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The development of biosensor arrays able to detect specific protein interactions is highly needed to precisely dissect fundamental biological systems and to broaden the scope of biomarker detection, especially to refine diagnostics of subtyped diseases such as cancer. Graphene-based field-effect transistor sensors (GFETs) are a promising emerging technology for such biomolecular sensing applications: their atomically-thin surface provides label-free sensitivity to biomolecules via direct electrostatic interactions, and their small footprint enables compact, rapid and parallelized assays. The selectivity of GFET sensors, however, requires functionalization of the graphene surface with a layer of bio-recognition species (“probe molecules”). In this presentation, we will describe our recent progress in immobilizing proteins as probe molecules on graphene field-effect transistors, specifically (1) monoclonal antibodies against a biomarker specific to MLL-translocated acute myeloid leukemia and (2) small Ras GTPase proteins regulated by various effector proteins. First, we will describe our GFET sensor design, based on on-chip arrays of GFETs made from CVD-grown graphene, mounted with a multi-channel delivery flow-cell enabling parallel assays on sub-ensembles of sensors. We will then present our investigation of protein immobilization on graphene. We found that antibodies and small proteins can spontaneously adhere to the graphene surface, but this adhesion is partially reversible under solution flow. To create stable anchor groups at the graphene surface which can then capture the protein probes, we propose to use covalent chemistry on the graphene surface. In particular, we developed a protocol based on electrochemically-driven aryldiazonium chemistry to increase the rate of formation and the density of anchor groups on the graphene surface. Using time-resolved electrical measurements, we observed a specific electrical signal associated with the irreversible immobilization of protein probes on the graphene surface. Finally, we will discuss the use of such protein-functionalized GFETs for the detection of specific probe-target interactions, for applications in fundamental biophysics and cancer diagnostics.

Field-effect transistor devices based on functionalized graphene (G-FETs) are a promising technology for biomolecular sensing applications due to the several advantages they present, including the label-free detection of biomolecules with direct electrical read-out, real-time detection and multiplexing capability. The development of highly selective and sensitive sensors for protein biomarkers is especially desirable to open novel technological avenues for the early detection and monitoring of biomarkers associated with cancer diseases. In this presentation, I’ll describe our recent progress on the design and development of a label-free immunosensor based on antibody-modified graphene field-effect transistors for protein biomarker detection. Specifically, monoclonal antibodies were selected to target a protein biomarker specific to MLL translocated acute myeloid leukemia and we tested approaches for their immobilization on graphene. We found that the antibodies can spontaneously adhere to the graphene surface but this adhesion is partially reversible under solution flow. In order to stabilize the immobilization of antibodies, we developed a protocol based on electrochemically-driven chemistry to form stable covalent anchor groups at the graphene surface which can then capture antibodies. We optimized the rate of formation of anchor groups at the surface in order to maximize the density of anchor groups while maintaining high electrical currents in the graphene. We were then able to record a specific electrical signature showing irreversible immobilization of antibodies on the graphene surface, thus allowing further optimization of target detection. Employing a combination of microfluidics and real-time electrical measurements, we then investigated the response of the sensors to non-specific interactions with graphene as well as their response to specific antibody-antigen interactions in phosphate buffer solution. Finally, the sensors response to different concentrations of target protein was characterized to assess their performance parameters.