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A fluorescent flavonoid for lysosome detection in live cells

under “wash free” conditions

Keti Assor Bertman1, Chathura S. Abeywickrama1, Hannah J. Baumann1, Nicolas Alexander1,

Lucas McDonald1, Leah P. Shriver,1,2 Michael Konopka1 and Yi Pang1,3*

1Department of Chemistry, 2Department of Biology and 3Maurice Morton Institute of Polymer

Science, University of Akron, Akron, Ohio 44325, USA.

*Corresponding Author

Supporting Information

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2018

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Figure S1.1 1H NMR spectra of 1 (300 MHz in CDCl3)

1

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Figure S1.2 1H NMR spectra of 2a (300 MHz in CDCl3)

2a

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Figure S1.3 1H NMR spectra of 2b (300 MHz in CDCl3)

2b

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Figure S2.1 Mass spectra of 1

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Figure S2.2 Mass spectra of 2a

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Figure S2.3 Mass spectra of 2b

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Figure S3.1 Absorbance (a) and fluorescence emission (b) spectra obtained for 2a (1x10-5 M) in

different solvents at room temperature.

300 350 400 450 500 550

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40A

bso

rban

ce

Wavelength (nm)

TOLUENE

DCM

ACN

DMSO

ETOH

MEOH

H2O

a

450 500 550 600 650 700

0.0

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

1.2x107

Em

iss

ion

Wavelength (nm)

TOLUENE

DCM

ACN

DMSO

ETOH

MEOH

H2O

b

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Figure S3.2 Absorbance (a) and fluorescence emission (b) spectra obtained for 2b (1x10-5 M) in

different solvents at room temperature.

350 400 450 500 550

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Ab

so

rba

nc

e

Wavelength (nm)

Toluene

DCM

Acetonitrile

DMSO

EtOH

MeOH

Water

a

450 500 550 600 650

0.0

4.0x106

8.0x106

1.2x107

1.6x107

Em

iss

ion

Wavelength (nm)

Toluene

DCM

Acetonitrile

DMSO

EtOH

MeOH

Water

b

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330 360 390 420 450

0.0

0.5

1.0

1.5

2.0

2.5

0 10 20 30 40 50 60

0.0

0.5

1.0

1.5

2.0

Ab

so

rba

nc

e a

t 3

98

nm

Concentration of 2a in M

Ab

so

rban

ce

AU

Wavelength (nm)

Conc. in M

1

5

10

20

30

40

50

60

450 500 550 600 650 700

0.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

3.5x106

4.0x106

0 10 20 30 40 50 60

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

3.5x106

4.0x106

Em

issio

n In

ten

sit

y

Concentration of 2a (M)

Emission at 518 nm

Emission at 573 nm

Em

issio

n I

nte

nsit

y

Wavelength (nm)

Conc. of 2a M

1

5

10

20

30

40

50

60

518

573

Figure S3.3 Absorbance (a) and fluorescence emission (b) spectra obtained for 2a in DCM in

different concentrations at room temperature.

a

b

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Figure S4 Fluorescent confocal microscopy images of MO3.13 cells (x100) treated with 1 (3 µM) for 30

minutes. Images show the fluorescence 1 (a), LysoTracker Red® (b), overlapped image (c), and bright field

(d). Laser 405 nm and 561 nm were used for the green and red channels respectively.

(d)

(c)

(a)

(b)

1

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Figure S5. Confocal microscope images of NHLF cells stained with 200 nM Lysotracker Green® (a-b) and 75 nM Lysotracker Red® (c-d) for 30 minutes with digitally enlarged magnification (a-d). Lysotracker green® and red® were excited with lasers 488 nm and 561 nm, respectively.

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Figure S6 Cell viability results (bar-chart) obtained for 2a (a) and 2b (b) by MTT cell viability assay.

a

b

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Figure S7. Confocal microscope images of MO3.13 cells stained with 2a and 2b at different concentrations

for 30 minutes at 100x magnification. Compounds 2a and 2b were excited at 405nm. No post-staining

washing was done for imaging. Filter region for emission is in the method and material section.

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Figure S8. Bright field from Confocal microscope time study images of MO3.13 cells stained with 3 M

2b at 100x magnification. Compound was excited with 405 nm laser.

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Figure S9. Confocal microscope time study images of MO3.13 cells stained with 0.2 M LysoTracker

Green® at 100x magnification. Compound was excited with 488 nm laser.

