Streptavidin-functionalized terahertz metamaterials for attomolar exosomal microRNA assay in pancreatic cancer based on duplex-specific nuclease-triggered rolling circle amplification

Exosomal microRNA (miRNA) is a promising non-invasive biomarker for liquid biopsies. Herein, we fabricated a terahertz (THz) metamaterial biosensor that comprises an array of gold (Au) discs surrounded by annular grooves for exosomal miRNA assays based on duplex-specific nuclease (DSN)-triggered rolling circle amplification (RCA). In this strategy, the target miRNA is captured by a probe P0 immobilized on magnetic beads (MBs); it then repeatedly releases a primer P1 under the action of DSN, which acts as a highly specific initiator of the subsequent RCA step utilizing biotin-dUTP. After target recycling and nucleic acid amplification, the biotinylated amplification products were captured by the streptavidin (SA)-functionalized THz metamaterials, and further conjugated to SA-modified AuNPs that permit formation of a trimeric complex of SA-biotinylated RCA products-AuNP. The complex population scales with the starting concentration of the target miR-21, resulting in a red shift of the resonance peak of the THz metamaterials. This biosensor can lead to highly specific and sensitive detection with one-base mismatch discrimination and a limit of detection (LOD) down to 84 aM. Significant distinctions are seen in the frequency shifts for exosomal miR-21 quantitation in clinical plasma samples between pancreatic cancer patients and healthy controls. The frequency shifts of the THz metamaterials are consistent versus the reverse transcription-polymerase chain reaction (RT-PCR) results, illustrating the applicability and accuracy of our assay in real clinical samples.