Benchmarking SERS Platforms for Label-Free G-Quadruplex Analysis

Abstract Efficient adsorption of DNA onto metal nanoparticle surfaces is essential for an SERS-based structural analysis. However, the negative surface charge of commonly used nanoparticles promotes electrostatic repulsion with DNA, limiting adsorption and SERS enhancement and often requiring the use of aggregating agents. As Raman enhancement depends not only on the nanoparticle charge but also on the nanoparticle morphology, composition, and surface chemistry, systematic evaluation of nanoparticles with diverse physicochemical properties is necessary to identify optimal conditions for DNA adsorption and SERS detection. Metallic nanoparticles differing in metal (silver or gold), surface charge (positive or negative), and shape (spheres or stars) were synthesized by chemical reduction. To eliminate residual synthesis species, silver and gold nanospheres were also prepared by pulsed-laser ablation in liquid (PLAL). Eight nanoparticle types were characterized by UV/vis/NIR absorption spectroscopy, electrophoretic and dynamic light scattering, and scanning electron microscopy. Their SERS performance for DNA structural analysis was evaluated using the human telomeric sequence d[(TTAGGG)4] (Tel24), which adopted an antiparallel G-quadruplex (G4) in the presence of sodium ions (100 mmol/L), enabling comparison of nonfolded and folded DNA. SERS spectra were recorded with 532 and 785 nm excitation. Circular dichroism spectroscopy was used to confirm the DNA structures. Negatively charged citrate-stabilized silver and gold nanospheres (AgNP_cit and AuNP_cit) and silver nanostars (AgNS_cit) produced the most intense and structurally informative SERS spectra. Spectral markers, including shifts of guanine bands associated with Hoogsteen hydrogen bonding, bands assigned to G-tetrads and d(TTA) loops, and changes in adenine and guanine ring-breathing band intensities, clearly distinguished nonfolded from G4 DNA. The adenine-to-guanine intensity ratio (IdA/IdG) proved to be particularly sensitive to G4 formation. Despite being free of residual surface species, PLAL-prepared nanoparticles (AgNP_PLAL and AuNP_PLAL) showed a substantially weaker SERS enhancement. Likewise, positively charged spermine-coated nanoparticles (AgNP_sp and AuNP_sp) performed poorly, despite favorable electrostatic interactions with DNA. Near-infrared excitation (785 nm) was superior to 532 nm as visible excitation often produced interfering signals or no detectable spectra. Overall, citrate-stabilized silver nanospheres exhibited the best performance, providing reliable and intense SERS detection of both nonfolded and G-quadruplex Tel24. These findings provided practical guidelines for selecting SERS substrates and excitation wavelengths for distinguishing nonfolded DNA from G4 structures.