Quantum dots CdSe/ZnS core-shell with maximum emissions of approximately 520 and 600

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1 Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 15 Electronic Supplementary Information Intracellular Zn + detection with Quantum Dot-based FLIM nanosensors Consuelo Ripoll, a Miguel Martin, b Mar Roldan, b Eva M. Talavera, a Angel Orte a,* and Maria J. Ruedas-Rama a,* a Dept. Physical Chemistry. Faculty of Pharmacy. University of Granada. Campus Cartuja, 1871 Granada (Spain). b GENYO. Pfizer-University of Granada-Junta de Andalucia Centre for Genomics and Oncological Research. Avda Ilustracion 114, PTS, 1816 Granada (Spain). Corresponding authors: AO: angelort@ugr.es, Tel ; MJRR: mjruedas@ugr.es, Tel Experimental Section... Methods of Analysis... Table S1. Table S Figure S1... Figure S... Figure S3... Figure S4... Figure S5... References. S S7 S9 S1 S11 S1 S13 S14 S15 S15 S1

2 Experimental Section Materials Quantum dots CdSe/ZnS core-shell with maximum emissions of approximately 5 and 6 nm (QD 5 and QD 6 ) and a lipophilic long chain surfactant capping of octadecylamine (ODA) were purchased from Mesolight (USA). 3-Mercaptopropionic acid (MPA) was purchased from Fluka. 1,4,7,1-tetraazacyclododecane (cyclen, 1), 1,4,8,11-Tetraazacyclotetradecane (cyclam, ), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Tris buffer, Bovine Serum Albumin (BSA), Ficoll4, and all inorganic salts were of analytical grade and used as obtained from Sigma-Aldrich (Spain). For cell culture, Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), penicillin, and streptomycin were obtained from Sigma. MitoTracker Deep Red dye was purchased from Life Technologies S.A. (Spain). The ph of solutions and buffers was adjusted using diluted NaOH (Sigma-Aldrich, Spain) and HCl (Sigma-Aldrich, Spain) (spectroscopic grade quality) dissolved in Milli-Q water. All chemicals were used as received without further purification, and stock solutions were kept at 4 ºC in a refrigerator and in the dark when not in use to avoid possible deterioration via exposure to light and heat. For microscopy experiments, all solutions were filtered with. μm filters (Whatman) before use. Synthesis of water-soluble MPA-capped CdSe/ZnS nanoparticles The lipophilic octadecylamine-capped QDs (QD-ODA) were modified using 3- mercaptopropionic acid (MPA) to achieve water solubility. The procedure for the surface-ligand exchange has been previously reported. 1 Briefly, 1 ml of QD-ODA dissolved in toluene was left to react overnight with ml of MPA, protected from light. After the ligand exchange, the particles were transferred to an aqueous phase by adding 1 M NaOH solution and shaking. The S

3 aqueous phase was separated, and the excess of MPA was removed from the water-soluble CdSe/ZnS QD-MPA nanoparticles by precipitation of the particles with acetone and centrifugation (1 min, 13, rpm), followed by the re-dissolution of the QD-MPA in 1 mm Tris buffer, ph 7.. Synthesis of QD-azacycle conjugates The azacycles cyclam and cyclen have four amino groups available for conjugation, with the carboxylic acid group capping the QD-MPA nanoparticles. This method has been previously used for QD modification, -3 achieving stable and water-soluble conjugates via amide formation using the EDC/NHS coupling reaction (Figure 1, main text). Upon optimization of the quantity of EDC, NHS and azacycle during the coupling reaction, the QD-azacycle conjugates, QD-1 and QD-, were prepared by mixing μl of QD-MPA with a solution of EDC (1 mm final concentration) in 1 mm Tris ph 7. for 1 minutes and then with a solution of NHS (5 mm final concentration) in 1 mm Tris ph 7.. After 5 min, the adequate amount of 1 or solution in 1 mm Tris ph 7. was added until reaching a final concentration of 1 mm. The mixture was stirred for 3 h at room temperature. The reacting mixture was then centrifuged at 13, rpm for 1 min. The supernatant containing the excess of reagents was removed, and the QD-azacycle conjugates in the residue were re-dissolved in 1 mm Tris ph 7.. Instruments Steady-state photoluminescence (PL) emission spectra were collected using a JASCO FP-65 spectrofluorometer equipped with a 45 W xenon lamp for excitation, with a temperature controller ETC-73T set at 5 C. All measurements were collected at 5 ºC using 5 1 mm cuvettes. S3

