Cu²⁺-encapsulated DNA nanosphere and filter enable sensitive and rapid analysis of miRNA-155

MicroRNAs (miRNAs) are critical regulators of gene expression, with aberrant levels linked to diseases such as breast cancer. Notably, they are challenging to detect due to their low abundance in complex sample matrices. In this study, a Cu^2⁺-encapsulated DNA nanosphere system capable of homogeneous, enzyme-free, one-pot detection of microRNA-155 (miRNA-155) in human plasma was developed. The system employed self-assembled DNA nanospheres loaded with copper ions as specific recognizers, coupled with a filter membrane-assisted reaction to separate free-Cu²⁺ from residual components. Moreover, dual quantum dots (QDs) were utilized for signal output, using competitive binding to enhance sensitivity for detecting ultra-low and subtle changes in miRNA levels in complex samples. The method achieved an excellent detection performance, with a limit of detection (LOD) at the low-aM level. Additionally, it demonstrated high specificity in distinguishing between different miRNAs and single nucleotide polymorphisms (SNPs). Validation using 38 clinical plasma samples achieved over 95% accuracy in identifying breast cancer patients, demonstrating 100% sensitivity and approximately 90% clinical consistency compared to imaging, pathology, and quantitative real-time polymerase chain reaction (qRT-PCR). The method required minimal sample pre-treatment and was completed within 1 h. Overall, the developed method offers a reliable tool for breast cancer diagnosis and establishes a mode for extending its application to other biomarkers in the clinical setting.