Supporting Information

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1 Supporting Information Ratiometric fluorescent polymeric thermometer for thermogenesis investigation in living cells Juan Qiao,,ǁ Yoon-Ho Hwang,,ǁ Chuan-Fang Chen, Li Qi *, Ping Dong, & Xiao-Yu Mu and Dong-Pyo Kim * Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, P.R. China National creative research center of applied microfluidic chemistry, Department of chemical engineering, Pohang University of Science and Technology, Pohang, Republic of Korea Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Beijing, P.R. China & Research Centre of Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P.R. China * Correspondence: Prof. Li Qi qili@iccas.ac.cn; Fax: Prof. Dongpyo Kim dpkim@postech.ac.kr; Fax: Contents 1. Experimental Details 2. Supporting Tables (Table S1 and Table S2) 3. Supporting Figures (Figures S1-S15) S-1

2 Characterization Molecular mass and 1 H NMR measurement of the polymer The gel permeation chromatography (GPC) was used for the molecular weight and PDI of the P(NIPAm-NBD-NSVB) determination. The experiment was carried out on a model L-2130 HPLC pump from Hitachi Co., a model 2410 refractive index detector from Waters Co., and a model 2487 ultraviolet detector from Waters Co. The columns used for the molecular determination was a combination of MZ-Gel SDplus columns (5 μm, porosity of 10 3, 10 4, and 10 5 Å) from MZ-Analysentechnik GmbH (Mainz, Germany) and the polystyrene standards (MW of 1.3 K, 3.3 K, 5.2 K, 13.0 K and 25.0 K) were used for calibration. THF was applied as eluent with the flow rate of 1.0 ml/min. The 1 H NMR investigaion of the products was carried out on a Bruker DMX-400 spectrometer in CDCl 3. Investigation of LCST and DLS of the polymer-tfauncs The UV-Visible Spectrophotometer (UV-2450, Shimadzu, Japan) was used for the determination of the LCST by monitoring the transmittance at 500 nm. Polymer-TfAuNCs with the concentration of (5.0 mg ml -1 :0.8 mm) was used for determination. The temperature of the sample was controlled from 25 to 45 ºC by a temperature controller (Shimadzu, Japan) with the heating rate 1 ºC per 1 min. The temperature with 50% transmittance was defined as the LCST. The DLS measurements was carried out by hydrodynamic diameter estimation using a Zetasizer Nano ZS (Malvern Instruments). Fluorescence imaging measurement of the polymer-tfauncs Fluorescence measurements were made on a fluorescence spectrophotometer (FL4500, Shimadzu, Japan) with a temperature controller (Shimadzu, Japan). The photo microgram instrument (lympus ix83) with an andor deep cooled single photon detector (DU-897U-CS0-#BV) was used for the obtaining of all S-2

3 the fluorescence imaging with metamorph premier as the software. The confocal laser scanning microscope software (lympus FV 10-ASW, Japan) was applied for the ratio images treatment. The cell sample temperature controlling was realized by the Incubation System for Microscope (WSKM, Tokai hit) ranging from C and the temperature resolution was C. Cell cytotoxicity investigation CCK-8 Kit, a kind of commercial available kit has been employed for the cyctotocity assay. HeLa cell with the concentration ( cells/well) were cultured in the 96-well plates in 5% C 2 atmosphere at 37 o C for 24 h. Then the polymer-tfauncs with different concentration were diluted with the serum-free medium (v:v=1:9) and incubated with the HeLa cells for 12 h. After washed with the PBS for three times, the cells were incubated with 10% CCK-8 in serum-free medium for 2 h. The control experiment was done by detecting the growth culture medium without the polymer-tfauncs. S-3

4 H H + N NHS DCC, DMAP ethyl acetate RT, 36 h N VBA NSVB Figure S1. Synthesis of the monomer N-succinimide p-vinylbenzoate (NSVB). S-4

5 Figure S2. 1 H NMR spectrum of the NSVB. S-5

6 N NSVB + HN N 2 H N N N NBDAA + HN NIPAm CH 3 HC C S CH 3 S C S DATB (CTA) CH 3 C CH CH 3 AIBN, dioxane, 60 o C CH 3 HC C S CH 3 HN NH NH N N N S C 2 N 2 P(NIPAm-NBD-NSVB) Figure S3. Synthesis of the thermal sensitive polymer P(NIPAm-NBD-NSVB). S-6

7 Q d : (Monomers + AIBN + CTA) in dioxane (0.2~1.0 ml/h) Q d Q c Rxn time : 16~21 min Q c : FC oil (3.0 ml/h) T-junction 80 o C il bath Cooling Figure S4. The schematic diagram of the droplet-based microfluidic reactor. S-7

8 Figure S5. 1 H NMR spectrum of the thermal sensitive polymer P(NIPAm-NBD-NSVB). S-8

9 Table S1 Synthesis of the thermal sensitive polymer P(NIPAm-NBD-NSVB) Polymer Reaction time (min) Flow rate of oil (ml/h) Flow rate of organic solution (ml/h) Temperature ( o C) MW (kda) PDI a P(NIPAm-NBD-NSVB) min a P(NIPAm-NBD-NSVB) min a P(NIPAm-NBD-NSVB) min b P(NIPAm-NBD-NSVB)-4 24 h a : Synthesis of the thermal sensitive polymer P(NIPAm-NBD-NSVB) in a droplet-based microfluidic reactor; b : Synthesis of the thermal sensitive polymer P(NIPAm-NBD-NSVB) in a glass bottle. Reaction conditions; NIPAm( 0.6 g, 5.0 mmol), NSVB (12.0 mg, 50 μmol ), NBD(13.8 mg, 50 μmol), DATB( 14.0 mg, 50 μmol), AIBN (0.8 mg, 5 μmol) were dissolved in 5 ml dioxane. S-9

