Programmable Modulation of Copper Naonoclusters. Electrochemiluminescence via DNA Nanocranes for Ultrasensitive

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1 Supporting information Programmable Modulation of Copper Naonoclusters Electrochemiluminescence via DNA Nanocranes for Ultrasensitive Detection of microrna Ying Zhou, Haijun Wang, Han Zhang, Yaqin Chai, Ruo Yuan 1 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 1 Corresponding authors at: Tel.: , fax: addresses: yqchai@swu.edu.cn (Y. Q. Chai), yuanruo@swu.edu.cn (R. Yuan). S1

2 Table of Contents Chemicals and apparatus. DNA sequences employed in the proposed biosensor. Preparation of amino-modified Fe 3 O 2. S3 S4 S5 Figure S1. Optical spectrum characterization of S 2 O 8 2 /O 2 ECL system. S7 Figure S2. Experimental condition optimization. Characterization of the ECL biosensor. S7 S8 ECL efficiency of the Cu NCs. S9 Figure S4. The HR-TEM image of dsdna-cunps. S10 Native PAGE characterization of TDN nanostructure. S11 Limit of detection calculation. S11 Table S1. Comparison of this work with the previous research. Application of the biosensor. S11 S11 S2

3 Chemicals and apparatus. Copper sulfate pentahydrate (CuSO 4 5H 2 O), ferric chloride hexahydrate (FeCl 3 6H 2 O), and cerium nitrate hexahydrate (Ce(NO 3 ) 3 6H 2 O) were acquired from Qiangshun Chemical Reagent Co. Ltd. (Shanghai, China). NaOH, NaAc, HNO 3, H 2 O 2 (30%), and NH 3 OH were provided by Kelong Chemical Inc. (Chengdu, China). mirna-155, 6-mercapto-1-hexanol (MCH), and adenosine triphosphate (ATP) were received from J&K Chemical Technology (Beijing, China). Phi29 DNA polymerase, 10 phi29 DNA polymerase reaction buffer, and the solution of deoxyribonucleoside triphosphate (dntps) mixture were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The ECL measurements were performed on a model MPI-E electrochemiluminescence analyzer (Xi An Remax Electronic Science & Technology Co. Ltd., Xi An, China) with a conventional three-electrode system used with Ag/AgCl (saturated KCl) as the reference electrode, a platinum wire as auxiliary electrode, and a modified glass carbon electrode (GCE, Φ = 4 mm) as the working electrode in the experiment. A series of optical filters were placed between the electrode and PMT in this ECL analyzer to obtain ECL spectrum, which was calculated with the maximum ECL intensity during the cyclic potential sweep with the optical filter range from 250 to 800 nm. Electrochemical measurement was carried out with a CHI660C electrochemistry workstation (Shanghai Chenhua Instruments, China). High-resolution transmission electron microscopy (HRTEM, FEI Tecnai G20, FEI, Hillsboro, OR, USA) at an acceleration voltage of 200 KV was used to characterize the nanomaterials. The photophysical characterizations were carried out with a UV-2550 spectrophotometer (UV-2450, Shimadzu, Japan), and a RF-5301PC fluorescence spectrophotometer (Shimadzu Co., Tokyo, Japan). S3

4 DNA sequences employed in the proposed biosensor (5 3 ). DNA 1 : HS-(T) 42 AAT CGT GAT AGG GGT TCT CTT CTC CGA GCC GGT CGA AAT AGT DNA 2 : AAG AGA ACC CCT ATC ACG ATT AGC ATT AA DNA 3 : HS-(T) 14 CAC TATr AGG AAG AGAT TATATATA CC TATATATA CC TATATATA CC TATATATA CC ACC TGG GGG AGT AT MiRNA-155: UUA AUG CUA AUC GUG AUA GGG GU TDN-24: A 1 : TATATATA AAA CTGGAGATACATGCACATTACGGC TTT CTATTAGAAGGTCTCAGGTGCGCG TTT GTAAGTAGACGGGACCAGTTCGCC A 2 : CCCGCCCGGCGC AAA CTGGAGATACATGCACATTACGGC TTT CTATTAGAAGGTCTCAGGTGCGCGTTTGTAAGTAGACGGGACCAGTTCGCC B: HOOC-CGCGCACCTGAGACCTTCTAATAG TTT GCGACAGTCGTTCAACTAGAATGC TTT GGGCTGTTCCGGGTGTGGCTCGTC C: HOOC-GGCCGAGGACTCCTGCTCCGCTGC TTT GGCGAACTGGTCCCGTCTACTTAC TTT CCGACGAGCCACACCCGGAACAGC D: HOOC-GCCGTAATGTGCATGTATCTCCAG TTT GCAGCGGAGCAGGAGTCCTCGGCC TTT GCATTCTAGTTGAACGACTGTCGC Apt 2 : TTGCGGAGGAAGGTTTTTTTTTTTTTTTTGCGCCGGGCGGG DNA sequences employed in the contrast test (5 3 ). AT8-dsDNA probe DNA 1 *: GGG ATATATAT GGG ATATATAT GGG ATATATAT GGG ATATATAT CCC DNA 1 : TT GCG CCG GGC GGG ATATATAT CCC ATATATAT CCC ATATATAT CCC ATATATAT CCC AT4-dsDNA probe DNA 2 *: GGG ATAT GGG ATAT GGG ATAT GGG ATAT GGG ATAT GGG ATAT GGG DNA 2 : TT GCG CCG GGC GGG ATAT CCC ATAT CCC ATAT CCC ATAT CCC ATAT CCC ATAT CCC AT16-dsDNA probe DNA 3 *: GGGG ATATATATATATATAT GGGGG ATATATATATATATAT GGGG DNA 3 : TT GCG CCG GGC GGG CCCC ATATATATATATATAT CCCCC ATATATATATATATAT CCCC ssdna: CCCGCCCGGCGCAA AAAAAAAAA-COOH S4