4 PL decay traces of QDs were recorded in the Single Photon Timing (SPT) mode using the FluoTime fluorometer (PicoQuant, GmbH, Germany) previously described. 4 In brief, the samples were excited using a 44 nm pulsed laser (LDH-P-C-44 PicoQuant, GmbH, Germany) with a 1 MHz repetition rate, which was controlled by a PDL-8-B driver (PicoQuant). The full width at half maximum of the laser pulse was ~ 8 ps. The PL was collected after crossing through a polarizer set at the magic angle and a nm bandwidth monochromator. PL decay histograms were collected using a TimeHarp board (PicoQuant), with a time increment per channel of 36 ps, at the emission wavelengths of 5, 54 and 56 nm for QD 5 and 59, 594 and 596 nm for QD 6. The histogram of the instrument response function (IRF) was determined using a LUDOX scatterer. Sample and IRF decay traces were recorded in triplicate until they typically reached 1 4 counts in the peak channel. PL lifetime images were recorded with a MicroTime fluorescence lifetime microscope system (PicoQuant, GmbH, Germany) based on single photon timing using the time-tagged timeresolved (TTTR) methodology, which permits reconstruction of the PL decay traces from the QD nanoparticles in the confocal volume. The excitation source was a 485-nm pulsed laser (LDH-P- C-485, PicoQuant), operated with a Sepia II driver (PicoQuant GmbH) set at a repetition rate of 1 MHz. The laser power at the microscope entrance was between. and 4.4 μw. The excitation beam passed through an achromatic quarter-wave plate (AQWP5M-6, Thorlabs, NJ), set at 45º from the polarization plane of the laser, and was directed by a dichroic mirror (51DCXR, AHF/Chroma, Germany) to the oil immersion objective (1.4 NA, 1 ) of an inverted confocal microscope (IX-71, Olympus). The PL emission was collected through the same objective and directed into a 75-μm pinhole by using a dichroic mirror after passing through a specific cutoff, i.e., a long pass filter (5LP, AHF/Chroma, Germany). The PL emitted photons were detected by using an avalanche photodiode (SPCM-AQR-14, Perkin S4

5 Elmer) after crossing an adequate bandpass filter (6/4, AHF/Semrock, Germany). Individual photons time tagging was performed within a TimeHarp module (PicoQuant), with a time resolution of 9 ps per channel. To image a region, a sample was raster-scanned with an x-y piezo-driven device (Physik Instrumente, Germany). The imaging data were normally acquired with a pixel resolution and a collection time of.6 ms per pixel. FLIM Imaging of QD-1 in buffered solutions FLIM imaging experiments of QD-1 at different Zn + concentrations were performed in solutions buffered with 1 mm Tris buffer ph 7.. The glass slides were washed twice with.5 ml of 1 mm Tris buffer at ph 7., followed by washing with ethanol and then finally being dried with a lens tissue. Then, 1 µl of QD-1 nanosensors were dissolved in 1 ml of Tris buffer at ph 7. and sonicated for 1 minutes. Subsequently, 4 µl of the buffer solution was placed on the slide and µl of the sonicated solution was added to the QD-1, leaving the sample ready for viewing under the microscope. This protocol ensured that the coating of the surface was not too crowded, was suitable for imaging, and avoided interactions between individual nanoparticles. Finally, surface areas between 36 and 1156 μm were raster-scanned for FLIM imaging with a spatial resolution of 14 to 7 nm/pixel. FLIM Imaging of QD-1 in HepG cells The Cell Culture Facility, University of Granada, provided the HepG 3 cell line. Cells were grown in DMEM supplemented with 1% (v/v) FBS, mm glutamine, 1 U/mL penicillin, and.1 μg/ml streptomycin at 37 ºC in a humidified 5% CO incubator. For the FLIM experiments, HepG cells were seeded onto mm diameter glass slides at a density of 115 cells/cm. The glass slides were washed with the DMEM medium and phosphate-buffered saline (PBS) before S5

6 adding the cells. The cells seeded onto the glass slides were incubated for h at 37 ºC with the addition of μl of the stock solution of QD-1 into 3 ml of the cell culture medium. After incubation, the cells were washed twice with the PBS buffer at ph 8. For the experiments in the presence of Zn +, the QD-loaded cells were later incubated at 37 ºC for 1 min in a 1 mm Zn + solution in PBS buffer ph 8. For the FLIM experiments, images of surface areas between 39 and 45 μm were collected with a spatial resolution of 1 to 7 nm/pixel. Cell Viability Assays To assay possible side toxicity on cells by QDs load, cell viability was studied by using CellTiter Blue viability assay (Promega). Cell sixtuplicates were plated in cell culture-treated black 96 well optical flat bottom plates at 1.x1 3 cells/well. After 48h of cell culture, 1,, 4 and 6 µl of QDs from a sonication-cleared stock solution were added directly to the wells, being µl QDs/well the concentration equivalent to the higher used in the other experiments. After h incubation, % v/v of CellTiter-Blue (Promega) reagent was added to the wells, incubated for hours at 37 C, and then fluorescence was directly read at 55/58-64nm in a Glomax - Multidetection System (Promega). Untreated cell controls, and wells with reagents only as background controls, were run together with treated cells. The absolute fluorescence arbitrary units were recorded and subsequently the data were expressed at percentage relative to untreated control cells. S6

