Supporting Information for: Nanoparticles self-assembled from multiple interactions: a. novel near-infrared fluorescent sensor for detection of

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1 Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 215 Supporting Information for: anoparticles self-assembled from multiple interactions: a novel near-infrared fluorescent sensor for detection of serum albumin in human sera and turn-on live-cell imaging Xiaopeng Fan, Qingyuan He, Shiguo Sun, Hongjuan Li, Yuxin Pei and Yongqian Xu* College of Science, orthwest A&F University, Yangling, Shaanxi, P.R. China, 7121, xuyq@nwsuaf.edu.cn 1. Experimental 1.1 Synthesis material and instruments All chemicals and reagents were used directly as obtained commercially unless otherwise noted. Water used was ultra filter deionized. Avidin, concanavalin, myoglobin, casein, lysozyme, RaseA, trypase, pepsin, thrombin, BSA, casein were purchased from SIGMA. MR spectra were recorded on a Varian 3 Gemini spectrometer. Mass spectrum was obtained in ESI mode on a HP11LC/MSD mass spectrometer. UV-Vis spectra were acquired on a Shimadzu 175 UV-visible spectrometer. Fluorescence spectra were obtained on a RF-531 fluorescence spectrometer. 1.2 Measurement Procedures The stock solutions of SQ-P with a concentration of M were prepared first by dissolving the appropriate amount of the dye in DMSO, respectively. For measurement of spectroscopic properties, 3 µl of each stock solution were diluted with PBS (.1 M, 3 ml, ph 7.2) to obtain aqueous solution of SQ-P ( M) under vigorous stirring at room temperature. Stock solutions of the various proteins

2 were prepared in deionized water and the concentrations were fixed at M. The quantum yield of fluorescence of the sample was measured using bis(3-ethylbenzothia zol-2-ylidene)squaraine in ethanol (Φ =.21) as a standard s1 and calculated using eq 1: I unk A std n Φ Φ unk unk = std I std A unk n std 2 (1) Where Φ unk is the fluorescence quantum yield of the sample, Φ std is the fluorescence quantum yield of the standard, I unk and I std are the integrated emission intensities of the sample and the standard, respectively, A unk and A std are the absorbance of the sample and the standard at the excitation wavelength, respectively, and n unk and n std are the refractive indexes of the corresponding solution. The preparation of deproteinized human blood samples is followed the reported literature. S2 Human blood samples were collected from healthy volunteers treated in the local Medical Hospital. All samples were obtained by venipuncture and collected in heparinized vacutainer tubes. Then, a 2 μl aliquot of the blood was deproteinized by mixing immediately with 4 μl of cold 1% Cl 3 CCOOH. After vortex mixing, the mixture was centrifuged at 8 rpm for 1 min. A total of 4 μl of the supernatant was collected. Then Cl 3 CCOOH was evaporated out. The obtained supernatant without Cl 3 CCOOH was ready for assays. 1.3 Atomic Force Microscopy (AFM) and Field emission scanning electron microscope (FESEM) Samples for the imaging were prepared by spin-casting the SQ-P in the absence and presence of its specific protein (BSA) at the specified concentrations. AFM (anoscope V) was performed in the ambient air condition in the tapping mode, a frequency near resonance. The scan rate was 1 Hz with a scan field of view of 5 nm 5 nm to 5μm 5μm. The microstructure of the samples was analyzed by field emission scanning electron microscopy (FESEM, S-48). All samples were dried and detected at room temperature with SE detection at 1. kv. DLS measurements were performed on a Delsa TM ano system (Beckman Coulter, Inc., CA, U.S.A.).

