Multiplexed lncRNA Analysis in Breast Cancer via Cascade Amplification Reaction through Fluorescent and Electrochemical Dual-Signal Output Modalities

Long noncoding RNAs (lncRNAs) have increasingly come to the forefront as promising blood biomarkers in the realm of cancer diagnosis and treatment, sparking intense interest in early cancer detection. Nevertheless, considering that cancer is a complex, multistage disease, the detection of a single lncRNA alone is insufficient to precisely mirror the progression of the disease. In the present study, we devised a sophisticated dual-mode sensing strategy that ingeniously combined fluorescent and electrochemical modalities through a cascade amplification reaction, which allowed for the concurrent identification of lncRNA MALAT1 and HOTAIR. In the presence of MALAT1 and HOTAIR, the corresponding DNAzyme activities were activated. Under the impetus of Mg²⁺, the DNAzymes cleaved specific sites on their substrates, thereby generating a copious amount of trigger sequences (T). Subsequently, these trigger sequences were isolated via magnetic separation and then participated in downstream toehold-mediated strand displacement (TMSD) reactions. With the assistance of fuel strands, the cyclic reactions gave rise to substantial fluorescent signals from labeled FAM and Cy3, as well as electrochemical responses from labeled methylene blue (MB) and ferrocene (Fc), thus facilitating the fluorescent/electrochemical dual-mode sensing. The limits of detection as low as 6.3 fM for MALAT1 and 15.2 fM for HOTAIR have been achieved. Significantly, as a proof of concept and preliminary feasibility exploration, this method has been applied to quantitatively assess the levels of MALAT1 and HOTAIR in multiple cancer cells and 13 whole blood samples from breast cancer patients, which demonstrated high levels of consistency with those obtained by real-time quantitative polymerase chain reaction (qRT-PCR) test kits.