S1

Electronic Supplementary Information

Intracellular Zn2+ 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, 18071

Granada (Spain).

b GENYO. Pfizer-University of Granada-Junta de Andalucia Centre for Genomics and

Oncological Research. Avda Ilustracion 114, PTS, 18016 Granada (Spain).

Corresponding authors: AO: angelort@ugr.es, Tel. +34-958243825; MJRR: mjruedas@ugr.es,

Tel. +34-958247887

Experimental Section……………………………................... S2

Methods of Analysis……………………………………......... S7

Table S1………………………………………………………. S9

Table S2……………………………………………………… S10

Figure S1……………………………………………………... S11

Figure S2……………………………………………………... S12

Figure S3……………………………………………………... S13

Figure S4……………………………………………………... S14

Figure S5……………………………………………………... S15

References……………………………………………………. S15

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

S2

Experimental Section

Materials

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

nm (QD520 and QD600) and a lipophilic long chain surfactant capping of octadecylamine (ODA)

were purchased from Mesolight (USA). 3-Mercaptopropionic acid (MPA) was purchased from

Fluka. 1,4,7,10-tetraazacyclododecane (cyclen, 1), 1,4,8,11-Tetraazacyclotetradecane (cyclam, 2),

1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide

(NHS), Tris buffer, Bovine Serum Albumin (BSA), Ficoll400, 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 0.2 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 2 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

S3

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 (10 min, 13,000 rpm), followed by the re-dissolution of the QD-MPA in 10 mM

Tris buffer, pH 7.2.

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,2-3

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-2, were prepared by mixing 200 μL of QD-MPA with a solution of EDC (100 mM final

concentration) in 10 mM Tris pH 7.2 for 10 minutes and then with a solution of NHS (50 mM

final concentration) in 10 mM Tris pH 7.2. After 5 min, the adequate amount of 1 or 2 solution in

10 mM Tris pH 7.2 was added until reaching a final concentration of 10 mM. The mixture was

stirred for 3 h at room temperature. The reacting mixture was then centrifuged at 13,000 rpm for

10 min. The supernatant containing the excess of reagents was removed, and the QD-azacycle

conjugates in the residue were re-dissolved in 10 mM Tris pH 7.2.

Instruments

Steady-state photoluminescence (PL) emission spectra were collected using a JASCO FP-6500

spectrofluorometer equipped with a 450 W xenon lamp for excitation, with a temperature

controller ETC-273T set at 25 °C. All measurements were collected at 25 ºC using 510 mm

cuvettes.

S4

PL decay traces of QDs were recorded in the Single Photon Timing (SPT) mode using the

FluoTime 200 fluorometer (PicoQuant, GmbH, Germany) previously described.4 In brief, the

samples were excited using a 440 nm pulsed laser (LDH-P-C-440 PicoQuant, GmbH, Germany)

with a 10 MHz repetition rate, which was controlled by a PDL-800-B driver (PicoQuant). The

full width at half maximum of the laser pulse was ~ 80 ps. The PL was collected after crossing

through a polarizer set at the magic angle and a 2 nm bandwidth monochromator. PL decay

histograms were collected using a TimeHarp 200 board (PicoQuant), with a time increment per

channel of 36 ps, at the emission wavelengths of 522, 524 and 526 nm for QD520 and 592, 594

and 596 nm for QD600. 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 2 104 counts in the peak channel.

PL lifetime images were recorded with a MicroTime 200 fluorescence lifetime microscope

system (PicoQuant, GmbH, Germany) based on single photon timing using the time-tagged time-

resolved (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

10 MHz. The laser power at the microscope entrance was between 0.2 and 4.4 μW. The

excitation beam passed through an achromatic quarter-wave plate (AQWP05M-600, Thorlabs,

NJ), set at 45º from the polarization plane of the laser, and was directed by a dichroic mirror

(510DCXR, AHF/Chroma, Germany) to the oil immersion objective (1.4 NA, 100×) 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 (500LP, AHF/Chroma, Germany). The PL

emitted photons were detected by using an avalanche photodiode (SPCM-AQR-14, Perkin

S5

Elmer) after crossing an adequate bandpass filter (600/40, AHF/Semrock, Germany). Individual

photons time tagging was performed within a TimeHarp 200 module (PicoQuant), with a time

resolution of 29 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 512 × 512 pixel resolution and a collection time of 0.60 ms per pixel.

FLIM Imaging of QD-1 in buffered solutions

FLIM imaging experiments of QD-1 at different Zn2+

concentrations were performed in

solutions buffered with 10 mM Tris buffer pH 7.2. The glass slides were washed twice with 0.5

mL of 10 mM Tris buffer at pH 7.2, followed by washing with ethanol and then finally being

dried with a lens tissue. Then, 10 µL of QD-1 nanosensors were dissolved in 1 mL of Tris buffer

at pH 7.2 and sonicated for 10 minutes. Subsequently, 40 µL of the buffer solution was placed on

the slide and 2 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 360 and 1156 μm2 were raster-scanned for FLIM imaging with a

spatial resolution of 14 to 27 nm/pixel.

