CEMs-SELEX: DNAzyme-powered de novo biomarker discovery and noninvasive cancer diagnosis.

The ability to interrogate complex biological environments without prior target knowledge is a key challenge in chemical sensing. Conventional methods are limited in discovering previously unidentified biomarkers. To overcome this, we developed CEMs-SELEX (cell extracellular mixtures-systematic evolution of ligands by exponential enrichment), a de novo selection platform that screens for functional DNAzymes responsive to disease-specific CEMs. Applied to tongue squamous cell carcinoma (TSCC), we identified C502, a DNAzyme activated by tumor secretions with high affinity (Kd = 24.49 nanomolar) and fast catalytic turnover (Kcat = 47.56 per second). Integrated multiomics and affinity pull-down identified annexin A2 (ANXA2) as its target, and molecular docking with mutagenesis elucidated the recognition mechanism. Engineering C502 into a DNA Walker enabled ultrasensitive detection of ANXA2 in clinical serum and saliva. In 80 individuals, the assay clearly differentiated patients with TSCC from healthy donors, highlighting its potential as a noninvasive diagnostic framework. Collectively, this work closes the loop from functional probe discovery to clinical assay, offering a generalizable platform for noninvasive cancer diagnostics.