Scalable production of highly sensitive nanosensors based on graphene functionalized with a designed G protein-coupled receptor

Abstract

We have developed a novel, all-electronic biosensor for opioids that consists of an engineered μ-opioid receptor protein, with high binding affinity for opioids, chemically bonded to a graphene field-effect transistor to read out ligand binding. A variant of the receptor protein that provided chemical recognition was computationally redesigned to enhance its solubility and stability in an aqueous environment. A shadow mask process was developed to fabricate arrays of hundreds of graphene transistors with average mobility of ∼1500 cm(2) V(-1) s(-1) and yield exceeding 98%. The biosensor exhibits high sensitivity and selectivity for the target naltrexone, an opioid receptor antagonist, with a detection limit of 10 pg/mL.

Figure 1

Fabrication process for high quality graphene field effect transistors (GFETs). (a) Schematic of the fabrication process (see Methods and main text for description). (b) Copper foil shadow mask placed in contact with graphene on catalytic copper foil. Narrow regions of graphene protected by the mask are eventually transferred onto source and drain contacts to form the transistor channel. (c) Example of a GFET device made by transferring graphene stripes onto prefabricated electrodes and photograph of an array of 192 GFET devices, with ∼99.5% device yield.

Figure 2

Performance characteristics of graphene field effect transistors (GFETs). (a) Representative set of 50 I–V_g curves, demonstrating the uniformity of the electrical characteristics. (b, c) Histograms of GFET mobility and Dirac voltage along with Gaussian fits (black curves).

Figure 3

Results of characterization by Raman spectroscopy and atomic force microscopy (AFM). (a) Raman spectrum of graphene before (red data) and after (black data) exposure to diazonium salt solution. The strongly enhanced D-band (near 1360 cm^–1) after diazonium treatment indicates the formation of carboxybenzene sites on the graphene surface. (b) AFM image of soluble μ-receptor proteins (white dots) decorating the graphene surface. The density of protein molecules is approximately 10 times greater on the graphene as compared to the SiO₂ substrate. Scale bar is 2 μm. (c) Histogram of the heights of proteins indicating that the 46 kDa μ-receptor monomer is ∼4 nm tall on the surface, with dimers and trimers of 8 and 12 nm, respectively.

Figure 4

Current–gate voltage (I–V_G) characteristic measurements after chemical treatment and naltrexone exposure. (a) I–V_G plots after successive functionalization steps. After functionalization with the solubilized μ-receptor, exposure to a solution of 1 μg/mL naltrexone in buffer leads to an increase in the Dirac voltage of 8.5 V (green curve to orange curve). (b) Magnified view of the Dirac voltage increase. (c) I–V_G plots after successive functionalization steps with the device now exposed to a solution of 100 pg/mL naltrexone in buffer (green curve to orange curve). The Dirac voltage increase is 1.8 V. (d) Magnified view of this shift in the Dirac voltage. (e) Sensor response (increase in Dirac voltage) as a function of naltrexone concentration. The signal is still discernible from the bare buffer response at 10 pg/mL naltrexone. The data are fit to a modified Hill–Langmuir equation (black curve; see main text for details). The buffer response (green line) is defined as the average response plus one standard error of 15 GFETs exposed to pure buffer without protein.