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The sensitive detection of the dysregulated expression of ten-eleven translocation 1 (TET1) dioxygenase, a key DNA 5-methylcytosine (5 mC) oxidation regulator in the expression of developmental genes, is of significant importance for the diagnosis of various genetic diseases and cancers. This study describes the establishment of a highly sensitive fluorescent TET1 bioassay based on the 5 mC-modified/Zn²⁺-dependent DNAzyme-containing hairpin probe and the autocatalytic and concatenated DNAzyme/catalytic hairpin assembly (CHA) signal amplification cascades. TET1 target molecules specifically recognize and cut the 5 mC sites in the hairpin probes to release active DNAzyme sequences, which bind and cleave the double-stem-loop substrate strands to trigger multiple concatenated signal amplification recycling cycles with the presence of the fuel strands and two fluorescently quenched signal hairpins. These DNA reaction cascades thus result in the unfolding of lots of signal hairpins to substantially recover fluorescence for highly sensitive TET1 assay with a calculated detection limit of 6.9 fM. Additionally, such bioassay shows high selectivity toward TET1 and its real applicability has been successfully demonstrated for cancer cell lysate and human serum samples.

01 Oct 2025·Talanta

Element-bridged dual-walker amplification for sensitive detection of multiple miRNAs by inductively coupled plasma mass spectrometry

Article

Author: Li, Daxiu ; Tian, Jian ; Tang, Yaqin ; Qin, Yao ; He, Tingting ; Tang, Yike ; Xiang, Yi

The simultaneous detection of multiple microRNAs (miRNAs) is critical for advancing precise disease diagnosis and treatment strategies. Herein, we introduce a novel method for the sensitive detection of multiple miRNAs using inductively coupled plasma mass spectrometry (ICP-MS), based on a platinum group element-bridged DNAzyme dual-walker amplification system. Upon binding to target miRNAs, the terminal of DNAzyme is released, triggering two DNAzyme-mediated ribonucleotide hydrolysis walking processes driven by Mg²⁺. These DNAzyme-walking sequences are recycled, enabling the continuous cleavage of substrate strands. After magnetic separation, the generated element pairs are quantified by mass spectrometry, enabling simultaneous and highly sensitive detection of multiple miRNAs. The method demonstrates a linear detection range from 5 fM to 10 pM with detection limits of 1.8 fM for miRNA-21, 1.1 fM for miRNA-199a, and 1.5 fM for miRNA-499, respectively.Furthermore, the approach exhibits remarkable specificity, making it suitable for detecting miRNAs in human serum. By demonstrating such a high sensitivity and specificity, this DNA walker-based sensing strategy holds significant potential for the development of versatile platforms to detect other miRNAs at varying expression levels.

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