10 2.0 ultrafiltration-2 Tf ultrafiltration Abs Wavelength (nm) Figure S6. Ultrafiltration of the TfAuNCs with the viva spin column ultrafiltration and membrane (Sartorius AG, Gottingen, Germany/3000 Da) at run min -1 for 10 min. Ultrafiltration-1 and ultrafiltration-2 are the UV-absorption spectra of the filtration for once and twice. Tf is the UV-absorption spectrum of the Tf protein solution. S-10

11 15.0 NBDAA in H 2 NBDAA in CHCl FL Temperature ( o C) Figure S7. The relative fluorescent intensities of NBDAA were response to the temperature variation in organic solvent and in aqueous solution. S-11

12 Figure S8. Ratio of the polymer-tfauncs investigation for temperature-responsive fluorescent spectra of the polymer-tfauncs at different concentration of TfAuNCs. P(NIPAm-NBD-NSVB) (5.0 mg ml -1 ) was reacted with different concentration of TfAuNCs: A: 1.3 mm, B: 1.1 mm, C: 0.8 mm, D: 0.5 mm. E: the graph of R vs amount of TfAuNCs at different temperature. S-12

13 Mean number (%) TfAuNCs polymer-tfauncs polymer Diameter (nm) Figure S9. The DLS diameters of the polymer, TfAuNCs and the polymer-tfauncs measured at 25 o C. S-13

14 A FL B FL Polymer-30 o C Polymer-31 o C Polymer-32 o C Polymer-34 o C Polymer-35 o C Polymer-36 o C Polymer-38 o C Polymer-39 o C ( ) TfAuNCs-30 o C TfAuNCs-34 o C TfAuNCs-37 o C TfAuNCs-39 o C Wavelength (nm) Figure S10. (A) The magnified fluorescence spectra of Figure 4A for the changing of the ratiometric signal of TfAuNCs with the temperature increasing. (B) The fluorescence spectra of polymer and TfAuNCs changing with the temperature increasing, respectively. S-14

15 Figure S11. A: Fluorescent ratio was response to the temperature variation in different concentration of sodium solutions and proteins. B: Fluorescent ratio was response to the temperature variation in different ph. C: The respond time of polymer-tfauncs recorded at 39 C. D: Viability of HeLa cells after treatment in the presence of different concentrations of P(NIPAm-NBD-NSVB)-TfAuNCs. Each data bar represents an average of three parallels, and error bars indicate one standard deviation from the mean. Different concentrations of P(NIPAm-NBD-NSVB)-TfAuNCs have been investigated and the concentrations of P(NIPAm-NBD-NSVB) 0.05 mg ml -1, 0.10 mg ml -1, 0.30 mg ml -1, 0.50 mg ml -1 and 0.80 mg ml -1. S-15

16 Figure S12. Schematic diagram of entry process of the polymer-tfauncs and the polymer into the HeLa cells. S-16

17 Figure S13. Representative images of HeLa cells treated with the polymer and polymer-tfauncs. Differential interference contrast (DIC) images (B, D and F) and confocal fluorescence images (A, C and E). A and B: HeLa cells; C and D: HeLa cells treated with the polymer for 3 h; E and F: HeLa cells treated with the polymer-tfauncs for 3 h. S-17

18 Figure S14. Flow CytoMetry results of intracellular polymer and polymer-tfauncs. The polymer-tfauncs were composed of P(NIPAm-NBD-NSVB)-TfAuNCs (5.0 mg ml-1: 0.8 mm). A: control Hela cells; B: Hela cells treated with the polymer for 3 h; C: Hela cells treated with the polymer-tfauncs for 3 h. The y axis and the x axis are the fluorescent intensity of cells stained by the polymer-tfauncs and the polymer, respectively. S-18

19 Figure S15. Flow Cytometric quantification of intracellular polymer-tfauncs in HeLa cells after incubation for different time (histograms). The y axis is the relative cell number, x axis is the fluorescent intensity of cells stained by the polymer-tfauncs. S-19

20 Table S2 A comparison of performance between the prepared polymer-tfauncs and the reported nano thermometers for intracellular temperature sensing Nano thermometers Temperature controller Entry into cells Temperature sensing range ( o C) Temperature sensing resolution ( o C) Excitation/Emission Ref. NaYF 4 :Er 3+,Yb 3+ nanoparticles An electric power dissipated with a resistor Incubation for 1.5 h 26~ Two-photon excitation F. Vetrone, et al, ACS Nano 2010, 4, CdSe-QDs An open oven Incubation for 2 h 30~ Single excitation /Single emission L. M. Maestro, et al, Nano Lett. 2010, 10, AuNCs Thermo FisherScientific NESLAB RTE7 system Incubation for 2 h 10~45 0.1~0.3 Single excitation /Single emission L. Shang, et al, Angew. Chem. Int. Ed. 2013, 52, Green fluorescent protein Incubation system for microscope Transfection 20~ Single excitation /Single emission J. S. Donner, et al, Nano Lett. 2012, 12, 2107 Hydrophilic fluorescent nanogel A stage plate heater (Tokai Hit) Microinjection 27~40 0.3~0.5 Single excitation /Single emission C. Gota, et al, J. Am. Chem. Soc. 2009, 131, Fluorescent polymeric thermometer A stage plate heater (Tokai Hit) Microinjection 29~39 0.2~0.6 Single excitation /Single emission K. kabe, et al, Nat. Commun. 2012, 3, 705. Ratiometric fluorescent polymer thermometer Incubation system for microscope (WSKM, Tokai Hit) Targetting/ Incubation for 3 h 32~39 0.3~0.5 Single excitation /Dual self-calibration emission This work S-20