Tailoring DNA\nSurface Interactions on Single-Layer\nGraphene: Comparative Analysis of Pyrene, Acridine, and Fluorenyl\nMethyl Linkers

This study investigates the effect of different linkers\nand solvents\non the immobilization of DNA probes on graphene surfaces, which are\ncrucial for developing high-performance biosensors. Quartz crystal\nmicrobalance with dissipation (QCM-D) measurements were used to characterize\nin situ and real-time the immobilization of ssDNA and hybridization\nefficiency on model graphene surfaces. The DNA probes immobilization\nkinetics and thermodynamics were systematically investigated for all\nthe pairings between three bifunctional linkers1-pyrenebutyric\nacid succinimidyl ester (PBSE), Fluorenylmethylsuccinimidyl carbonate\n(FSC), and Acridine Orange (AO) succinimidyl esterand three\norganic solvents (DMF, DMSO, and 10% DMF/ethanol). The linker’s\nspatial orientation and effective surface modification for DNA probe\nattachment were also evaluated based on footprints and DNA probe surface\ncoverage. Graphene surfaces functionalized with PBSE in DMF achieved\nthe highest DNA probe surface density (up to 1.31 × 10<sup>13</sup> molecules cm<sup>–2</sup>) and fastest kinetic, <i>p</i> values above 4, and hybridization efficiencies of at least 70%,\nwith 20 to 30% of ssDNA directly adsorbed nonspecifically on the functionalized\ngraphene surface, which has significant implications for the design\nof sensitive biosensors. The efficiency of the ethanolamine-NHS blocking\nreaction was estimated to be 80%. The surface packing density of the\nlinker was estimated at 25% of the entire surface coverage for PBSE,\nand about 22 and 13% for AO and FSC, respectively. Overall, the surface\ncoverage achieved for probe DNA was in the same order of magnitude\nas that obtained on flat gold surfaces (≥10<sup>13</sup> molecules\ncm<sup>–2</sup>), typically used in biosensors. These findings\nhighlight the importance of the selected conditions for graphene surface\nmodification to achieve high DNA probe surface density on graphene\nmaterials. These results underscore the critical role of interface\nengineering in achieving target functional outcomes in biosensing\ntechnology.