7 Methods of Analysis Time resolved PL decay traces collected from experiments in solution were deconvoluted from the instrument response function and fitted using the FluoFit 4.4 package (PicoQuant). The experimental decay traces were fitted to three-exponential functions via a Levenberg-Marquard algorithm-based nonlinear least-squares error minimization deconvolution method. The quality of fits was judged by the value of the reduced chi-squared, χ, and visual inspection for random distribution of the weighted residuals and the autocorrelation functions. To compare the PL decay times of the QD-MPA and QD-azacycle with different Zn + concentrations, it was necessary to determinate their intensity-weighted average PL lifetime, τ ave, using equation 1: 5 ave a i i a i i (eq. 1) where τ i represents the decay times and a i the corresponding pre-exponential factors. The FLIM images were analysed using the SymphoTime software (PicoQuant). The FLIM images were reconstructed by sorting all photons corresponding to a single pixel into a temporal histogram by the TTTR methodology. The PL decay traces in each pixel of the regions of interest (pixels containing QD emission and at least 1 photons per pixel) were fitted to a twoexponential function through an iterative reconvolution method based on the maximum likelihood estimator (MLE), which yields the best parameter fitting for low count rates. 6 The short decay time was fixed at 1.5 ns, accounting for the short components and for the cell auto fluorescence for the experiments with cells. The second decay time was left as an adjustable parameter. The instrument response function for the iterative reconvolution analysis was reconstructed from images with a high total count rate, using the dedicated routine in the SymphoTime software. To achieve a higher count rate in each pixel, thus improving the S7

8 reliability of the fits, spatial rebinning of 5 5 pixels and temporal binning of four channels in the SPT scale (for a final 116 ps/channel temporal resolution) were employed. The image could then be redrawn using an arbitrary colour scale illustrating just the values of the second, large decay time in each pixel. Frequency distributions of this decay time in the regions of interest were constructed. S8

9 Table S1. Decay times and normalized pre-exponentials of QD 6 -MPA and QD 6-1 at different Zn + concentrations. PL decay traces collected at ex = 44 nm and em = 596 nm. (a 1 ) τ 1 (ns) QD-MPA (.4) 3.1 (a ) τ (ns) (.49) 1.64 (a 3 ) τ 3 (ns) (.11).1 τ ave χ² (ns) a QD-1 (.8) (.48) 5.54 (.44) QD mm Zn + (.8) (.48) 5.66 (.44) QD-1 +. mm Zn + (.8) (.48) 5.71 (.44) QD mm Zn + (.8) (.48) 5.89 (.44) QD mm Zn + (.9). (.48) 5.48 (.43) QD-1 +. mm Zn + (.9).43 (.49) 5.94 (.4) QD mm Zn + (.1).94 (.49) 6.14 (.39) QD mm Zn + (.14) 1.47 (.47) 6.34 (.39) QD-1 +. mm Zn + (.15).99 (.48) 6.65 (.37) QD mm Zn + (.15) 3.55 (.48) 6.9 (.37) QD-1 +1 mm Zn + (.18) 4.15 (.49) 7.4 (.33) QD-1 + mm Zn + (.18) 4.7 (.5) 7.48 (.3) a Associated errors in τ ave, obtained through error propagation of the fitting errors of the adjustable parameters, were always between.13 and.15 ns. S9

10 Table S. Decay times and normalized pre-exponentials of QD 5 -MPA and QD 5 - at different Zn + concentrations. PL decay traces collected at ex = 44 nm and em = 56 nm. (a 1 ) τ 1 (ns) QD-MPA (.36).14 (a ) τ (ns) (.44) 8.98 (a 3 ) τ 3 (ns) (.) 1.8 τ ave χ² (ns) a QD- (.3) (.47) 6.69 (.3) QD- +.1 mm Zn + (.7) 17.3 (.47) 6.9 (.6) QD- +.5 mm Zn + (.8) 17.8 (.49) 7.34 (.3) QD mm Zn + (.3) (.47) 7.47 (.1) QD- +. mm Zn + (.31) (.48) 7.87 (.1) QD- +.4 mm Zn + (.31) (.5) 8.8 (.19) QD- + 1 mm Zn + (.37) (.49) 8.56 (.14) QD mm Zn + (.33). (.53) 9.13 (.14) QD- + mm Zn + (.36) (.51) 9.6 (.13) a Associated errors in τ ave, obtained through error propagation of the fitting errors of the adjustable parameters, were always between.13 and.15 ns. S1