Figure S10. Bright field from Confocal microscope time study images of MO3.13 cells stained with 0.2

M LysoTracker Green® at 100x magnification. Compound was excited with 488 nm laser. Morphology

changes with time as follows: 1 – Round cell (dead cell); 2a – Healthy cell, 2b – Morphology changed

significantly, 2c – Shrinking and round up cell; 3a – Healthy cell, 3b – Healthy cell, 3c – Shrinking and

round cell; 4a – Healthy cell, 4b – Healthy cell, 4c – Shrinking cell.

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1a 1b0.0

0.2

0.4

0.6

0.8

1.0

2b

Ov

erl

ap

Co

eff

ec

ien

t (a

ve

rag

e)

Probe2a

Probe Average Overlap coefficient

Error +/-

2a 0.7812 0.033

2b 0.8476 0.025

Figure S11. Mander's overlap coefficient calculations for 2a and 2b in colocalization experiments with

LysoTracker Red DND 99 in NHLF and MO3.13 cells.

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300 350 400 450 500 550 600 650

0.00

0.05

0.10

0.15

0.20

0.25

2 4 6 8 10 120.00

0.05

0.10

0.15

0.20

0.25

Ab

so

rba

nc

e @

41

0 n

m

pH

Ab

so

rba

nc

e

Wavelength (nm)

pH

1

2

3

4

5

6

7

8

9

10

11

12

410

Figure S12. UV-vis absorption of 2a at different pH at room temperature.

pH Quantum yield

1 0.075

2 0.0085

3 0.0051

4 0.004

5 0.0041

6 0.0057

7 0.0059

8 0.019

9 0.15

10 0.21

11 0.49

12 0.61

Figure S13. Quantum yield values for 2a at different pH in room temperature.

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350 400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0

535E

xc

ita

tio

n /

Em

iss

ion

(N

orm

ali

ze

d)

Wavelength (nm)

Ex. @ -125oC

Ex. @ 20oC

Em. @ -125oC

Em. @ 20oC

405419 480

a

561

450 500 550 600 650

0.0

3.0x105

6.0x105

9.0x105

1.2x106

1.5x106

1.8x106 b

-120 -100 -80 -60 -40 -20 0 20

480

490

500

510

520

530

540

Em

issio

n m

axim

a (

nm

)

Temperature (oC)

Em

iss

ion

Wavelength (nm)

-125oC

-112oC

-94oC

-75oC

-60oC

-40oC

-20oC

0oC

20oC

480

535

Figure S14. (a) Normalized emission of 2a in ethanol solvent at different temperature. (b) emission of 2a

at various temperature. The emission at 480 nm is likely from the flavonoid’s normal form (N*), while the

emission at ~535 nm from the tautomeric form (T*). The inset plots emission maxima vs. temperature °C.

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Figure S15. Molecular HOMO and LUMO orbitals of 2a in CH2Cl2 and water solvents. The

orbitals were generated by using Gaussian 09 software with DFT at B3LYP/6-31(d,p) setting, and

with geometry optimization followed by energy minimization. In the LUMO orbitals, the C=O

group (indicated by a double arrow) exhibits a slightly larger electron density in H2O than in

CH2Cl2, attributing to stronger solvent effect in water.

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350 375 400 425 450 475 500

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

2a

Zn2+

Cd2+

Na+

K+

Mg2+

Ca2+

Ni2+

Fe3+

Cu2+

Ba2+

Ag+

Al3+

Co2+

Fe2+

Cu+

Ab

so

rban

ce

Wavelength (nm)

450 500 550 600 650 700

0.0

2.0x106

4.0x106

6.0x106

8.0x106

535

Wavelength (nm)

Em

iss

ion

2a

Zn2+

Cd2+

Na+

K+

Mg2+

Ca2+

Ni2+

Fe3+

Cu2+

Ba2+

Ag+

Al3+

Co2+

Fe2+

Cu+

575 nm

Figure S16. Absorption (a) and Fluorescence emission (b) responses of probe 2a (10 µM in

acetonitrile) upon addition of different metal ions (10 µM in water) at room temperature. The

emission spectra were obtained by exciting metal species added probe at 400nm.

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0

1x106

2x106

3x106

4x106

5x106

6x106

Zn2+

Cu+

Fe2+

Co2+

2a

Res

po

nse

Metals

2a Cd2+

Na+K

+Mg

2+Ca

2+Cu

2+Ba

2+Ag

+Al

3+Ni

2+Fe

3+

Figure S17. Emission intensity of probe 2a (10 µM in acetonitrile) at 575 nm upon addition of

different metal ions (10 µM in water) at room temperature. The emission spectra were obtained

by exciting metal species added probe at 400nm.

