A Protease-Responsive and Protein-Gated Gold Nanocluster Platform for Non-Invasive Urinary Colorimetric Diagnosis of Glioblastoma.

BACKGROUND

Glioblastoma multiforme (GBM) diagnosis remains challenging because conventional imaging may have limited ability to distinguish tumor progression or recurrence from treatment-related changes, and robust non-invasive biomarkers are still lacking. This study developed a protease-responsive gold nanocluster platform for urinary colorimetric detection of GBM via a synthetic biomarker strategy.

METHODS

We synthesized glutathione-capped AuNCs (GSH-AuNCs) and conjugated them with a substrate-responsive peptide (SRP) and a bulky NeutrAvidin (NAv) protein to form the AuNC-SRP-NAv complex. The NAv acts as a steric shield to lock the peroxidase-like activity of the AuNCs. The complex's physicochemical properties, stability, and protease-triggered activation (MMP-9) were characterized. In vivo biodistribution, renal clearance, and diagnostic performance were evaluated in orthotopic GBM-bearing mice.

RESULTS

The AuNC-SRP-NAv complex (diameter ~18.6 nm) exhibited robust stability and a signal-off state in systemic circulation. Upon encountering GBM-overexpressed MMP-9, the SRP was cleaved, releasing ultra-small AuNC fragments (<6 nm) that rapidly translocated to the urine. In tumor-bearing mice, these urinary AuNCs catalyzed the oxidation of TMB, producing a distinct blue color change within 20 minutes, while healthy controls showed no significant signal. Long-term safety assays (35 days) confirmed excellent biocompatibility with no systemic toxicity.

CONCLUSION

The AuNC-SRP-NAv platform provides a simple, rapid, and non-invasive liquid biopsy approach for GBM. By integrating nanozyme catalysis with protease-responsive renal clearance, this system offers a promising alternative for early-stage tumor monitoring at the point of care.