FLIM Imaging of QD-1 in HepG2 cells

The Cell Culture Facility, University of Granada, provided the HepG2 30 cell line. Cells were

grown in DMEM supplemented with 10% (v/v) FBS, 2 mM glutamine, 100 U/mL penicillin, and

0.1 μg/mL streptomycin at 37 ºC in a humidified 5% CO2 incubator. For the FLIM experiments,

HepG2 cells were seeded onto 20 mm diameter glass slides at a density of 11250 cells/cm2. The

glass slides were washed with the DMEM medium and phosphate-buffered saline (PBS) before

S6

adding the cells. The cells seeded onto the glass slides were incubated for 2 h at 37 ºC with the

addition of 2 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 Zn2+

, the QD-loaded cells were later incubated at 37 ºC for 10 min in a 1 mM Zn2+

solution in PBS buffer pH 8. For the FLIM experiments, images of surface areas between 390

and 2450 μm2 were collected with a spatial resolution of 10 to 70 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 1.2x103 cells/well. After 48h of cell culture, 1, 2, 4 and

6 µl of QDs from a sonication-cleared stock solution were added directly to the wells, being 2 µl

QDs/well the concentration equivalent to the higher used in the other experiments. After 2h

incubation, 20% v/v of CellTiter-Blue™ (Promega) reagent was added to the wells, incubated

for 2 hours at 37°C, and then fluorescence was directly read at 525/580-640nm 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.

S7

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, χ2, 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 Zn2+

concentrations, it was necessary to

determinate their intensity-weighted average PL lifetime, τave, using equation 1:5

iiiiave aa 2 (eq. 1)

where τi represents the decay times and ai 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 100 photons per pixel) were fitted to a two-

exponential 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

S8

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.

S9

Table S1. Decay times and normalized pre-exponentials of QD600-MPA and QD600-1 at different

Zn2+

concentrations. PL decay traces collected at ex = 440 nm and em = 596 nm.

(a1) τ 1

(ns)

(a2) τ2

(ns)

(a3) τ3

(ns)

τave

(ns)a

χ²

QD-MPA (0.40) 23.12

(0.49) 10.64

(0.11) 2.12

18.40 1.133

QD-1 (0.08) 19.11

(0.48) 5.54

(0.44) 1.45

9.21 1.128

QD-1 + 0.001 mM Zn2+

(0.08) 19.55

(0.48) 5.66

(0.44) 1.49

9.53 1.134

QD-1 + 0.002 mM Zn2+

(0.08) 19.81

(0.48) 5.71

(0.44) 1.48

9.62 1.093

QD-1 + 0.005 mM Zn2+

(0.08) 19.88

(0.48) 5.89

(0.44) 1.46

9.71 1.173

QD-1 + 0.01 mM Zn2+

(0.09) 20.02

(0.48) 5.48

(0.43) 1.40

10.19 1.119

QD-1 + 0.02 mM Zn2+

(0.09) 20.43

(0.49) 5.94

(0.42) 1.49

10.38 1.165

QD-1 + 0.05 mM Zn2+

(0.12) 20.94

(0.49) 6.14

(0.39) 1.48

11.69 1.126

QD-1 + 0.1 mM Zn2+

(0.14) 21.47

(0.47) 6.34

(0.39) 1.43

12.87 1.129

QD-1 + 0.2 mM Zn2+

(0.15) 22.99

(0.48) 6.65

(0.37) 1.48

14.13 1.196

QD-1 + 0.5 mM Zn2+

(0.15) 23.55

(0.48) 6.92

(0.37) 1.58

14.35 1.122

QD-1 +1 mM Zn2+

(0.18) 24.15

(0.49) 7.24

(0.33) 1.70

15.43 1.167

QD-1 +2 mM Zn2+

(0.18) 24.70

(0.50) 7.48

(0.32) 1.71

16.03 1.137

a Associated errors in τave, obtained through error propagation of the fitting errors of the adjustable parameters,

were always between 0.13 and 0.15 ns.

S10

Table S2. Decay times and normalized pre-exponentials of QD520-MPA and QD520-2 at different

Zn2+

concentrations. PL decay traces collected at ex = 440 nm and em = 526 nm.

(a1) τ 1

(ns)

(a2) τ2

(ns)

(a3) τ3

(ns)

τave

(ns)a

χ²

QD-MPA (0.36) 20.14

(0.44) 8.98

(0.20) 1.28

15.83 1.226

QD-2 (0.23) 17.36

(0.47) 6.69

(0.30) 1.37

12.00 1.207

QD-2 + 0.01 mM Zn2+

(0.27) 17.30

(0.47) 6.92

(0.26) 1.37

12.55 1.150

QD-2 + 0.05 mM Zn2+

(0.28) 17.80

(0.49) 7.34

(0.23) 1.46

13.02 1.137

QD-2 + 0.15 mM Zn2+

(0.32) 17.86

(0.47) 7.47

(0.21) 1.70

13.42 1.231

QD-2 + 0.20 mM Zn2+

(0.31) 18.31

(0.48) 7.87

(0.21) 1.65

14.24 1.109

QD-2 + 0.40 mM Zn2+

(0.31) 18.99

(0.50) 8.28

(0.19) 1.65

14.32 1.159

QD-2 + 1 mM Zn2+

(0.37) 19.14

(0.49) 8.56

(0.14) 1.73

14.90 1.123

QD-2 + 1.5 mM Zn2+

(0.33) 20.02

(0.53) 9.13

(0.14) 2.02

15.11 1.069

QD-2 + 2 mM Zn2+

(0.36) 19.67

(0.51) 9.06

(0.13) 2.00

15.16 1.167

a Associated errors in τave, obtained through error propagation of the fitting errors of the adjustable parameters,

were always between 0.13 and 0.15 ns.

