Supporting Information

A near-infrared fluorescent probe for endogenous hydrogen peroxide

real-time imaging in living cells and zebrafish

Xin Huang, Zhipeng Li, Zixin Liu, Chengchu Zeng and Liming Hu*

College of Life Science and Bioengineering, Beijing Key Laboratory of Environmental and Oncology, Beijing University

of Technology, Beijing, 100124, China. E-mail: huliming@bjut.edu.cn; Fax: +86 10 67392001; Tel: +86 10 67396211

CONTENTS

1. Apparatus, Chemicals and Reagents

2. Spectroscopic properties of probe the probe Cy-H2O2

3. 1H NMR, 13C NMR and HRMS

4. The cytotoxicity of the probe Cy-H2O2

5. Zebrafish imaging

1. Apparatus, Chemicals and Reagents

Fluorescence spectra measurement was performed on L-55 fluorescence spectrophotometer

(PE), in a 1 cm quartz cell. HRMS Spectrometer recorded by means of the electronic spray

ionization (ESI). NMR spectra was recorded on a Varian INOVA-400 MHz spectrometer (at 400

MHz for 1H NMR and 100 MHz for 13C NMR) using Tetramethylsilane (TMS) as internal

standard. IVIS Lumina II bio-luminescent fluorescent small animal live imaging system is product

of the United States Caliper Life Sciences. Bio-imaging was performed using a Leica SPE

confocal laser scanning microscope with an excitation wavelength of 730 nm.

All reagents were obtained from commercial sources and used as received without further

purification. The different solutions of various testing species were respectively prepared from

NaCl, MgCl2, NaNO3, NH4Cl, CaCl2, FeSO4, KMnO4, K2Cr2O7, NaH2PO4, Na2HPO4, NaHCO3,

CH3COOK, KI, CuSO4, Proline, Alanine, N-acetylcysteine, Vitamin C, HgS, GSH, NaS2O3,

Na2SO3, NaClO, ROO•, t-BuOOH, ONOO⁻, •OH, NO· and H2O2. All solvents used in

spectroscopic test were of spectroscopic grade. Distilled, deionized water was used throughout the

experiment.

2. Spectroscopic properties of Cy-H2O2

Stock solution of H2O2 were prepared in PBS (pH=7.4) solution. The solution of Cy-H2O2

(10 mM) was prepared in distilled water. During the experiments, different amounnxts of H2O2,

0.5 mL PBS and 1 μL probes were mixed and filled up with PBS solution to 1 mL in volumetric

tubes. All experiments were performed at 25°C. 1 mL aliquots of the mixed solution above-

mentioned were pipetted into 1 cm cuvettes for spectral measurements. 5 nm band passes were

used for both excitation and emission wavelengths. An excitation wavelength of 730 nm was used

for the acquisition of emission spectra.

Fig. S1 The time-dependent fluorescence changes (λex = 730 nm), acquired from a mixture of probe Cy-H2O2 (10

μM) and H2O2 (5 μM) in PBS (pH =7.4) solution at room temperature. With the increase of 790 nm intensity, the

sample was collected with 1 acquisition every second.

Fig. S2 The time-dependent fluorescence changes (λex = 730 nm), acquired from a mixture of probe Cy-H2O2 (10

μM) and H2O2 (7 μM) in PBS (pH =7.4) solution at room temperature. With the increase of 790 nm intensity, the

sample was collected with 1 acquisition every second.

Fig. S3 The time-dependent fluorescence changes (λex = 730 nm), acquired from a mixture of probe Cy-H2O2 (10

μM) and H2O2 (10 μM) in PBS (pH =7.4) solution at room temperature. With the increase of 790 nm intensity, the

sample was collected with 1 acquisition every second.

Fig. S4 The time-dependent fluorescence changes (λex = 730 nm), acquired from a mixture of probe Cy-H2O2 (10

μM) and H2O2 (20 μM) in PBS (pH =7.4) solutio n at room temperature. With the increase of 790 nm intensity, the

sample was collected with 1 acquisition every second.

Fig. S5 The time-dependent fluorescence changes (λex = 730 nm), acquired from a mixture of probe Cy-H2O2 (10

μM) and H2O2 (0μM, 5 μM, 7 μM, 10 μM and 20 μM) in PBS (pH =7.4) solution at room temperature. With the

increase of 790 nm intensity, samples were collected with 1 acquisition every second.

Fig S6. Fluorescence intensity increase of probe Cy-H2O2 (10 μM) upon the addition of H2O2 (0-100 μM), λem =

790 nm. The system was in PBS (pH = 7.4). Inset: Changes in the absorption curve, measured at 730 nm. The

equilibration time prior to luminescence measurement was 10 minutes.

3. 1H NMR, 13C NMR and HRMS

Fig. S7. Mass spectrum of Cy-piperazine

Fig. S8-1. 1H NMR spectrum of Cy-piperazine in CDCl3.

