Supporting Information

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1 Supporting Information Living Cell Multilifetime Encoding Based on Lifetime-Tunable Lattice-Strained Quantum Dots Li Zhang, Chi Chen, Wenjun Li, Guanhui Gao, Ping Gong, Lintao Cai* Guangdong Key Laboratory of Nanomedicine, CAS Key Laboratory of Health Informatics, Shenzhen Bioactive Materials Engineering Lab for Medicine, Institute of Biomedicine and Biotechnology. Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, , P. R. China These authors contributed equally to this work. * lt.cai@siat.ac.cn. S-1

2 1. Methods 1.1. Preparation of stock solution Cadmium chloride (CdCl 2 ) stock solution was prepared by dissolving g CdCl 2 (99.996%, Alfa Aesar) in 10 ml deionized water. 3-mercaptopropionic acid (MPA) stock solution was prepared by dissolving g MPA (99%, Alfa Aesar) in 10 ml deionized water. Shell stock solution was prepared by adding 1 ml CdCl 2 (0.1 M) and 1 ml MPA (0.2 M) in a 50 ml centrifuge tube and diluted to 40 ml with deionized water, then the ph of the solution was adjusted to 11 by 1.0 M NaOH solution while stirring. Sodium hydrogen telluride (NaHTe) was freshly made before each synthesis by dissolving g Sodium borohydride (NaBH 4, 99%, Sigma-Aldrich) in 1.0 ml deionized water and then g tellurium powder (60-mesh, %, Alfa Aesar) was added into the NaBH 4 solution. This reaction was conducted in room temperature for four hours Synthesis of CdTe magic-core The route to obtain CdTe magic-core was adapted from previously literature. 1 For a 25 ml synthesis, 5 ml of CdCl 2 (0.1 M) and 4.5 ml of MPA (0.2 M) were mixed in a 50 ml centrifuge tube and diluted to 25 ml with deionized water, then the ph of the solution was adjusted to After degassed for one hour with argon, 100 μl of freshly as-prepared NaHTe (0.2 M) was injected into the above mixture solution. This solution was then incubated in refrigerator at 4 C for 18 h to promote nanocrystal growth. Then as-prepared CdTe magic-core were purified by adding ethanol and S-2

3 centrifugation at rpm for three times, then dried under vacuum for one night. This procedure was repeated for more synthesis Synthesis of CdTe/CdS core/shell QDs Firstly, as-prepared CdTe magic-core powder was dissolved in 10 ml deionized water. Then 2 ml of the shell stock solution was added into the above solution under various time intervals (30, 90, 180, 270, 360 min) at 80 o C. Finally, as-prepared CdTe/CdS core/shell QDs (emission at 520 nm) were purified by adding ethanol and centrifugation at rpm for three times, dried under vacuum for one night. This procedure was repeated for more synthesis Synthesis of peptides functionalized lattice-strained CdTe/CdS core/shell QDs Firstly, as-prepared CdTe/CdS core/shell QDs (emission at 520 nm) powder was dissolved in 10 ml deionized water. For the synthesis of biotin, TAT and CPP30 (PG-fldtlvvlhr-GPG-GYRRTTPSYYRMYLR, Capital letters; l-amino acid. Lower case letters; d-amino acid) functionalized lattice-strained CdTe/CdS QDs with different NIR-emitting wavelength and fluorescence lifetime were synthesized by further aging the CdTe/CdS core/shell QDs in the presence of solution of Shell stock solution/funcational molecules (with molar ratio MPA : funcational molecules =500:1) under 80 o C and various time intervals (generally, 3 h for CPP30-LS-QDs-620, 6 h for CPP30-LS-QDs-670, 9 h for CPP30-LS-QDs-700, 12 h for CPP30-LS-QDs-742). The original QDs were purified with an Amicon Ultra-4 centrifugal filter device (YM-3, Merck Millipore) via centrifugation at 8000 rpm for S-3

