Nanozyme sensor arrays for detecting versatile analytes from small molecules to proteins and cells

Supporting Information Nanozyme sensor arrays for detecting versatile analytes from small molecules to proteins and cells Xiaoyu Wang, a, Li Qin, a, Min Zhou, a Zhangping Lou, a and Hui Wei a,b,* a Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing National Laboratory of Microstructures, Nanjing University, Nanjing, Jiangsu 210093, China. b State Key Laboratory of Analytical Chemistry for Life Science and State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Collaborative Innovation Center of Chemistry for Life Sciences, Nanjing University, Nanjing, Jiangsu 210023, China. * E-mail: weihui@nju.edu.cn; Web: http://weilab.nju.edu.cn S1

Table of contents Supplementary Figure 1. Representative TEM images of Pt, Ru, and Ir nanoparticles. Supplementary Figure 2. (a) XPS spectrum of Pt 4f in Pt NPs. (b) XPS spectrum of Ru 3p in Ru NPs. (c) XPS spectrum of Ir 4f in Ir NPs. Supplementary Figure 3. Zeta potentials of Pt, Ru, and Ir nanozymes. Supplementary Figure 4. (a-c) Typical absorption spectra of different reaction systems of OPD, OPD + H 2O 2, OPD + nanozymes, and OPD + H 2O 2 + nanozymes after catalytic oxidation in ph 4.5, 0.2 M acetate buffer at 37 C. (d-f) Typical absorption spectra of 1 mm TMB, 2 mm OPD, and 1 mm ABTS after catalytic oxidation with 10 mm H 2O 2 in ph 4.5 acetate buffer at 37 C, in the presence of 10 μg/ml of Pt, Ru, and Ir nanozymes. Supplementary Figure 5. (a) Kinetic curves of A 450 for monitoring the catalytic reation of 2 mm OPD with various concentration of Pt nanozymes in the presence of 10 mm H 2O 2. (b) Kinetic curves of A 450 for monitoring the catalytic reation of 2 mm OPD with various concentration of H 2O 2 in the presence of 5 μg/ml Pt nanozymes. (c, d) ph and temperaturedependent peroxidase mimicking activities of Pt nanozymes. Supplementary Figure 6. The steady-state kinetics assays of Pt nanozyme. Supplementary Figure 7. The steady-state kinetics assays of Ru nanozyme. Supplementary Figure 8. The steady-state kinetics assays of Ir nanozyme. Supplementary Figure 9. Chemical structure of six biothiols studied in this work. Supplementary Figure 10. Typical absorption spectra for monitoring the catalytic oxidation of OPD in the presence of Pt nanozyme with various concentration of biothiols. Supplementary Figure 11. Typical absorption spectra for monitoring the catalytic oxidation of OPD in the presence of Ru nanozyme with various concentration of biothiols. Supplementary Figure 12. Typical absorption spectra for monitoring the catalytic oxidation of OPD in the presence of Ir nanozyme with various concentration of biothiols. Supplementary Figure 13. Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 5 μm of biothiols. Supplementary Figure 14. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 10 μm of biothiols. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 10 μm of biothiols. Supplementary Figure 15. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 100 μm of biothiols. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 μm of biothiols. Supplementary Figure 16. Canonical variable 1 of the sensor arrays plotted versus different concentrations of Cys. Supplementary Figure 17. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 100 μm of biothiols in the presence of 1% FBS without pretreament. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 μm of biothiols in the presence of 1% FBS. Supplementary Figure 18. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 20 μm of biothiols in the presence of 1% FBS. The sample pretreatment procedures were conducted by a centrifugal filter unit. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 20 μm of biothiols in the presence of 1% FBS. S2

