Supporting Information Simultaneous Visualization of Multiple mRNAs and Matrix Metalloproteinases in Living Cells Using a Fluorescence Nanoprobe Wei Pan, Huijun Yang, Na Li, Limin Yang, and Bo Tang*[a] chem_201500365_sm_miscellaneous_information.pdf
Experimental details: Materials.
Tamoxifen,
β-Estradiol
Bovine
insulin,
3-(4,5-dimethyl-thiazol-2-yl)
-2,5-
diphenyltetrazolium bromide (MTT), Dynasore and Chlorpromazine were purchased from Sigma Chemical Company; Ethylisopropylamiloride (EIPA) was purchased from J&K Scientific Ltd., Filipin was purchased from Cayman Chemical Company; Ilomastat was purchased from American Focus Biomolecules Company; Hydrogen tetrachloroaurate(III) (HAuCl4·4H2O, 99.99 %), DTT, DMSO, Trisodium citrate (C6H5Na3O7·2H2O), MgCl2 and KCl were purchased from China National Pharmaceutical Group Corporation (Shanghai, China). MMP-2 (Recombinant Human Matrix Metalloproteinase-2) and MMP-7 (Recombinant Human Matrix Metalloproteinase-7) were purchased from ProSpec Company. Cell culture products, unless mentioned otherwise, were purchased from GIBCO. All the chemicals were of analytical grade and used without further purification. Sartorius ultrapure water (18.2 MΩ cm) was used throughout the experiments. DNA oligonucleotides were synthesized and purified by TAKARA Biotechnology (Dalian, China) and Sangon Biotechnology Co., Ltd (Shanghai, China). The peptides were synthesized and purified by Apeptide Co., Ltd. (Shanghai, China). The human breast cancer cell line MCF-7 was purchased from KeyGEN biotechnology Company (Nanjing, China), the normal immortalized human mammary epithelial cell line MCF10A was purchased from Shanghai Bioleaf Biotechnology Company (Shanghai, China)；Human hepatocellular liver carcinoma cell line HepG2 and human hepatocyte cell line HL-7702 were obtained from the Committee on Type Culture Collection of the Chinese Academy of Sciences. Instruments. Transmission electron microscopy (TEM) was carried out on a JEM-100CX II electron microscope. Absorption spectra were measured on a pharmaspec UV-1700 UV-visible spectrophotometer (Shimadzu, Japan). Fluorescence spectra were obtained with FLS-920 Edinburgh Fluorescence Spectrometer with a Xenon lamp and 1.0 cm quartz cells at the slits of 3.0/3.0 nm. All pH measurements were performed with a pH-3c digital pH-meter (Shanghai LeiCi Device Works, Shanghai, China) with a combined glass-calomel electrode. The images for selectivity were performed with Caliper IVIS Lumina III. Absorbance was measured in a microplate reader (Synergy 2, Biotek, USA) in the MTT assay. Confocal fluorescence imaging was performed with a TCS SP5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens (×20).
