Supporting Information

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1 Supporting Information Ultrasensitive Electrochemiluminescence Biosensor for MicroRNA Detection by 3D DNA Walking Machine Based Target Conversion and Distance-Controllable Signal Quenching and Enhancing Ziqi Xu, Linli Liao, Yaqin Chai, Haijun Wang, Ruo Yuan Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing , People s Republic of China. Corresponding author. Tel.: ; Fax: address: hjwang@swu.edu.cn (H. J. Wang); yuanruo@swu.edu.cn (R. Yuan) S-1

2 Table of Contents Reagents and Materials ---- S3 Instrumentation ---- S3 Optimal conditions of the ECL biosensor ---- S4 PAGE Analysis ---- S4 Characterization of 3 O S6 ECL energy transfer between AuNPs and CdS:Mn QDs ---- S6 REFERENCES ---- S7 S-2

3 Reagents and Materials. Nt.BsmAI nicking endonuclease, Cut Smart Buffer, Lambda exonuclease and Lambda exonuclease reaction buffer were purchased from New England Biotechnology Co., Ltd. (Beijing, China); Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), 3-Mercaptopropionic acid (MPA), N-(3-dimethylaminopropyl)-N -ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were supplied by J & K Technology Co., Ltd. (Beijing, China). Trisodium citrate, sodium borohydride, cadmium nitrate tetrahydrate, manganese acetate tetrahydrate and potassium ferricyanide were obtained from Sichuan East Chemical (Group) Co., Ltd. (Chongqing, China). NH 2 -Fe 3 O 4 was provided by Tianjin BaseLine ChromTech Research Centre (Tianjin, China). 0.1 M Phosphate-buffered solution (PBS) (ph 7.0) and 1% HAuCl 4 4 H 2 O were produced according to the previous work. 1 The DNA probes in our work (Table S-1) were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Table S-1. The DNA Probes in Our Work primer primer sequence (5 to 3 ) Walker S1 S2 Protecting Support mirna-141 SH-TTT TTT GTA CGC TAG ACT TGA CCC TCC GGC GAG ACG GTA AAG ATG GCT TTT TT NH 2 -AT GCG CCG CCG SH-TTT CCG GAG GAG CTA CCT ACG ATC AAT CCA ACC ACA CGC TCC TCC GGC GGC GGC GC AAA AAA GCC ATC TTT ACC AGA CAG TGT TA SH-TTT TTT ATT CAT TTT ACC GTC TCG CCG GAG GAG CGT GTG GTT GGA TTG ATC GTA GGT AGC TCC TCC GG UAA CAC UGU CUG GUA AAG AUG G Instrumentation. Electrochemical impedance spectroscopy (EIS) was monitored by a CHI 660A electrochemistry workstation (Shanghai CH Instruments, China), using a S-3

4 traditional three electrode electrochemical system with a modified glassy carbon electrode (GCE, Φ = 4 mm) as the working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode (SCE) as the reference electrode. ECL emission was conducted by MPI-E ECL analyzer (Xi an Remax Electronic Science & Technology Co. Ltd., Xi an, China), with potential scan being set from -1.5 V to 0 V and photomultiplier tube being set at 800V. Polyacrylamide gel electrophoresis (PAGE) was actualized by a DYY-8C electrophoretic device (Beijing WoDeLife Sciences Instrument Company, Ltd.). Optimal conditions of the ECL biosensor. In the 3D DNA walking machine, the ratio between walker probe and support probe was a main factor for its efficiency which was optimized. As shown in Figure S1, when the ratio between walker probe and support probe shifted from 1:5 to 1:25, the ECL response increased and reached the maximum at 1:20. Hence, 1:20 was chosen as the appropriate experimental condition. Figure S1. Influence of the ratio between walker probe and support probe towards ECL signal (ratio between walker probe and support probe 1:5, 1:10, 1:15, 1:20, 1:25). PAGE Analysis. The 16% PAGE was carried out to corroborate the interactions among these DNA sequences. As shown in Figure S2, a single distinct band could be observed, which corresponded to walker probe (Lane 1). Then, multiple separate bands were observed from the mixture of walker probe, support probe, protecting probe and mirna-141 (Lane 2). As S-4