Supplementary Figure 19. Kinetic curves of A 450 for monitoring the catalytic oxidation of 2 mm OPD with 40 mm H 2O 2 in the presence of 1 μg/ml of (a) Pt, (b) Ru, and 0.25 μg/ml of (c) Ir nanozymes with various proteins. Supplementary Figure 20. Dependence of colorimetric response (A/A 0) in the presence of Pt, Ru, and Ir nanozymes on the concentration of (a, b, c) Cyt and (d, e, f) HSA. Supplementary Figure 21. Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 20 nm of proteins. Supplementary Figure 22. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 50 nm of proteins. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 50 nm of proteins. Supplementary Figure 23. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 100 nm of proteins. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 nm of proteins. Supplementary Figure 24. 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 2 μm of proteins. Supplementary Figure 25. Canonical variable 1 of the sensor array plotted versus different concentrations of HSA. Supplementary Figure 26. Concentration-dependent response of the as-prepared nanozyme sensor arrays. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards various concentrations of Cyt. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against six different concentrations of Cyt. (c) Canonical variable 1 of the sensor array plotted versus different concentrations of Cyt. Supplementary Figure 27. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 100 nm of proteins in the presence of human urine. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 nm of proteins in the presence of human urine. Supplementary Figure 28. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards the mixture of HSA and GSH. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against the mixture of HSA and GSH. Supplementary Table 1. Kinetics parameters of Pt, Ru, and Ir nanozymes as well as HRP. Supplementary Table 2. Identification of 30 unknown biothiol samples. Supplementary Table 3. Basic physical properties of protein analytes. Supplementary Table 4. Kinetics parameters of Ir, Ir-GSH, Ir-CYS, and Ir-HSA. Supplementary Table 5. Identification of 45 unknown protein samples. S3

Supplementary Figure 1. Representative TEM images of (a, b) Pt nanoparticles, (c, d) Ru nanoparticles, and (e, f) Ir nanoparticles. S4

Supplementary Figure 2. (a) XPS spectrum of Pt 4f in Pt NPs. (b) XPS spectrum of Ru 3p in Ru NPs. (c) XPS spectrum of Ir 4f in Ir NPs. S5

Supplementary Figure 3. Zeta potentials of Pt, Ru, and Ir nanozymes. Each error bar shows the standard deviation of three independent measurements. We measured the zeta potentials of Pt, Ru, and Ir nanoparticles because the surface charges would affect the interaction between nanozymes and analytes. As shown in Figure S2, all the three nanozymes showed negative zeta potentials. S6

Supplementary Figure 4. (a-c) Typical absorption spectra of different reaction systems of OPD, OPD + H 2O 2, OPD + nanozymes, and OPD + H 2O 2 + nanozymes after catalytic oxidation in ph 4.5, 0.2 M acetate buffer at 37 C. (d-f) Typical absorption spectra of 1 mm TMB, 2 mm OPD, and 1 mm ABTS after catalytic oxidation with 10 mm H 2O 2 in ph 4.5 acetate buffer at 37 C, in the presence of 10 μg/ml of Pt, Ru, and Ir nanozymes. S7

Supplementary Figure 5. (a) Kinetic curves of A 450 for monitoring the catalytic reation of 2 mm OPD with various concentration of Pt nanozymes in the presence of 10 mm H 2O 2. (b) Kinetic curves of A450 for monitoring the catalytic reation of 2 mm OPD with various concentration of H 2O 2 in the presence of 5 μg/ml Pt nanozymes. (c, d) ph and temperaturedependent peroxidase mimicking activities of Pt nanozymes. Each error bar shows the standard deviation of three independent measurements. As shown in Figure S5, the Pt nanozymes exhibited H 2O 2 concentrations, catalyst concentrations, and ph-dependent peroxidase mimicking activity, which is similar with natural peroxidases. However, the catalytic activity of Pt nanozymes slightly increased as the temperature increased, demonstrating the higher thermal stability of Pt nanozymes than natural peroxidases. S8

Supplementary Figure 6. The steady-state kinetics assays of Pt nanozyme. Plots of the velocity of the reaction versus different concentrations of H 2O 2 (a, 0.5 mm TMB) or TMB (b, 50 mm H 2O 2). Double reciprocal plots of the velocity versus varying concentration of (c) H 2O 2 and (d) TMB. Each error bar shows the standard deviation of three independent measurements. S9

Supplementary Figure 7. The steady-state kinetics assays of Ru nanozyme. Plots of the velocity of the reaction versus different concentrations of H 2O 2 (a, 0.5 mm TMB) or TMB (b, 50 mm H 2O 2). Double reciprocal plots of the velocity versus varying concentration of (c) H 2O 2 and (d) TMB. Each error bar shows the standard deviation of three independent measurements. S10

