Key Laboratory for Material Chemistry of Energy Conversion and Storage, Ministry of Education,

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1 Supporting Information Portable, Self-Powered and Light-Addressable Photoelectrochemical Sensing Platforms using ph Meter Readouts for High-Throughput Screening of Thrombin Inhibitor Drugs Juan Wang, a Mengmeng Song, b Chengguo Hu,* b Kangbing Wu* a a Key Laboratory for Material Chemistry of Energy Conversion and Storage, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan , China b Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan , China * To whom correspondence should be addressed: Dr. Chengguo Hu, cghu@whu.edu.cn; Prof. Kangbing Wu, kbwu@hust.edu.cn (K. Wu). TABLE OF CONTENT A. Supporting Experimental Section Preparation of L-cys derived C 60 materials Construction of P-LAPECS P-LAPECS for Thrombin Detection and Inhibitor Screening Photoelectrochemical Test B. Supporting Figures Schematic representation for the preparation of bioprobe Schematic procedures for the construction of the P-LAPECS Photos of the detection mode of P-LAPECS S-1

2 SEM images and photocurrents of two materials Bioprobe for CEA detection Comparison of two materials Condition optimization Inhibitory efficiency of three kinds of inhibitors A. Supporting Experimental Section Preparation of L-cys derived C 60 materials The C 60 -L-cys nanohybrid was prepared according to a previous literature 1 with slight modification. In brief, 0.4 g L-cys was dissolved in 1.2 ml NaOH solution (7.1 mol/l) and then 8.0 ml ethanol was added into the solution. Next, the above mixture solution was added into a C 60 toluene solution (1 mg/ml, 4 ml) and reacted for 5 days under gentle shaking at room temperature on a multifunctional rotary table. The resulting mixture solution was washed with water on a mixed cellulose ester membrane (MCE, pore size 0.1 µm, Shanghai Xinya Purifier Devices Factory, China) by filtration until the filtrate became neutral, and then redispersed in 4 ml water. The C 60 -L-cys/CR nanohybrid was obtained by drying C 60 -L-cys and grinding with CR at a mass ratio of 1:3 for 1.0 h. Moreover, the C 60 /CR/L-cys composite was prepared by grinding the solid mixture of C 60, CR and L-cys at a mass ratio of 1:3:5 for 1.0 h. The resulting milled mixtures were dissolved in water and thoroughly washed with water by filtration until the filtrate became colorless, and then redispersed in water. The concentration of C 60 in all the C 60 -based nanohybrid solutions is calculated to be 1.0 mg/ml without considering the mass loss of C 60 during synthesis. Construction of P-LAPECS S-2

3 Preparation and partition of GPIFE. The GPIFE was prepared by a simple seeded growth method according to a previous report. 2 Then, a punched tape was used to divide the GPIFE into eight identical sensing zones (the diameter of each zone is 3 mm). The partitioned GPIFE was electrochemically cleaned by potential scanning between -0.2 to 1.6 V in 0.5 M H 2 SO 4 until reproducible cyclic voltammograms were obtained, which was rinsed with copious water and dried in a nitrogen stream. Fabrication of P-LAPECS. First, 8.0 µl of peptide (0.1 mg/ml, activated by 10 mm TCEP) was dropped onto each zone of the partitioned GPIFE and incubated in a humid atmosphere for 12 h at room temperature, followed by rinsing with PBS buffer. Then, each sensing area of the peptide modified GPIFE (peptide/gpife) was covered with 8.0 µl of 5.0 mm MCH in water and kept at room temperature for 3.0 h to block any non-specific reaction sites, which was then rinsed with the washing buffer. Finally, the resulting MCH/peptide/GPIFE was further blocked by 0.1 M MEA for 1 h. The obtained P-LAPECS (MEA/MCH/peptide/GPIFE) was used as the light-addressable sensing platform for the portable detection of thrombin and screen of thrombin inhibitors. P-LAPECS for Thrombin Detection and Inhibitor Screening Thrombin Detection. Taking advantage of the existence of eight separated sensing areas, P-LAPECS can realize thrombin and its inhibitor detection on a single electrode. For thrombin activity detection, thrombin at different concentrations in buffer solutions (8.0 µl, 50 mm Tris-HCl, 100 mm NaCl, ph=8.4) was dropped on the different sensing areas of P-LAPECS (1-4) and incubated at 37 o C for 1.0 h. After the washing step, the above four areas of thrombin/mea/mch/peptide/gpife were covered with 8.0 µl of the C 60 /CR-L-cys@SA solution and incubated at 37 o C for 1.5 h, producing the probe/thrombin/mea/mch/peptide/gpife. S-3

