Supporting Information

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1 Supporting Information Stretchable and Photocatalytically Renewable Electrochemical Sensor Based on Sandwich Nanonetworks for Real-Time Monitoring of Cells Ya-Wen Wang, Yan-Ling Liu, Jia-Quan Xu, Yu Qin, Wei-Hua Huang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan , China These authors contributed equally to this work * whhuang@whu.edu.cn, Tel: Supporting Information includes Experimental Methods and Figure S1-S9. S-1

2 Table of Contents 1. Experimental Methods 1.1 Fabrication of Au NTs/TiO 2 NWs/Au NTs/PDMS film 1.2 Characterization of Au NTs/TiO 2 NWs/Au NTs nanocomposite 1.3 Cell culture and imaging 1.4 Real-time monitoring NO released from HUVECs and 5-HT released from P815 cells 2. Supplementary Figures 3. References 1. Experimental Methods 1.1 Fabrication of Au NTs/TiO 2 NWs/Au NTs/PDMS film Au NTs/TiO 2 NWs/Au NTs/PDMS film fabrication. Ag NWs (35-45 nm in diameter, 10 um in length) solution was diluted into a concentration of 2.0 mg ml -1 in isopropyl alcohol (IPA), and TiO 2 NWs (100 nm in diameter, 20 um in length) powder was added into ethanol and ultrasonic dispersedfor 30 min to get a concentration of 1.0 mg ml -1 TiO 2 NWs/ethanol solution before use. To obtain Ag NWs/PDMS film, appropriate amount of Ag NWs solution was drop-cast on the surface of poly(dopamine)-coated PDMS films and kept standing for 2 min, then the film was spinned at a rate of 400 rpm for 5 s to throw away the excessive Ag NWs solution and transferred onto a heater to evaporate the IPA. Then, TiO 2 NWs solution was spin-coated on the Ag NWs/PDMS film in the same way to get TiO 2 NWs/Ag NWs/PDMS film. Following, Ag NWs solution was again spinned on the TiO 2 NWs/Ag NWs/PDMS film to get the sandwich structure materials Ag NWs/TiO 2 NWs/Ag NWs/PDMS film, and different ratios between Ag NWs and TiO 2 NWs can be obtained by changing the spin-coating times of Ag NWs solution and TiO 2 NWs S-2

3 solution. And the electrode used for electrochemical detection was spinned 3 times of TiO 2 NWs and 1 time of Ag NWs respectively. Finally, the above-prepared Ag NWs/TiO 2 NWs/Ag NWs/PDMS film was replaced to Au NTs/TiO 2 NWs/Au NTs/PDMS film under a mild galvanic replacement reaction in the [Au(en) 2 ]Cl 3 solution according to our previous work Characterization of Au NTs/TiO 2 NWs/Au NTs nanocomposite. Morphology properties. Scanning electron microscopy (SEM) images of Au NTs/TiO 2 NWs/Au NTs were obtained by using a filed-emission microscope (ZEISS SIGMA) and Energy dispersive X-ray spectroscopy (EDX) images were obtained by using an INCAPentalFETx3 Oxford EDX spectrometer. Sheet resistance was measured with a ST-21L portable square resistance meter. Stretching experiments of Au NTs/TiO 2 NWs/Au NTs/PDMS were conducted using a linear sliding motor, and the corresponding resistance was recorded by a multimeter. All electrochemical measurements were handled on a CHI 660A electrochemical workstation. A three-electrode system was used in the experiment including a Au NTs/TiO 2 NWs/Au NTs/PDMS (1 cm 0.5 cm) working electrode, Ag/AgCl reference electrode and Pt counter electrode. Flexibility and stretch deformation tests. Two ends of Au NTs/TiO 2 NWs/Au NTs/PDMS film were contacted with a copper conductive wire and the contacts were insulated with PDMS. The final electrode active area was 1.0 cm 1.0 cm. For flexibility test, the Au NTs/TiO 2 NWs/Au NTs/PDMS film was wrapped on various cylindrical with different curvatures (radius was from 0.5 mm to 12 mm) and folded in half. For stretchability, the ends of a rectangular Au NTs/TiO 2 NWs/Au NTs/PDMS film were quoined by two fixtures connected to a digital force gauge, and stretched along with pre-set parameters. The relative change in the resistance was measured by a multimeter. Characterization of renewable performance of the electrode under UV light. 5-hydroxytryptamine (5-HT), poly-l-lysine (PLL), cell culture medium (CCM) and collagen were chosen to passivate electrode. 2 For 5-HT passivation, electrode was immersed in 0.5 mm 5-HT solution and applied an oxidation potential of +0.6 V (vs. Ag/AgCl) for 600 s. For PLL, CCM and collagen passivation, electrodes were immersed in 0.02% PLL solution, CCM and 0.05% collagen for 48 h. After S-3