1
Synthesis of oligonucleotides. All DNA oligonucleotides used to prepare and test mRNA targets were synthesized and HPLC purified by TAKARA Biotechnology (Dalian, China) and Sangon Biotechnology Co., Ltd (Shanghai, China). The sequences of the involved oligonucleotides are listed in Table S1. The thiolated oligonucleotides were reduced with Tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HCl) before they were assembled on the surface of gold nanoparticles. MB structure. The potential secondary structure of MB was predicted by using UNAfold on www.idtdna.com. It indicated that the “stem and loop” conformation has formed for the two MBs. Preparation of gold nanoparticles. The gold nanoparticles (AuNPs) of 20 nm were prepared according to the sodium citrate reduction method reported before.1 All glassware was cleaned in aqua regia (HCl/HNO3, 3:1), rinsed with triply distilled H2O, and oven-dried before the experiments. After that, 100 mL HAuCl4 (0.01 %) was heated to boiling with vigorous stirring, then 1.8 mL trisodium citrate (1 %) was added under stirring. The color of the solution turned from pale yellow to colorless and finally to burgundy. Boiling was continued for an additional 10 min. After the heating source was removed, the colloid was stirred until the solution reached room temperature. Then it was filtered through a 0.45 µm Millipore membrane filter. Transmission electron microscopy (TEM) images indicated the particle sizes are 20 ± 2 nm (100 particles sampled). The prepared AuNPs were stored at 4 °C. Preparation of the nanoprobe. Equimolar MBs (labeled by Alexa Fluor 405 and Cy5) were mixed and then added to a solution of Au NPs (1 nM) with a final concentration of 25 nM for each MB and shaken overnight. SDS solution (10 %) was added to the mixture and the final concentration of SDS was 0.1 %. After 12 hours, phosphate buffer (0.1 M; pH = 7.4) was added to the mixture to achieve 0.01 M phosphate concentration and the NaCl concentration of the mixture was slowly increased to 0.1 M over an eight-hour period. Then the resulted solution was centrifuged (13500 g, 30 min) and resuspended in phosphate buffered saline (PBS) for three times. After that, two kinds of peptides were added into the nanaocarrier (1nM) with a final concentration of 750 nM each. The mixture was shaken slowly for two days. Then the nanoprobe solution was centrifuged (13500 g, 30 min) and resuspended in phosphate buffered saline (PBS) for three times. Then the nanoprobe was sterilized using a 0.22 µm acetate syringe filter and resuspended in PBS with a concentration of 3 nM as stock solution stored at 4 °C. The nanoprobe
2
was diluted to certain concentration for use in all subsequent experiments. The concentration of AuNPs was determined by measuring their extinction at 524 nm (ε = 2.7 × 108 L mol-1 cm-1). Quantitation of each MB and peptide loaded on the nanoprobe. The MBs and peptides loaded on AuNPs were quantitated according to the published protocol.2 The DTT was added (final concentration 20 mM) to the probe solution (1 nM ). After incubated 24 h with shaking at room temperature, the MBs and peptides were released. Then the released MBs and peptides were separated via centrifugation and the fluorescence was measured with a fluorescence spectrometer. The fluorescence of Alexa Fluor 405 labeled MB was excited at 405 nm and measured at 420 nm; the fluorescence of FITC labeled peptide was excited at 476 nm and measured at 525 nm; the fluorescence of RhB labeled peptide was excited at 554 nm and measured at 575 nm and the fluorescence of Cy5 labeled MB was excited at 648 nm and measured at 688 nm. The fluorescence was converted to molar concentrations of MB and peptide by interpolation from a standard linear calibration curve that was prepared with known concentrations of MB or peptide with identical buffer pH, ionic strength and DTT concentrations (Figure S3). By dividing molar concentrations of each MB and peptide by the original nanoprobe concentration, the amount of MBs and peptides per nanoprobe was calculated. Response experiment. For multiplexed analyte detection, the nanoprobe (1 nM) was incubated with the two complementary mRNA targets, and two MMP targets respectively with increasing concentrations of the DNA targets (0, 5, 10, 15, 20, 30, 40, 50, 90, 100, 120, 150, 200 nM) or MMP-2, MMP-7 (10-7，10-6，10-5, 10-4, 10-3, 10-2, 10-1 μg/mL). After incubation for 1h at 37 °C, the fluorescence was monitored at appropriate excitation wavelengths. All experiments were repeated at least three times. Specificity experiment. The complementary DNA targets, MMP-2 and MMP-7 for every MB or peptide and other targets were spiked in 1 mL hybridization buffer containing 1 nM nanoprobe for 1 h at 37 °C. The DNA targets were 200 nM and MMPs were 0.4 µg/mL. All the samples were added into 96 well plate and the images were taken by Caliper IVIS Lumina III. Kinetics. The nanoprobe (1 nM) was incubated with two perfectly matched targets (200 nM), and two MMPs (0.4 µg/mL), respectively, then the fluorescence intensity was determined with increasing time (0, 5, 10, 15, 20, 30, 40, 50, 60 minutes or 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes). The fluorescence of Alexa Fluor 405 was excited at 405 nm and measured at 420 nm; the fluorescence of FITC was excited at 476 nm and measured at 525 nm; the