5 TDN-12 A12: CCCGCCCGGCGCAA T CACTACGTCAGA T AGCTTGCATCAC T GTCACCAGAGTA B12: HOOC-ACGAGCGAGTTG T GTGATGCAAGCT T ATGCGAGGGTCC C12: HOOC-CAACTCGCTCGT T ACTACACTGTGC T TACTCTGGTGAC D12: HOOC-TCTGACGTAGTG T GCACAGTGTAGT T GGACCCTCGCAT TDN-18 A18: CCCGCCCGGCGCAA TT GACATTCCTAAGTCTGAA TT GATTACAGCTTGCTACAC TT CGAAGAGCCGCCATAGTA B18: HOOC-TATCACCAGGCAGTTGCA TT GTGTAGCAAGCTGTAATC TT ATGCGAGGGTCCAATACG C18: HOOC-TGCAACTGCCTGGTGATA TT TACGACACTACGTGGGAA TT TACTATGGCGGCTCTTCG D18: HOOC-TTCAGACTTAGGAATGTC TT TTCCCACGTAGTGTCGTA TT CGTATTGGACCCTCGCAT TDN-30 A30: CCCGCCCGGCGCAA TTT CTATGCCTGGAGATACATGCACATTACGGC TTT CGATCCCTATTAGAAGGTCTCAGGTGCGCG TTT CCAACGGTAAGTAGACGGGACCAGTTCGCC B30: HOOC- CGCGCACCTGAGACCTTCTAATAGGGATCG TTT GCGACAGTCGTTCAACTAGAATGCCCGCAA TTT GGGCTGTTCCGGGTGTGGCTCGTCGGACAC C30: HOOC- ATATGGCCGAGGACTCCTGCTCCGCTGCGG TTT GGCGAACTGGTCCCGTCTACTTACCGTTGG TTT GTGTCCGACGAGCCACACCCGGAACAGCCC D30: HOOC- GCCGTAATGTGCATGTATCTCCAGGCATAG TTT CCGCAGCGGAGCAGGAGTCCTCGGCCATAT TTT TTGCGGGCATTCTAGTTGAACGACTGTCGC Preparation of amino-modified Fe 3 O 2. The magnetite microspheres (Fe 3 O 4 NPs) were firstly prepared according to the following method 1. Typically, FeCl 3 6H 2 O (0.7 g), polyethylene glycol (0.5 g) and NaAc (1.8 g) were respectively dissolved in 20 ml of ethylene glycol with stirring for 30 min to form a clear solution. Then, the obtained solution was transferred to a 30 ml Teflon-lined stainless steel S5

