Immobilization of Variant Receptor on GFETs

The immobilization of C-terminus-containing variant receptor, wsMOR-G2_Cter, on GFETs was done through the linker 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE, Sigma-Aldrich, St. Louis, MO) as indicated in Fig. 1b. GFET arrays were incubated in 1 mM PBASE in N, N-dimethylformamide (DMF, Fisher Scientific, Hampton, NH) for 20 h. A long incubation time is essential to achieve maximum coverage of PBASE on graphene. The aromatic pyrenyl group in PBASE binds to the graphene basal plane through π − π stacking [19 ]–[21 (link)]. Next the GFETs were exposed to 2μg/ml of wsMOR-G2_Cter in 10 mM sodium phosphate (NaPi) buffer (pH=8.0, 0.01% SDS, sodium dodecyl sulfate) for 3 h. SDS is present in the buffer since it is needed during the protein purification process and it is not easily removed as we discussed previously. [3 (link)] The succinimide group in the PBASE molecule conjugates with an amine group in the protein to form a stable amide bond and immobilize the protein. A series of G-protein solutions with known concentrations in 10 mM NaPi buffer solution (pH=8.0, 0.01% SDS) were then added onto the GFET surfaces for 1h to allow for G-protein coupling. The arrays were completely rinsed by deionized water to wash off any unbounded molecules and then N₂ blow-dried after each exposure to the solution.