DNAzyme-Powered Rolling Circle Amplification for the Synthesis of Hairpin DNA-Templated Fluorescent Copper Nanoclusters: A Label-Free miRNA Sensing Platform

Specific and sensitive detection of microRNA (miRNAs) is significant for the early diagnosis of cancer. Herein, we developed a cascade amplification strategy that couples DNAzyme with rolling circle amplification (RCA). The amplified DNA products serve as templates for synthesizing fluorescent copper nanoclusters (CuNCs), thereby enabling the label-free and sensitive detection of miRNA-21. First, miRNA-21 is amplified into abundant single-stranded DNA (ssDNA) fragments through a DNAzyme-assisted primary amplification module. Subsequently, the output DNA fragments trigger RCA, generating repetitive hairpin DNA (HP-DNA) structures with polythymine (T) sequences in the loop region. Finally, the amplified products act as templates for synthesizing fluorescent CuNCs, owing to the crowded microenvironment provided by the HP-DNA. The synthesized CuNCs exhibited significantly enhanced photoluminescence (PL) intensity compared to those templated by conventional poly-T single-stranded DNA. Concurrently, the cascade DNAzyme-RCA amplification is significantly more sensitive than traditional RCA, achieving a 4.1 pM detection limit. In addition, the biosensor was used to analyze the expression level of miRNA-21 in human serum and various cell lysates. The proposed detection method features simple design, low cost, and high sensitivity and eliminates the need for fluorophore labeling, holding great potential for clinical diagnostics.