Supporting Information. Triple Quenching of a Novel Self-Enhanced Ru(II) Complex by. Hemin/G-Quadruplex DNAzymes and Its Potential Application to

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1 Supporting Information Triple Quenching of a Novel Self-Enhanced Ru(II) Complex by Hemin/G-Quadruplex DNAzymes and Its Potential Application to Quantitative Protein Detection Min Zhao, a Ni Liao, a,b Ying Zhuo,,a Ya-Qin Chai, a Ji-Peng Wang, a Ruo Yuan,a a Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing ,China. b College of Biological and Chemical Engineering, Panzhihua University, Panzhihua , China. Corresponding authors at: Tel.: , fax: addresses: yingzhuo@swu.edu.cn (Y. Zhuo), yuanruo@swu.edu.cn (R. Yuan). S-1

2 Table of Contents for Supporting Information Figure S1 A schematic diagram of the prepared procedure of PTCA-PEI-Ru(II) compounds. Figure S2 The photos of (a) PEI, (b) Ru(II), (c) PEI-Ru(II), (d) PTCA and (e) PTCA-PEI-Ru(II) under the natural light (up) and UV light of 365 nm (down). Figure S3 SEM image of HPtNPs. Figure S4 FTIR spectrum of PTCA-PEI-Ru(II) compounds (KBr pellet). Figure S5 (A) Effect of NADH concentration towards the ECL intensity of BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE. (B) ECL-time profiles of BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE testing in 3 ml PBS buffer under air-saturated PBS (red line) and N 2 -saturated PBS (green line), respectively. (C) ECL-time profiles of BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE testing in 3 ml PBS buffer without (red line) and with (blue line) hemin. Figure S6 (A) The relation between ECL intensity and ph by using PTCA-PEI-Ru(II)/GCE detecting in different ph PBS buffers. (B) The relation between ECL intensity and NADH concentration by using BSA/TBA I/ AuNPs/PTCA-PEI-Ru(II)/GCE detecting in 3 ml PBS (ph 8.0) containing different concentration NADH. All ECL signals were obtained through setting photomultiplier tube at 800 V and applied potential between 0.2 V and 1.25 V with a scan rate of 100 mv/s. Table S1 Comparison of the proposed method with some reported methods for thrombin detection. Table S2 Comparison of sensitivity of the proposed method with the one using single or dual quenching mechanism for thrombin detection. S-2

3 Detail Preparation Process of PTCA-PEI-Ru(II) Figure S1 A schematic diagram of the prepared procedure of PTCA-PEI-Ru(II) compounds. Photos of Preparation Process of the Self-enhanced PTCA-PEI-Ru(II) Figure S2 The photos of (a) PEI, (b) Ru(II), (c) PEI-Ru(II), (d) PTCA and (e) PTCA-PEI-Ru(II) under the natural light (up) and UV light of 365 nm (down). SEM Characterization of HPtNPs S-3

4 Figure S3 SEM image of HPtNPs. The morphology of HPtNPs was investigated in Figure S3 which demonstrated a well-defined shape and hollow spheres structure with size of about 100 nm, indicating that the HPtNPs were prepared successfully. FTIR Spectra Characterization of PTCA-PEI-Ru(II) Compounds The FTIR spectrum of the self-enhanced PTCA-PEI-Ru(II) compound was displayed in Figure S4. The very broad stretching band ν(n-h) at cm -1 is assigned to the structure of PEI. The peaks at 1650 cm -1 st(c=o) assigned to the structure of PTCA. The peaks at cm -1, 1458 cm -1 assigned to the typical absorption peaks of aromatic of PTCA and Ru(II). Additionally, the peak at 1087 cm -1 assigned to the N-C from the PEI and Ru(II). Therefore, the FTIR data indicated that PTCA-PEI-Ru(II) were successfully cross-linked as one molecule. S-4

5 Figure S4 FTIR spectrum of PTCA-PEI-Ru(II) compounds (KBr pellet). Feasibility Analyses of Triple Quenching toward PTCA-PEI-Ru(II) The effect of NADH concentration have been investigated in the section of the optimization of experimental conditions for aptasensor, we could see that the ECL intensity was increasing with the NADH concentration before reaching the saturation concentration. In other words, the ECL intensity was decreased with the decreasing NADH concentration (Figure S5(A)). When the TBA II bioconjugates was immobilized on the thrombin/bsa/tba I/AuNPs/PTCA-PEI-Ru(II)/GCE via specific binding of aptamer-protein, the hemin/g-quadruplex DNAzymes in TBA II bioconjugates mimicked NADH oxidase to oxidize NADH to NAD + for consuming the NADH. It caused the decrease of the ECL intensity due to the consumption of the NADH. In order to investigate the effect of O 2 towards PTCA-PEI-Ru(II), an exploratory experiment has been designed by comparing the BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE was measured in 3 ml air-saturated PBS and N 2 -saturated PBS. Specifically, the N 2 -saturated PBS was with high purity nitrogen for S-5

6 at least 20 m in to deaerate O 2. And the result is displayed in the Figure S5(B). It can be seen that the red curve (under air-saturated condition) was much lower than the green curve (under N 2 -saturated condition), suggesting O 2 could quench the ECL intensity. It is attributed that the O 2 oxidizes the excited state of the PTCA-PEI-Ru(II)* to form the PTCA-PEI-Ru(III), making the ECL emission reduce. In order to confirm the effect of hemin towards the ECL responses, the BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE was testing in 3 ml PBS without and with 0.05 mm hemin, respectively. As exhibited in Figure S5(C), we could see that the ECL intensity with hemin (blue line) was significantly lower than that without hemin (red line). It was attributable to the quenching effect of hemin toward Ru(II) complex. Therefore, the triple quenching paths were feasibility in this siganl-off mode for thrombin detection. Figure S5 (A) Effect of NADH concentration towards the ECL intensity of BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE. (B) ECL-time profiles of BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE testing in 3 ml PBS buffer under air-saturated PBS (red line) and N 2 -saturated PBS (green line), respectively. (C) ECL-time profiles of BSA/TBA I/AuNPs/PTCA-PEI-Ru(II)/GCE testing in 3 ml PBS buffer without (red line) and with (blue line) hemin. S-6

