In Situ DNA-Templated Synthesis of Silver Nanoclusters for Ultrasensitive and Label-Free Electrochemical Detection of MicroRNA

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1 Supporting Information for In Situ DNA-Templated Synthesis of Silver Nanoclusters for Ultrasensitive and Label-Free Electrochemical Detection of MicroRNA Cuiyun Yang, Kai Shi, Baoting Dou, Yun Xiang,* Yaqin Chai, Ruo Yuan Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing , PR China (Y.X.). * Corresponding author. Tel.: (Y.X.). S1

Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering,

2 Figure S1. Typical TEM image of the AgNCs formed on the dsdna polymers with C-rich loop regions after HCR assembly of H 1 and H 2 in the presence of 10 pm mirna-199a. The image was taken by a Tecnai G2 F20 S-TWIN microscope at an acceleration voltage of 200 kv. Table S1. Comparison of different methods for mirna detection. Target Detection method DL Signal amplification strategy Let-7a fluorescence 0.2 fm qrt-pcr 1 Let-7a fluorescence 500 fm MiRNA-21 chronoamperometry 0.06 pm MiRNA-21 DPV 5.36 fm Let-7b chemiluminescence 10 fm MiRNA-141 fluorescence 100 fm hybridization assisted realtime PCR bio-barcoded gold nanoparticles and HRP catalyzed H 2 O 2 isothermal amplification reaction DSN-assisted target recycling amplification with DNAzymes DSN-assisted target recycling amplification MiRNA-15a ECL 21.7 fm CdTe QDs-based ERET 7 MiRNA-199a DPV 0.64 fm TAPNR and HCR dual signal amplification Ref This work S2

Target Detection method DL Signal amplification strategy Let-7a fluorescence 0.2 fm qrt-pcr 1 Let-7a fluorescence 500 fm MiRNA-21 chronoamperometry 0.06 pm MiRNA-21 DPV 5.

3 Figure S2. Typical fluorescence spectra of HCR assembly of H 1 and H 2 (a) with and (b) without the C-rich loop regions in the presence of 10 pm mirna-199a. Fluorescence spectra were recorded on a RF-5301PC fluorescence spectrophotometer (Shimadzu, Tokyo, Japan). The excitation wavelength was set at 281 nm, and the emission spectra were recorded in the range from 575 nm to 750 nm with both excitation and emission slits of 5 nm. The HCR self-assembly of H 1 and H 2 with/without the C-rich loop regions followed by further incubation with AgNO 3 and NaBH 4 was monitored by using fluorescent spectrophotometer. According to Figure S2, HCR self-assembly of H 1 and H 2 without the C-rich loop regions shows minimal fluorescent emission (curve b) while the H 1 and H 2 with the C-rich loop regions exhibits strong fluorescent emission at 600 nm, which is consistent with previous reports 8,9 on the fluorescent emission of AgNCs synthesized by DNA-templates. S3

The excitation wavelength was set at 281 nm, and the emission spectra were recorded in the range from 575 nm to 750 nm with both excitation and emission slits of 5 nm.

4 Figure S3. Chronocoulometric curves for (a) SH-CP/MCH and (b) MCH modified electrodes in the presence of 50 µm RuHex. The chronocoulometric intercept at t = 0 represents the charges of the redox marker confined near the electrode surface. The density of SH-CP on the AuE surface was measured by using the Chronocoulometric method. According to the result in Figure S3, the surface coverage of the SH-CP on the AuE was estimated to be molecules per cm 2 by the Cottrell s equations listed below. 10,11 Qds Qdl Γ Ru= (1) nfa Γ DNA=ΓRu(z m) NA (2) Where Γ Ru is the amount of the redox marker confined near the electrode surface (mol/cm 2 ), Q ds and Q dl are the surface charges (C), n is the number of electrons in the reaction, F is the Faraday constant (C/equiv), A is the area of the working AuE (cm 2 ), Γ DNA is the surface density of DNA, z is the charge of the redox marker, m is the numbers of the bases in DNA, and N A is Avogadro s number. REFERENCES (1) Zhang, J. Y.; Li, Z. P.; Wang, H.; Wang, Y. C.; Jia, H. X.; Yan, J. L. Ultrasensitive Quantification of Mature MicroRNAs by Real-Time PCR Based on Ligation of a Ribonucleotide-Modified DNA Probe. Chem. Commun. 2011, 47, S4

The density of SH-CP on the AuE surface was measured by using the Chronocoulometric method. According to the result in Figure S3, the surface coverage of the SH-CP on the AuE was estimated to be 8.

5 (2) Yu, C. Y.; Yin, B. C.; Ye, B. C. A Universal Real-Time PCR Assay for Rapid Quantification of MicroRNAs via the Enhancement of Base-Stacking Hybridization. Chem. Commun. 2013, 49, (3) Yin, H. S.; Zhou, Y. L.; Zhang, H. X.; Meng, X. M.; Ai, S. Y. Electrochemical Determination of MicroRNA-21 Based on Graphene, LNA Integrated Molecular Beacon, AuNPs and Biotin Multifunctional Bio Bar Codes and Enzymatic Assay System. Biosens. Bioelectron. 2012, 33, (4) Yu, Y. Y.; Chen, Z. G.; Shi, L. J.; Yang, F.; Pan, J. B.; Zhang, B. B.; Sun, D. P. Ultrasensitive Electrochemical Detection of MicroRNA Based on an Arched Probe Mediated Isothermal Exponential Amplification. Anal. Chem. 2014, 86, (5) Deng, H. M.; Ren, Y. Q.; Shen, W.; Gao, Z. Q. An Ultrasensitive Homogeneous Chemiluminescent Assay for MicroRNAs. Chem. Commun. 2013, 49, (6) Yin, B. C.; Liu, Y. Q.; Ye, B. C. One-Step, Multiplexed Fluorescence Detection of MicroRNAs Based on Duplex-Specific Nuclease Signal Amplification. J. Am. Chem. Soc. 2012, 134, (7) Cheng, Y.; Lei, J. P.; Chen, Y. L.; Ju, H. X. Highly Selective Detection of MicroRNA Based on Distance-Dependent Electrochemiluminescence Resonance Energy Transfer Between CdTe Nanocrystals and Au Nanoclusters. Biosens. Bioelectron. 2014, 51, (8) Morishita, K.; MacLean, J. L.; Liu, B. W.; Jiang, H.; Liu, J. W. Correlation of Photobleaching, Oxidation and Metal Induced Fluorescence Quenching of DNA- Templated Silver Nanoclusters. Nanoscale 2013, 5, (9) Zhang, L. B.; Zhu, J. B.; Zhou, Z. X.; Guo, S. J.; Li, J.; Dong, S. J.; Wang, E. K. A New Approach to Light up DNA/Ag Nanocluster-Based Beacons for Bioanalysis. Chem. Sci. 2013, 4, (10) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Electrochemical Quantitation of DNA Immobilized on Gold. Anal. Chem. 1998, 70, (11) Zhang, J.; Song, S. P.; Wang, L. H.; Pan, D.; Fan, C. H. A Gold Nanoparticle-Based Chronocoulometric DNA Sensor for Amplified Detection of DNA. Nat. Protoc. 2007, 2, S5

Biosens. Bioelectron. 2012, 33, 247 253. (4) Yu, Y. Y.; Chen, Z. G.; Shi, L. J.; Yang, F.; Pa

Supporting Information. Rational design of Au nanorods assemblies for highly sensitive and selective SERS detection of prostate specific antigen

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