Supporting Information for Prussian Blue as a Highly Sensitive and Background-Free Resonant Raman Reporter Yongmei Yin, Qiang Li, Sisi Ma, Huiqiao Liu, Bo Dong, Jie Yang, and Dingbin Liu* Dr. Y. Yin, Q. Li, S. Ma, H. Liu, B. Dong, Dr. J. Yang, Prof. Dr. D. Liu State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, College of Chemistry, Research Center for Analytical Sciences, and Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin 300071 (China) Prof. Dr. D. Liu Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071 (China) *E mail: liudb@nankai.edu.cn. S1
Supplementary experimental section Materials and Instrumentation. Tetrachloroauric acid (HAuCl4), sodium citrate, hydroxylamine hydrochloride, ferric chloride (FeCl3), potassium hexacyanoferrate(iii) (K3[Fe(CN)6]), potassium hexacyanoferrate(ii) trihydrate (K4[Fe(CN)6] 3H2O), folic acid (FA), poly-l-lysine hydrobromide (PLL), bovine serum albumin (BSA), cysteine, homocysteine, glutathione, N-hydroxysucinimide (NHS), 1-ethyl-3-(3- dimethyllaminopropyl) carbodiimide hydrochloride (EDC-HCl), Rhodamine 6G (Rh6G), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-aminothiophenol (4-ATP), and 2-mercaptopyridine (2-MPY), 4-mercaptobenzonitrile, poly(ethylene glycol) methyl ether maleimide and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Malachite green isothiocyanate (MGITC) dye was purchased from Life Technologies. Human IgG and Goat Anti- Human IgG Antibody were purchased from Biocell Biotechnol. Co., Ltd. (Zhengzhou, China). NHS-activated magnetic beads (NHS-MBs) with a diameter of around 1 µm were purchased from Fisher Scientific. HeLa cells and HepG2 cells were supplied by American Type Culture Collection. The 96-well polystyrene plate was purchased from R&D Systems. Phosphate buffered saline (PBS, 10, ph 7.4) was purchased from Mediatech, Inc. and was diluted for 10-fold when used. All solutions were prepared by using de-ionized water (Milli-Q grade, Millipore) with a resistivity of 18.2 MΩ-cm (at 25 C). RPMI 1640 and fetal bovine serum (FBS) were from GIBCO. TEM images, HAADF-STEM imaging, and EDX elemental mapping were obtained by FEI Tecnai G2 F20 S-TWIN at 200 kv. UV-vis absorption spectra were collected with U-3900 spectrophotometer (Hitachi). SERRS was carried out with a Renishaw micro-raman system including research grade Leica DMLM microscope. Dynamic light scattering (DLS) and zeta potential (ζ) were performed on a Zeta Sizer Nano ZS (Malvern Zetasizer 3000HS and He/Ne laser at 632.8 nm at scattering angles of 90 at 25 C). Powder X-ray diffraction (XRD) was measured by X-Ray Powder Diffractometer (BrukerD8FOCUS). IR spectra were collected by FT-IR Spectrometer (Bio-rad, FTS6000). MTT results were collected by a microplate reader (BioTek, Synergy S2
2/SLFPAD). Preparation of 30 nm AuNPs. Au NPs with a diameter of 30 nm was first prepared by a two-step method. 1 Briefly, 10 ml of sodium citrate (40 mm) was added into 100 ml of an aqueous solution of HAuCl4 3H2O (100 mm) under vigorous stirring at 120 C. The solution rapidly turned red, indicating the formation of 13 nm Au NPs (10 nm). 2 The Au NPs seeds (0.1 nm) and 1.0 ml of 0.2 M aqueous hydroxylamine hydrochloride were added into 125 ml purified water under magnetic stirring. Subsequently, 10 ml of 2.5 mm aqueous chloroauric acid (HAuCl4 3H2O) was added dropwise and the solution was further stirred for 30 min at 30 C. The final solution turned into red and 5% aqueous sodium citrate solution (2 ml) was added as stabilizer. The UV-vis spectrum was collected and the maximal absorbance peak appeared at 525 nm. Preparation of anti IgG-immobilized MBs (Ab-MBs). NHS-activated magnetic beads (NHS-MBs, 200 μl) were washed three times with 0.01 M imidazole-hcl buffer (ph 7.0) and then suspended to a final volume of 200 μl in above buffer solution. Anti- IgG antibody with a final concentration of 10 μg/ml was added into the above NHS- MBs (10 μg/ml or 10 5 beads/ml) solution and the mixture was allowed to stand overnight at room temperature. Finally, the resulting Ab-MBs were separated from the solution magnetically and re-suspended in 1% BSA solution (100 μl) for 2 h, then washed twice with the above buffer solution. Preparation of Au@PB@PLL@FA NPs. FA was firstly activated by the following methods. 