4 20 min for several times, and then were redissolved in PBS buffer with the same volume. This procedure was repeated for more synthesis. 2. Characterization The UV-vis-NIR absorption spectra, fluorescence spectra and time-resolved fluorescence decay curves of as-prepared QDs were recorded on a PerkinElmer Lambda 750 absorption spectrophotometer and a FSP 920 fluorometer, respectively. The lifetime data was fitted with 2nd and 3rd order exponential decay according to the Equation S1 below. F( t) α e n i 1 i t / τi (S1) Where τ i is the lifetime, а i is the pre-exponential factor and the F(t) is in log scale. And the average lifetime was calculated from the Equation S2 below. τ average α τ 2 i i α τ i i (S2) The X-ray powder diffraction (XRD) patterns were performed on Bruker D8 Advance X-ray diffractometer, which was equipped with a Cu Kα X-ray source. High resolution transmission electron microscopy (HRTEM) images were taken on a FEI Tecnai G2 F20 S-Twin transmission electron microscope operating at 200 kv. The samples for HRTEM images were prepared using the 200-mesh Au gird. 3. Photoluminescence Quantum Yield Determination The PL QY of as-prepared functionalized QDs was determined using Rhodamine 101 in ethanol HCl as a standard (λ ex =490 nm, Φ s =1). 1 The PL QY was calculated according to the following Equation S3: S-4

5 Φ x As Φ s Ax lnt ln t x s η η x s 2 (S3) Where Φ is the PL QY, A is the absorbance at the excitation wavelength, lnt is the area under the emission peak, and η is the refractive index of the solvent. The subscripts s and x denote the respective values of the standard and functionalized QDs. 4. Specificity Tests of QDs Modified with Peptides Firstly, the streptavidin microbeads were mixed with biotin functionalized LS-QDs and QDs without biotin modification in PBS buffer, respectively. Then the microbeads were collected via centrifugation at 1000 rpm for 2 min for several times, and dispersed in PBS buffer. Finally, the samples were dropped on a slide and imaged with an inverted microscope (Leica DMI 6000). 5. Stability Measurements LS-QDs-670, TAT-LS-QDs-670, and CPP30-LS-QDs-670 were selected to evaluate the stability. The colloid stabilities were evaluated by incubating the purified samples in water at 37 o C, and the photoluminescence intensities and average lifetimes were recorded at different time for a month. The ph stabilities were evaluated by dissolving the samples in buffers with different ph values (ph: 4~10) and average lifetimes were calculated. The photo-stabilities were evaluated by continuously exciting the samples with 480 nm laser for up to 120 min. The interferences by coexisting ions and biomolecules were tested by mixing the samples with different concentrations of potential interfering ions (K +, Mg 2+, Mn 2+, and HCO 3- ) and biomolecules (Bovine Serum Albumin and glucose) and average lifetimes were calculated. S-5

6 6. Cell Culture MCF-7 cells (Human breast cancer cell line), HeLa cells (human cervix cancer cell line),c6 cells (human brain glioma cell line) and BEND3 cells (mouse brain cerebral cortex cell line) were obtained from American Type Culture Collection (ATCC). Cells were maintained in DMEM high glucose (Hyclone) supplemented with 10%(v/v) fetal bovine serum (Gibco), 1%(v/v) antibiotic/antimycotic(hyclone), cultured at 37 o C-humidified atmosphere containing 95% air/5% CO Cytotoxicity Measurements The cytotoxicities of samples were evaluated by using the Cell Counting Kit-8 (CCK-8, Dojingdo, Japan) assay. The MCF-7 cells (5000 cells/well) were seeded into 96-well plates (Corning) and incubated for 24 h. Cells were washed with PBS (Hyclone) buffer twice, then incubated for 24 h with various concentrations of the samples (0.1, 1, 5, 10, 20 and 50 μg/ml). 10 μl of CCK-8 solution was added and further incubated for 4 h. The absorbance was measured at 450 nm with the Multiskan GO microplate reader (Thermo Scientific). 8. Cellular Uptake and Imaging MCF-7, HeLa, C6 and BEND3 cells were cultured in DMEM high glucose, with 10% fetal bovine serum (FBS) at 37 C (5% CO 2 ) and were grown in 8-well chambers (Thermo) overnight, then 40 μl of CPP30-LS-QDs-670, TAT-LS-QDs-620 and LS-QDs-700 solution was added, incubated for 2 h. Cells were washed twice with PBS buffer. Fluorescence microscopic imaging was taken by confocal laser scan microscopy (Leica TCS SP5) and images were collected using a Plan-apochromat S-6