5 protecting probe firstly paired with walker probe, mirna-141 was added to pair with protecting probe for releasing of walker probe which would further pair with support probe. Consequently, bands from top to bottom were corresponded to the hybridized dsdna of walker probe and support probe (walker-support dsdna), the remained hybridized dsdna of walker probe and protecting probe, the hybridized dsdna of protecting probe and mirna-141, indicating the successful hybridization among these DNA sequences. Subsequently, the walker probe was mixed with support probe, and a clear band at high position was observed (Lane 3), indicating the formation of walker-support dsdna. Then 5 U Nt.BsmAI nicking endonuclease was added into the above mixture, resulting in the appearance of multiple bands (Lane 4). The band at the low position corresponded to the single stranded DNA (intermediate DNA) released from the walker-support dsdna. Besides, the band at the high position is similar with the band of walker probe in Lane 1, indicating the successful releasing of walker probe. The above results confirmed the interactions among these DNA sequences. S-5

6 Figure S2. 16% PAGE of different samples. Lane 1: 2 µm walker probe; Lane 2: a mixture included 2 µm walker probe, 2 µm protecting probe, 2 µm support probe and 2 µm target mirna-141; Lane 3: mixture of 2 µm walker probe and 2 µm support probe; Lane 4: the mixture of 2 µm walker probe and 2 µm support probe with 5 U Nt.BsmAI nicking exonuclease. Characterization of Au@Fe 3 O 4. The transmission electron microscope (TEM) and UV-Vis absorption spectrum (UV) were performed to confirm the synthesis Fe 3 O As shown in Figure S3. A, AuNPs possessed a singular absorbance peak at 520 nm (curve a) and the Fe 3 O 4 had no obvious absorbance peak (curve b). After AuNPs reacted with Fe 3 O 4 through Au-N bond, the absorbance peak of AuNPs was red shifted to 560 nm (curve c), indicating the successful synthesis of Fe 3 O Furthermore, TEM was also employed to characterize the Fe 3 O AuNPs with an average diameter of 4.2 ± 1.0 nm could be observed in Figure S3. B. And Figure S3.C showed that a lot of AuNPs were adhered on the surface of Fe 3 O 4, indicating the successful synthesis of Fe 3 O Figure S3. (A) UV-vis absorption spectrum of AuNPs (a), Fe 3 O 4 (b), Fe 3 O (c). (B) TEM image of AuNPs. (C) TEM image of Fe 3 O ECL energy transfer between AuNPs and CdS:Mn QDs. For demonstration purposes, the UV of AuNPs and photoluminescence spectrum (PL) of CdS:Mn QDs were S-6

7 stuied. As shown in Figure S4, the maximum emission wavelength of CdS:Mn QDs was observed at 510 nm, while a maximum absorption wavelength of AuNPs was observed at 520 nm. Apparently, there was a large spectral overlap between the emission spectrum of CdS:Mn QDs and the absorption spectrum of AuNPs, indicating good potentiality of energy transfer between AuNPs and CdS:Mn QDs. All the characterization results were consistent with the previous works. 2-3 Figure S4. PL spectrum of the prepared CdS:Mn QDs (curve a), UV-vis absorption spectrum of the prepared AuNPs (curve b). REFERENCES (1) Yang Z. H.; Zhuo Y.; Yuan R.; Chai Y. Q. Anal. Chem. 2016, 88, (2) Yildiz H. B.; Tel-Vered R.; Willner I. Adv. Funct. Mater. 2008, 18, (3) Wu K. F.; Rodríguez-Córdoba W. E.; Yang Y.; Lian T. Q. Nano Lett. 2013, 13, S-7