10 mm of FA was dissolved in 5 ml DMF and the solution was mixed with EDC-HCl (50 mm) and NHS (50 mm). The mixture was allowed to react at room temperature overnight. The PLL-coated Au@PB NPs (0.4 nm) were added into the asprepared NHS-activated FA solution dropwise under stirring. The solution was further stirred for 4 h at room temperature, followed by centrifugation at 7000 rpm for 6 min. The supernatant was removed and the resulted NPs were washed with DMF and ethanol S3
for 3 times. Subsequently, poly(ethylene glycol) methyl ether melamine (1 μm) was added to react with the excessive free amines in PLL and to improve the colloidal stability of the particles in biological samples. Cytotoxicity assays. The cytotoxicity of various concentrations of the SERRS tags was evaluated using MTT assay. To do this, HeLa or HepG2 cells (2 10 4 cells/well) were grown in a 96-well plate in 100 μl of RPMI 1640 supplemented with FBS. After 24 h seeding, cells were incubated with various concentrations (from 0.5 to 32 nm) of the FA-modified SERRS tags for another 24 h. The cell viability was measured by directly adding 10 μl of the MTT (5 mg/ml) solution to the incubated cells in each well. After 4 h incubation at 37 C, the amount of formazan dyes was measured by a microplate reader. S4
Supplementary Figures S1-25 Figure S1. Molecular structures of several typical conventional Raman reporters. S5
Figure S2. Raman spectra of HepG2 cells and several types of conventional reporters (molecular structures are shown in Figure S1) coated on 0.1 nm of 30 nm sized AuNPs. Both cells and conventional reporters show multiple bands in the fingerprint region (<1800 cm -1 ) while no signals can be observed in the cellular Raman-silent region (1800-2800 cm -1 ). S6
Figure S3. TEM images of the as-prepared Au NPs etched by potassium hexacyanoferrate(iii) (0.5 mm) for a) 0 min, b) 5 min, c) 30 min, d) 180 min, and e) 720 min. f) Particle size of Au NPs etched by potassium hexacyanoferrate(iii) for different time. The error bars represent the standard deviations of independent measurements of the particle sizes shown in the TEM images. S7
Figure S4. UV-vis spectra of the as-prepared Au NPs etched by potassium hexacyanoferrate(iii) (0.5 mm) for a) 0 min, b) 5 min, c) 30 min, d) 180 min, and e) 720 min. With etching, the maximal absorption bands blue-shifted gradually from 525 to 521 nm, indicating the decrease of particle size. In order to be observed easily, the spectra were obtained by scanning Au NPs with different concentration, while the maximal absorbance had no significant variation when same level of Au NPs were etched by potassium hexacyanoferrate(iii) for different time. S8
Figure S5. Raman intensity at 2156 cm -1 for Au@CN NPs prepared by treating the asprepared Au NPs with potassium hexacyanoferrate(iii) (0.5 mm) for different time. All samples were measured in capillaries with 633 nm laser excitation. The error bars represent the standard deviations of three repeating experiments. S9
