Cysteamine

A monolayer graphene film was produced by CVD (Supplementary Note 1) and transferred onto a clean SiO₂/Si substrate to fabricate a FET device by photolithography. The device was thermally annealed to improve the contacts between graphene and electrodes (50 nm Au/5 nm Cr), and then Au NPs were deposited onto graphene by vacuum evaporation (Covap, Angstrom Corp.) and thermal annealing at 200 °C for 30 min. The resulting graphene/Au NPs was submerged in 10⁻² M cysteamine (Sigma-Aldrich) solution. The assembly of cysteamine on Au NPs was kept overnight at room temperature, followed by rinsing with ethanol for several times to remove extra cysteamine. The cysteamine-modified device was immersed in 10⁻⁵ M PP (Sigma-Aldrich) and N,N-dimethylformamide (DMF) solution for 15 h, by adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich) and N-hydroxysuccinimide (Sigma-Aldrich) as the catalysts. The resulting FET sensor, with immobilized PP, was rinsed with DMF, acetone, ethanol, and distilled water in sequence to remove extra residues. The graphene/Au/Cys-PP was dipped in Cd²⁺, Zn²⁺, or Mg²⁺ solution for 6 h before tests. Owing to the coordination reaction with PP, Cd²⁺, Zn²⁺, or Mg²⁺ was bound to the surface of FET to charge-dope graphene. A solution chamber, which was employed in all solution-based measurements, was fabricated by a three-dimensional printer.

dSTORM imaging was performed using an Elyra PS.1 microscope (Carl Zeiss Microscopy) equipped with a Plan-Apochromat 100×/1.46 oil objective and a liquid cooled EMCCD camera (Andor Technology). Imaging was carried out in MEA imaging buffer as previously described (36 (link)). In short, fresh stock solutions (1 M cysteamine in 360 mM HCl, 10% glucose in PBS, 70 mg/ml glucose oxidase in PBS, and 20 mg/ml catalase in PBS) were prepared the day before imaging and stored at 4°C and mixed directly before imaging to final concentrations of 0.124 M cysteamine (Sigma), 44.8 mM HCl, 8.6% glucose, 1.08 mg/ml glucose oxidase from Aspergillus niger (Sigma), and 0.0773 mg/ml catalase from bovine liver (Sigma) in PBS. Imaging was performed in 12.8 × 12.8-μm areas in an inclined total internal reflection fluorescence microscope mode (37 (link)). Single molecule fluorescence detection on the EMCCD camera was acquired with 100 × 100-nm pixel size, 20-ms Exposure time, and 100 Gain. 20,000 image frames were acquired for each channel. Both channels were imaged sequentially in 500 frame sequences and the appropriate filters and lasers for each dye were used (642 nm for Alexa Fluor 647 and 488 nm for Atto 488). The images were analyzed with the ImageJ plugin SMLocalizer (38 (link)).

N-Isopropylacrylamide (NIPAm), N,N,N′,N′-tetramethylethylenediamine (TEMED), N-tert-butylacrylamide (TBAm), phosphate-buffered saline (PBS), and N,N′-methylenebisacrylamide (BIS) were purchased from Alfa Aesar (USA). Ammonium persulfate (APS) was purchased from Acros (USA), glutaraldehyde (GA) was produced by Amresco (USA), and silica gel, acrylic acid (AAc), 3-aminopropyltriethoxysilane (APTES), HSA, lysozyme, pepsin, bovine serum albumin (BSA), cysteamine, ethanolamine, ethanol, and toluene were obtained from Merck (Germany). Acetone was obtained from VWR Chemicals (USA), and N-(3-aminopropyl) methacrylamide hydrochloride (APM) was purchased from Polysciences, Inc. (USA).

The imaging solution for dSTORM was prepared as reported. Briefly, the following stock solutions were prepared:•

0.1 M Tris supplemented with 20 mM NaCl, pH 8, filtered by 0.02 μm filter (VWR, cat. no. 516-1501), stored at 4°C (2× imaging solution of dSTROM)

•

25% glucose, stored at 4°C (2.5× imaging solution of dSTORM)

•

1 M cysteamine (Merck, cat. no. 30070) in 0.36 M HCl, stored at 4°C for no more than 1 week (20× imaging solution of dSTORM)

•

GOD buffer (24 mM PIPES, 4 mM MgCl₂, 2 mM EGTA) at pH 6.8 and filtered by 0.02 μm filter, stored at 4°C

•

20 mg/mL glucose oxidase from Aspergillus niger (Merck, cat. no. G2133) in GOD buffer, centrifuge filtered with 0.22 μm filter (Merck, cat. no. UFC30GV0S), flash-frozen in liquid nitrogen, stored in −80°C (40× imaging solution of dSTORM)

•

5 mg/mL catalase (Merck, cat. no. C40) in GOD buffer, centrifuge filtered with 0.22 μm filter (Merck, cat. no. UFC30GV0S), flash-frozen in liquid nitrogen, stored in −80°C (125× imaging solution of dSTORM)

The final working imaging solution for dSTORM contains 0.5 mg/mL glucose oxidase, 40 μg/mL catalase, 50 mM cysteamine, and 10% glucose in 50 mM Tris supplemented with 10 mM NaCl at pH 8. This solution was prepared freshly and immediately before imaging.