Surface-Enhanced Raman Scattering Active Gold Nanoparticles. with Enzyme-Mimicking Activities for Measuring Glucose and

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1 Surface-Enhanced Raman Scattering Active Gold Nanoparticles with Enzyme-Mimicking Activities for Measuring Glucose and Lactate in Living Tissues Yihui Hu, Hanjun Cheng, Xiaozhi Zhao, Jiangjiexing Wu, Faheem Muhammad, Shichao Lin, Jian He, Liqi Zhou, Chengping Zhang, Yu Deng, Peng Wang, Zhengyang Zhou, *, Shuming Nie,,# and Hui Wei *,, Department of Biomedical Engineering, College of Engineering and Applied Sciences, Collaborative Innovation Center of Chemistry for Life Sciences, Nanjing National Laboratory of Microstructures, Nanjing, Jiangsu , China Department of Urology and Department of Radiology, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu , China Department of Materials Science and Engineering, College of Engineering and Applied Sciences, Jiangsu Key Laboratory of Vehicle Emissions Control, Center of Modern Analysis, and State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu , China # Department of Biomedical Engineering, Emory University, Atlanta, Georgia 30322, United States weihui@nju.edu.cn; Tel: ; Fax: ; Web: weilab.nju.edu.cn zyzhou@nju.edu.cn 1

2 Table of contents Figure S1. (A) TEM image of citrate-protected AuNPs, (B) TEM image of citrate-protected AgNPs, and (C) SERS performances of AuNPs and AgNPs without or with 1 mm H2O2 treatment. Error bars indicate standard deviations of three independent measurements. Figure S2. (A) Schematic illustration of the synthetic approach to MIL-101, AuNPs@MIL-101 nanozyme, and AuNPs@MIL-101@oxidase integrative nanozyme, (B) Seed-mediated growth method to synthesize l-aunps@mil-101. Note, the models are not drawn to scale. Figure S3. TEM images of MIL-101 in water (A and B), AuNPs@MIL-101@GOx in water (C and D) and AuNPs@MIL-101@LOx in water (E and F). Figure S4. Powder XRD patterns of MIL-101 and AuNPs@MIL-101. Figure S5. UV visible absorption spectra of MIL-101 and AuNPs@MIL-101. Figure S6. Zeta-potential of MIL-101, AuNPs@MIL-101, AuNPs@MIL-101@GOx, and AuNPs@MIL-101@LOx. Figure S7. (A) TEM image of m-aunps@mil-101 with a large size of 500 nm, (B) TEM image of m- AuNPs@MIL-101 with a small size of 220 nm, and (C) Raman intensity of MG at 1615 cm 1 from SERS-active m-aunps@mil-101 with different sized MIL-101 matrix in 50 mm Tris-HCl buffer (ph=7.0) with 10 mm H2O2 and 1.25 mm LMG. Error bars indicate standard deviations of three independent measurements. Figure S8. UV visible absorption spectra of original oxidase solutions and the corresponding supernatants after assembling onto AuNPs@MIL-101. Figure S9. Plots of the velocity (v) of the reaction versus different concentrations of H2O2 (A, 0.5 mm TMB) or TMB (C, 10 mm H2O2) for 100 μg/ml m-aunps@mil-101 nanozymes catalyzed oxidation of H2O2 and TMB in 0.10 M acetate buffer (ph 4.0). Double reciprocal plots of activity of m- AuNPs@MIL-101 nanozymes versus varying concentration of H2O2 (B) or TMB (D). Error bars indicate standard deviations of three independent measurements. Figure S10. Raman intensity of MG at 1615 cm 1 from SERS-active m-aunps@mil-101 in 50 mm Tris-HCl buffer (ph=7.0) with 10 mm H2O2 and 1.25 mm LMG (set of ten replicates). 2

3 Figure S11. Raman intensity of MG at 1615 cm 1 from m-aunps@mil-101 and m-aunps@mil- 101@GOx catalyzed cascade reactions for glucose sensing in 50 mm Tris-HCl buffer (ph=7.0) with 1.25 mm LMG and 100 μm glucose. Error bars indicate standard deviations of three independent measurements. Figure S12. (A) SERS spectra of AuNPs@GOx + glucose, MIL-101@GOx + glucose, and AuNPs@MIL-101@GOx + glucose, respectively. (B) SERS spectra of AuNPs@LOx + lactate, MIL- 101@LOx + lactate, and AuNPs@MIL-101@LOx + lactate, respectively. Figure S13. Optimizing the reaction time and incubation temperature for detecting glucose (A and C) and lactate (B and D) with the integrative nanozymes. The concentrations of the integrative nanozymes and glucose (or lactate) were 100 μg/ml and 500 μm, respectively. Figure S14. Plots of Raman intensity at 1615 cm 1 versus the loading amount of oxidases for detecting glucose (A) and lactate (B). The concentrations of the integrative nanozymes and glucose (or lactate) were 100 μg/ml and 100 μm, respectively. Figure S15. Glucose and lactate metabolism in living brains following ischemia and reperfusion with and without ATX treatment. Table S1. Optimal amount of oxidase assembled onto m-aunps@mil-101. Table S2. Comparison of Km and Vmax between m-aunps@mil-101 and MSN-AuNPs. 3

