Supporting Information for

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1 Supporting Information for Label-free electrochemiluminescent aptasensor with attomolar mass detection limits based on Ru(phen) double-strand DNA composite film electrode Xue-Bo Yin*, You-Ying Xin, and Yue Zhao Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin , China xbyin@nankai.edu.cn Contents: Evidence of the direct oxidation of TPA at the ds-dna-assembled electrode. Figure S1 S12. Table S1. 1

2 The effect of incubation time for intercalation of Ru(phen) 2+ 3 on the ECL signal. The incubation of Ru(phen) 2+ 3 has an significant effect on the signal intensity of the present ECL aptasensor. The effect of incubation time for intercalation of Ru(phen) 2+ 3 on the hybrid of 20-mer ss-dna and the anti-thrombin apatamer. Figure R1 shows the dependence of ECL signal on the incubation time. We can find the intercalation time of 6 h can give a 93 % of the maximum ECL signal and the time beyond 7 h shows the maximum signal. The results was validated by Kuwabara et al s work. They studied the DNA-binding mode of different antitumor agents using the intercalated Ru(phen) 2+ 3 as ECL probe. The incubation for 8 h in Ru(phen) 2+ 3 solution was selected for the ECL test. [S1] Figure S1. The dependence of ECL signal of ds-dna on the incubation time for intercalation of Ru(phen) 3 2+ for the hybrid of 20-mer ss-dna and the anti-thrombin apatamer. Evidences of the direct oxidation of TPA at the ds-dna-assembled electrode. Figure S2-S5 presents the evidence of the oxidation behavior of TPA on the composite film electrode. Previous work indicated that TPA oxidations were characterized as an irreversible electron transfer. [S2] Kanoufi et al [S3] studied TPA oxidation in PBS at different ph levels and found that the TPA oxidation peak observed by cyclic voltammetry decreased in intensity and shifted towards a more positive potential as the ph decreased from 11 to 7.5. The oxidation of TPA in the Ru(bpy) 2+ 3 /TPA system (ph 7.5) was mainly achieved through the catalytic homogeneous electron transfer between Ru(bpy) 3+ 3 and TPA because of the high TPA oxidation peak potential (about 1.15 V vs. Ag/AgCl). [S3] To investigate the role of the DNA composite film in TPA oxidation, the oxidation of TPA at bare gold electrode was tested as a comparison (Figure S3). We found a very small oxidation current in PBS (ph 7.5) containing 20 mm TPA at about 1.0 V. TPA can be directly oxidized in PBS solution at ph 11 and the oxidation potential is about 0.8 V vs. Ag/AgCl. [S3] But the oxidation current was obviously lower than that obtained using the Ru(phen) ds-dna composite film electrode at ph 7.5d as shown in Figure S1b, even if PBS at ph 7.5 was used as electrolyte. 2

3 Figure S4 shows the CVs of Ru(phen) ds-dna composite film electrode in 50 mm PBS (ph 7.5) before and after being immersed in 0.2 M PBS containing 20 mm TPA solution. The increased oxidation peak in Figure S4b validates that backbone of DNA can adsorb TPAH + via electrostatic interaction between TPAH + and phosphate ions in the backbone. The direct oxidation of TPA was further validated by the ECL of Ru(phen) ds-dna composite film electrode in PBS containing 20 mm TPA. An ECL peak was observed at about 0.88 V in Figure S5b, which is lower than the oxidation potential of Ru(phen) To validate the source of this ECL emission, CV and the related ECL were recorded at a scan potential ranging from 0.0 to 0.9 V as shown in Figure S5c and d. Similarly, a peak appeared at 0.88 V. The ECL emission is attributed to the reaction between the electrogenerated TPA + active radical and Ru(phen) 2+ 3 because an ECL route based on the oxidation of the electrogenerated TPA + 2+ to Ru(bpy) 3 was proposed. [S2] Typical examples of CVs of the Ru(phen) ds-dna composite film electrode before and after reacting with 5 pm thrombin at various scan rates are shown in Figure S7 and S8. The anodic peak currents in both cases were proportional to the square root of the scan rate in the range of mv/s, showing that the TPA oxidation was controlled by diffusion through the composite film. During the electrochemical procedure, TPA was transferred from the bulk solution to the electrode surface quickly. Because of ir effects, the oxidation peak potential was shifted positively with increase in the scan rate. 3

4 Figure S2. Cyclic voltammograms of Ru(phen) ds-dna composite film electrode in 0.2 M PBS (ph 7.5) without a) and with b) 20 mm TPA solution. Scan rate: 50 mv/s. Figure S3. Cyclic voltammograms of the bare gold electrode in 0.2 M PBS containing 20 mm TPA at a) ph 7.5 and b) ph 11. Scan rate: 50 mv/s. Figure S4. Cyclic voltammograms of Ru(phen) ds-dna composite film electrode in 0.2 M PBS (ph 7.5) before a) and after b) being immersed in 50 mm PBS containing 20 mm TPA solution. Scan rate: 50 mv/s. 4