Supplementary Figure 8. The steady-state kinetics assays of Ir nanozyme. Plots of the velocity of the reaction versus different concentrations of H 2O 2 (a, 0.5 mm TMB) or TMB (b, 50 mm H 2O 2). Double reciprocal plots of the velocity versus varying concentration of (c) H 2O 2 and (d) TMB. Each error bar shows the standard deviation of three independent measurements. S11

Supplementary Figure 9. Chemical structure of six biothiols studied in this work. S12

Supplementary Figure 10. Typical absorption spectra for monitoring the catalytic oxidation of OPD in the presence of Pt nanozyme with various concentration of biothiols. (a) Cys, (b) DTT, (c) MA, (d) ME, and (e) MS. S13

Supplementary Figure 11. Typical absorption spectra for monitoring the catalytic oxidation of OPD in the presence of Ru nanozyme with various concentration of biothiols. (a) Cys, (b) DTT, (c) MA, (d) ME, and (e) MS. S14

Supplementary Figure 12. Typical absorption spectra for monitoring the catalytic oxidation of OPD in the presence of Ir nanozyme with various concentration of biothiols. (a) Cys, (b) DTT, (c) MA, (d) ME, and (e) MS. S15

Supplementary Figure 13. Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 5 μm of biothiols. Each error bar shows the standard deviation of five measurements. A and A 0 were the absorption of oxopd in the presence and absence of biothiols in the nanozyme reaction systems, respectively. S16

Supplementary Figure 14. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 10 μm of biothiols. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 10 μm of biothiols. The canonical scores were calculated by LDA for the identification of six biothiols. S17

Supplementary Figure 15. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 100 μm of biothiols. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 μm of biothiols. The canonical scores were calculated by LDA for the identification of six biothiols. S18

Supplementary Figure 16. Canonical variable 1 of the sensor arrays plotted versus different concentrations of Cys. S19

Supplementary Figure 17. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 100 μm of biothiols in the presence of 1% FBS without pretreament. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 μm of biothiols in the presence of 1% FBS. The canonical scores were calculated by LDA for the identification of six biothiols and serum. S20

Supplementary Figure 18. (a) Colorimetric response patterns ((A 0-A)/A 0) of nanozyme sensor arrays towards 20 μm of biothiols in the presence of 1% FBS. The sample pretreatment procedures were conducted by a centrifugal filter unit. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 20 μm of biothiols in the presence of 1% FBS. The canonical scores were calculated by LDA for the identification of six biothiols and serum. S21

Supplementary Figure 20. Dependence of colorimetric response (A/A 0) in the presence of Pt, Ru, and Ir nanozymes on the concentration of (a, b, c) Cyt and (d, e, f) HSA. Each error bar shows the standard deviation of three independent measurements. S23

Supplementary Figure 21. Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 20 nm of proteins. Each error bar shows the standard deviation of five measurements. A and A 0 were the absorption of oxopd in the presence and absence of proteins in the nanozyme reaction systems, respectively. S24

Supplementary Figure 22. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 50 nm of proteins. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 50 nm of proteins. The canonical scores were calculated by LDA for the identification of nine proteins. S25

Supplementary Figure 23. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 100 nm of proteins. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 nm of proteins. The canonical scores were calculated by LDA for the identification of nine proteins. S26

Supplementary Figure 24. 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 2 μm of proteins. The canonical scores were calculated by LDA for the identification of nine proteins. S27

Supplementary Figure 25. Canonical variable 1 of the sensor array plotted versus different concentrations of HSA. S28

Supplementary Figure 26. Concentration-dependent response of the as-prepared nanozyme sensor arrays. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards various concentrations of Cyt. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against six different concentrations of Cyt. The canonical scores were calculated by LDA for the identification of nine proteins. (c) Canonical variable 1 of the sensor array plotted versus different concentrations of Cyt. S29

Supplementary Figure 27. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards 100 nm of proteins in the presence of human urine. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against 100 nm of proteins in the presence of human urine. The canonical scores were calculated by LDA for the identification of nine proteins and urine. S30