4 (A) (B) 50 nm 50 nm (C) Normalized Intensity / a.u AuNPs AgNPs 0.0 untreated H 2 O 2 treated untreated H 2 O 2 treated Figure S1. (A) TEM image of citrate-protected AuNPs, (B) TEM image of citrate-protected AgNPs, and (C) SERS performances of AuNPs and AgNPs without or with 1 mm H2O2 treatment. Error bars indicate standard deviations of three independent measurements. To compare the SERS performances of the citrate-protected AuNPs and AgNPs without and with 1 mm H2O2 treatment, the SERS spectra of 1.25 mm LMG in 50 mm Tris-HCl buffer (ph=7.0) with 10 mm H2O2 were measured. As shown in Figure S1, after the H2O2 treatment, the SERS signal from AgNPs decreased to 43% while the SERS signal from AuNPs still retained 90%. This demonstrated the superior stability of AuNPs compared with AgNPs. Therefore, AuNPs were chosen to prepare AuNPs@MIL-101 in this work. 4

5 (A) h Cr 3+ HAuCl 4 citric acid oxidase MIL-101 AuNPs@MIL-101 AuNPs@MIL-101@oxidase (B) HAuCl 4 NH 2 OH HCl Seeds of m-aunps@mil-101 l-aunps@mil-101 Figure S2. (A) Schematic illustration of the synthetic approach to MIL-101, AuNPs@MIL-101 nanozyme, and AuNPs@MIL-101@oxidase integrative nanozyme, (B) Seed-mediated growth method to synthesize l-aunps@mil-101. Note, the models are not drawn to scale. 5

6 (A) (B) 0.5 μm 200 nm (C) (D) 0.5 μm 200 nm (E) (F) 0.5 μm 200 nm Figure S3. TEM images of MIL-101 in water (A and B), in water (C and D) and in water (E and F). MIL-101 was synthesized via a hydrothermal method. 1 TEM images showed that MIL-101 had octahedron plate-like morphology with a mean size of ~ 500 nm. After the incorporation of AuNPs into the MIL-101 framework, the obtained AuNPs@MIL-101 exhibited a little irregular morphology with a mean size of ~ 500 nm (Figure 3). The further assembly of oxidases onto AuNPs@MIL-101 did not significantly affect its structure and morphology (Figure S3C-S3F). 6

7 Figure S4. Powder XRD patterns of MIL-101 and The XRD pattern of the prepared MIL-101 matched well with the literature ones, further confirming the successful preparation. The XRD pattern of the not only exhibited the characteristic diffraction peaks of MIL-101, but also had the diffraction peaks at 38.1, 44.4, and These peaks were assigned to the Au (111), Au (200) and Au (220) lattice planes, indicating the high crystalline nature of the embedded AuNPs within the MIL-101 frameworks. 2 7

8 2.0 Absorbance/ a.u MIL Wavelength/ nm Figure S5. UV visible absorption spectra of MIL-101 and Compared with the absorption spectrum of MIL-101, the absorption spectrum of exhibited a peak at around 570 nm. The peak could be attributed to the collective surface plasmonic resonance of the embedded AuNPs, which would act as excellent substrates for SERS enhancements. 8

9 Figure S6. Zeta-potential of MIL-101, and 9

10 (A) (B) 100 nm 100 nm (C) 4000 Intensity/ a.u m-aunps@mil-101 (small) m-aunps@mil-101 (large) Figure S7. (A) TEM image of m-aunps@mil-101 with a large size of 500 nm, (B) TEM image of m- AuNPs@MIL-101 with a small size of 220 nm, and (C) Raman intensity of MG at 1615 cm 1 from SERS-active m-aunps@mil-101 with different sized MIL-101 matrix in 50 mm Tris-HCl buffer (ph=7.0) with 10 mm H2O2 and 1.25 mm LMG. Error bars indicate standard deviations of three independent measurements. We have synthesized the AuNPs@MIL-101 with different sized MIL-101 matrix and studied their SERS performances. As shown in Figure S7, the size and morphology of the protecting matrix (MIL-101) has little impact on the SERS performances. 10

11 Absorbance/ a.u supernatant of GOx after assebmly original of GOx supernatant of LOx after assebmly original of LOx Wavelength/ nm Figure S8. UV visible absorption spectra of original oxidase solutions and the corresponding supernatants after assembling onto AuNPs@MIL-101. The amount of oxidase assembled onto the AuNPs@MIL-101 was estimated by measuring the absorption spectra before and after the assembly (Figure S8). As shown in Table S1, substantial amount of oxidase was successfully assembled onto the AuNPs@MIL