S11

0 20 40 60 80100

1000

10000

100000

Co

un

ts

Time (ns)

-0,2

0,0

0,2 Autocorrelation Function

-0,2

0,0

0,2

-2

0

2

Residuals

-2

0

2

0 20 40 60 80100

1000

10000

100000

Co

un

ts

Time (ns)

-0,2

0,0

0,2 Autocorrelation Function

-0,2

0,0

0,2

-2

0

2

Residuals

-2

0

2

QD520-2QD520-1

0 20 40 60 80100

1000

10000

100000

Co

un

ts

Time (ns)

-0,2

0,0

0,2 Autocorrelation Function

-0,2

0,0

0,2

-2

0

2

Residuals

-2

0

2

QD600-1

0 20 40 60 80100

1000

10000

100000C

ou

nts

Time (ns)

-0,2

0,0

0,2 Autocorrelation Function

-0,2

0,0

0,2

-2

0

2

Residuals

-2

0

2

QD600-2

ave = 12.00 ns

ave = 15.83 ns

ave = 18.40 ns

ave = 9.21 ns

ave = 18.73 ns

ave = 13.17 ns

ave = 15.83 ns

ave = 8.61 ns

Figure S1. PL decay traces of QD-MPA (black) and QD-azacycle conjugates (red, QD-1 and

QD-2) for QD520 and QD600. 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 QD600-MPA, shown for the QD600-1 and QD600-2 figures, correspond to

different batch preparations.

S12

Blank

Na(

I) 10

0mM

K(I)

100

mM

Ca(

II) 5

mM

Mg(

II) 1

mM

Mn(

II) 0

.5m

M

Ni(I

I) 0.

5mM

Co(

II) 0

.01m

M

Fe(II)

0.0

01m

M

Fe(II)

0.0

05m

M

Cu(

II) 0

.001

mM

Ficoll 0

.5%

BSA 0

.5 m

g/m

L

Zn(II)

1m

M

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

av

e/

0 av

e

A

a)

Blank

Na(

I) 10

0mM

K(I)

100

mM

Ca(

II) 1

mM

Mg(

II) 0

.5m

M

Mn(

II) 0

.05m

M

Ni(I

I) 0.

05m

M

Co(

II) 0

.001

mM

Fe(II)

0.0

002m

M

Fe(II)

0.0

01m

M

Cu(

II) 0

.000

1mM

Ficoll 0

.5%

BSA 0

.5 m

g/m

L

Zn(II)

1m

M

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

av

e/

0 av

e

b)a)

b)

Figure S2. Interference study of QD600-1 (a) and QD600-2 (b) conjugates as Zn2+

nanosensors at

pH 7.2. 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).

S13

5.47 6.49 7.204

6

8

10

12 a

ve (

ns

)

pH

Figure S3. Average PL lifetime of QD600-1 conjugates buffered with 10 mM Tris solutions at

different pH values in the absence (black) and presence (red) of 0.1 mM Zn2+

.

S14

Figure S4. Dual-channel fluorescence microscopy images of HepG2 cells incubated with

nanosensor QD520-1 (green channel) and MitoTracker Deep Red dye (red channel), in PBS pH

8.0 buffer. The scale bar (white line) represents 10 μm. A dual-colour excitation scheme was

employed using a 470-nm laser (LDH-P-C-470, 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 10 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 (600DCXR, AHF/Chroma, Germany) separated the fluorescence

emission into two channels: channel 1 for the QD520-1 emission (using a 520/35, Omega Filters)

and channel 2 for the MitoTracker Deep Red emission (using a 685/70, 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 QD520-1 nanosensors were used in

these experiments for a better spectral compatibility with the MitoTracker Deep Red (avoiding

spectral crosstalk) and the dual-colour instrumentation.

S15

CONTR

OL

QD-1

x0.

5

QD-1

x1

QD-1

x2

QD-1

x3

0

20

40

60

80

100

120

Su

rviv

al

rate

(%

)

QD-1 Dosage

Figure S5. Survival rate of 143B cells upon 2-hour incubation with QD600-1 conjugates at

different dosages: 0.5, 1, 2, and 3 times the concentration of QD600-1 used in the cell FLIM

imaging experiments. Error bars are expressed as s.e.m. from 6 repetitions.

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4. 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

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A. M., An Experimental Comparison of the Maximum Likelihood Estimation and Nonlinear

Least-Squares Fluorescence Lifetime Analysis of Single Molecules. Anal. Chem. 2001, 73, 2078-

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