A Label-Free Graphene Oxide-Enhanced Piezoelectric Acoustic Biosensor for DLX1 Detection

Distal-less homeobox 1 (DLX1) has emerged as a promising urinary biomarker for prostate cancer. This study developed a label-free piezoelectric acoustic biosensor for selective DLX1 detection using a quartz crystal microbalance (QCM). The QCM gold electrode was sequentially functionalized with L-cysteine, graphene oxide (GO), and an amine-terminated DLX1 capture probe covalently immobilized through EDC–NHS-mediated amide bond formation. Stepwise surface functionalization was characterized by X-ray photoelectron spectroscopy (XPS), supported by contact angle measurements, X-ray diffraction, and field-emission scanning electron microscopy. XPS provided multi-element evidence for Au–S thiolate formation, GO deposition, amide coupling, probe immobilization, and Watson–Crick hybridization with the synthetic DLX1 target. Under optimized conditions, the biosensor exhibited a linear response to DLX1 concentrations and achieved a limit of detection of 81.19 nM. Non-complementary sequences, including PCA3 and SARS-CoV-2, produced frequency shifts below 9 Hz, confirming high selectivity. Comparative experiments showed that GO-mediated covalent immobilization was essential for reliable detection, whereas direct DNA attachment to bare Au generated anomalous positive frequency shifts, which were attributed to weak physisorption. The proposed platform offers a sensitive and selective strategy for quantitative DLX1 detection and may support future point-of-care nucleic acid diagnostics.