Tailoring DNA Surface Interactions on Single-Layer Graphene: Comparative Analysis of Pyrene, Acridine, and Fluorenyl Methyl Linkers

This study investigates the effect of different linkers and solvents on the immobilization of DNA probes on graphene surfaces, which are crucial for developing high-performance biosensors. Quartz crystal microbalance with dissipation (QCM-D) measurements were used to characterize in situ and real-time the immobilization of ssDNA and hybridization efficiency on model graphene surfaces. The DNA probes immobilization kinetics and thermodynamics were systematically investigated for all the pairings between three bifunctional linkers─1-pyrenebutyric acid succinimidyl ester (PBSE), Fluorenylmethylsuccinimidyl carbonate (FSC), and Acridine Orange (AO) succinimidyl ester─and three organic solvents (DMF, DMSO, and 10% DMF/ethanol). The linker's spatial orientation and effective surface modification for DNA probe attachment were also evaluated based on footprints and DNA probe surface coverage. Graphene surfaces functionalized with PBSE in DMF achieved the highest DNA probe surface density (up to 1.31 × 10¹³ molecules cm^-2) and fastest kinetic, <i>p</i> values above 4, and hybridization efficiencies of at least 70%, with 20 to 30% of ssDNA directly adsorbed nonspecifically on the functionalized graphene surface, which has significant implications for the design of sensitive biosensors. The efficiency of the ethanolamine-NHS blocking reaction was estimated to be 80%. The surface packing density of the linker was estimated at 25% of the entire surface coverage for PBSE, and about 22 and 13% for AO and FSC, respectively. Overall, the surface coverage achieved for probe DNA was in the same order of magnitude as that obtained on flat gold surfaces (≥10¹³ molecules cm^-2), typically used in biosensors. These findings highlight the importance of the selected conditions for graphene surface modification to achieve high DNA probe surface density on graphene materials. These results underscore the critical role of interface engineering in achieving target functional outcomes in biosensing technology.