Figure S6. TEM images of Au@PB core-shell NPs with various shell thicknesses prepared by adding different amounts of PB precursors. The thickness of PB shell increased with the addition of PB precursors, a) 0 μm, b) 25 μm, c) 50 μm, and d) 100 μm. At low concentration of the precursors, the PB shell appears to be comprised of patchy protrusions around the surface of the Au NP cores. When increased the concentration of the precursors to be 100 μm, the PB shell evolves into a relatively well-defined core-shell nanostructures. S10
Figure S7. Infrared spectra of Au NPs, Au@CN NPs, PB NPs, and Au@PB NPs. The bands at approximately 2090 cm -1 are characteristics of CN stretching. S11
Figure S8. Raman intensity of Au@PB NPs (red) and 4-mercaptobenzonitrilemodified AuNPs (black) at the same concentration of NPs (0.1 nm). Au@PB NPs show 5-fold brightness higher than the 4-mercaptobenzonitrile-modified AuNPs by measuring the intensity of the peaks in Raman-silent region. The spectra were taken in capillaries and measured under the same conditions with 633 nm laser excitation. S12
Figure S9. Raman spectra of a) MGITC-capped AuNPs and b) Au@PB NPs with various colloidal concentrations. S13
Figure S10. Raman spectra of PB NPs (black), Au@CN NPs (red) and Au@PB NPs (blue). The amounts of PB and Au in Au@PB NPs is the same to the PB NPs and the Au@CN NPs respectively. All spectra were taken in capillaries and measured under the same conditions with 633 nm laser excitation. S14
Figure S11. TEM images of PB NPs. The typical cubic structures of PB NPs were observed with an edge length of approximately 20 nm. S15
Figure S12. Dynamic light scattering (DLS) data for a) Au NPs, b) Au@CN NPs, c) Au@PB NPs, d) Au@PB@PLL NPs, and e) Au@PB@PLL@Ab NPs. f) Particle sizes of the above samples were measured by DLS. The error bars represent the standard deviations of three samples measured independently. S16
Figure S13. Zeta potentials of Au NPs, Au@CN NPs, Au@PB NPs, Au@PB@PLL NPs, and Au@PB@PLL@Ab NPs. The error bars represent the standard deviations of three independent measurements. S17
Figure S14. The hydrodynamic diameters of a) Au@PB@PLL NPs and b) Au@PB@PLL@FA NPs. The size increase suggested the successful conjugation of PLL with FA. S18
Figure S15. Long-term stability of the FA-modified SERRS tags (0.1 nm) in PBS with different ph values ranging from 5.1 to 7.4. Since tumor microenvironment and some organelles (e.g., lysosome) are acidic, it is essential to investigate the long stability of the SERRS tags against low ph values. Results indicate that the CN signals in the SERRS tags keep consistent for over 3 months in aqueous solutions with ph values ranging from 5.1 to 7.4; any decomposition of the CN-bridged networks on surfaces of Au NPs can be reflected by the change of the CN signal intensity. The vertical axis (I/I0) represented the quotient of Raman intensity of the tested samples to that of original samples. The error bars represent the standard deviations of three independent measurements. S19
Figure S16. Stability study of the FA-modified SERRS tags (0.1 nm) in different physiologically thiolated compounds such as bovine serum albumin (BSA), cysteine, homocysteine, glutathione as well as (RPMI 1640 medium). No precipitation was observed in these solutions. Moreover, no obvious change in the characteristic CN vibrational bands were observed, indicating excellent stability in these samples. The vertical axis (I/I0) represented the quotient of Raman intensity of the tested samples to that of original samples. The error bars represent the standard deviations of three independent measurements. S20
Figure S17. Cytotoxicity of the FA-modified SERRS tags against HeLa cells (2 10 4 cells/well) at different concentrations after 24 h incubation. The error bars represent the standard deviations of six independent measurements. S21
Figure S18. Cytotoxicity of the FA-modified SERRS tags against HepG2 cells (2 10 4 cells/well) at different concentrations after 24 h incubation. The error bars represent the standard deviations of six independent measurements. S22