6 autoclave, which was heated at 200 ºC for 8 h. The Fe 3 O 4 precipitate was collected by magnet separation and washed with ethanol and ultrapure water several times. On the other hand, to prepare the ceria precursor, 0.5 g Ce(NO 3 ) 3 6H 2 O and 0.5 g NaOH were separately dissolved in 10 ml of ethanol under vigorous stirring for 24 h at 50 ºC. Subsequently, hydrogen peroxide (30% H 2 O 2, ml) was taken into the mixture solution and then stirred for 2 h. The precipitate was washed and collected by centrifugation. Second, 0.5 g of above-mentioned precipitate solution was adjusted to ph 0.1 by adding HNO 3 (65%). The solution was heated at 40 ºC for 2 h to obtain transparent light-yellow solution g Fe 3 O 4 nanoparticles were dispersed in a mixture containing 15 ml ultrapure water and 1.5 ml ceria precursor through an ultrasonic treatment process for 15 min. Subsequently, 0.5 M NH 3 H 2 O solution was added to adjust the ph to 6.8. The final mixed solution was agitated vigorously by stirring for 4 h at a constant temperature of 60 ºC. After the reaction was completed, the Fe 3 O 2 were collected through an external magnet and washed thoroughly with ultrapure water and ethanol before drying at 60 ºC overnight. Finally, the Fe 3 O 2 were modified amidogen via the oxidative self-polymerization of dopamine. 200 mg of Fe 3 O 2 were dispersed in 40 ml of 10 mm dopamine hydrochloride solution, and followed by the addition of 40 ml tris-hcl buffer solution (10 mm, ph 8.5). The above mixture was stirred for 10 h at 40 ºC. The amino-modified Fe 3 O 2 were washed with water and collected by magnetic separation. S6

7 Figure S1. a typical S 2 O 8 2 /O 2 ECL system. Experimental Condition Optimization. In the assay, the concentrations of phi29 (c phi29 ) was of great significance, which because it directly influenced the quantitative determination of bisensor. As shown in Figure S2A, a consecutive increased ECL intensity was observed when concentration at the range of 20 to 100 U ml -1. And then a plateau was reached at 100 U ml -1, thus the optimized c phi29 was 100 U ml -1 in the work. On the other hand, the ECL property of Cu NCs played an important role in the performance of the biosensor. Thus the experiment condition of Cu NCs should be controlled by optimizing concentration of Cu 2+. Figure S2B showed different ECL intensities of proposed biosensors in the different concentration of Cu 2+. When c Cu2+ was greater than 100 µm, the ECL signal reached a relatively stable value owing that no more Cu NCs generated. Thus the optimum concentration of Cu 2+ was 100 µm in the synthetic process. Furthermore, the optimum concentration of Mn 2+ and the optimum operation time of DNA walker were respectively investigated to enhance Mn 2+ -DNAzyme assisted target transfer efficiency. Figure S2C showed different ECL intensities of the product DNA (Apt 1 ) in the different reaction concentration of Mn 2+. The ECL intensity enhanced gradually with the augment of time and then reached to a plateau after 10 S7

8 mm, suggesting that 10 mm was enough for cleaving DNA. Finally, optimum reaction time of target recycling strategy was carried out and an incremental ECL intensity was accordingly observed with the increase of time (Figure S2D). When time was greater than 60 min, the ECL signal reached a relatively stable value owing to completion of Mn 2+ -DNAzyme assisted target recycle. Thus the optimized time was 60 min in the work. Figure S2. (A) Optimum concentration of phi29; (B) Optimum concentration of Cu 2+ ; (C) Optimum concentration of Mn 2+ ; (D) Optimum reaction time of target recycling strategy. The ECL signal was measured in 0.1 M PBS (ph 7.4) containing 5 mm S 2 O 8 2. Characterization of the ECL biosensor. The electrochemical performance of the elaborated sensor was investigated by CV measurements in PBS with 5 mm [Fe(CN) 6 ] 3-/4-. As can be seen from Figure S3A, a pair of well-defined redox peaks of [Fe(CN) 6 ] 3-/4- could be observed for the bare GCE (curve a). After the modification of Fe 3 O 2, the peak currents showed an enhancement due to its excellent S8

9 conductivity and electron transfer ability (curve b). When TDNs was immobilized onto the electrode surface via amido bond, the redox currents decreased obviously (curve c). After the resultant electrode was blocked with MCH (curve d) and respectively incubated with Apt 1 (curve e), ATP (curve f), the CV responses declined in succession for the hindrance of biomacromolecule. The electrochemical impedance spectroscopy (EIS) was further used for evaluation the interfacial changes of the electrode during the fabrication procedure. As shown in Figure S3B, the bare GCE (curve a) showed a small semicircle. While Fe 3 O 2 was successfully modified on the GCE (curve b), a smaller semicircle can be observed, because the Fe 3 O 2 accelerated the electrons transference. When tetrahedral DNA nanostructure (TDNs) was successfully immobilized on the electrode (curve c), an obviously increased impedance value can be observed. After the proposed electrode was blocked with MCH (curve d) and incubated with Apt 1 (curve e) and ATP (curve f), because of the obstruction of biomacromolecule. Figure S3. (A) CV profiles and (B) EIS profiles of (a) bare GCE, (b) GCE/Fe 3 O 2, (c) GCE/Fe 3 O 2 /TDNs, (d) GCE/Fe 3 O 2 /TDNs/MCH, (e) GCE/Fe 3 O 2 /TDNs /MCH/Apt 1, (f) GCE/Fe 3 O 2 /TDNs/MCH/Apt 1 /ATP. ECL efficiency of the Cu NCs. The relative ECL quantum efficiency was defined as the number of photons per electron transferred, which was estimated using the S9