4 passivation, the electrode was renewed by UV light irradiation at room temperature for 1 h. A high-pressure mercury lamp (primary wavelength of 365 nm, 12mW/cm 2 ) was used as the UV sources to regenerate electrode. Preparation of the Au NTs/TiO 2 NWs/Au NTs electrode for electrochemical detection. To ensure good connection with the electrochemical workstation, Ag NWs/PDMS film as stretchable conducting wire was pressed onto the Au NTs/TiO 2 NWs/Au NTs/PDMS films to further reduce the possibility of connection failure during mechanical deformation. PDMS pre-polymer was carefully coated on the edge of the film and then thermally cured at 75 C during 2 h to insulate the contact and define a chamber ( cm) to hold analytical test solutions or cell culture. Calibration of Au NTs/TiO 2 NWs/Au NTs electrode for NO. NO was obtained via disproportionation of NaNO 2 in H 2 SO 4 solution and stocked in deaerated PBS solution (1.8 mm at 20 C). 1 Calibration for NO was carried out by steadily dropping NO standard solution into PBS buffer solution stored in the PDMS chamber on Au NTs/TiO 2 NWs/Au NTs/PDMS film. A three electrode system was used in the experiment including Au NTs/TiO 2 NWs/Au NTs/PDMS, Ag/AgCl reference electrode and Pt counter electrode. The potential on the electrode for amperometric oxidation of NO was kept at +0.8 V. Calibration of Au NTs/TiO 2 NWs/Au NTs electrode for 5-HT. 5-hydroxytryptamine powder was dissolved in PBS to get 5mM 5-HT stock solution, different concentrations of standard solution was obtained by diluting the stock solution gradually. Calibration for 5-HT was performed by gentle dropping 5-HT standard solution into PBS solution stored in the PDMS chamber on Au NTs/TiO 2 NWs/Au NTs/PDMS film. A three electrode system was used in the experiment including Au NTs/TiO 2 NWs/Au NTs/PDMS, Ag/AgCl reference electrode and Pt counter electrode. The potential on the electrode for amperometric oxidation of 5-HT was hold at V. 1.3 Cell culture and imaging. Cell culture. Human umbilical vein endothelical cells (HUVECs) were conventionally cultured S-4

5 using RPMI 1640 culture medium with mg/ml HEPES, mg/ml L-glutamine, 0.85 mg/ml NaHCO 3, 12% fetal bovine serum, penicillin and streptomycin (100 U) in culture flask at 37 C in a humidified incubator (95% air with 5% CO 2 ). And mastocytoma cells (P815, DBA/2) were cultured using DMEM (Invitrogen, 12430) culture medium with 10% FBS (Gibco), 1% Sodium Pyruvate 100mM Solution (Invitrogen, ) in culture flask at 37 C in a humidified incubator (95% air with 5% CO 2 ). Fluorescent staining and cell imaging. The viability of cultured HUVECs and P815 cells was assessed by fluorescent live/dead cell markers Calcein-AM and PI. Cells were washed three times with PBS before cell staining, and then Calcein-AM (2 µg ml -1 ) and PI (3 µg ml -1 ) diluted in PBS was added and maintained for 15 min in a humidified incubator. The stained cells were washed three times with PBS before imaging to reduce fluorescence background. 1.4 Real-time monitoring NO released from HUVECs and 5-HT released from P815 cells Cell detection. For NO release detection, electrode was sterilized by water exposed to UV light overnight, followed by PBS washing, CCM washing and seeding HUVECs with an approximate density of cells/cm 2. After being cultured 12 h on electrode, the cells were used for NO release detection and the electrode was stretched to 10% with a speed of mm/s. For 5-HT release detection, the electrode was pre-treated in the same way. P815 cells was seeded on the electrode with an approximate density of cells/cm 2, and cultured in DMEM culture medium with 1 µm 5-HT to facilitate the uptake and storage by mast cells. After being cultured 12 h on electrode, the cells were used for 5-HT release detection and the electrode was stretched to 10% with a speed of mm/s. S-5