3
fluorescence of RhB was excited at 554 nm and measured at 575 nm and the fluorescence of Cy5 was excited at 648 nm and measured at 688 nm. Cell culture. All the cells were cultured in Dulbecco’s modified Eagles medium (DMEM) and were supplemented with 10 % fetal bovine serum and 100 U/ml 1 % antibiotics penicillin/streptomycin and maintained at 37 °C in a 100 % humidified atmosphere containing 5 % CO2 at 37 °C. MTT assay. MTT assay was performed to study the cytotoxicity of the nanoprobe. MCF-7 cells (1×106 cells/well) were dispersed within replicate 96-well microtiter plates to a total volume of 200 µL well-1. Plates were maintained at 37 °C in a 5 % CO2 / 95 % air incubator for 24 h. After the original medium has been removed, the MCF-7 cells were incubated with naked-Au NPs (1 nM), nanoprobe (1 nM) for 3 h, 6 h, 12 h, 24 h. Then the cells were washed with PBS for three times and 100 µL MTT solutions (0.5 mg mL-1 in PBS) were added to each well. After 4 h, the remaining MTT solution was removed, and 150 µL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with a synergy 2 microplate reader. Confocal fluorescence imaging. For the cellular uptake experiment of the nanoprobe, chlorpromazine was used to inhibit clathrin-mediated endocytosis, filipin was used to inhibit cavolin-mediated endocytosis, dynasore was used to inhibit both pathways above, and ethylisopropylamiloride (EIPA) was used to inhibit macropinocytosis. The cells were preincubated with endocytosis inhibitors (chlorpromazine 10 µM, filipin 5 µM, dynasore 100 µM and EIPA 50 µM) for 30 min, respectively. The nanoprobe (1 nM) was added and incubated for 4 h. The internalization pathways of the nanoprobe in living cells were investigated using confocal laser scanning microscopy (CLSM). The imaging experiments were performed by CLSM with 405 nm, 488 nm, 543nm and 633 nm exciation. The range of the emission wavelength was 420460 nm, 500-550nm, 550-600 nm, 650-700 nm, respectively. In comparative experiment of cancer cells and normal cells, all cells were plated on chamber slides for 24 h. Then the nanoprobe (1 nM) was respectively delivered into MCF-10A, MCF-7, HepG2 and HL-7702 cells in DMEM culture medium at 37 °C in 5 % CO2 for 3 h. The cells were examined by CLSM with different laser transmitters. In another experiments HL-7702 cells were treated with the medium in which HepG2 cells had been incubated for 2 days, and MCF10A cells were treated with the medium in which MCF-7 cells had been incubated for 2 days.
4
After two days, nanoprobe (1 nM) was added and 4 h later images were taken by CLSM with 405 nm, 488 nm, 543 nm and 633 nm excitation. In the experiments for the expression levels of tumor mRNA, one group of MCF-7 cells was treated with β-Estradiol (10-8 mol/L) and the other group of MCF-7 cells was treated with tamoxifen (10-6 mol/L) for 48 h. One group of MCF-7 cells without treatment was served as control. Other steps performed as described above using the nanoprobe (1 nM). Then the cells were monitored by CLSM. In the experiments for inhibiting MMP-2 and MMP-7, one group of MCF-7 was treated with Ilomastat for 48 h another group of MCF-7 cells without treatment was served as control. Other steps performed as described above using the nanoprobe (1 nM). Then the cells were monitored by CLSM. References: 1 K. C. Grabar, R. G. Freeman, M. B. Hommer, M. J. Natan, Anal. Chem. 1995, 67, 735-743. 2 L. M. Demers, C. A. Mirkin, R. C. Mucic, R. A. Reynolds, R. L. Letsinger, R. Elghanian, G. Viswanadham, Anal. Chem., 2000, 72, 5535-5541.
5
Supporting Table: Table S1. DNA sequences and peptides employed in this work. Sequence MB1
5’-Alexa Fluor 405- ACGACGCCAGGGAGAACAGAAACCGTCGTAAAAAA-(CH2)3-SH-3’
MB2
5’-Cy5-CAGTGTCTTATGCGGATAGTGAAACACTGAAAAAA(CH2)3-SH-3’
Peptide1
FITC-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys
Peptide2
RhB-Val-Pro-Leu-Ser-Leu-Thr-Met-Gly-Cys
TK1 perfectly matched target
5’-GTTTCTGTTCTCCCTGG-3’
TK1 single-base mismatched target
5’-GTTTCTGTGCTCCCTGG-3’
GalNAc-T target
5’-TTCACTATCCGCATAAG-3’
GalNAc-T single-base mismatched target
5’-TTCACTATGCGCATAAG-3’
a
Underlined letters represent the stem sequence; bLetters in red represent the mismatched site.
