PNA-SERS biosensor for label-free detection of SARS-CoV-2 genomic sequences in saliva

The COVID-19 pandemic has emphasized the need for rapid, sensitive, and accessible molecular diagnostics. In this study, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS) biosensor for the direct detection of SARS-CoV-2 RNA in biological fluids. The proposed sensor is based on a thiolated Peptide Nucleic Acid (PNA) probe immobilized on colloidal gold nanoparticles (AuNPs) deposited on functionalized glass substrates. A stable and selective hybridization with target sequences is provided by the intrinsic characteristics of PNA molecules, such as neutral backbone, high sequence affinity and enzymatic resistance. Whereas AuNPs enables strong signal enhancement and excellent reproducibility, without requiring complex nanofabrication techniques. Overall, the biosensor fabrication relies entirely on standard laboratory procedures and commercially available reagents, making it cost-effective and easily scalable. The detection of the target RNA occurs through label-free SERS, responsible for amplifying the vibrational fingerprint of nucleobases. Multivariate analysis through principal component analysis (PCA) and regression (PCR) further enhances spectral discrimination and detection sensitivity. The sensor exhibits a limit of detection of 110 pM, falling within the clinically relevant range of salivary SARS-CoV-2 RNA concentrations. Detection performance was assessed in both buffer and artificial saliva, demonstrating the potential of the platform for use with real biological samples. Moreover, the device demonstrates high selectivity, effectively distinguishing between fully matched, mismatched, and random sequences.

This work highlights the potential of PNA-SERS biosensors for rapid, amplification-free viral RNA detection and offers a promising approach for point-of-care diagnostics in infectious diseases.