3 1.4 Cell culture and fluorescence image HeLa cells were seeded on 35 mm glass-bottomed dishes (EST) and incubated in RPMI-164 in an incubator (37 C, 5% CO 2 and 2% O 2 ) for 24 h. The cells were rinsed slightly 3 times with fresh RPMI-164 and incubated in RPMI-164 medium spiked with or without sensor (5 μm) for 3 min, respectively. After washing with fresh RPMI-164, the cells treated with sensor were further incubated in fresh RPMI- 164 containing of 5 μm hydrazine for.5 h. Cells were then analyzed by Laser Scanning Confocal Microscope (A1R). 1.5 Calculation of detecting limit Detecting limit DL = K S b1 /S, where K=3, S b1 is the standard derivation of the blank solution and S is the slope of the calibration curve. S3 1.6 Synthesis of SQ-P and SQ-B S O OMe S CF3SO3 - H2 HCl TEA CH2Cl2 S O H S CF3SO3 - S O H S CF3SO3 - SQ SQ-P SQ-B The compound SQ was synthesized according to the procedure reported previously. S4 SQ-P was synthesized by using a modified procedure (Santos et al, 25). 1- aminomethylpyrene hydrochloride (.38 mmol, 12 mg) and triethylamine (.76 mmol, 16 μl) were dissolved in 13 ml anhydrous CH 2 Cl 2. The solution was refluxed under nitrogen atmosphere for 2 h and cooled to room temperature. SQ (.338 mmol,2 mg) was added to above solution under nitrogen atmosphere, and the resulting solution was further at room temperature for 48 h. The solvent was removed under reduced pressure and the residue was applied to silica gel chromatograph (by using as CH 2 Cl 2 /MeOH eluent) to get solid SQ-P (25 mg, 93 %). 1 H MR (5 MHz, DMSO-d 6 ) δ 9.44 (t, J = 5.6 Hz, 1H), 8.49 (d, J = 9.3 Hz, 1H), (m, 4H), (m, 2H), (m, 2H), 8.1 (d, J = 7.9 Hz, 1H),

4 7.84 (d, J = 7.8 Hz, 1H), 7.7 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.41 (s, 1H), 7.33 (d, J = 2.8 Hz, 2H), 7.24 (m,, 1H), 6.35 (s, 1H), 5.67 (d, J = 5.5 Hz, 2H), 5.51 (s, 1H), 4.3 (d, J = 7.1 Hz, 2H), 3.58 (d, J = 7. Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H),.25 (t, J = 7.1 Hz, 3H). 13 C MR (126 MHz, DMSO-d 6 ) δ , , , , , , 14.21,139.97, , 13.81, 13.51, 13.26, , , , , , , , 125.5, , , 125.8, 124.4, , , 123.3, , , 113.3, , 86.53, 86.41, 45.26, 41.7, 4.38, 12.6, MS (ESI+) found (M) +, calcd for C 41 H 32 3 OS 2, For comparison, a similar compound SQ-B was also synthesized, where the pyrene group was replaced by phenyl group. The synthesis of the compound SQ-B is similar to that of SQ-P by using 1-aminomethylphenyl hydrochloride to replace 1- aminomethylpyrene hydrochloride. 1 H MR (5 MHz, DMSO-d 6, ppm) δ 9.29 (t, J = 6.1 Hz, 1H), 8. (d, J = 7.8 Hz, 1H), 7.9 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.56 (dd, J = 17.7, 8.3 Hz, 2H), (m, 5H), (m, 3H), 6.32 (s, 1H), 5.72 (s, 1H), 4.92 (d, J = 6.1 Hz, 2H), (m, 2H), 4.18 (q, J = 6.8 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H), 1.8 (t, J = 6.3 Hz, 3H). 13 C MR (126 MHz, DMSO-d 6 ) δ , , 161.7, , , , 14.28, 129.1, 128., , , , , , , 123.6, , , , 86.6, 86.3, 46.86, 41.75, 4.97, 12.6, Absorbance a) 337 nm.6 μm 4 μm 1μM 16 μm 3 μm 48 μm 84 μm 12 μm 192 μm 32 μm 448 μm FL Intnesity (a.u.) μm 2 μm 4 μm 6 μm 8 μm 1 μm 12 μm 14 μm 16 μm 2 μm Wavelength/nm Wavelength/nm

