Nucleic Acid Amplification Circuit-Based Hydrogel (NACH) Assay for One-Step Detection of Metastatic Gastric Cancer-Derived Exosomal miRNA

Gastric cancer (GC) is recognized as the fifth most prevalent malignant tumor worldwide. It is characterized by diverse clinical symptoms, treatment responses, and prognoses. In GC prognosis, the promotion of epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, thereby triggering the dissemination of tumor cells. This study proposes a nucleic acid amplification circuit-based hydrogel (NACH) assay for identifying exosomal miRNA derived from metastatic GC. The NACH assay employs the rolling circle amplification method and targets miRNA-21, a tumor-related oncogene, and miRNA-99a, which promotes EMT. Specific amplification probes for each target are immobilized within the hydrogel, enabling a streamlined, one-step amplification reaction. The NACH assay exhibits a detection limit of 1 fm for miRNA-21 and miRNA-99a, thereby enabling rapid and highly sensitive on-site detection. Performance evaluation using exosomal miRNA extracted from cell culture media, mouse plasma, and human plasma revealed fluorescence intensity patterns similar to those obtained in qRT-PCR. Furthermore, deploying a custom-developed portable fluorometer for the NACH assay allows for diagnostic performance assessment and point-of-care testing using clinical samples from GC patients. These findings emphasize the potential of the NACH assay to be used as a robust tool for the genetic diagnosis of GC based on exosome detection. The Nucleic Acid Amplification Circuit-Based Hydrogel (NACH) assay is developed to detect metastatic gastric cancer (GC)-related exosomal miRNAs biomarkers. This assay can analyze exosomal miRNA expression levels in a one-step, and its performance has been demonstrated using blood samples from GC patients at both early and metastatic stages. The NACH assay shows potential as a diagnostic tool for liquid biospy. image