Mechanistic Insights into Label-Free SERS Discrimination of Structurally Similar Small-Molecule Biomarkers: A DFT Study of 4-HBA and 4-HPAA on Au Clusters

In this study, density functional theory (DFT) calculations were employed to investigate the interactions between structurally similar small molecules, 4-hydroxybenzoic acid (4-HBA) and 4-hydroxyphenylacetic acid (4-HPAA), and Au-enhanced substrates in the context of surface-enhanced Raman scattering (SERS). The calculated molecular electrostatic potential (ESP) results indicate that both molecules exhibit pronounced electronegativity at the carboxyl and hydroxyl groups, suggesting that these regions may serve as potential active sites for interactions with the Au substrate. Based on the possible binding sites and binding configurations, 4-HBA/4-HPAA–Au6 complex models were constructed, and their binding energies were calculated. The binding energy calculations confirm that thermodynamically stable molecule–gold cluster complexes are formed under different binding sites and binding modes. Through analysis of the frontier molecular orbitals (FMO) and charge density difference (CDD) of the isolated molecules and their complexes, charge transfer (CT) excitation between 4-HBA/4-HPAA and the Au6 cluster near different active sites was identified. Through theoretical Raman/SERS spectral analysis of the complexes, we found that pronounced selective enhancement, frequency shifts, and spectral broadening of characteristic vibrational modes occur under different docking configurations. Finally, based on the observed characteristic frequency variations of the two molecules, a theoretically reproducible, highly sensitive, and high-throughput label-free detection strategy is proposed for the identification of structurally similar small-molecule biomarkers, 4-HBA and 4-HPAA, in physiological matrices. This study not only deepens the understanding of the interaction mechanisms between molecules and enhancement substrates in the SERS effect but also demonstrates the theoretical feasibility of label-free identification of 4-HBA and 4-HPAA in physiological samples using SERS. In addition, this work provides a promising label-free sensing strategy for the detection of structurally similar small-molecule biomarkers in physiological matrices.