6
Supporting Figures:
a
a
b
b
Figure S1. TEM images of AuNPs (a) and the nanoprobe (b). Scale bars are 50 nm.
AuNPs nanoprobe
Absorbance
2
1
0
500
600
700
Wavelength Figure S2. UV-vis spectra for AuNPs and the nanoprobe.
7
b
Fluorenscence
Fluorescence
a
0
30 60 90 Concentration (nM)
0
120
100
30 60 90 Concentration (nM)
120
d
Fluorescence
Fluorenscence
c
20 40 60 80 Concentration (nM)
0
20 40 60 80 Concentration (nM)
100
0
Figure S3. Standard linear calibration curves of fluorescent dyes. a) Alexa Fluor 405, b) FITC, c) RhB and d) Cy5.
8
MB1 Co TK TKM GT M-7 M-2
MB2 Co GT GTM TK M-7 M-2 peptide1 Co M-2 M-7 GT TK peptide2 Co M-7 M-2 GT TK
Figure S4. Selectivity of each recognition unit of the nanoprobe over several DNA targets and MMP targets. The fluorescence was recorded when the nanoprobe was mixed with the perfectly matched target, single-base mismatched target and other three targets, respectively. Co, TK, TKM, GT, GTM, M-2 and M-7 represent for control, TK1 target, TK1 single-base mismatched target, GalNAc-T target, GalNAc-T single-base mismatched target, MMP-2 target and MMP-7 target.
9
Cell viability / %
90
60
30
0
3h
6h 12h Time / h
24h
Figure S5. Growth inhibition assay (MTT). MCF-7 cells were incubated with unmodified Au NPs (1 nM), nanoprobe (1 nM) for 3h, 6 h, 12 h, 24 h. Black bars stand for the control, red bars stand for the unmodified Au NPs; blue bars stand for the nanoprobe.
10
Experimental details: Materials.
Tamoxifen,
β-Estradiol
Bovine
insulin,
3-(4,5-dimethyl-thiazol-2-yl)
-2,5-
diphenyltetrazolium bromide (MTT), Dynasore and Chlorpromazine were purchased from Sigma Chemical Company; Ethylisopropylamiloride (EIPA) was purchased from J&K Scientific Ltd., Filipin was purchased from Cayman Chemical Company; Ilomastat was purchased from American Focus Biomolecules Company; Hydrogen tetrachloroaurate(III) (HAuCl4·4H2O, 99.99 %), DTT, DMSO, Trisodium citrate (C6H5Na3O7·2H2O), MgCl2 and KCl were purchased from China National Pharmaceutical Group Corporation (Shanghai, China). MMP-2 (Recombinant Human Matrix Metalloproteinase-2) and MMP-7 (Recombinant Human Matrix Metalloproteinase-7) were purchased from ProSpec Company. Cell culture products, unless mentioned otherwise, were purchased from GIBCO. All the chemicals were of analytical grade and used without further purification. Sartorius ultrapure water (18.2 MΩ cm) was used throughout the experiments. DNA oligonucleotides were synthesized and purified by TAKARA Biotechnology (Dalian, China) and Sangon Biotechnology Co., Ltd (Shanghai, China). The peptides were synthesized and purified by Apeptide Co., Ltd. (Shanghai, China). The human breast cancer cell line MCF-7 was purchased from KeyGEN biotechnology Company (Nanjing, China), the normal immortalized human mammary epithelial cell line MCF10A was purchased from Shanghai Bioleaf Biotechnology Company (Shanghai, China)；Human hepatocellular liver carcinoma cell line HepG2 and human hepatocyte cell line HL-7702 were obtained from the Committee on Type Culture Collection of the Chinese Academy of Sciences. Instruments. Transmission electron microscopy (TEM) was carried out on a JEM-100CX II electron microscope. Absorption spectra were measured on a pharmaspec UV-1700 UV-visible spectrophotometer (Shimadzu, Japan). Fluorescence spectra were obtained with FLS-920 Edinburgh Fluorescence Spectrometer with a Xenon lamp and 1.0 cm quartz cells at the slits of 3.0/3.0 nm. All pH measurements were performed with a pH-3c digital pH-meter (Shanghai LeiCi Device Works, Shanghai, China) with a combined glass-calomel electrode. The images for selectivity were performed with Caliper IVIS Lumina III. Absorbance was measured in a microplate reader (Synergy 2, Biotek, USA) in the MTT assay. Confocal fluorescence imaging was performed with a TCS SP5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens (×20).