6 2. Supplementary Figures Figure S1. a) CVs of 1 mm K 3 [Fe(CN) 6 ] in 1 mol/l KCl on Au NTs/TiO 2 NWs/Au NTs electrode with different ratios between Au NTs and TiO 2 NWs. Ag NWs at the top and bottom of the sandwich structure were spinned 1 time respectively, and then replaced to Au NTs. ST was the spinning times of TiO 2 NWs. b) The renewable efficiency of Au NTs/TiO 2 NWs/Au NTs electrode with different ratios between Au NTs and TiO 2 NWs. Despite the good photocatalytic degradation properties, TiO 2 nanomaterials have poor electrochemical activity owing to low conductivity, therefore different densities of TiO 2 NWs in Au NTs/TiO 2 NWs/Au NTs/PDMS film were optimized. As shown in Figure S1a, this composite materials possessed excellent ability of electrochemical sensing with a pair of reversible redox peak at 0.17 V and 0.22 V, but the redox current was gradually decreased with the increasing of TiO 2 NWs owing to the low conductivity and poor electrochemical activity of the TiO 2 NWs. While, with the increasing of the spinning times, the photocatalytic efficiency gradually increased to 98.7%, displaying that the composite materials possessed excellent photocatalytic ability. Considering the balance of electrochemical performance and photocatalytic ability, TiO 2 NWs were spinned 3 times in Au NTs/TiO 2 NWs/Au NTs electrode, which possessed high photocatalytic activity (96.2%) but also retained good electrochemical sensing in the meanwhile. In addition, we also constructed other structure composite materials based on Au NTs and TiO 2 NWs at the same time, TiO 2 NWs/Au NTs/PDMS film and Au NTs/TiO 2 NWs/PDMS film. Results displayed that this TiO 2 NWs/Au NTs/PDMS film possessed bad electrochemical activity, and Au NTs/TiO 2 NWs/PDMS film possessed good electrochemical activity but the resistance changed a lot after under tensile stress. Therefore, they were not appropriate to be used for stretchable electrochemical sensor. S-6

7 Figure S2. a) R/R 0 as a function of radius of curvature.the curve shows the bending properties of the composite nanomaterials while the radius of curvature is ranged from 0.5 mm to 12 mm. b) R/R 0 as a function of tensile strain. The curve shows the tensile properties of the composite nanomaterials while the materials are stretched from 0 to 50%. Figure S3. CVs of 1 mm K 3 [Fe(CN) 6 ] on Au NTs/TiO 2 NWs/Au NTs electrode before fouling (black line), after fouling (red line) and after photocatalysis-induced cleaning under UV (blue line). The passivation mediums were a) PLL, b) CCM and c) collagen, respectively. Figure S4. a-d) Fluorescent microscopic images of Au NTs/TiO 2 NWs/Au NTs/PDMS film a) before, and b) after being immersed in phycoerythrin-labeled mouse antihuman CD 45 (PE-CD 45) solution, and then c) washed by water and d) irradiated under UV light. e-h) SEM images of Au NTs/TiO 2 NWs/Au NTs/PDMS film e) before, and f) after being immersed in CCM and CD 45 solution, and then g) washed by water and h) irradiated under UV light. S-7

8 i) CVs of 1 mm K 3 [Fe(CN) 6 ] on Au NTs/TiO 2 NWs/Au NTs electrode before fouling (black line) and after fouling (red line), and then washed by water (blue line) and photocatalysis-induced cleaned under UV light (green line). Phycoerythrin-labeled mouse antihuman CD 45 was used to investigate the adsorption and photocatalytic removal of proteins on the sensor surface. The results from both fluorescent imaging and SEM imaging show that proteins are strongly adsorbed on the surface (Figure S4b, f) and difficult to be completely washed out by water or buffer solution (Figure S4c, g), while can be efficiently removed under UV irradiation (Figure S4d, h). The adsorption and removal of proteins on sensor surface are further demonstrated by electrochemical characterizations (Figure S4i). Figure S5. The calibration curve of Au NTs/TiO 2 NWs/Au NTs electrode to a) NO and b) 5-HT. S-8

9 Figure S6. Microscopic images of HUVECs culture on Au NTs/TiO 2 NWs/Au NTs/PDMS film for a) 12 h, b) 24 h, c) 36 h, d) 60 h, e) 72 h and f) stained by Calcein-AM (green) and PI (red). Figure S7. Amperomatic response of the original electrode (blue line), fouled electrode (black line) and renewable electrode to 0.2 µm NO solution. Figure S8. Photograph showing the set-up for electrochemical monitoring of cells cultured on the stretchable sensors submitted to stretching strains. Figure S9. The microscopic images of the Calcein-AM (green) and PI (red) stained P815 cells cultured on Au NTs/TiO 2 NWs/Au NTs. 3. References S-9

10 (1) Liu, Y. L.; Jin, Z. H.; Liu, Y. H.; Hu, X. B.; Qin, Y.; Xu, J. Q.; Fan, C. F.; Huang, W. H. Angew. Chem. Int. Ed. 2016, 55, (2) Xu, J. Q.; Liu, Y. L.; Wang, Q.; Duo, H. H.; Zhang, X. W.; Li, Y. T.; Huang, W. H. Angew. Chem. Int. Ed. 2015, 54, S-10