Angiopep2 (Ang-2) peptide linked DNA walker assay for sensitive and accurate detection of glioma cell

Liquid biopsy has emerged as a promising alternative for glioma detection; however, the blood-brain barrier restricts the abundance of circulating tumor cells (CTCs) in peripheral blood, posing a significant challenge for sensitive CTCs-based diagnostics of glioma. To address this limitation, we present an Angiopep2 (Ang-2) peptide linked DNA walker assay for colorimetric glioma cell detection with high sensitivity and specificity. This system integrates Ang-2 and a cholesterol labeled tracker (cholesterol-tracker) as dual-targeting ligands, coupled with a DNA walker-mediated signal amplification strategy for enhanced sensitivity and specificity. Compared to conventional immunoassays relying on monoclonal antibodies, our platform demonstrates superior performance due to its preferential binding to intact glioma cells and the signal amplification induced by the λ-exonuclease-driven DNA walking mechanism. The biosensor achieved an exceptionally low detection limit of 0.12 cells/mL and exhibited strong anti-interference capabilities across multiple cell lines. Furthermore, the biosensor maintained ≥96 % of initial signal retained after 21 days at 4 °C (96.5 % ± 0.35, n = 3), with only marginal signal attenuation observed. Clinical validation using constructed serum samples revealed excellent agreement in glioma concentration calculated by the proposed sensing platform and added glioma cell concentrations. Collectively, our findings demonstrate that this sensing platform represents a rapid, sensitive, specific, and portable technology for the detection of U251 glioblastoma cells, with potential applications in clinical diagnostics of glioma.