1
Synthesis of oligonucleotides. All DNA oligonucleotides used to prepare and test mRNA targets were synthesized and HPLC purified by TAKARA Biotechnology (Dalian, China) and Sangon Biotechnology Co., Ltd (Shanghai, China). The sequences of the involved oligonucleotides are listed in Table S1. The thiolated oligonucleotides were reduced with Tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HCl) before they were assembled on the surface of gold nanoparticles. MB structure. The potential secondary structure of MB was predicted by using UNAfold on www.idtdna.com. It indicated that the “stem and loop” conformation has formed for the two MBs. Preparation of gold nanoparticles. The gold nanoparticles (AuNPs) of 20 nm were prepared according to the sodium citrate reduction method reported before.1 All glassware was cleaned in aqua regia (HCl/HNO3, 3:1), rinsed with triply distilled H2O, and oven-dried before the experiments. After that, 100 mL HAuCl4 (0.01 %) was heated to boiling with vigorous stirring, then 1.8 mL trisodium citrate (1 %) was added under stirring. The color of the solution turned from pale yellow to colorless and finally to burgundy. Boiling was continued for an additional 10 min. After the heating source was removed, the colloid was stirred until the solution reached room temperature. Then it was filtered through a 0.45 µm Millipore membrane filter. Transmission electron microscopy (TEM) images indicated the particle sizes are 20 ± 2 nm (100 particles sampled). The prepared AuNPs were stored at 4 °C. Preparation of the nanoprobe. Equimolar MBs (labeled by Alexa Fluor 405 and Cy5) were mixed and then added to a solution of Au NPs (1 nM) with a final concentration of 25 nM for each MB and shaken overnight. SDS solution (10 %) was added to the mixture and the final concentration of SDS was 0.1 %. After 12 hours, phosphate buffer (0.1 M; pH = 7.4) was added to the mixture to achieve 0.01 M phosphate concentration and the NaCl concentration of the mixture was slowly increased to 0.1 M over an eight-hour period. Then the resulted solution was centrifuged (13500 g, 30 min) and resuspended in phosphate buffered saline (PBS) for three times. After that, two kinds of peptides were added into the nanaocarrier (1nM) with a final concentration of 750 nM each. The mixture was shaken slowly for two days. Then the nanoprobe solution was centrifuged (13500 g, 30 min) and resuspended in phosphate buffered saline (PBS) for three times. Then the nanoprobe was sterilized using a 0.22 µm acetate syringe filter and resuspended in PBS with a concentration of 3 nM as stock solution stored at 4 °C. The nanoprobe
2
was diluted to certain concentration for use in all subsequent experiments. The concentration of AuNPs was determined by measuring their extinction at 524 nm (ε = 2.7 × 108 L mol-1 cm-1). Quantitation of each MB and peptide loaded on the nanoprobe. The MBs and peptides loaded on AuNPs were quantitated according to the published protocol.2 The DTT was added (final concentration 20 mM) to the probe solution (1 nM ). After incubated 24 h with shaking at room temperature, the MBs and peptides were released. Then the released MBs and peptides were separated via centrifugation and the fluorescence was measured with a fluorescence spectrometer. The fluorescence of Alexa Fluor 405 labeled MB was excited at 405 nm and measured at 420 nm; the fluorescence of FITC labeled peptide was excited at 476 nm and measured at 525 nm; the fluorescence of RhB labeled peptide was excited at 554 nm and measured at 575 nm and the fluorescence of Cy5 labeled MB was excited at 648 nm and measured at 688 nm. The fluorescence was converted to molar concentrations of MB and peptide by interpolation from a standard linear calibration curve that was prepared with known concentrations of MB or peptide with identical buffer pH, ionic strength and DTT concentrations (Figure S3). By dividing molar concentrations