7 63X/1.4 oil immersion objective by sequential line scanning, with excitation 488 nm along with a bright field image. 9. Fabrication and Recognition of Living Cells Multilifetime Encoding MCF-7 cells were seeded in 6-well flat(corning) over night, CPP30-LS-QDs-742, CPP30-LS-QDs-700 and CPP30-LS-QDs-670 solution were added and incubated for another 2 h, after washed with PBS buffer, FLIM was taken by Time-Resolved Fluorescence Microscopy (PicoQuant MicroTime 100, excitation, 485 nm, emission, 500 nm long-pass). To fabricate and recognize the living cells multilifetime encoding, these three types of CPP30-LS-QDs with different molar ratio were incubated with MCF-7 cells. To present the process of targeted living cells multilifetime encoding, MCF-7 and C6 cells were cocultured in the same plate overnight, then incubated with well-defined mixed CPP30-LS-QDs encoded solution, FLIM was taken according to the protocol as described before. S-7

8 Figure S1. (a) HRTEM (scale bar: 5 nm), TEM image (scale bar: 20 nm) and size distribution of CdTe ultrasmall core. (b) Absorption and photoluminescence spectrum of ultrasmall CdTe core. Figure S2. HRTEM (scale bar: 5 nm), TEM image (scale bar: 50 nm) and size distribution of CPP30-LS-QDs-670. S-8

9 Figure S3. XPS data of (a) the Te 3d lever and (b) the Cd 3d lever of CdTe ultrasmall core (violet), CPP30-LS-QDs-742 (red) and nonlattice-strained QDs-726 (black). Figure S4. XRD patterns for CdTe ultrasmall core (violet), CPP30-LS-QDs-742 (red) and nonlattice-strained QDs-726 (black). Bulk diffraction peaks for zinc blende (ZB) ZnS (top) and ZB CdS (bottom) are indexed. S-9

10 Figure S5. Fluorescence microscopy imaging of streptavidin microbeads. (a) Transmission grey image and (b) fluorescence image of streptavidin microbeads labelled with biotin functionalized QDs. (c) Transmission grey image and (d) fluorescence image of streptavidin microbeads mixed with LS-QDs without biotin(scale bar: 100 um). Figure S6. Hydrodynamic size distributions of CPP30-LS-QDs-670, TAT-LS-QDs-670, and LS-QDs-670 measured by DLS (From top to the bottom). S-10

11 Figure S7. (a) Photo, (b) colloid, and (c) lifetimestabilities of LS-QDs-670 (black), TAT-LS-QDs-670 (blue) and CPP30-LS-QDs-670 (red). d) The lifetime stabilities of CPP30-LS-QDs-670 in buffers with different ph values (from 3.0 to 9.0). Figure S8. (a) Cell viability data of MCF-7 cells incubated with LS-QDs (black), TAT-LS-QDs-670 (blue) and CPP30-LS-QDs-670 (red) after 24 hours in various concentration (0.1, 1, 5, 10, 20 and 50 μg/ml). (b) Interference study of CPP30-LS-QDs-670 at ph 7.0. S-11

12 Figure S9. The cellular uptake and specific tumor cells imaging of lattice-strained QDs-700 (scale bar: 25 μm). S-12

13 Figure S10. (a) The FLIM images and (b) lifetime distribution of each CPP30-LS-QDs encoded MCF-7 cells, respectively (from left to right: CPP30-LS-QDs-742, CPP30-LS-QDs-700, CPP30-LS-QDs-670, scale bar: 30 μm). Table S1. Details fitted parameter and average lifetime values of LS-QDs-520 and CPP30-LS-QDs with different fluorescence emission spectra (620, 670, 700, and 742). a 1 (%) τ 1 (ns) LS-QDs CPP30-LS-QDs CPP30-LS-QDs CPP30-LS-QDs CPP30-LS-QDs a 2 (%) τ 2 (ns) x 2 τ average (ns) Table S2. Details fitted parameter and average lifetime values of nonlattice-strained QDs with different fluorescence emission spectra (520, 616, 670, 690 and 726). a 1 τ 1 a 2 τ 2 a 3 τ 3 x 2 τ average (%) (ns) (%) (ns) (%) (ns) (ns) QDs / / QDs / / QDs / / QDs QDs S-13

14 References (1) Deng, Z.; Schulz, O.; Lin, S.; Ding, B.; Liu, X.; Wei, X.; Ros, R.; Yan, H.; Liu, Y. Aqueous Synthesis of Zinc Blende CdTe/CdS Magic-Core/Thick-Shell Tetrahedral-Shaped Nanocrystals with Emission Tunable to Near-Infrared. J. Am. Chem. Soc. 2010, 132, S-14