Rapid and Specific Detection of Gastric Cancer EVs Using a Cas12a-Powered Aptasensor with a Novel Targeting Aptamer.

Gastric cancer remains a predominant contributor to global cancer-related mortality, characterized by a pronounced disparity in five-year survival rates between early stage (>90%) and advanced-stage (<30%) disease. This disparity underscores the urgent necessity for accessible early detection methods. Present diagnostic approaches, such as serum biomarkers and endoscopy, either lack adequate sensitivity or are invasive. Extracellular vesicles (EVs) represent promising biomarkers for liquid biopsy; however, a major limitation is the lack of probes that can specifically identify EVs derived from gastric cancer, as most existing markers are broad-spectrum and exhibit low specificity. To address this limitation, we isolated high-purity EVs from gastric cancer cells and utilized a combined immunomagnetic bead-based SELEX strategy to identify a novel aptamer, H-EV-4-1, which demonstrates high affinity (Kd = 13.32 ± 2.69 nM) and specificity for gastric cancer EVs. Subsequently, this aptamer was incorporated into a CRISPR-Cas12a-based biosensor. The aptamer was hybridized with a biotinylated oligonucleotide (H1-biotin) and immobilized on magnetic beads. Upon binding of the target EVs, the aptamer was displaced, thereby exposing H1-biotin to activate the Cas12a/crRNA complex. This activation induced the trans-cleavage of a fluorescent reporter, producing a quantifiable signal. This aptasensor facilitates the rapid, highly sensitive, and specific detection of gastric cancer EVs, presenting a promising platform for the development of noninvasive, point-of-care early diagnostic tools.