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1 Supporting Information Ultrasensitive Homogeneous Electrochemical Detection of Transcription Factor by Coupled Isothermal Cleavage Reaction and Cycling Amplification based on Exonuclease III Lihua Lu, Huijuan Su, and Feng Li* College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao , People s Republic of China * Corresponding author. Tel/Fax: lifeng@qust.edu.cn S-1

2 Table of Content Cell lysate preparation...s3 Figure S1 DPV peak current of MB with the treated ITO electrode or with the un-treated ITO electrode...s3 Figure S2 DPV peak current of MB with oxidation current signal or with reduction current signal...s3 Figure S3 DPV peak current of MB under different ph...s4 Figure S4 Nondenaturing PAGE imaging of ON1/ON2 and ON1/ON2 + NF-κB p50...s4 Figure S5 DPV peak current change in the presence of wild-typed dsdna ON1/ON2, mutant dsdna ON1-mut1/ON2-mut1 or ON1-mut2/ON2-mut2...S5 Figure S6 Responses of DPV peak current to 7 samples under the same detection conditions... S6 Table S1 Performance comparison of the transcription factor detection reported in recent years...s6 Table S2 Results of the recovery test of NF-κB p50 in 10-fold diluted human serum...s7 References...S7 S-2

3 Cell lysate preparation The SK-BR-3 cell line were purchased from American Type Culture Collection (Manassas, VA USA). Breast cancer cells were trypsinized and resuspended in TE buffer (10 mm Tris-HCl, ph = 7.4, 1 mm EDTA). After incubation on ice for 10 min, the lysate was centrifuged and the supernatant was collected.. Figure S1 DPV peak current of MB with the treated ITO electrode or with the un-treated ITO electrode. Figure S2 DPV peak current of MB (A) with oxidation current signal or (B) with reduction current signal. S-3

4 Figure S3 DPV peak current of MB under different ph. Figure S4 Nondenaturing PAGE imaging of ON1/ON2 and ON1/ON2 + NF-κB p50: lane 1, ON2; lane 2, Hairpin DNA ON4; lane 3, ON1/ON2; lane 4, ON1/ON2 + NF-κB p50. S-4

5 Figure S5 DPV peak current change ( ip= ip ip,0, in which ip is the DPV peak current in the presence of NF-κB p50, and ip,0 is the DPV peak current in the absence of NF-κB p50) in the presence of (a) wild-typed dsdna ON1/ON2, (b) mutant dsdna ON1-mut1/ON2-mut1, (c) mutant dsdna ON1-mut2/ON2-mut2. Figure S6 Responses of DPV peak current to 7 samples under the same detection conditions. The concentrations of ON1/ON2, NF-κB p50 and Exo III were 1 µm, 0.5 nm and 30 U/mL, respectively; the reaction time was 1 hour. S-5

6 Table S1 Performance comparison of the transcription factor detection reported in recent years. Method Linear range Detection Ref. limit Homogeneous electrochemical strategy for transcription factor detection by using exonuclease nm 0.01 nm This work III-assisted cycling amplification Homogenous luminescent switch-on probe for the nm 30 nm 1 detection of nanomolar transcription factor NF-kappaB Detection of transcription factors with graphene nm 0.2 nm 2 oxide fluorescence switch-based multifunctional G-quadruplex-hairpin probe Detection of transcription factors using near-infrared Not given 0.14 pm 3 fluorescent solid-phase rolling circle amplification Detection of transcription factors using an pm 25 pm 4 Ag + -stabilized self-assembly triplex DNA molecular switch Electrochemiluminescent detection of transcription nm nm 5 factors with hybridization chain reaction amplification Detection of transcription factors using 10 fm 1 nm 6.03 fm 6 transcription-mediated isothermally exponential amplification-induced chemiluminescence Electrochemical approach for nuclear factor kappa B nm 0.13 nm 7 detection based on triplex DNA and gold nanoparticles Determination of transcription uuclear factor-kappa Not given 0.2 nm 8 B using an electrochemical, DNA-based nanoswitch Detection of transcription factors using nm 93 pm 9 target-converted helicase-dependent amplification assay Detection of transcription factors using pm pm 10 DNA-templated copper nanoparticles as fluorescent indicator and hairpin DNA cascade reaction as signal amplifier Electrochemical biosensor for nuclear factor kappa nm nm 11 B using a gold nanoparticle-assisted dual signal amplification method Detection of transcription factors by isothermal pm 3.8 pm 12 exponential amplification-based colorimetric assay Impedimetric aptasensor for nuclear factor kappa B nm 7 pm 13 with peroxidase-like mimic coupled DNA nanoladders as enhancer Fluorescence detection of transcription factors based on kisscomplex formation and the T7 RNA Polymerase amplification method pm 0.23 pm 14 S-6

7 Table S2 Results of the recovery test of NF-κB p50 in 10-fold diluted human serum. Sample No. Added (nm) Found (nm) Recovery (%) ± ± ± ±2.9 References (1) Ma, D. L.; Xu, T.; Chan, D. S.; Man, B. Y.; Fong, W. F.; Leung, C. H. Nucleic Acids Res. 2011, 39, e67. (2) Zhu, D.; Wang, L.; Xu, X.; Jiang, W. Biosens. Bioelectron. 2016, 75, 155. (3) Yin, J.; Gan, P.; Zhou, F.; Wang, J. Anal. Chem. 2014, 86, (4) Zhu, D.; Zhu, J.; Zhu, Y.; Wang, L.; Jiang, W. Chem. Commun. 2014, 50, (5) Xiong, Y.; Lin, L.; Zhang, X.; Wang, G. Rsc Adv. 2016, 6, (6) Ma, F.; Yang, Y.; Zhang, C. Y. Anal. Chem. 2014, 86, (7) Min, S.; Mei, Y.; Hao, L.; Liang, Z.; Li, G. Electrochim. Acta 2012, 60, (8) Liang, Z.; Duan, A.; Li, X.; Liu, F.; Liu, L.; Wang, K.; Liu, X. Anal. Lett. 2014, 47, (9) Cao, A.; Zhang, C. Y. Anal. Chem. 2013, 85, (10) Liang, S.; Zhang, X.; Wang, G. Biosens. Bioelectron. 2016, 82, (11) Ye, Z.; Zhang, B.; Yang, Y.; Wang, Z.; Zhu, X.; Li, G. Microchim. Acta 2014, 181, (12) Zhang, Y.; Hu, J.; Zhang, C. Anal. Chem. 2012, 84, (13) Peng, K.; Zhao, H.; Xie, P.; Hu, S.; Yuan, Y.; Yuan, R.; Wu, X. Biosens. Bioelectron. 2016, 81, 1-7. (14) Zhang, K.; Wang, K.; Zhu, X.; Xie, M. Chem. Commun. 2017, 53, S-7