5 Figure S5. Cyclic voltammograms (a, c) and ECL profiles (b, d) of Ru(phen) thrombin aptamercomplementary ss-dna (0.1 fm) composite film electrode in 0.2 M PBS (ph 7.5) containing 20 mm TPA at different scan ranges: a) and b) V; c) and d) V. Scan rate: 50 mv/s. Figure S6. Cyclic voltammograms in 0.2 M PBS (ph 7.5) containing 20 mm TPA solution using: a) bare gold electrode; b) the 27-mer aptamer assemble gold electrode; c) the gold electrode modified with ds- DNA between 27-mer aptamer and its complementary 20-mer ss-dna. The integrated peak area was used to calculate the TPA oxidation efficiency on the different electrodes. 5

6 Figure S7. (A) Cyclic voltammograms of Ru(phen) ds-dna composite film electrode in 0.2 M PBS (ph 7.5) containing 20 mm TPA at different scan rates: a) 0.01; b) 0.05; c) 0.1; d) 0.15; e) 0.2, and f) 0.3 V/s. (B) The relationship between the anodic peak currents and the square root of the scan rate. Figure S8. (A) Cyclic voltammograms of Ru(phen) ds-dna composite film electrode after reacting with 5 pm thrombin at different scan rates: a) 0.01; b) 0.05; c) 0.1; d) 0.15; e) 0.2; f) 0.25, and g) 0.3 V/s in 0.2 M PBS (ph 7.5) containing 20 mm TPA. (B) The relationship between the anodic peak currents and the square root of the scan rate. 6

7 Table S1. Method detection limits (DL) for thrombin using different aptamer-based techniques. Method Notes DL a Sample volume Relative/pM Absolute/attomole /μl Ref. ECL b Ru(phen) 2+ 3 as intercalator into ds This DNA work ECL Sandwich type with Ru(bpy) 2+ 2 as 10 nm [S6] label ECL ECL quench with ferrocene as femtomole 500 [S7] quencher ECL Ru(bpy) 2+ 3 doped silica nanoparticle [S8] as probe EC b Sandwich with antibody as capturing probe and aptamer as detection probe 0.5 nm [S9] EC EC impedance amplified with [S10] guanidine to denature thrombin EC Sandwich type with Quantum dot [S11] labeling EC Gold nanoparticles bio bar codes with enzyme linked aptamer assay 0.1 nm [S12] EC Horseradish peroxidase label to 3.5 nm 70 femtomole 20 [S13] aptamer via biotin-avidin reaction EC Sandwich type using glucose 10 nm [S14] dehydrogenase as label EC EC impedance spectroscopy 0.5 nm 25 femtomole 50 [S15] EC Ferrocene labeling with square wave [S16] voltammetry EC Methylene blue labeling to aptamer 6.4 nm [S17] EC Enhanced impedance with Au 0.1 [S18] nanoparticle CL b Aptamer-Au nanoparticle conjugate with dot-blot assay femtomole 1 ml [S19] FL b Self-assembled signal aptamer 5 nm pl [S20] aggregate for programmable sensing thrombin FL Self-assembled signaling aptamer 1 nm [S21] aggregate for thrombin recognition a Some of the absolution detection limits are obtained based on the relative detection limits and the sample consumption. b ECL: electrochemiluminescence; EC: electrochemistry; CL: Colorimetric. FL: fluorescence method. 7

8 Figure S9. ECL profiles of the aptasensor based on the intercalation of Ru(phen) 3 2+ for determination of 50 pm thrombin under continuous cyclic voltammeter for 5 cycles. Figure S10. Specificity of ECL aptasensor constructed by using anti-lysozyme aptamer. Lysozyme (lyso) is at 1pM level while the interferents including four proteins and two amino acids at their 50 pm level: bovine hemoglobin (BHB), bovine serum albumin (BSA), thrombin, arginine (arg) and histidine (his). The error bars show the standard deviation of four replicate determinations. Figure S11. The ECL response (ΔI ECL ) to the logarithm of concentrations of thrombin in 1:5 diluted fetal calf serum. The error bars show the standard deviation of four replicate determinations. Scan rate: 50 mv/s. 8

9 Figure S12. Calibration curve of lysozyme between the decreased ECL intensity and the logarithm of concentrations of lysozyme using the ECL aptasensor. The error bars show the standard deviation of four replicate determinations. 9

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