Fig. S8-2. 1H NMR spectrum of Cy-piperazine in CDCl3.

Fig. S9. 13C NMR spectrum of Cy-piperazine in CDCl3.

710.3708

+MS, 21.7min #1291

0.0

0.5

1.0

1.5

2.0

2.5

3.0

5x10Intens.

300 400 500 600 700 800 900 1000 1100 m/z

Fig. S10. Mass spectrum of the probe

Fig. S11-1. 1H NMR spectrum of probe in CDCl3.

Fig. S11-2. 1H NMR spectrum of probe in CDCl3.

Fig. S12. 13C NMR spectrum of probe in CDCl3.

Compound Cy-H2O2: The pale blue solid was obtained in 74% yield (52.54 mg). 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 8.9 Hz, 1H), 8.42 – 8.23 (m, 3H), 7.84 (d, J = 13.7 Hz, 2H), 7.38 (s, 1H), 7.36 (s, 1H), 7.35 (s, 1H), 7.34 (d, J = 2.6 Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.17 (s, 1H), 7.07 (s, 1H), 7.05 (s, 1H), 5.96 (s, 1H), 5.92 (s, 1H), 3.81 – 3.70 (m, 4H), 3.62 (s, 3H), 2.57 (t, J = 6.4 Hz, 4H), 1.93 – 1.82 (m, 4H), 1.75 – 1.66 (m, 7H), 1.25 (s, 6H), 0.88 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 189.03, 170.59, 170.07, 164.44, 151.26, 143.11, 141.61, 140.24, 137.28, 131.43, 129.91, 128.64, 126.70, 124.26, 122.14, 109.81, 101.81, 98.38, 54.00, 53.53, 48.51, 31.99, 29.71, 28.10, 27.23, 25.57, 25.43. HRMS (ESI): m/z calcd. for C44H48N5O4

+ [M]+ 710.3701, found 710.3708.

4. The cytotoxicity of the probe Cy-H2O2.

Before the bio-imaging employment, we had study the cytotoxicity. We selected HeLa cells to

measure the cytotoxicity of this probe. The Methyl Thiazolyl Tetrazolium (MTT) assay was used

to measure the cytotoxicity of Cy-H2O2 in HeLa cells. HeLa cells were seeded into a 96-well cell-

culture plate. Cells were dosed with Cy-H2O2 at final concentrations ranging from 6.25 μM to 100

μM in each well of the plates. The cell viability was 98.2%, 100.3%, 98.9%, 96.7% and 97.1%.

The results are shown in Figure S10, the Cy-H2O2 exhibited almost non-biotoxicity of the probe

Cy-H2O2 to cells.

Figure S13. MTT assay of HeLa cells were treated in the presence of Cy-H2O2 (6.25, 12.5, 25, 50 and 100 μM)

and incubated for 48 h. The standard deviation is calculated based on three sets of parallel experiments.

5. Zebrafish imaging

Imaging of exogenous H2O2 in zebrafish

The feasibility of in vivo imaging of H2O2 by the probe Cy-H2O2 was evaluated with zebrafish

larva as a vertebrate model. Fluorescent image of a zebrafish incubated with the probe (10 μg/mL)

for 60 min, and then incubated with different concentrations of H2O2 solution for 60 min. Later,

the larvae was washed with E3 medium for three times and then imaged on an OLYMPUS IX71

fluorescence microscope (Fig. S14).

Fig. S14. Microscopic images of zebrafish larvae. Fluorescent image of a zebrafish incubated with the probe (10

μg/mL) for 60 min, then incubated with different concentrations of H2O2 solution for 60 min. Scale bar: 1.0 mm.

(automatic exposure mode was adopted for the bright field images; the exposure time for the Red channel is 100

ms; Red channel: λex = 647 nm, λem = 663–738 nm).

Imaging of endogenous H2O2 in zebrafish as a result of drug induced oxidative damage

In this study, we used the APAP-induced organ injury model. 5-day-old larvae were cultured on

6-well microplate plates with Cy-H2O2 (10 μg/mL) E3 media suspension for 1 h, and then, 5-day-

old larvae cultured with different concentrations of APAP E3 media at different time (6 h, 12 h, 24

h, 48 h). Later, fishes were washed three times by the E3 medium, except for the remaining APAP.

Later, all of the fishes were imaged on OLYMPUS IX71 fluorescence microscope, the results see

in Fig. S15.

Fig. S15. Microscopic images of zebrafish larvae. Fluorescent image of a zebrafish incubated with the probe (10

μg/mL) for 60 min, then incubated with different concentrations of APAP solution for different time (6-48 hours).

Scale bar: 1 mm. (automatic exposure mode was adopted for the brightfield images; the exposure time for the red

channel is 100 ms; red channel: λex = 647 nm, λem = 663–738 nm).