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1 Supporting Information for Building Electromagnetic Hot Spots in Living Cells via Target-Triggered Nanoparticle Dimerization Wen Zhou, 1,2 Qiang Li, 1 Huiqiao Liu, 1 Jie Yang, 1 Dingbin Liu 1,2 * 1. College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, and Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin , China 2. Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin , China Corresponding Author:* liudb@nankai.edu.cn 1
2 Supplementary Figures Figure S1. Native polyacrylamide gel electrophoresis (PAGE) image for the hybridization products of the LNA sequences to target mir-21. Lane M, DNA size marker; Lane 1, LNA1 (3 μm) ; Lane 2: LNA2 (3 μm) ; Lane 3, mir-21 (3 μm); Lane 4, LNA1 (1.5 μm) plus LNA2 (1.5 μm); Lane 5, LNA1 (1.5 μm) hybridized with mir-21 (1.5 μm); Lane 6, LNA2 (1.5 μm) hybridized with mir-21 (1.5 μm); Lane 7, LNA1 (1 μm) and LNA2 (1 μm) hybridized with mir-21 (1 μm). Figure S2. The procedure for fabricating the asymmetrically functionalized AuNP probes. 2
3 Figure S3. UV-vis spectra of the as-prepared 40 nm AuNPs, the same sized AuNPs asymmetrically functionalized with PEG, and those asymmetrically co-modified with PEG and the mixture of LNA sequences and RRs on both sides of the AuNP surfaces. The wavelengths with maximal absorbance are 528, 530, and 533 nm respectively for the three types of AuNPs. Figure S4. Zeta potentials for the as-prepared 40 nm AuNPs, the same sized AuNPs asymmetrically functionalized with PEG, and those asymmetrically co-modified with PEG and the mixture of LNA sequences and RRs on both sides of the AuNP surfaces. The error bars represent the standard deviations of three independent measurements. 3
4 Figure S5. Hydrodynamic diameters of the as-prepared 40 nm AuNPs, the same sized AuNPs asymmetrically functionalized with PEG, and those asymmetrically co-modified with PEG and the mixture of LNA sequences and RRs on both sides of the AuNP surfaces. Figure S6. Calibration curves used for quantification of (a) Raman reporters (RRs) and (b) LNA sequencesloading on the asymmetrically modified AuNPs. Each AuNP with a diameter of 40 nm was coated with 8542±768 RRs and 122±57 LNAs per nanoparticle, respectively. The error bars represent the standard deviations of three parallel measurements. 4
5 Figure S7. Six TEM images of AuNPs distribution for the same sample. The scale bar is 200 nm. The dimers are in the red circles. Figure S8. The 40 nm AuNPs modified with the mixture of PEGs, LNA sequences, and RRs without orientation were incubated with 10 nm target mir-21. The TEM image shows the formation of AuNP aggregates in an uncontrollable way. 5
6 Figure S9. Spectra of the absorption cross section (σabs) of AuNP dimers with gap distances 1, 1.8, 2, 3, 4, and 5 nm. Figure S10. Raman spectra of the AuNP probes (namely probe 1 and probe 2, 120 pm) which were incubated with 10 nm mir-21 for varying time. 6
7 Figure S11. Raman intensity at 2101 cm -1 for the AuNP dimers treated with 37 o C and 60 o C reversibly. Figure S12. Raman spectra of the AuNP probes (namely probe 1 and probe 2, 120 pm) incubated with various concentrations of mir-21. 7
8 Figure S13. Raman spectra of the AuNP probes (namely probe 1 and probe 2, 120 pm) incubated with various concentrations of mir-155. Figure S14. The Raman intensities of different sizes of AuNPs as the SERS substrates in the presence of 10 nm of mir-21. The error bars represent the standard deviations of three independent measurements. 8
9 Figure S15. Raman signal stability of the AuNP dimers dispersed in various samples. The error bars represent the standard deviations of three independent measurements. Figure S16. Cell viability of the LNA1/C C-modified AuNPs probes against MCF-7 cells at various concentrations (from 0 to 400 pm) after 24 h incubation. The error bars represent the standard deviations of three independent measurements. 9
10 Figure S17. Cell viability of the LNA1 /C N-modified AuNPs probes against MCF-7 cells at various concentrations (from 0 to 400 pm) after 24 h incubation. The error bars represent the standard deviations of three independent measurements. Figure S18. Live-dead staining of MCF-7 cells that had taken with the nanoprobes for 12 h and then irradiated with a 633 nm laser at 10 mw for 0, 12, and 25 min (red, dead cells; green, live cells). The scale bar in all images is 50 µm. 10
11 Figure S19. Bright-field (BF), Raman mapping in 2101 cm -1 channel, and merged images of living MCF-7 cells after incubation with the probe 1 and probe 2 (50 pm) for a) 3 h, b) 6 h, c) 9 h, d)12 h, and e) 15 h. The scale bar in all BF images is 10 µm. f) Raman signal ratios at various incubation time. We define the signal ratios by calculating the measurable signal points out of the total mapping points in each cell. The measurable signal points were transformed from the Raman signals with intensity above three times the standard variation in the mean background signal. The error bars represent the standard deviations of Raman intensity from three different cells. The maximal signal for mir-21 imaging was obtained at 12 h. 11
