Control of Antibody Orientation on Graphene Using Porphyrin Linker Molecules for High-Performance Graphene-Based Immuno-Biosensors

The appropriate orientation of antibodies on the graphene surface is critical for the high performance of graphene biosensors. In this article, we show that a predominantly vertical orientation of antibodies to the graphene surface can be achieved by functionalizing the surface with tetrakis (4-carboxyphenyl) porphyrin (TCPP). Unlike conventional pyrene-based linkers, such as 1-pyrene butanoic acid succinimidyl ester (PBASE), which rely on a single flexible chain, the rigid porphyrin macrocycle of TCPP forms an extended π-π network with graphene and provides sites for uniform and directional antibody coupling. Applying this system to a graphene field effect transistor platform for exosome detection, we achieved 2 orders of magnitude improvement in the detection limit compared to PBASE-functionalized sensors. Computational and experimental analysis revealed that the antibody can highly exhibit vertical orientation on TCPP-functionalized graphene, while mixed orientations on PBASE-functionalized graphene confirm that the sensitivity enhancement is directly caused by the optimized orientation. The high efficiency of the TCPP method was further validated by successfully detecting pancreatic cancer exosome biomarkers in clinical samples. This biofunctionalization method could enable ultrasensitive detection of disease biomarkers on a graphene surface.