DMSN@AuNPs-enhanced QCM immunosensor array for non-invasive and highly sensitive detection of Cyfra21-1 in saliva.

Early detection of oral cancer is essential for improving clinical outcomes, and salivary biomarkers offer a non-invasive approach for early screening. Cyfra21-1, a cytokeratin fragment detectable in saliva, is a clinically relevant biomarker for oral squamous cell carcinoma (OSCC) assessment and monitoring, supporting point-of-care testing with a clinically meaningful threshold. In this study, we developed quartz crystal microbalance (QCM) immunosensor array for sensitive detection of Cyfra21-1 in saliva. The immunosensors were established based on a sandwich assay, employing nanoparticle-based probes for signal amplification. Three strategies were systematically investigated: (1) covalent immobilization of the primary antibody (Ab1) on the QCM surface via a cysteamine/glutaraldehyde (CysA/GA) self-assembled monolayer, using AuNPs-labeled secondary antibodies (AuNPs@Ab2) as signal probes; (2) oriented immobilization of Ab1 via staphylococcal protein A (SPA), and AuNPs@Ab2 for signal amplification; and (3) signal enhancement through dendritic mesoporous silica nanoparticles loaded with gold nanoparticles (DMSN@AuNPs@Ab2), combined with SPA-based Ab1 immobilization. The SPA-based QCM sensor demonstrated higher sensitivity than the CysA/GA approach, and the DMSN@AuNPs@Ab2 strategy further enhanced the signal. Under optimal conditions, the sensor demonstrated a linear response to Cyfra21-1 from 1 to 100 ng/mL with a detection limit of 0.9 ng/mL, covering clinically reported salivary Cyfra21-1 levels in healthy individuals (∼3.8 ng/mL) and oral cancer patients (17.46 ± 1.46 ng/mL). Furthermore, we also designed a multi-channel QCM array for simultaneous Cyfra21-1 detection over 0.1-100 ng/mL with a detection limit of 0.2 ng/mL, and validated the optimized strategy (Strategy III) in spiked artificial saliva and clinical saliva, highlighting its strong potential for non-invasive clinical diagnostics.