5 FL Intensity(a.u.) c) R 2 = BSA/μM Fig. S1 Spectroscopic response of SQ-P to BSA. (a) UV-Vis absorption change of SQ-P (5 μm) upon addition of BSA (-448 μm). ( Fluorescence spectral changes of SQ-P (5 μm) upon addition of BSA (-2 μm) (λ ex = 6 nm, slit width of excitation and emission is 1 and 1 nm, respectively). Fluorescence measurements were performed 1 min after adding BSA to the SQ-P solution. (c) Plot of the fluorescence intensity at 668 nm to BSA concentrations (-2 μm). All experiments were performed in 1 mm PBS buffer (ph 7.2). FL/Intensity μm PBS Buffer BSA/ μm Wavelength/nm Fig. S2. Fluorescence spectral changes of SQ-B (5 μm) upon addition of BSA (-113 μm) (λ ex = 6 nm, slit width of excitation and emission is 1 and 5 nm, respectively). Fluorescence measurements were performed 1 min after adding BSA to the SQ-B solution.

6 I/I 1 SQ-P SQ-B a) FL Intensity (a. u.) SQ-P SQ-B [BSA]/ M Fig. S3. (a) Plots of the fluorescence intensities of SQ-P at 668 nm and SQ-B at 674 nm to BSA concentrations. ( The fluorescence spectra of SQ-P and SQ-B in the absence of BSA, where I SQ-B /I SQ-P at 66 nm is about 13.7-fold. All experiments were performed in 1 mm PBS buffer (ph 7.2), λ ex = 6 nm, slit width of excitation and emission is 1 and 1 nm, respectively SQ-B+HSA SQ-P+HSA I/I Concentration ( M) Fig. S4. Plots of the fluorescence intensities of SQ-P at 674 nm and SQ-B at 674 nm to HSA concentrations. All experiments were performed in 1 mm PBS buffer (ph 7.2).

7 2 Ex: 337 nm M BSA Fig. S5. Fluorescence spectral changes of SQ-P (5 μm) upon addition BSA (2 μm) (λ ex = 337 nm) in PBS buffer (1 mm, ph=7.2). FL Intensity (a. u.) probe 2-pepsin 3-trypsin 4-RaseA 5-lysozyme 6-casein 7-myoglobin 8-BSA 9-HSA Fig. S6 Selectivity test of SQ-P with other non-targeted proteins. SQ-P (5 µm) was tested with non-targeted proteins at 2 µm. Bars represent fluorescence intensity at 684 nm.

8 1 a) M Cys 2 M 4 M 6 M 8 M 1 M 2 M 3 M M GSH 2 M 4 M 6 M 8 M 1 M 2 M 3 M c) M Hcy 2 M 4 M 6 M 8 M 1 M 2 M 3 M d) M S 2-2 M 4 M 6 M 8 M 1 M 2 M 3 M Fig. S7. Fluorescence spectral changes of SQ-P (5 μm) upon addition of thiolcontaining compounds (2 μm) (λ ex = 6 nm) in PBS buffer (1 mm, ph=7.2). a) Cys. GSH. c) Hcy. d) S a) 1 M Cys 2 M 3 M M GSH 2 M 3 M c) 1 M HCy 2 M 3 M 1 d) M S 2-2 M 3 M

9 Fig. S8. Fluorescence spectral changes of SQ-P (5 μm) upon addition of thiolcontaining compounds (2 μm) (λ ex = 6 nm) in PBS:CH 3 C=1:1 (1 mm, ph=7.2) (1 mm, ph=7.2), where SQ-P exists in monomer state. a) Cys. GSH. c) Hcy. d) S 2-. Absorbance a) 1 M Cys 2 M 3 M Absorbance M GSH 2 M 3 M Absorbance c) 1 M Hcy 2 M 3 M Absorbance d) 1 M S 2-2 M 3 M Fig. S9. Absorption changes of SQ-P (5 μm) upon addition of thiol-containing compounds (2 μm) (λ ex = 6 nm) in PBS:CH 3 C=1:1 (1 mm, ph=7.2) (1 mm, ph=7.2), where SQ-P exists in monomer state. a) Cys. GSH. c) Hcy. d) S 2-. Absorption a) L 3 L 6 L 9 L 12 L 15 L 18 L 21 L 24 L 27 L 3 L 36 L 42 L 48 L 54 L L 3 L 6 L 9 L 12 L 15 L