Note (1): The slight fluorescence enhancement should not be a concern for cell application, as

intracellular Zn2+ concentration is typically in a much lower concentration (e.g. nanomolar

range).

Note (2): It should be noted that the probe 2a has two emission peaks at ~535 nm (from N*) and

575 nm (from T*) in aqueous (see Figure S16). The tautomer emission (T*) wavelength at 575

nm was used to generate the plot here, since the emission wavelength at 575 nm appeared to be

more consistent in comparison with the wavelength at ~535 nm.

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350 400 450 500 550 600

0.00

0.05

0.10

0.15

0.20

Ab

so

rba

nc

e

Wavelength (nm)

1a

2a + 1 eq. F-

2a + 1 eq. Cl-

2a + 1 eq. Br-

2a + 1 eq. HS-

2a + 1 eq. PO4

3-

2a + 1 eq. OH-

(a)

450 500 550 600 650 700

0.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

Em

iss

ion

Wavelength (nm)

2a

2a + 1 eq. F-

2a + 1 eq. Cl-

2a + 1 eq. Br-

2a + 1 eq. HS-

2a + 1 eq. PO4

3-

2a + 1 eq. OH-

(b)

Figure S18. Absorption (a) and Fluorescence emission (b) responses of probe 2a (10 µM in

acetonitrile) upon addition of different 1 eq. of anions ions (10 µM in water) at room temperature.

The emission spectra were obtained by exciting metal species added probe at 400nm.

Tetrabutylammonium (R4N+) salts were used to prepare anion solutions.

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350 400 450 500 550

0.00

0.05

0.10

0.15

0.20A

bs

orb

an

ce

Wavelength (nm)

2a

2a + 1 eq. Glutamic acid

2a + 1 eq. Aspartic acid

2a + 1 eq. Leucine

2a + 1 eq. Tryptophan

2a + 1 eq. Valine

2a + 1 eq. Arginine

2a + 1 eq. Tyrosine

2a + 1 eq. Cystine

2a + 1 eq. Methionine

(a)

450 500 550 600 650 700

0.0

6.0x105

1.2x106

1.8x106

2.4x106

Em

iss

ion

Wavelength (nm)

2a

2a + 1 eq. Glutamic acid

2a + 1 eq. Aspartic acid

2a + 1 eq. Leucine

2a + 1 eq. Tryptophan

2a + 1 eq. Valine

2a + 1 eq. Arginine

2a + 1 eq. Tyrosine

2a + 1 eq. Cystine

2a + 1 eq. Methionine

(b)

Figure S19. Absorption (a) and Fluorescence emission (b) responses of probe 2a (10 µM in

acetonitrile) upon addition of different 1 eq. different amino acids (10 µM in water) at room

temperature. The emission spectra were obtained by exciting metal species added probe at 400nm.

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350 400 450 500 550 600

0.00

0.05

0.10

0.15

0.20A

bs

orb

an

ce

Wavelength (nm)

2a

2a + 10 uL H2O

2(35% w/w)

2a + 10 uL ROS (4.5% w/w)

2a + 1eq. NaNO2

(a)

450 500 550 600 650 700

0

1x106

2x106

3x106

4x106

5x106

Em

iss

ion

Wavelength (nm)

2a

2a + 10 uL H2O

2(35% w/w)

2a + 10 uL ROS (4.5% w/w)

2a + 1eq. NaNO2

(b)

Figure S20. Absorption (a) and Fluorescence emission (b) responses of probe 2a (10 µM in

acetonitrile) upon addition of different reactive oxygen and nitrogen species at room temperature.

The emission spectra were obtained by exciting probe at 400 nm, after addition of reactive oxygen

species (ROS) or reactive nitrogen species. Two different ROS were used. Potassium

peroxomonosulphate compound (~ 4.5 % active oxygen) and hydrogen peroxide (30% w/w) were

prepared at 10 mM concentration. the reactive nitrogen species (HNO2) was prepared from

H+/NaNO2 solutions at 10 mM concentration (2HNO2 NO2 + NO + H2O).

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Figure S21. Confocal microscope images of NHLF cells stained with 2 µM 2a for 30 minutes (a)

and sequential sating with pHrodoTM Red Avidin (5 µg/ml) for 1 hour at 0⁰ C and 30 minutes at

37⁰ C (b). Figure (c) represents the overlapped image obtained for two dyes and figure (d)

represents the bright field. Probe 2a was excited with 405 nm laser and pHrodoTM Red Avidin was

excited with 561 nm laser.