7 Optimization of Experimental Conditions for Aptasensor One of the key factors that affected the assay performance is the ph of working buffer. In order to obtain optimal assay conditions, we performed an experiment to investigate the ph of working buffer towards the effect of ECL responses of PTCA-PEI-Ru(II) compound. Namely, 5 μl PTCA-PEI-Ru(II) was dropped onto the surface of the clean GCE and dried in air to obtain PTCA-PEI-Ru(II)/GCE. Then the PTCA-PEI-Ru(II)/GCE was further detected in the different ph working buffer and recorded the ECL responses. As shown in Figure S6(A), the ph influence dependence of the ECL response was investigated over a ph range of The results show that the ECL intensity increased with increasing the ph before ph 8.0 and the ECL intensity tended to decline after more than ph 8.0. Thus, the ph 8.0 of PBS was used as the optimum buffer solution in the further study. The concentration of NADH played a key role during the detection process. Herein, before incubation with the target thrombin, we investigated the effect of NADH concentration to the ECL intensity. For this purpose, the effect of the NADH concentration on the ECL signal output of the prepared BSA/TBA I/ AuNPs/PTCA-PEI-Ru(II)/GCE was investigated by testing in 3 ml PBS (ph 8.0) containing different concentration NADH. As displayed in Figure S6(B), the ECL intensity rapidly increased with the increasing NADH concentration range from 0.3 to 0.9 mm, and tended to reach a plateau thereafter. Thus, 0.9 mm NADH in 3 ml ph 8.0 PBS was employed throughout the detection process. S-7

8 Figure S6 (A) The relation between ECL intensity and ph by using PTCA-PEI-Ru(II)/GCE detecting in different ph PBS buffers. (B) The relation between ECL intensity and NADH concentration by using BSA/TBA I/ AuNPs/PTCA-PEI-Ru(II)/GCE detecting in 3 ml PBS (ph 8.0) containing different concentration NADH. All ECL signals were obtained through setting photomultiplier tube at 800 V and applied potential between 0.2 V and 1.25 V with a scan rate of 100 mv/s. Comparison of the Proposed Method for Thrombin Detection Table S1 Comparison of the proposed method with some reported methods for thrombin detection. Detection method Linear range LOD Ref Luminescence energy transfer 0.375nM ~11.25 nm nm 1 Organic field-effect transistors 100 pm~100 nm 100 pm 2 Fluorescence 0.1nM ~ 200 nm 0.04 nm 3 Electrochemical 5 pm ~ 1 nm 5 pm 4 Tunable resistive pulse sensing 0.1nM ~ 1000 nm 5 ECL 10-5 nm ~ 0.1 nm 3.5 fm This work S-8

9 Comparison of the Quenching Method for Thrombin Detection Table S2 Comparison of sensitivity of the proposed method with the one using single or dual quenching mechanism for thrombin detection. ECL system Quenching type Quenching mechanism Detection mode Linear range Ru(bpy) 2+ 3 /TPrA Fc Single quenching Signal-on 0.2 nm ~ 0.2 μm Ru(bpy) 2+ 3 /TPrA Fc Single quenching Signal-on 0.1 pm ~ 10 μm Ru-PtNPs/TPrA Fc Single quenching Signal-off 5 pm ~ 50 nm Ru(bpy) 2+ 3 /PEI Hemin, Dual quenching Signal-off 0.1 pm ~ Au@CeO 2 10 nm PTCA-PEI-Ru/ Hemin, O 2, triple quenching Signal-off 10 fm ~ NADH consuming 0.1 nm NADH LOD Ref 60 pm 6 50 fm pm 30 fm fm This work Reference [1] Yuan, F.; Chen, H. Q.; Xu, J.; Zhang, Y. Y.; Wu, Y.; and Wang, L. Chem. Eur. J. 2014, 20, [2] Hammock, M. L.; Knopfmacher, O.; Naab, B. D.; Tok, J. B. -H.; and Bao, Z. N. ACS nano, 2013, 7, [3] Wang, L.; Zhu, J. B.; Han, L.; Jin, L. H.; Zhu, C. X.; Wang, E. K.; and Dong, S. J. ACS nano, 2012, 6, [4] Liu, S. F.; Lin, Y.; Wang, L.; Liu, T.; Cheng, C. B.; Wei, W. J.; and Tang, B. Anal. Chem. 2014, 86, [5] Billinge, E. R.; Broom, M.; and Platt, M. Anal. Chem. 2014, 86, [6] Li, Y.; Qi, H. L.; Peng, Y. G.; Gao, Q.; Zhang, C.X. Electrochem. Commum. 2008, S-9

10 10, [7] Wang, X. Y.; Dong, P.; Yun, W.; Xu, Y.; He, P. G.; Fang, Y. Z. Biosens. Bioelectron. 2009, 24, [8] Liao, Y. H.; Yuan, R.; Chai, Y. Q.; Mao, L.; Zhuo, Y.; Yuan, Y. L.; Bai, L. J.; Yuan, S. R. Sensors and Actuators B, 2011, 158, [9] Hong, L. R.; Chai, Y. Q.; Zhao, M.; Liao, N.; Yuan, R.; Zhuo, Y. Biosens. Bioelectron. 2015, 63, S-10