10 c) R 2 = d) in PBS with 3.3% supernatant in PBS Volume ( L) Concentration ( M) Fig. S1 Spectroscopic response of SQ-P to human blood serum. (a) UV-Vis absorption change of SQ-P (5 μm) upon addition of blood sample (-54 μl). ( Fluorescence spectral changes of SQ-P (5 μm) upon addition of blood sample (-15 μl) (λ ex = 6 nm). Fluorescence measurements were performed 1 min after adding blood sample to the SQ-P solution. (c) Plot of the fluorescence intensity at 668 nm to blood sample volumes (-15 μl). (d) Plot of the fluorescence intensity at 668 nm to addition of HSA with and without 3.3% deproteinized blood sample. All experiments were performed in 1 mm PBS buffer (ph 7.2). a) Fig. S11 AFM images of SQ-P. a) SQ-P alone. SQ-P and BSA. The solution concentrations used for film preparation are 5 µm for SQ-P and 2 µm for BSA in PBS buffer and the scale is 1 µm.

11 a) c) 15. um 5. um d) 15. um 5. um Fig. S12 FESEM images of SQ-P. (a) and (c): SQ-P alone. ( and (d): SQ-P and BSA. The solution concentrations used for film preparation are 5 µm for SQ-P and 2 µm for BSA in PBS buffer. Differential Intensity (%) Diameter (nm) Differential Intensity (%) Diameter (nm) Fig. S13. DLS analysis of SQ-P (5 μm) solution (1 mm PBS buffer, ph 7.2) in the absence (left) and presence (right) of 2 μm BSA. DLS data of BSA protein alone (6 μm) is also obtained. The intensity of large particles (~ 7 nm) assigned to large aggregates (oligomer) reduced after adding serum albumin, suggesting that the equilibrium between monomer and aggregates shifts along the direction of monomer.

12 HOOC O 2 S O 2 S H 2 Dansylproline (DP) Dansylamide (DSA) a) Wavelength/nm DSA.6 mm.16 mm.26 mm.36 mm.46 mm DP.6 mm.16 mm.26 mm.36 mm.46 mm.56 mm.66 mm.76 mm.86 mm.96 mm 1.6 mm 1.26 mm 1.46 mm 1.66 mm 1.86 mm 2.6 mm 2.26 mm 2.46 mm 2.66 mm 2.86 mm Wavelength/nm Fig. S14. Fluorescence of SQ-P (5 μm) in phosphate buffer solution (1 mm, ph7.2) with different concentration of DSA (a) and DP ( in the presence of 25 μm BSA Before After Fig. S15. Fluorescence images of BSA stained by SQ-P after electrophoresis. (1) BSA 1 μg, (2) 11 μg, (3) 2 μg before and after washing.

13 ppm Fig. S16. 1 H MR of SQ-P in DMSO-d 6 solvent. The signals at 3.3 and 2.5 ppm are attributed to H 2 O and DMSO, respectively ppm Fig. S C MR of SQ-P in DMSO-d 6 solvent. The signal at ppm is attributed to DMSO.

14 [SSG]--H-1 #2-8 RT:.-.3 AV: 7 L: 6.56E4 T: ITMS + c ESI sid=35. Full ms [5.-2.] Relative Abundance m/z Fig. S18. MS spectrum of SQ-P. Fig. S19. 1 H MR of SQ-B in DMSO-d 6 solvent. The signals at 3.3 and 2.5 ppm are attributed to H 2 O and DMSO, respectively.

15 Fig. S2. 13 C MR of SQ-B in DMSO-d 6 solvent. The signal at ppm is attributed to DMSO. S1 S. Das, K. G. Thomas, R. Ramanathan, M. V. George and P. V. Kamat, J. Phys. Chem. 1993, 97, S2 D. Tian, Z. Qian, Y. Xia and C. Zhu, Langmuir, 212, 28, S3 A. Hakonen, Anal. Chem., 29, 81, S4 Y. Xu, Z. Li, A. Malkovskiy, S. Sun and Y. Pang, J. Phys. Chem. B, 21, 114, 8574.