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Figure S22. Confocal microscope images of NHLF cells stained with 2 µM 2b for 30 minutes (a)

and sequential sating with pHrodoTM Red Avidin (5 µg/ml) for 1 hour at 0⁰ C and 30 minutes at

37⁰ C (b). Figure (c) represents the overlapped image obtained for two dyes and figure (d)

represents the bright field. Probe 2b was excited with 405 nm laser and pHrodoTM Red Avidin was

excited with 561 nm laser.

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Figure S23. Figures (a) and (c) represents overlapped fluorescent confocal images obtained for

pHrodoTM Red Avidin in the presence of probes 2a and 2b at 2 µM concentration. Figure (b) and

(d) represents the digitally zoomed in regions from images (a) and (c) to highlight the overlapped

regions, respectively.

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450 500 550 600 650 700 750

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

0 2 4 6 8 100.0

4.0x105

8.0x105

1.2x106

Em

issio

n @

512 n

m

Amount of 10 % BSA (w/v) added (in l)

Em

iss

ion

Wavelenght (nm)

2a

2a + 2 l BSA

2a + 4 l BSA

2a + 6 l BSA

2a + 8 l BSA

2a + 10 l BSA

(a)

450 500 550 600 650 700 750

0.0

6.0x104

1.2x105

1.8x105

2.4x105

(b)

0 2 4 6 8 10

0.0

8.0x104

1.6x105

2.4x105

Em

issio

n @

512 n

m

Amount of 10 % BSA (w/v) added (in l)

Em

iss

ion

Wavelenght (nm)

2a

2a + 5 l BSA

2a + 10 l BSA

Figure S24.1 Fluorescence emission spectra obtained for 2a (5 x 10-6 M) upon titration with 10%

BSA (w/v) at room temperature. Figure (a) represents the emission spectra obtained in de-ionized

water and figure (b) represents spectra obtained at pH 4.3.

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450 500 550 600 650 700 750

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

(c)

0 2 4 6 8 100.0

4.0x105

8.0x105

1.2x106

Em

issio

n @

493 n

m

Amount of 10 % BSA (w/v) added (in l)

Em

iss

ion

Wavelenght (nm)

2b

2b + 2 l BSA

2b + 4 l BSA

2b + 6 l BSA

2b + 8 l BSA

2b + 10 l BSA

450 500 550 600 650 700 750

0.0

6.0x104

1.2x105

1.8x105

(d)

0 2 4 6 8 10

0.0

4.0x104

8.0x104

1.2x105

1.6x105

Em

issio

n @

493 n

m

Amount of 10 % BSA (w/v) added (in l)

Em

iss

ion

Wavelenght (nm)

2b

2b + 5 l BSA

2b + 10 l BSA

Figure 24.2 Fluorescence emission spectra obtained for 2b (5 x 10-6 M) upon titration with 10%

BSA (w/v) at room temperature. Figure (c) represents the emission spectra obtained in de-ionized

water and figure (d) represents spectra obtained at pH 4.3.

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500 550 600 650 700

0.0

4.0x106

8.0x106

1.2x107

1.6x107

2.0x107

0 1 2 3 4 5 6 7 8 9 100.0

5.0x106

1.0x107

1.5x107

2.0x107

Em

issio

n @

506 n

m

Amount of 10 % BSA (w/v) added (in l)

(e)

Em

iss

ion

Wavelenght (nm)

LG

LG + 2 l BSA

LG + 4 l BSA

LG + 6 l BSA

LG + 8 l BSA

LG + 10 l BSA

500 550 600 650 700

0.0

4.0x106

8.0x106

1.2x107

1.6x107

2.0x107

0 2 4 6 8 100.0

5.0x106

1.0x107

1.5x107

2.0x107

Em

iss

ion

at

50

6 n

m

Amount of 10 % BSA (w/v) added (in l)

(f)

Em

iss

ion

Wavelenght (nm)

LG

LG + 5 l BSA

LG + 10 l BSA

Figure 24.3 Fluorescence emission spectra obtained for Lysotracker Green DND-26 (5 x 10-6

M) upon titration with 10% BSA (w/v) at room temperature. Figure (e) represents the emission

spectra obtained in de-ionized water and figure (f) represents spectra obtained at pH 4.3.