11 Counts Counts Counts Counts 1 1 Autocorrelation Function,, -,,, -, - - Residuals 1 1 Autocorrelation Function, 1 1 ave = ns ave = ns QD 5 - ave = 1. ns QD 5-1 ave = 8.61 ns Time (ns) Time (ns), -,,, -, - - Residuals 1 Autocorrelation Function,, 1 Autocorrelation Function,, -,, -,, 1, -, - Residuals 1, -, - Residuals 1-1 ave = 18.4 ns QD 6-1 ave = 9.1 ns Time (ns) QD Time (ns) - ave = ns ave = ns Figure S1. PL decay traces of QD-MPA (black) and QD-azacycle conjugates (red, QD-1 and QD-) for QD 5 and QD 6. The calculated intensity-weighted average PL lifetimes are also indicated. Residuals and autocorrelation functions from the tri-exponential fits are also shown. The PL decay trace of QD 6 -MPA, shown for the QD 6-1 and QD 6 - figures, correspond to different batch preparations. S11

12 b) a) ave / ave b) a) ave / ave Blank Na(I) 1mM K(I) 1mM Ca(II) 1mM Mg(II).5mM Mn(II).5mM Ni(II).5mM Co(II).1mM Fe(II).mM Fe(II).1mM Cu(II).1mM Ficoll.5% BSA.5 mg/ml Zn(II) 1mM.. Blank Na(I) 1mM K(I) 1mM Ca(II) 5mM Mg(II) 1mM Mn(II).5mM Ni(II).5mM Co(II).1mM Fe(II).1mM A Fe(II).5mM Cu(II).1mM Ficoll.5% BSA.5 mg/ml Zn(II) 1mM Figure S. Interference study of QD 6-1 (a) and QD 6 - (b) conjugates as Zn + nanosensors at ph 7.. The average PL lifetimes of the corresponding QD-azacycle conjugates in the presence of foreign species were normalized by the average PL lifetime of the blank (in the absence of interfering species). S1

13 1 ave (ns) ph Figure S3. Average PL lifetime of QD 6-1 conjugates buffered with 1 mm Tris solutions at different ph values in the absence (black) and presence (red) of.1 mm Zn +. S13

14 Figure S4. Dual-channel fluorescence microscopy images of HepG cells incubated with nanosensor QD 5-1 (green channel) and MitoTracker Deep Red dye (red channel), in PBS ph 8. buffer. The scale bar (white line) represents 1 μm. A dual-colour excitation scheme was employed using a 47-nm laser (LDH-P-C-47, PicoQuant) and a 635-nm laser (LDH-P-635, PicoQuant), both operated simultaneously with a Sepia II driver (PicoQuant GmbH) set at a repetition rate of 1 MHz. A dual-band dichroic mirror was used to direct the excitation beams to the objective and collect the fluorescence emission. After focusing through the pinhole, an emission dichroic mirror (6DCXR, AHF/Chroma, Germany) separated the fluorescence emission into two channels: channel 1 for the QD 5-1 emission (using a 5/35, Omega Filters) and channel for the MitoTracker Deep Red emission (using a 685/7, Omega Filters). The fluorescence photons were detected by two SPCM-AQR-14 avalanche photodiode detectors. The image is of the two detection channels merged together. Only QD 5-1 nanosensors were used in these experiments for a better spectral compatibility with the MitoTracker Deep Red (avoiding spectral crosstalk) and the dual-colour instrumentation. S14

15 Survival rate (%) CONTROL QD-1 x.5 QD-1 x1 QD-1 Dosage QD-1 x QD-1 x3 Figure S5. Survival rate of 143B cells upon -hour incubation with QD 6-1 conjugates at different dosages:.5, 1,, and 3 times the concentration of QD 6-1 used in the cell FLIM imaging experiments. Error bars are expressed as s.e.m. from 6 repetitions. References 1. Ruedas-Rama, M. J.; Hall, E. A. H., A quantum dot-lucigenin probe for Cl. Analyst 8, 133, Chan, W. C. W.; Nie, S., Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science 1998, 81, Ruedas-Rama, M. J.; Hall, E. A. H., Azamacrocycle Activated Quantum Dot for Zinc Ion Detection. Anal. Chem. 8, 8, Ruedas-Rama, M. J.; Orte, A.; Crovetto, L.; Talavera, E. M.; Alvarez-Pez, J. M., Photophysics and Binding Constant Determination of the Homodimeric Dye BOBO-3 and DNA Oligonucleotides. J. Phys. Chem. B 1, 114, Lakowicz, J. R., Principles of Fluorescence Spectroscopy. 3rd ed.; Springer: Maus, M.; Cotlet, M.; Hofkens, J.; Gensch, T.; De Schryver, F. C.; Schaffer, J.; Seidel, C. A. M., An Experimental Comparison of the Maximum Likelihood Estimation and Nonlinear Least-Squares Fluorescence Lifetime Analysis of Single Molecules. Anal. Chem. 1, 73, S15