of each MB and peptide by the original nanoprobe concentration, the amount of MBs and peptides per nanoprobe was calculated. Response experiment. For multiplexed analyte detection, the nanoprobe (1 nM) was incubated with the two complementary mRNA targets, and two MMP targets respectively with increasing concentrations of the DNA targets (0, 5, 10, 15, 20, 30, 40, 50, 90, 100, 120, 150, 200 nM) or MMP-2, MMP-7 (10-7，10-6，10-5, 10-4, 10-3, 10-2, 10-1 μg/mL). After incubation for 1h at 37 °C, the fluorescence was monitored at appropriate excitation wavelengths. All experiments were repeated at least three times. Specificity experiment. The complementary DNA targets, MMP-2 and MMP-7 for every MB or peptide and other targets were spiked in 1 mL hybridization buffer containing 1 nM nanoprobe for 1 h at 37 °C. The DNA targets were 200 nM and MMPs were 0.4 µg/mL. All the samples were added into 96 well plate and the images were taken by Caliper IVIS Lumina III. Kinetics. The nanoprobe (1 nM) was incubated with two perfectly matched targets (200 nM), and two MMPs (0.4 µg/mL), respectively, then the fluorescence intensity was determined with increasing time (0, 5, 10, 15, 20, 30, 40, 50, 60 minutes or 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes). The fluorescence of Alexa Fluor 405 was excited at 405 nm and measured at 420 nm; the fluorescence of FITC was excited at 476 nm and measured at 525 nm; the
3
fluorescence of RhB was excited at 554 nm and measured at 575 nm and the fluorescence of Cy5 was excited at 648 nm and measured at 688 nm. Cell culture. All the cells were cultured in Dulbecco’s modified Eagles medium (DMEM) and were supplemented with 10 % fetal bovine serum and 100 U/ml 1 % antibiotics penicillin/streptomycin and maintained at 37 °C in a 100 % humidified atmosphere containing 5 % CO2 at 37 °C. MTT assay. MTT assay was performed to study the cytotoxicity of the nanoprobe. MCF-7 cells (1×106 cells/well) were dispersed within replicate 96-well microtiter plates to a total volume of 200 µL well-1. Plates were maintained at 37 °C in a 5 % CO2 / 95 % air incubator for 24 h. After the original medium has been removed, the MCF-7 cells were incubated with naked-Au NPs (1 nM), nanoprobe (1 nM) for 3 h, 6 h, 12 h, 24 h. Then the cells were washed with PBS for three times and 100 µL MTT solutions (0.5 mg mL-1 in PBS) were added to each well. After 4 h, the remaining MTT solution was removed, and 150 µL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with a synergy 2 microplate reader. Confocal fluorescence imaging. For the cellular uptake experiment of the nanoprobe, chlorpromazine was used to inhibit clathrin-mediated endocytosis, filipin was used to inhibit cavolin-mediated endocytosis, dynasore was used to inhibit both pathways above, and ethylisopropylamiloride (EIPA) was used to inhibit macropinocytosis. The cells were preincubated with endocytosis inhibitors (chlorpromazine 10 µM, filipin 5 µM, dynasore 100 µM and EIPA 50 µM) for 30 min, respectively. The nanoprobe (1 nM) was added and incubated for 4 h. The internalization pathways of the nanoprobe in living cells were investigated using confocal laser scanning microscopy (CLSM). The imaging experiments were performed by CLSM with 405 nm, 488 nm, 543nm and 633 nm exciation. The range of the emission wavelength was 420460 nm, 500-550nm, 550-600 nm, 650-700 nm, respectively. In comparative experiment of cancer cells and normal cells, all cells were plated on chamber slides for 24 h. Then the nanoprobe (1 nM) was respectively delivered into MCF-10A, MCF-7, HepG2 and HL-7702 cells in DMEM culture medium at 37 °C in 5 % CO2 for 3 h. The cells were examined by CLSM with different laser transmitters. In another experiments HL-7702 cells were treated with the medium in which HepG2 cells had been incubated for 2 days, and MCF10A cells were treated with the medium in which MCF-7 cells had been incubated for 2 days.