12 Velocity/ 10-8 M S -1 (A) (B) /Velocity/ 10 8 M -1 S V max = MS -1 K m = mm H 2 O 2 concentration/ mm / H 2 O 2 concentration/ mm -1 (C) (D) Velocity/ 10-8 M S /Velocity/ 10 8 M -1 S V max = MS -1 K m = mm TMB concentration/ mm /TMB concentration/ mm -1 Figure S9. Plots of the velocity (v) of the reaction versus different concentrations of H2O2 (A, 0.5 mm TMB) or TMB (C, 10 mm H2O2) for 100 μg/ml m-aunps@mil-101 nanozymes catalyzed oxidation of H2O2 and TMB in 0.10 M acetate buffer (ph 4.0). Double reciprocal plots of activity of m- AuNPs@MIL-101 nanozymes versus varying concentration of H2O2 (B) or TMB (D). Error bars indicate standard deviations of three independent measurements. 12

13 4000 Intensity/ a.u Sample number Figure S10. Raman intensity of MG at 1615 cm 1 from SERS-active m-aunps@mil-101 in 50 mm Tris-HCl buffer (ph=7.0) with 10 mm H2O2 and 1.25 mm LMG (set of ten replicates). To investigate the reproducibility of the SERS measurements, ten independent measurements were carried out with m-aunps@mil-101 in 50 mm Tris-HCl buffer (ph=7.0) containing 10 mm H2O2 and 1.25 mm LMG. As shown in Figure S10, the Raman intensity of MG at 1615 cm 1 of the ten measurements had a mean value of and a standard deviation of (5.68%), demonstrating the satisfactory reproducibility. 13

14 8000 Intensity/ a.u m-aunps@mil-101 m-aunps@mil-101@gox Figure S11. Raman intensity of MG at 1615 cm 1 from m-aunps@mil-101 and m-aunps@mil- 101@GOx catalyzed cascade reactions for glucose sensing in 50 mm Tris-HCl buffer (ph=7.0) with 1.25 mm LMG and 100 μm. Error bars indicate standard deviations of three independent measurements. Compared with AuNPs@MIL-101@GOx, it was found that AuNPs@MIL-101 almost have no detectable SERS signal for glucose sensing, demonstrating assembling oxidase is necessary for the efficient enzymatic cascade reactions. 14

15 (A) (B) Intensity/ a.u Intensity/ a.u Raman shift/ cm -1 Raman shift/ cm -1 Figure S12. (A) SERS spectra of AuNPs@GOx + glucose, MIL-101@GOx + glucose, and AuNPs@MIL-101@GOx + glucose, respectively. (B) SERS spectra of AuNPs@LOx + lactate, MIL- 101@LOx + lactate, and AuNPs@MIL-101@LOx + lactate, respectively. 15

16 Figure S13. Optimizing the reaction time and incubation temperature for detecting glucose (A and C) and lactate (B and D) with the integrative nanozymes. The concentrations of the integrative nanozymes and glucose (or lactate) were 100 μg/ml and 500 μm, respectively. 16

17 Intensity/ a.u. (A) GOx loading/ mg/g Intensity/ a.u. (B) LOx loading/ mg/g Figure S14. Plots of Raman intensity at 1615 cm 1 versus the loading amount of oxidases for detecting glucose (A) and lactate (B). The concentrations of the integrative nanozymes and glucose (or lactate) were 100 μg/ml and 100 μm, respectively. 17

18 ATX treatment Cerebral ischemia Reperfusion improve ischemis/hypoxia anaerobic respiration aerobic respiration glucose lactate glucose lactate glucose lactate Figure S15. Glucose and lactate metabolism in living brains following ischemia and reperfusion with and without ATX treatment. Due to the blockage of cerebral blood flow as well as increased anaerobic respiration during ischemia, the concentration of glucose greatly reduced while the concentration of lactate increased. Previous studies reported that ATX has protective effects against ischemic stroke. ATX may prevent the glucose and lactate fluctuation levels by potential vasodilation of microvessels. 18

19 Table S1. Optimal amount of oxidase assembled onto GOx LOx (mg/g)

20 Table S2. Comparison of Km and Vmax between and MSN-AuNPs. Nanozymes Substrate Km/ mm Vmax/ 10-8 M. s -1 References m-aunps@mil-101 TMB This work m-aunps@mil-101 H2O This work MSN-AuNPs TMB MSN-AuNPs H2O Note: MSN is the abbreviation for mesoporous silica nanoparticles. 20

21 References (1) Ferey, G.; Mellot-Draznieks, C.; Serre, C.; Millange, F.; Dutour, J.; Surble, S.; Margiolaki, I. A Chromium Terephthalate-Based Solid with Unusually Large Pore Volumes and Surface Area. Science 2005, 309, (2) Hu, Y.; Liao, J.; Wang, D.; Li, G. Fabrication of Gold Nanoparticle-Embedded Metal-Organic Framework for Highly Sensitive Surface- Enhanced Raman Scattering Detection. Anal. Chem. 2014, 86, (3) Tao, Y.; Ju, E.; Ren, J.; Qu, X. Bifunctionalized Mesoporous Silica-Supported Gold Nanoparticles: Intrinsic Oxidase and Peroxidase Catalytic Activities for Antibacterial Applications. Adv. Mater. 2015, 27,