Supplementary information. Quantifying the Degree of Aggregation from Fluorescent. Dye-Conjugated DNA Probe by Single Molecule

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1 Supplementary information Quantifying the Degree of Aggregation from Fluorescent Dye-Conjugated DNA Probe by Single Molecule Photobleaching Technology for the Ultra-sensitive Detection of Adenosine Xingbo Shi, 1,2,* Yu He, 1 Wenli Gao, 1 Xiaoying Liu, 4 Zhongju Ye, 3 Hua Liu, 3 and Lehui Xiao 3,* 1 Hunan Provincial Key Laboratory of Food Science and Biotechnology, College of Food Science and Technology, Hunan Agricultural University, Changsha, 41128, China; 2 State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 4182, China; 3 State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 371, China; 4 College of Science, Hunan Agricultural University, Changsha, 41128, China. shixingbo123@aliyun.com, lehuixiao@nankai.edu.cn Fax: Tables of content Experimental Section. Supporting Tables and Figures. Reference. S-1

2 Experimental section Materials All oligonucleotides were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China) and are listed in Table S1. All oligonucleotide stock solutions were prepared in borate buffer (ph 8.) and stored at -2 C. Adenosine, guanosine, thymidine, ATP, ADP, and AMP were purchased from J&K Scientific Ltd. (Beijing, China). Cytidine and uridine were purchased from Tokyo Chemical Industry (Tokyo, Japan). All other reagents were analytical grade and used without further purification. Ultrapure water was used in all experiments. Sample preparation Solutions of adenosine with different concentrations were prepared using a 4% HCl stock solution. Adenosine identification solution was prepared by mixing Cy5-Probe1 and Cy5-N-Probe2 together in a molar ratio of 8:1. For the adenosine measurement, the target adenosine solution was mixed together with the identification solution in a volume ratio of 1:2 for 1 h at 37 o C, where the final concentration in the reaction solution is in the range of 1-8 nm. The concentration of Cy5-Probe1 and Cy5-N-Probe2 in the corresponding reaction solution was adjusted to 8 and 1 nm, respectively. The sample was then deposited on the glass slide surface and covered with a coverslip. To avoid water evaporation, the chamber between the glass slide and the coverslip was sealed using nail polish oil. The prepared sample was then incubated for additional 5 min at 37 C in a water bath before the microscopic observation. For the bio-sample assay, we replaced water with cattle serum. The else experimental procedures were similar with the process mentioned above. Analysis of adenosine in serum samples A Waters e2695 HPLC system (Milford, Massachusetts, USA) equipped with an e2998 diode array detector was used to analyze the concentration of adenosine in adult cattle serum samples. Ten microliters of a sample solution were injected into a symmetry-c18 column (4.6 mm 25 mm, 5. µm) and eluted using 6% acetonitrile in water with a flow rate of 1 ml/min at 25. The concentration of adenosine could be detected by monitoring the 256 nm chromatographic absorption peaks at 6.66 min. All mobile phases were degassed prior to injection by using an ultrasonicator and were filtered with.45 µm filter. Single molecule imaging measurements To observe the signals from single molecules, an upright microscope (Olympus BX51, Japan) with a 1 oil immersion objective (NA = 1.45, UPLSAPO, Olympus, Tokyo, Japan) and an electron multiplied charge coupled device (EMCCD, ixon3 DU-897D, Andor Technology, North Ireland) were used. The EM gain and working temperature were set at 5 and -75 C, respectively. The exposure time was set in the S-2

3 range of.1-.5 s. A mercury lamp (1 W) was used as the light source. To obtain enough data before the dye being photobleached, the intensity of the light was weakened by holding a neutral-density filter (ND25) in the light path and the aperture diaphragm was set at 1/2. To accurately calculate the number of single molecules in the dye aggregates, at least 5 consecutive frames were acquired. Several imaging tips should be noticed. (1) The concentration of the sample should be in nm, so that each fluorescent spots can be separated in the fluorescence image. (2) The lower the illumination power, the better for single molecule imaging for a long time. (3) The coverslips should be soaked in chromic acid solution for 3 min, and washed by pure water three times. To further remove the background fluorescence, the coverslips are recommended to be pre-bleached by ultraviolet light. Data image processing All of the data were processed using Image J software (National Institutes of Health, USA). The fluorescence intensity time traces of the dye aggregates were analysed by the plot profile plugin in the Image J software. For each DOA value, at least 3 aggregates were observed. All assays were performed in triplicate. S-3

4 Supporting Tables and Figures Table S1. Comparison of different methods for the of adenosine. Method Materials LOD/M Linear range/m References Graphene oxide (GO), FAM-DNA N-Methyl mesoporphyrin IX, DNA Exonuclease III, graphene oxide, DNA Gold nanoparticles, FAM-DNA Glutaraldehyde, DNA SYBR Green I, dttp, DNA CdTe quantum dots, Fe 3 O 4 nanoparticles, DNA Cy5-DNA [1] [2] [3] [4] [5] [6] [7] This work S-4

5 Table S2. Recovery results for adenosine (n = 3) with our biosensor. Samples Concentration of adenosine Added/nM Desired DOA Measured DOA( mean+sd) Concentration of adenosine Found/ nm ( mean+sd) Recovery (%) ± ± ± ± ± ± S-5

6 Table S3. Recovery results for adenosine (n = 3) in cattle serum samples. Cattle serum Sample content (determined by HPLC) /nm Concentration of added adenosine / nm Total adenosine concentration of spiked sample / nm Desired DOA for spiked sample Measured DOA for spiked sample Measured total adenosine concentration of spiked sample / nm Recovery (%) ± ± ± ±.32 S-6

7 nm 1 nm 2 nm Frequency nm nm nm nm DOA nm nm Figure S1 The distribution of DOA from the individual fluorescent spots under different adenosine concentrations (from 1 to 8 nm). S-7

8 Figure S2. The relationship between DOA and adenosine concentration when the concentrations of Cy5-N-Probe2 and Cy5-Probe1 in the reaction solution were 1 and 8 nm, respectively. The final concentrations of adenosine in the reaction solution were 1, 2, 3, 4, 5, 6, 7 and 8 nm. The curve (r 2 =.99) was fitted using the formula described in the manuscript. The inset shows the linear relationship between the reciprocal of DOA and the adenosine concentration over the range of -8 nm. References 1. Bai, Y.;Feng, F.;Zhao, L., et al., Analyst 214, 139 (8), Fu, B.;Cao, J.;Jiang, W., et al., Biosens. Bioelectron. 213, 44, Hu, P.;Zhu, C.;Jin, L., et al., Biosens. Bioelectron. 212, 34 (1), Zhang, J.-Q.;Wang, Y.-S.;Xue, J.-H., et al., J. Pharmaceut. Biomed. 212, 7, Huang, D.-W.;Niu, C.-G.;Zeng, G.-M., et al., Biosens. Bioelectron 211, 29 (1), Liao, D.;Jiao, H.;Wang, B., et al., Analyst 212, 137 (4), Song, Y. Y.;Tang, T.;Wang, X., et al., Sens. Actuator B-Chem. 217, 247, S-8