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Figure S19. Bright-field (BF) images of multiple HeLa cells and single-cell merge images of each BF image and Raman mapping. The cells were treated with the FAmodified SERRS tags (1 nm) for 1, 2, 4, 8, and 12 h (a-e). Following the introduction of the SERRS tags, the probes adsorbed onto cell surfaces via specific interaction between FA and its receptor. As a consequence, the mapping pictures revealed that the interaction between the SERRS tags and HeLa cells was mainly localized at the cell surface. When further incubation of the SERRS tags with HeLa cells, the CN signals in the cytoplasm elevated gradually while those signals on cell membranes decreased simultaneously, confirming the time-dependent endocytosis governed by FA receptors. Along with the metabolism process, exocytosis of the probe caused the gradually reduced Raman signals. S24
Figure S20. Signal statistics of the multiple HeLa cells treated with the FA-modified SERRS tags (1 nm) for 1, 2, 4, 8, and 12 h. Incubating Hela cells with the proposed SERRS tags for 4h, the average number of positive CN signals at 2156 cm -1 is maximal. When the incubation time is less than 4h, the probes adsorbed onto cell surfaces via specific interaction between FA and its receptor while positive signals are weak. After 4h incubation, the number of CN signals decreased due to metabolism process. The error bars represent the standard deviations of independent measurements of CN signals in single cells shown in the large-view BF images. S25
Figure S21. TEM images of sectioned HepG2 cells incubated with the SERRS tags (1 nm) for 3 h. Negligible SERRS tags can be observed in either the cytosol or the lysosomes. S26
Figure S22. SERRS imaging of single HeLa cell treated with the FA-modified SERRS tags (1 nm). a) Bright-field, SERRS mapping, and merged image of single HeLa cell. b) Representative SERRS spectra from five different points on the HeLa cell. S27
Figure S23. TEM images of sectioned HeLa cells incubated with the SERRS tags (1 nm) for 3 h. The SERRS tags can be notably observed in the cytoplasm. S28
Figure S24. SERRS imaging of multiple cancer cells. Bright-field (large views), and merged images of cells and Raman mapping (single cells), were collected and compared: a) HeLa cell (high expression of FA receptors) treated with the SERRS tags (1 nm) shows notably enhanced Raman signals with a peak at 2156 cm -1 ; b) HepG2 cell (low S29
expression of FA receptors) treated with the tags exhibits negligible CN signals; c) HeLa cell treated with Au@PB NPs (in the absence of FA) shows insignificant CN signals; d) HeLa cell was treated with free FAs to block the FA receptors, then incubation of the SERRS tags with the FA-blocked cells displays ignorable CN signals. Figure S25. Signal statistics of the multiple HeLa cells. a) HeLa cell (high expression of FA receptors) treated with the SERRS tags (1 nm) shows notably enhanced Raman signals with a peak at 2156 cm -1 ; b) HepG2 cell (low expression of FA receptors) treated with the tags exhibits negligible CN signals; c) HeLa cell treated with Au@PB NPs (in the absence of FA) shows insignificant CN signals; d) HeLa cell was treated with free FAs to block the FA receptors, then incubation of the SERRS tags with the FA-blocked cells displays ignorable CN signals. The error bars represent the standard deviations of independent measurements of CN signals in single cells shown in the large-view BF images. S30
References: (1) Dai, Q.; Liu, X.; Coutts, J.; Austin, L.; Huo, Q., J. Am. Chem. Soc. 2008, 130, 8138-8139. (2) Liu, D.; Chen, W.; Sun, K.; Deng, K.; Zhang, W.; Wang, Z.; Jiang, X., Angew. Chem., Int. Ed. 2011, 50, 4103-4107. S31