Supplementary Figure 28. (a) Colorimetric response patterns (A/A 0) of nanozyme sensor arrays towards the mixtures of 200 nm HSA and 200 μm GSH at different ratios. Each error bar shows the standard deviation of five measurements. (b) 2D canonical score plot for the first two factors of colorimetric response patterns obtained against the mixtures of HSA and GSH. The canonical scores were calculated by LDA for the identification of nine proteins. As we know, cell lysate consists of biothiols, proteins, etc. Before cells discrimination assays, we examined the ability of sensor arrays to differentiate the mixture of GSH and HSA. As shown in Figure S28, these mixtures with different ratios were clustered into different groups with no errors or misclassifications, suggesting that the sensor arrays could discriminate the mixture of GSH and HSA. The result above demonstrated the possibility for discrimination of cells using their lysate solutions. S31

Supplementary Table 1. Kinetics parameters of Pt, Ru, and Ir nanozymes as well as HRP. Catalyst Substrate K m (mm) V max (Ms -1 ) Ref. Pt TMB 0.863 1.74 10-6 This work Pt H 2 O 2 7.92 5.356 10-7 This work Ir TMB 0.33 1.27 10-6 This work Ir H 2 O 2 44.28 1.37 10-6 This work Ru TMB 0.0907 5.97 10-7 This work Ru H 2 O 2 17.56 2.19 10-6 This work HRP TMB 0.434 10 10-8 [1] HRP H 2 O 2 3.7 8.71 10-8 [1] S32

Supplementary Table 2. Identification of 30 unknown biothiol samples. No. Concentration Source Data Identification Verification 1 5 μm 0.40076 0.14568 0.47921 DTT DTT 2 5 μm 0.44114 0.28571 0.51422 ME ME 3 5 μm 0.49659 0.39299 0.62822 MA MA 4 5 μm 0.02729 0.17647 0.42135 MS MS 5 5 μm 0.17599 0.23279 0.48999 Cys Cys 6 5 μm 0.07913 0.05757 0.37941 GSH GSH 7 10 μm 0.75347 0.23117 0.43923 DTT DTT 8 10 μm 0.70891 0.2772 0.58316 ME ME 9 10 μm 0.77887 0.29745 0.61538 MA MA 10 10 μm 0.3011 0.13124 0.2749 MS MS 11 10 μm 0.33366 0.2751 0.54691 Cys Cys 12 10 μm 0.08574 0.10906 0.27386 GSH GSH 13 20 μm 0.88816 0.36878 0.61096 DTT DTT 14 20 μm 0.84605 0.47059 0.65738 ME ME 15 20 μm 0.86184 0.56109 0.74466 MA MA 16 20 μm 0.54474 0.39367 0.51439 MS MS 17 20 μm 0.35379 0.3033 0.50628 Cys Cys 18 20 μm 0.03684 0.22059 0.36026 GSH GSH 19 50 μm 0.91682 0.45618 0.72695 DTT DTT 20 50 μm 0.89834 0.45379 0.59856 DTT ME 21 50 μm 0.89249 0.61705 0.78922 MA MA 22 50 μm 0.61826 0.56895 0.59647 MS MS S33

23 50 μm 0.58418 0.44178 0.60838 Cys Cys 24 50 μm 0.35091 0.29652 0.49341 GSH GSH 25 10 μm in serum 0.45787 0.16095 0.48904 DTT DTT 26 10 μm in serum 0.54021 0.34993 0.6137 ME ME 27 10 μm in serum 0.6783 0.42749 0.65903 MA MA 28 10 μm in serum 0.27809 0.09538 0.34795 GSH MS 29 10 μm in serum 0.3378 0.29429 0.49726 Cys Cys 30 10 μm in serum 0.2522 0.20614 0.35065 GSH GSH The samples in red were mis-identified. S34

Supplementary Table 3. Basic physical properties of protein analytes. Protein MW (kda) pi α-amylase (Amy) 50 5.0 Bovine serum albumin (BSA) 66.3 4.8 Trypsin (Try) 24 10.5 Cytochrome c (Cyt) 12.3 10.7 Transferrin (Tfn) 76 5.9 Human serum albumin (HSA) 69.4 5.2 Hemoglobin (Hem) 64.5 6.8 Lysozyme (Lys) 14.4 11 Glucose oxidase (GOx) 160 4.2 Nine kinds of proteins with different characteristics including molecular weight and isoelectric point (pi) were chosen to study the discrimination ability of the nanozyme sensor arrays. S35