4 Thrombin Inhibitor Screening. For the inhibitor screening experiments, different inhibitors at a desired concentration was added into the thrombin buffer solution to react with thrombin (10 nm) for 30 min. The resulted thrombin mixture was dropped onto the different rest zones of the above P-LAPECS (6-8) to conduct the same procedures followed afterwards as the above thrombin detection. Moreover, one sensing area of P-LAPECS (5) was covered by buffer instead of peptide and underwent all the followed steps, which act as the blank control zone for self-calibration of P-LAPECS. Photoelectrochemical Test. When using an electrochemical workstation as the test instrument, the sensor was immersed in 10.0 ml of 0.1 M PBS (ph 7.4) containing 0.1 M AA and photocurrent responses were collected by irradiating the sensing areas one by one with the portable red laser light. To achieve the portable detection of LAPECS, a handled ph meter and a simple electrochemical cell were adopted. As show in Figure S3, the fabricated sensor was adhered to the surface of glass slide and a punched PVC plate was used to seal the slide (the Pt wire was inserted into the PVC plate). After the addition of electrolyte solution (0.1 M AA/0.1 M PBS (ph 7.4)) into the punched hole, a simple detecting pool was formed. Then, two electrode clips of the ph meter were used to connect GPIFE and Pt wires, respectively. Finally, the ph meter was adjusted to the mv state and used to record the potential value of each sensing area under the irradiation with the red laser light. B. Supporting Figures S-4

5 Figure S1. Schematic representation for the preparation of C 60 /CR-L-cys@SA bioprobe. Figure S2. Schematic procedures for the construction of the P-LAPECS and the detection of thrombin and its potential inhibitors. Figure S3. Photos of the detection mode of P-LAPECS with light off (A) and light on (B). S-5

6 Figure S4. SEM images of C 60 /CR (A) and C 60 /CR-L-cys (B). The materials are dropped on the surface of silicon slice and dried under an infrared lamp. Insets of A and B are their corresponding TEM images. (C) Photocurrents of C 60 /CR and C 60 /CR-L-cys, which were dropped on the surface of GPIFE for overnight absorption and then washed by water. Figure S5. (A) Construction of C 60 /CR-L-cys based PEC bioprobe of rabbit IgG. (B) Schematic procedures for the construction of the sensor for the detection of rabbit IgG. Here, CEA was employed as the interference for signal comparison. (C) Photocurrents of 1 ng/ml rabbit IgG (target, a) and 1 µg/ml CEA (interference, b) on the constructed sensor. S-6

7 Figure S6. (A) Photocurrents of different materials: a. C 60 /CR (the free CR was washed by centrifugation); b. C 60 /CR (the free CR was washed by filtration). The concentrations of a and b were calculated to be 1.0 mg/ml for C 60. The materials are dropped on the surface of ITO and illuminated by a green laser pen of 30 mw at 530 nm for identical comparison with our previous work. 3 (B) UV-vis spectra of the above two materials (a was in 5 times diluted concentration, b was in 50 times diluted concentration). Inset of Part B shows the photos of solutions a and b. Figure S7. (A) Cyclic voltammogram (CV) plots in 1.0 M KCl containing 5.0 mm Fe(CN) 3-/4-6 of different electrodes during the fabrication of P-LAPECS (peptide 2 as the thrombin substrate). (B) Photocurrent of the P-LAPECS (peptide 2 as the thrombin substrate) incubated with 0 (b) and 1 nm (c) thrombin and P-LAPECS incubated with 0 (a) and 1 nm (d) thrombin. (C) Photocurrents of the P-LAPECS (peptide 1 as the thrombin substrate) incubated with thrombin (10 nm) at 37 C for different periods: 0, 10, 20, 30, 40, 50, 60, 70, 90 and 120 min (from bottom to top). (D) Changes in photocurrent intensity as a function of incubation time. S-7

8 Figure S8. Inhibitory efficiency of AEBSF (A), PMSF (B) and benzamidine (C) on the activity of thrombin. The concentration of thrombin is 10 nm. References (1) Lu, Q.; Hu, H.; Wu, Y.; Chen, S.; Yuan, D.; Yuan, R. Biosens. Bioelectron. 2014, 60, (2) Wang, J.; Sun, J.; Hu, C.; Liu, Z.; Hu, S. J. Electroanal. Chem. 2015, 759, 2-7. (3) Hu, C. G.; Zheng, J. N.; Su, X. Y.; Wang, J.; Wu, W. Z.; Hu, S. S. Anal. Chem. 2013, 85, S-8