12 Figure S20. Bright-field (BF), Raman mapping in 2221 cm -1 channel, and merged images of living MCF-7 cells after incubation with the probe 1 and probe 2 (50 pm) for a) 3 h, b) 6 h, c) 9 h, d)12 h, and e) 15 h. The scale bar in all BF images is 10 µm. f) Raman signal ratios at various incubation time. We define the signal ratios by calculating the measurable signal points out of the total mapping points in each cell. The measurable signal points were transformed from the Raman signals with intensity above three times the standard variation in the mean background signal. The error bars represent the standard deviations of Raman intensity from three different cells. The maximal signal for mir-155 imaging was also obtained at 12 h. 12
13 Figure S21. SERS imaging of mir-155 in living MCF-7 cells. a) Raman spectra of MBN (C N-terminated reporter)-coated AuNPs (50 pm) and those of MCF-7 cells before and after treating with either probe 1 or probe 2, as well as that added with both probes. The spectra of the probe-treated single MCF-7 cells were collected on a Raman confocal microscope with a 633 nm laser (1 mw), an exposure time of 10 s, and a 100 objective lens. In comparison, the spectrum of the untreated cells was recorded under the same conditions except the use of higher laser power (10 mw). Bright-field (BF), Raman mapping at 1580 cm -1 (blue channel) and 2221 cm -1 (green channel), merged images and statistic Raman intensities for the cells treated with b) probe 1, c) probe 2, and d) the mixture of probe 1 and probe 2 at 37 o C for 12 h. The concentration of AuNP probes was identified to be 50 pm. The scale bar in all the BF images is 10 µm. The mapping images were acquired at an interval of 1 μm with an exposure time of 0.1 s (633 nm excitation, 10 mw, 100 objective lens). 13
14 Figure S22. Quantification of mir-21 in diverse cell lines using RT-PCR. a) PCR amplification of different concentrations of mir-21. b) PCR amplification curves of the standard samples. c) PCR amplification of mir-21 extracted from three different cell lines. The cell number for each cell line is d) The average amounts of mir-21 in each HEK-293, HeLa, and MCF-7 cell are quantified to be 110±1, 2926±18, and 9128±779, respectively through RT-PCR analysis of the cell extracts. 14
15 Figure S23. Bright-field (BF), Raman mapping in 2101 cm -1 channel, and merged images of a) living HeLa cells and b) MCF-7 cells after incubation with the probe 1 and probe 2 (50 pm) for 12 h. The scale bar in all BF images is 10 µm. c) The measureble Raman signal ratios of the HeLa and MCF-7 cells. We define the signal ratios by calculating the measurable signal points out of the total mapping points in each cell. The measurable signal points were transformed from the Raman signals with intensity above three times the standard variation in the mean background signal. The error bars represent the standard deviations of Raman intensity from three different cells. 15
16 Figure S24. Comparison of SERS imaging of mir-21 in living MCF-7 cell using the dimer probes and uncontrolled aggregates. Bright-field (BF), Raman mapping in 1580 cm-1 (blue channel) and 2101 cm-1 (red channel), merged images, and statistic Raman intensity for the cells treated with a) the uncontrolled aggregate probes and b) the dimer probes. The concentration of AuNP probes was identified to be 50 pm. The scale bar in all the BF images is 10 µm. TEM images of 60 nm thick MCF-7 cell sections were derived from the cells incubated with c) the uncontrolled aggregate probes and d) the dimer probes. The insets show the enlargement of the AuNP aggregates and the dimers distributed in the cells. 16
17 Figure S25. Multiplexed imaging of mir-21 and mir-155 in living cells at various incubation time. Bright-field (BF), Raman mapping in 2101 and 2221 cm -1 channels, and merged images of living MCF-7 cells after incubation with the mixture of probe 1 and probe 2 (hybridization with mir-21) and probe 1 and probe 2 (hybridization with mir-155) for a) 3 h, b) 6 h, c) 9 h, d)12 h, and e) 15 h. The concentration of the probes is 50 pm. f) Raman signal ratios at various incubation time. We define the signal ratios by calculating the measurable signal points out of the total mapping points in each cell. The measurable signal points were transformed from the Raman signals with intensity above three times the standard variation in the mean background signal. The error bars represent the standard deviations of Raman intensity from three different cells. The maximal signals for both mir-21 and mir-155 imaging were obtained at 12 h. 17
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