10 following equations 2-4 : (1) The relative ECL quantum efficiency compared with that of 1 mm [Ru(bpy) 3 ] 2+ (0.1 M (TBA)PF 6 /acetonitrile) with Φ 0 5%, I is ECL intensity, i is current value, and x is the sample. Figure S4. HR-TEM images of A) AT4-Cu NPs, B) AT16-Cu NPs. Native PAGE characterization of TDN nanostructure. The prepared samples were transformed to 12% nondenaturing polyacrylamide gels for analysis to verify the formation of TDN nanostructure. Electrophoresis was performed in 1 TBE at a 120 V constant voltage for 100 min. After staining with ethidium bromide, the gel imaging was implemented with a Bio-Rad Gel Doc XR+ System (Bio-Rad, Hercules, CA). As depicted in Figure S4, lane 1, 2, 3 and 4 were respectively TDN nanostructure, DNA tripolymer, DNA biopolymer and single stranded DNA (ssdna). First, the bright strand in lane 1 posed a highest position corresponding to TDN nanostructure, because TDN as a DNA tetramer had a largest molecular weight and the most complex spatial structures comparing with other strands. Subsequently, the strands in lane 2 and 3 successively were DNA tripolymer and DNA biopolymer, and the dim strand (lane 4) in last place was ssdna. This result demonstrated that the TDN S10

11 nanostructure was generated by the reported method. Figure S5. Native PAGE images of different samples. Lane 1: TDN nanostructure; Lane 2: DNA tripolymer; Lane 3: DNA biopolymer; Lane 4: ssdna. Limit of Detection Calculation. The limit of detection was calculated based on corresponding literatures 5, an ECL measurement for blank samples was executed with three parallel tests first, which exhibited average ECL intensity (I B ) of with standard deviation (S B ) of With signal-to-noise ratio value (k) of 3, the smallest detectable signal (I L ) could be calculated as: I L = I B + ks B = (2) Thus the limit of detection was calculated to be 36 am by the linear equation. Table S1. Comparison of this work with the previous research for mirna detection. Measurement protocol Detection target Detection limit References surface-enhanced raman scattering mirna am 6 photoluminescence mirna fm 7 electrochemistry mirna fm 8 electrochemistry mirna pm 9 electrochemiluminescence mirna am present work Application of the biosensor. The recovery experiment was performed to assess the analytical reliability and application potential of the newly developed biosensor for the mirna-155 assay. Briefly, the 100-fold diluted healthy human serum sample S11

12 (acquired from Xinqiao Hospital of Third Military Medical University, China) was used to prepare various concentrations of mirna-155. Then, the biosensor incubated with the above samples was investigated to assess the influence of the serum samples for mirna-155 detection. Table S2 showed the experimental result, it could be seen that the recovery varied from 90.4% to 109%, indicating the biosensor endowed remarkable potential for clinical study. Table S2. Clinical Serum Samples Analysis. Sample number Added / (fm) Found / (fm) Recovery / % REFERENCES (1) Wang, Q.; Li, Y.; Liu, B.; Dong, Q.; Xu, G.; Zhang, L.; Zhang, J. J. Mater. Chem. A 2015, 3, (2) Ishimatsu, R.; Matsunami, S.; Kasahara, T.; Mizuno, J.; Edura, T.; Adachi, C.; Nakano, K.; Imato, T. Angew. Chem. Int. Ed. 2014, 53, (3) Wallace, W. L.; Bard, A. J. J. Phys. Chem. 1979, 83, (4) Wang, F.; Lin, J.; Zhao, T. B.; Hu, D. D.; Wu, T.; Liu, Y. J. Am. Chem. Soc. 2016, 138, (5) Enustun, B. V.; Turkevich, J. J. J. Am. Chem. Soc. 1963, 85, (6) Li, X. X.; Ye, S. J.; Luo, X. L. Chem. Commun. 2016, 52, (7) Xu, F.; Shi, H.; He, X.; Wang, K.; He, D.; Guo, Q.; Qing, Z.; Yan, L.; Ye, X.; Li, D.; Tang, J. Anal. Chem. 2014, 86, (8) Dong, H.; Jin, S.; Ju, H.; Hao, K.; Xu, L.-P.; Lu, H.; Zhang, X. Anal. Chem. 2012, 84, (9) Liu, Y.; Wei, M.; Li, Y.; Liu, A.; Wei, W.; Zhang, Y.; Liu, S. Anal. Chem. 2017, 89, S12

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