of Chemosensors and Biosensors in China—2nd Edition)
Versions Notes

Abstract

MicroRNAs are closely associated with various physiological and pathological processes, making their in situ fluorescence imaging crucial for functional studies and disease diagnosis. Current methods for the in situ fluorescence imaging of microRNA predominantly rely on linear signal amplification, resulting in relatively weak imaging signals. This study introduces a Y-shaped cascade assembly (YCA) method for high-brightness microRNA imaging in cells. Triggered by target microRNA, catalytic hairpin assembly forms double-stranded DNA (H). Through annealing and hybridization, a Y-shaped structure (P) is created. These components assemble into DNA nanofluorescent particles with multiple FAM fluorophores, significantly amplifying fluorescence signals. Optimization experiments revealed that a 1:1 ratio of P to H and an assembly time of 60 min yielded the best results. Under these optimal conditions, the resulting fluorescent nanoparticles exhibited diameters of 664.133 nm, as observed by DLS. In Huh7 liver cancer cells, YCA generated DNA nanoparticles with a fluorescence intensity increase of 117.77%, triggered by target microRNA-21, producing high-intensity fluorescence images and enabling qualitative detection of microRNA-21. The YCA in situ imaging method offers excellent imaging quality and high efficiency, providing a robust and reliable analytical tool for the diagnosis and monitoring of microRNA-related diseases.
Keywords: in situ cellular detection; microRNA; fluorescence imaging; cascade assembly in situ cellular detection; microRNA; fluorescence imaging; cascade assembly

Share and Cite

MDPI and ACS Style

Liu, Y.; Fan, X.; Zhou, X.; Zhang, Z.; Yang, Q.; Yang, R.; Li, Y.; Zheng, A.; Zhou, L.; Zhang, W.; et al. High-Intensity In Situ Fluorescence Imaging of MicroRNA in Cells Based on Y-Shaped Cascade Assembly. Chemosensors 2025, 13, 343. https://doi.org/10.3390/chemosensors13090343

AMA Style

Liu Y, Fan X, Zhou X, Zhang Z, Yang Q, Yang R, Li Y, Zheng A, Zhou L, Zhang W, et al. High-Intensity In Situ Fluorescence Imaging of MicroRNA in Cells Based on Y-Shaped Cascade Assembly. Chemosensors. 2025; 13(9):343. https://doi.org/10.3390/chemosensors13090343

Chicago/Turabian Style

Liu, Yan, Xueqing Fan, Xinying Zhou, Zhiqi Zhang, Qi Yang, Rongjie Yang, Yingxue Li, Anran Zheng, Lianqun Zhou, Wei Zhang, and et al. 2025. "High-Intensity In Situ Fluorescence Imaging of MicroRNA in Cells Based on Y-Shaped Cascade Assembly" Chemosensors 13, no. 9: 343. https://doi.org/10.3390/chemosensors13090343

APA Style

Liu, Y., Fan, X., Zhou, X., Zhang, Z., Yang, Q., Yang, R., Li, Y., Zheng, A., Zhou, L., Zhang, W., & Li, J. (2025). High-Intensity In Situ Fluorescence Imaging of MicroRNA in Cells Based on Y-Shaped Cascade Assembly. Chemosensors, 13(9), 343. https://doi.org/10.3390/chemosensors13090343

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