Abstract
Sensitive detection of disease-specific biomarkers with high accuracy is crucial for early diagnosis, therapeutic monitoring, and understanding underlying pathological mechanisms. Traditional methods, such as immunohistochemistry and enzyme-linked immunosorbent assays (ELISA), face limitations due to the complex and expensive production of antibodies. In this context, aptamers, short oligonucleotides with advantages like easy synthesis, low cost, high specificity, and stability, have emerged as promising alternatives for biomolecular sensing. In this study, we introduce a surface-enhanced Raman spectroscopy (SERS) aptasensor for the multiplexed detection of human α-thrombin and vascular endothelial growth factor (VEGF-165). By leveraging aptamer-based biorecognition elements, our approach capitalizes on the small gaps created by aptamers, generating intense electromagnetic hotspots that significantly amplify the SERS signal. This enables simultaneous detection of human α-thrombin and VEGF-165 with remarkable sensitivity (100 fM and 1 pM, respectively). Notably, we also employ a digital protocol for analyzing the obtained vibrational spectra, marking the first-time utilization of this method for such aptasensors and offering precise quantification even at ultralow concentration regimes. We envision the reported aptasensor aptamer-based spectroscopical tools for a wide range of applications in biology and medicine.