Cascade amplification with an exonuclease-III (exo-III)-triggered target-reconstructed DNAzyme walker for ultrasensitive multiplex analysis of cervical cancer radiotherapy prognosis related microRNAs by ICP-MS

An Exo-III-triggered target-reconstructed DNAzyme walker enables ultrasensitive multiplex miRNA detection. • Cascade amplification integrates Exo-III recycling, polymerase/endonuclease cycling, and DNAzyme walking. • Orthogonal Pt, Ir, Rh tags allow simultaneous ICP-MS quantification of three miRNAs without spectral cross-talk. • Detection limits reach 0.15 fM (miR-21), 0.32 fM (miR-1246), and 0.26 fM (miR-20a) with excellent linearity. • The method discriminates single-base mismatches and performs well in diluted human serum (92–108% recovery). Cervical cancer remains a leading cause of cancer-related mortality among women, and the prognosis of patients after radiotherapy varies considerably due to differences in tumor radiosensitivity and treatment response. Reliable biomarkers for post-radiotherapy prognosis are therefore critically needed. MicroRNAs (miRNAs) have emerged as promising non-invasive indicators, yet accurate detection is hindered by their low abundance and the lack of multiplexing capability in conventional methods. Herein, we report a highly sensitive and specific platform for simultaneous detection of three cervical cancer radiotherapy-associated miRNAs (miR-21, miR-1246, and miR-20a) by integrating an Exo-III-triggered target-reconstructed DNAzyme walker with inductively coupled plasma mass spectrometry (ICP-MS). The strategy employs a structurally locked DNA walker that is sequentially activated through Exo-III-mediated target recycling and DNA polymerase/endonuclease-assisted cyclic amplification, ultimately unleashing a DNAzyme walker to cleave element-labeled substrates. Using orthogonal platinum group element tags (Pt, Ir, Rh), the released tags are quantified by ICP-MS without spectral cross-talk. The method achieves detection limits as low as 0.15 fM, 0.32 fM, and 0.26 fM for miR-21, miR-1246, and miR-20a, respectively, with excellent single-nucleotide discrimination and negligible cross-reactivity. Successful spike-recovery experiments in diluted human serum validate its practical feasibility. This cascade amplification strategy offers a robust, multiplex, and ultrasensitive tool for non-invasive liquid biopsy-based prognostic assessment of post-radiotherapy cervical cancer, and can be readily adapted for other disease-related miRNA panels. Download: Download high-res image (121KB) Download: Download full-size image