4
After two days, nanoprobe (1 nM) was added and 4 h later images were taken by CLSM with 405 nm, 488 nm, 543 nm and 633 nm excitation. In the experiments for the expression levels of tumor mRNA, one group of MCF-7 cells was treated with β-Estradiol (10-8 mol/L) and the other group of MCF-7 cells was treated with tamoxifen (10-6 mol/L) for 48 h. One group of MCF-7 cells without treatment was served as control. Other steps performed as described above using the nanoprobe (1 nM). Then the cells were monitored by CLSM. In the experiments for inhibiting MMP-2 and MMP-7, one group of MCF-7 was treated with Ilomastat for 48 h another group of MCF-7 cells without treatment was served as control. Other steps performed as described above using the nanoprobe (1 nM). Then the cells were monitored by CLSM. References: 1 K. C. Grabar, R. G. Freeman, M. B. Hommer, M. J. Natan, Anal. Chem. 1995, 67, 735-743. 2 L. M. Demers, C. A. Mirkin, R. C. Mucic, R. A. Reynolds, R. L. Letsinger, R. Elghanian, G. Viswanadham, Anal. Chem., 2000, 72, 5535-5541.
5
Supporting Table: Table S1. DNA sequences and peptides employed in this work. Sequence MB1
5’-Alexa Fluor 405- ACGACGCCAGGGAGAACAGAAACCGTCGTAAAAAA-(CH2)3-SH-3’
MB2
5’-Cy5-CAGTGTCTTATGCGGATAGTGAAACACTGAAAAAA(CH2)3-SH-3’
Peptide1
FITC-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys
Peptide2
RhB-Val-Pro-Leu-Ser-Leu-Thr-Met-Gly-Cys
TK1 perfectly matched target
5’-GTTTCTGTTCTCCCTGG-3’
TK1 single-base mismatched target
5’-GTTTCTGTGCTCCCTGG-3’
GalNAc-T target
5’-TTCACTATCCGCATAAG-3’
GalNAc-T single-base mismatched target
5’-TTCACTATGCGCATAAG-3’
a
Underlined letters represent the stem sequence; bLetters in red represent the mismatched site.
6
Supporting Figures:
a
a
b
b
Figure S1. TEM images of AuNPs (a) and the nanoprobe (b). Scale bars are 50 nm.
AuNPs nanoprobe
Absorbance
2
1
0
500
600
700
Wavelength Figure S2. UV-vis spectra for AuNPs and the nanoprobe.
7
b
Fluorenscence
Fluorescence
a
0
30 60 90 Concentration (nM)
0
120
100
30 60 90 Concentration (nM)
120
d
Fluorescence
Fluorenscence
c
20 40 60 80 Concentration (nM)
0
20 40 60 80 Concentration (nM)
100
0
Figure S3. Standard linear calibration curves of fluorescent dyes. a) Alexa Fluor 405, b) FITC, c) RhB and d) Cy5.
8
MB1 Co TK TKM GT M-7 M-2
MB2 Co GT GTM TK M-7 M-2 peptide1 Co M-2 M-7 GT TK peptide2 Co M-7 M-2 GT TK
Figure S4. Selectivity of each recognition unit of the nanoprobe over several DNA targets and MMP targets. The fluorescence was recorded when the nanoprobe was mixed with the perfectly matched target, single-base mismatched target and other three targets, respectively. Co, TK, TKM, GT, GTM, M-2 and M-7 represent for control, TK1 target, TK1 single-base mismatched target, GalNAc-T target, GalNAc-T single-base mismatched target, MMP-2 target and MMP-7 target.
9
Cell viability / %
90
60
30
0
3h
6h 12h Time / h
24h
Figure S5. Growth inhibition assay (MTT). MCF-7 cells were incubated with unmodified Au NPs (1 nM), nanoprobe (1 nM) for 3h, 6 h, 12 h, 24 h. Black bars stand for the control, red bars stand for the unmodified Au NPs; blue bars stand for the nanoprobe.
10
