A growing body of research indicates that PIWI-interacting RNAs (piRNAs) encapsulated in circulating exosomes represent promising novel molecular biomarkers suitable for tumor liquid biopsy. Nonetheless, available techniques for the direct in situ identification of exosomal piRNAs are still inadequate. Here, we developed a spherical nucleic acid-based probe called piR-651 that readily enters exosomes after a 2 h incubation period and permits in situ detection of piR-651 with a limit of detection of 5 × 107 particles/μL. Using this probe, we devised a liquid biopsy strategy enabling the in situ analysis of piR-651 in plasma-derived exosomes. The developed assay successfully differentiated piR-651 expression between plasma samples from 21 breast cancer patients and 22 healthy volunteers. Analysis of the receiver operating characteristic curve revealed an area under the curve of 0.9931, while the optimal cutoff value provided a diagnostic sensitivity of 85.7% and specificity of 100%. In addition, the probe supports convenient in situ visualization of piR-651 within living cells. To address the challenges of reduced sensitivity and slow reaction kinetics associated with detecting bulky PIWI-interacting RNA complexes, we carefully optimized both the architecture and operational mechanism of the piR-651 probe. The probe was fabricated by conjugating high-density Anchor-Report DNA duplexes onto 13-nm gold nanoparticles using the butanol dehydration approach. This innovative gold nanoparticle nucleic acid probe design can be extended to create probes for other large-volume nucleic acid targets, thereby facilitating the advancement of further liquid biopsy platforms reliant on molecular biomarkers for cancer diagnosis.