Supplementary Table 4. Kinetics parameters of Ir, Ir-GSH, Ir-CYS, and Ir-HSA. Catalyst Substrate K m (mm) V max (Ms -1 ) Ref. Ir TMB 0.33 1.27 10-6 This work Ir H 2 O 2 44.28 1.37 10-6 This work Ir-GSH TMB 0.20 0.77 10-6 This work Ir-GSH H 2 O 2 104.0 1.55 10-6 This work Ir-Cys TMB 0.14 0.57 10-6 This work Ir-Cys H 2 O 2 174.8 1.89 10-6 This work Ir-HSA TMB 0.155 0.61 10-6 This work Ir-HSA H 2 O 2 134.3 1.71 10-6 This work S36

Supplementary Table 5. Identification of 45 unknown protein samples. No. Concentration Source Data Identification Verification 1 20 nm 0.3856 0.5772 0.51703 Lys Lys 2 20 nm 0.2825 1.3728 1.10401 BSA BSA 3 20 nm 0.3845 0.9789 0.72384 Tra Tra 4 20 nm 0.4089 0.9360 0.64781 Amy Amy 5 20 nm 0.2692 1.3494 1.20742 HSA HSA 6 20 nm 0.6272 0.8853 0.69951 Tfn Try 7 20 nm 0.1429 0.5382 0.37105 Cyt Cyt 8 20 nm 0.8776 0.9828 0.83029 GOx GOx 9 20 nm 0.2105 0.6591 0.47141 Hem Hem 10 50 nm 0.4255 0.6361 0.53059 Lys Lys 11 50 nm 0.3198 1.3129 1.65106 BSA BSA 12 50 nm 0.3430 0.9312 0.92072 Tfn Tra 13 50 nm 0.5365 0.6686 0.56804 Amy Amy 14 50 nm 0.3727 1.2939 1.53558 HSA HSA 15 50 nm 0.7209 1.0801 1.32959 Try Try 16 50 nm 0.2024 0.6713 0.60237 Cyt Cyt 17 50 nm 1.0381 1.0341 0.93945 GOx GOx 18 50 nm 0.7067 0.8608 1.03933 Hem Hem 19 100 nm 0.4857 0.5853 0.52569 Lys Lys 20 100 nm 0.4052 1.3618 2.06324 BSA HSA 21 100 nm 0.3841 0.9288 0.99605 Tfn Tra 22 100 nm 0.6418 0.5805 0.62846 Amy Amy 23 100 nm 0.3952 1.4465 2.05534 HSA HSA 24 100 nm 0.5985 0.9071 1.0751 Try Try 25 100 nm 0.1933 0.5079 0.71937 Cyt Cyt 26 100 nm 1.0297 1.1320 1.1581 GOx GOx 27 100 nm 0.5935 1.0982 1.71542 Hem Hem 28 100 nm 0.8923 0.6877 0.60709 Lys Lys 29 100 nm 0.5320 1.5614 1.97817 BSA HSA 30 100 nm 0.5923 1.0275 1.3472 Tfn Tra 31 100 nm 0.8567 0.4395 0.8015 Amy Amy 32 100 nm 0.5907 1.5668 2.03274 HSA HSA 33 100 nm 0.2227 0.9924 1.09823 Try Try 34 100 nm 0.3665 0.7955 0.84584 Cyt Cyt 35 100 nm 1.2820 1.2216 1.23806 GOx GOx S37

36 100 nm 0.9526 1.5183 1.83492 Hem Hem 37 1 μm 0.5852 0.605 0.58333 Lys Lys 38 1 μm 0.5513 1.4776 2.08333 BSA Cyt 39 1 μm 0.4370 0.9365 1.40741 Tfn Tra 40 1 μm 0.8339 0.6573 1.00926 Amy Amy 41 1 μm 0.6104 1.3147 1.85648 HSA HSA 42 1 μm 0.4873 0.8580 0.89815 Try Try 43 1 μm 0.6028 1.4601 2.05093 Cyt Cyt 44 1 μm 0.6732 0.9191 1.06481 GOx GOx 45 1 μm 0.5840 2.1756 3.23611 Hem Hem The samples in red were mis-identified. S38