Development of a highly sensitive C1QC-targeting aptamer and its application in a modified antibody-aptamer sandwich biosensor for C1QC biomarker detection

Cancer remains a leading cause of mortality worldwide, with China bearing a particularly high burden. Early detection through reliable biomarkers is essential for improving patient outcomes. This study reports the development and validation of a highly specific aptamer-based biosensor for detecting the complement protein C1QC, a promising cancer biomarker. Using a multi-step selection process involving Magnetic beads-SELEX, qPCR, flow cytometry, high-throughput sequencing, surface plasmon resonance (SPR), pull-down, and western blotting, we identified Apt 13, a slow-dissociation aptamer with high specificity and affinity for C1QC (K D = 48.7 nM). SPR confirmed the formation of a sandwich complex between Apt 13, C1QC, and Anti_C1QC, confirming their distinct binding sites. We developed two biosensor platforms based on this aptamer: the enzyme-linked antibody-aptamer sandwich (ELAAS) and chemiluminescent antibody-aptamer sandwich (CLAAS) biosensors. The ELAAS biosensor, designed for visual colorimetric detection, demonstrated a limit of detection (LOD) of 98 ng/mL. In contrast, the CLAAS system, utilizing acridinium ester-modified antibodies, achieved an enhanced LOD of 0.375 ng/mL. Receiver Operating Characteristic (ROC) analysis on serum samples from 75 hepatocellular carcinoma (HCC) patients and 20 healthy controls yielded an AUC of 0.942, with 86.67 % sensitivity and 95.00 % specificity, demonstrating CLAAS’s high diagnostic accuracy in differentiating HCC from controls. Further testing in HCC patient serum samples revealed significantly elevated C1QC levels compared to healthy controls, underscoring C1QC’s potential as a HCC cancer biomarker. Apt 13 presents a promising tool for the development of innovative, sensitive, and cost-effective diagnostic technologies for C1QC detection and may offer future potential for targeted therapies.