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1 Supporting Information Real-Time Monitoring of Insulin Using a Graphene Field-Effect Transistor Aptameric Nanosensor Zhuang Hao, a,b Yibo Zhu, a Xuejun Wang, a Pavana G. Rotti, c,d Christopher DiMarco, a Scott R. Tyler, c Xuezeng Zhao, b John F. Engelhardt, c,d James Hone, a and Qiao Lin *,a a Department of Mechanical Engineering, Columbia University, New York, New York 10027, United States b Department of Mechanical Engineering, Harbin Institute of Technology, Harbin, Heilongjiang , China c Department of Anatomy and Cell Biology and d Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa 52242, United States *Corresponding author: qlin@columbia.edu S-1
2 1. Device Fabrication Protocol As the fabrication protocol shown in Figure S1, the wafer (285 nm SiO 2 /Si) employed as the substrate of the device was cleaned successively with acetone, IPA and deionized water, then dried by N 2. The device was fabricated via a bilayer lift-off process. Two layers of resist (sacrificial layer LOR 3A and photoresist S1811) were sequentially spin-coated on the wafer using spin coater (Spin coater WS B, Laurell). The planar source, drain and gate electrodes consisting of a Cr/Au structure (5 nm/45 nm) were defined on the SiO 2 surface using standard photolithography (SÜSS MA6 mask aligner, SÜSS Microtech) and metal deposition (Angstrom evovac deposition system, Angstrom Engineering) techniques. Then the device was kept in remover PG overnight at room temperature for resist removal. The sample was exposure to oxygen plasma (Plasma etch system, Diener) to remove the remaining residue on the SiO 2 surface. Figure S1. Schematic of the fabrication protocol of a GFET nanosensor. (a) Clean the wafer surface with oxygen plasma. (b) Spin coating the surface with photoresist LOR 3A and S1811. (c) Standard photolithography. (d) Deposition of Cr/Au. (e) Photoresist removal. (f) Graphene transfer and patterning. (g) Microfluidic package of the GFET device. Figure S2. Transfer of monolayer graphene onto arbitrary substrates. S-2
3 The synthesized CVD graphene was then transferred (Figure S2) onto the substrate. To pattern the graphene sheet into a rectangle shape stretching over drain/source electrodes, the photoresist S1811 layers was spin coated on graphene surface in turn for photolithography. After exposure and the resist developing with developer AZ 300 MIF, graphene sheet in the unprotected area was etched using oxygen plasma to create the GFET unit with a regular conducting channel. 2. Surface Functionalization Protocol To immobilize the aptamer onto the graphene channel, the sensor was first immersed in 5 mm PASE solution for 2 hours at room temperature and sequentially rinsed with dimethylformamide (DMF) to remove any free PASE. The device was then rinsed with PBS followed by incubation with 100 nm aptamer IGA3 solution overnight at room temperature. After rinsing with PBS, 100 mm ethanolamine was added onto the graphene channel for 1 h to deactivate and block the excess reactive groups remaining on the graphene surface. A polydimethylsiloxane (PDMS)-based open chamber (~30 μl) was used to hold sample solutions and was finally bonded to the device. (a) Device 1 (b) graphene Device (c) Device (d) Device 2 S-3
4 1626 (e) Device (f) Device 3 (g) (h) Figure S3. Measurements with PASE and aptamer functionalized graphene. (a, c, e) Raman spectrum of PASE functionalized graphene with different devices. (b, d, f) Transfer characteristics measured with PBS buffer on the bare graphene channel, PASE functionalized graphene channel and aptamer functionalized graphene channel respectively with different devices. (g, h) 3D-image of the graphene surface (before and after aptamer functionalization) taken by AFM. To test the successful functionalization of PASE and aptamer IGA3, we characterized the graphene channels of different devices with Raman Spectrum and AFM. The G-band splitting (1626, 1628, 1626 cm -1, respectively) was observed in the Raman spectra (Figure S3 a, c, e), implying the PASE was coupled to graphene. Transfer characteristic curves, corresponding to different graphene functionalization steps and displaying an ambipolar behavior with different V Dirac were plotted in Figure S3 b, d, f. The results, PASE induced p-type doping and single strand DNA aptamer induced n-type doping to graphene, were consistent with previous works, indicating the successful functionalization. 3. Control Experiments The real-time monitoring control experiment was also conducted with glucagon. The value of Δ /Δ, max, calculated with the time-resolved selective response, was less than 0.3. The results indicated that the graphene based aptameric nanosensor showed high specificity to insulin over glucagon. S-4
5 0.8 Buffer (PBS) Glucagon Insulin 150 nm 1000 nm Δ /Δ,max nm 500 nm 39 nm 80 nm 500 nm nm 1000 nm Time (s) Figure S4. Time-resolved responses of the IGA3 aptamer-based graphene nanosensor toward insulin (39, 80, 150, 500 nm and 1μM) and glucagon (10, 100, 500 nm and 1μM). 4. Components of KRB Buffer The components of the base KRB buffer are listed in Table S1. Table S1 Components of the base KRB buffer Components HEPES 5.96 NaCl 6.72 NaHCO 3 2 Mass (g) KCl MgCl 2 6 H 2 O BSA CaCl 2 2 H 2 O First, dissolve all the chemicals with 1 L DI water. After adding CaCl 2 2 H 2 O, stir the solution as the CaCl 2 2 H 2 O may not completely dissolve until the solution ph is adjusted. Finally, check the ph of the solution and adjust to 7.3 to 7.5 using either 1 N NaOH or 1 N HCl, if necessary. S-5
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