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Updated: Jan 25, 2026

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics
Published on: June 17, 2022
Specific discrimination and universal signal amplification for RNA detection by coupling toehold exchange with RCA
Wen Yu1, Juqiong Li1, Chen Zuo1
1Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, PR China.
Abstract:
Herein, we combined toehold exchange with ligation-free rolling circle amplification (RCA) by programming nucleolytic conversion of hairpin probe into sensors, allowed for both high specific recognition and universal signal amplification for RNA detection. The rational engineered HP ensured highly specific recognition based on toehold exchange and allowed the pre-formed circular template for RCA to be shared for different RNAs detection. Generally, detecting different RNA requires different circular template for signal amplification. In this paper, the circular template for RCA was independent of the sequences of the target, so the signal amplification system was an universal one for different RNAs detection. Taking miRNA let-7d as a model target, this method showed a wide linear range from 1 fM to 1 nM with a detection limit of 0.46 fM and exhibited a remarkable selectivity even in distinguishing homologous miRNAs with 1-nt or 2-nt difference. To evaluate the potential of the method, it was applied to analysis the let-7d concentration in human serum, total RNA, and cell lysates. In conclusion, we believe this method exhibits potential for both specific discrimination and universal signal amplification for RNA analysis in complex matrices.
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