Supporting Information. Electrochemiluminescence Peptide-Based Biosensor with Hetero-

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1 Supporting Information Electrochemiluminescence Peptide-Based Biosensor with Hetero- Nanostructures as Co-reaction Accelerator for the Ultra-sensitive Determination of Tryptase Fang-Fang Wu, Ying Zhou, Han Zhang, Ruo Yuan, Ya-Qin Chai Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing , PR China *Corresponding author. Tel.: ; Fax: address: S-1

2 Table of Contents Experimental section The amino acid sequence of vasoactive intestinal peptide (VIP)... S-3 The cleavage site of VIP... S-3 Table S1. DNA sequences used in this work... S-3 The preparation process of AAPHNs... S-4 Results and discussion Stepwise characterization of the sensing interface.... S-4 Comparison of the effect of different nanomaterials on the proposed biosensor.... S-5 Optimization of experimental conditions... S-7 Table S2 Comparison of the proposed method with some reported methods for proteins detection.... S-8 Calibration curve of TPS detection in human serum.... S-8 References... S-9 S-2

3 Experimental Section The Amino Acid Sequence of Vasoactive Intestinal Peptide (VIP): Three-letter code: Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys- Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH 2 (One-letter code: KPRRPYTDNYTRLRKQMAVKKYLNSILN-NH 2 ) The Cleavage Site of VIP: Table S1. DNA Sequences Used in this Work Oligonucleotide S1 S2 S3 R1 Sequences (from 5' to 3') AAT CGG AAA TCC GTT CTT ATC AAC GCA AGC GGA CGA GTG CTG GGA CCG CAA TCT ATC ACG TCA ATT TCC GAT TGA TAA GAA CGG CGC TTG CGT TCA GCA CTC GTC ATT GCG GTC CTG ACG TGA TAG GAC GAG TGC TTT TTG AAT CGG AAA TTT TT-(CH 2 ) 6 -COOH S-3

4 The Preparation Process of AAPHNs Figure S1. The preparation process of AAPHNs. Results and Discussion Stepwise Characterization of the Sensing Interface. The stepwise modified electrode process of the ECL-PB biosensors were performed by CV in 5.0 mm [Fe(CN) 6 ] 3 /4 solution containing 0.1 M KCl with scan potential from 0.2 to 0.6 V (vs. SCE). and the results were shown in Figure S2. As shown in Figure S2, a pair of reversible redox peaks was observed on the bare glassy carbon electrode (curve a). The peak current value was significantly enhanced on the electrode modified with AAPHNs (curve b), because the AAPHNs as a conductive material can accelerate the electron transfer. After VIP-DNANTs-Dox-Lu was incubated onto the surface of the modified electrode, the peak current was significantly reduced (curve c) owing to its nonelectroactive character. After blocking with BSA, the CV response remained declined (curve d). Finally, the current response increased after the modified electrode was immersed in the TPS solution (curve e) because partial VIP were released from the electrode surface due to TPS specific cleavage. S-4

5 Figure S2. The CVs of (a) bare GCE, (b) AAPHNs/GCE, (c) VIP-DNANTs-Dox-luminol /AAPHNs/GCE, (d) BSA/VIP-DNANTs-Dox-luminol/ AAPHNs/GCE, (e) TPS/BSA/VIP-DNANTs-Dox-luminol/AAPHNs/GCE, in 5.0 mm [Fe(CN) 6 ] 3-/4-. Comparison of the Effect of Different Nanomaterials on the Proposed Biosensor. In order to explore the superiority of Au-Ag-Pt hetero-nanostructures (AAPHNs), a contrast experiment was carried out. Figure S3 depicted the ECL responses of the Au nanorods (Figure S3A), Au-Ag nanocomposites (Figure S3B), Au-Pt nanocomposites (Figure S3C) and the AAPHNs (Figure S3D) modified biosensors, respectively. It can be seen from Figure S3 that under the same experimental conditions, only the AAPHNs exhibited superior co-reaction catalysis and the biosensor constructed by it showed better stability compared to the other three nanomaterials. Compared with the four graphs, it can be concluded that the effect of Au nanorods (Figure S3A) on the luminol - dissolved oxygen system is weak. When Ag (Figure S3B) and Pt (Figure S3C) were introduced separately, the ECL intensities S-5

6 were significantly enhanced, indicating that both Ag and Pt were effective in promoting the ECL luminescence system. However, it is noteworthy that the catalytic properties of Au-Ag alloy and Au-Pt alloy are not stable, and the ECL intensity decreases rapidly with the change of detection time. AAPHNs (Figure S3D) combine the catalytic properties of Ag and Pt to obtain efficient ECL emission; and also the biosensor constructed by it showed excellent stability due to the synergistic effect of Au, Ag, Pt. Figure S3. ECL responses of (A) Au nanorods, (B) Au-Ag nanocomposites, (C) Au-Pt nanocomposites and (D) AAPHNs modified biosensors, respectively. S-6

7 Optimization of Experimental Conditions The incubation time is the key factors which influence the performance of the proposed method. In order to achieve optimal assay conditions, the ECL intensity of the proposed biosensor was investigated at different incubation time for vasoactive intestinal peptide (VIP) and different cleavage time for trypsin (TPS). As can be seen from Figure S4A, in the VIP incubated process, the ECL responses increased with the increasement of the incubation time in the range of 1-7 h. The maximum values of ECL responses obtained at the incubation time of 7 h and then the ECL intensity remained at a.u. Therefore, in the process of VIP incubation the best incubation time is 8h. Furthermore, as can be seen from Figure S4B, the biosensor was incubated for 5, 10, 20, 30, 40 and 50 min in PBS containing 0.1 ng/ml TPS. As a result, the current response was rapidly changed within the first 30 min and then tended to level off, indicating an equilibration state was reached. Therefore, the incubation time of 30 min was adopted in the cleavage process. Figure S4. Effects of (A) incubation time for VIP and (B) cleavage time for TPS. S-7

8 Table S2 Comparison of the Proposed Method with Some Reported Methods for Proteins Detection. Target Detection method Linear range / (ng/ml) Detection limit / (ng/ml) Ref AFP CV AFP SERS PSA CV PSA ECL MMP-3 Fluorescence TPS ECL This work Abbreviation: AFP: α-fetoprotein, SERS: surface-enhanced Raman scattering, PSA: prostate specific antigen, ECL: electrochemiluminescence, MMP-3: matrix metalloproteinase-3, TPS: trypsin. Calibration Curve of TPS Detection in Human Serum. Figure S5. Calibration curve of TPS detection in human serum. S-8

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