Supplementary materials Near infra-red photoluminescent ... · 1 Supplementary materials Near...

1

Supplementary materials

Near infra-red photoluminescent graphene nanoparticles greatly expands use

in noninvasive biomedical imaging

Md Nurunnabia Zehedina Khatun

a Gerald R. Reeck

b Dong Yun Lee

c,* and Yong-kyu Lee

a,*

aDepartment of Chemical and Biological Engineering, Korea National University of

Transportation, Chungju, 380-702, Republic of Korea

bDepartment of Biochemistry, Kansas State University, Manhattan, KS 66506, USA

cDepartment of Bioengineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul,

133-791, Republic of Korea

Experimental:

Methods

Materials. Pitch carbon fiber was purchased from Fibre Glast Development Corporation (Carr

Drive Brookville, OH). Sulfuric acid, Nitric acid, Sodium hydroxide, sodium carbonate was

purchased from Sigma-Aldrich (St. Louis, MO). Cell culture reagents, including fetal bovine

serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), penicillinestreptomycin,

trypsin/EDTA, and Dulbecco’s phosphate buffer saline (PBS) were purchased from Gibco BRL

(Carlsbad, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2 and 5-diphenyl tetrazolium bromide (MTT)

were obtained from Amresco Inc. (Solon, OH, USA).

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

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Synthesis of Photoluminescent Graphene Nanoparticles. The synthesis process is represented

in the schematic diagram in figure S1. The amount of carbon fiber (CF), sulfuric acid, nitric acid

and water with other reaction parameter is presented in Table S1. In brief, half portion of sulfuric

acid and the entire nitric acid were taken in a 3 neck round bottom flask. The three neck flask

was placed on a heating mantle with continuous flow of N2 gas. On the other beaker CF was

dissolved in a mixture of remaining sulfuric acid and DI water. The CF containing solution was

sonicated by ultrasonicator (Ningbo Haishu Sklon Electronic Instrument Co., Ltd.,

sonicator83062) for 30 min. Temperature was controlled from 65 to 110 °C depending on the

desired photoluminescent graphene (Tablse S1). The solution was sonicated for further 60 min

by bath sonicator at room temperature for homogeneous dispersion. The sonicated CF containing

solution was injected to the three neck flask and stirred by mechanical stirrer for 12 hr. During

stirring temperature was maintained by heating mantle. After completion of reaction required

amount of water was added. Sodium hydroxide and sodium carbonate was added as specified

amount to make the acidic solution neutralization. Reaction solution was slowly stirred at 0-4 °C

for a certain period to remove the precipitated salts. The graphene nanoprticles containing

solution was collected by decantation and freeze dried for 48 hr.

Characterization of photoluminescent graphene nanoparticles. The size distribution and

morphologies of near infrared graphene nanoprticles were characterized using dynamic light

scattering (DLS) (ELS-Z2, Otsuka Electronics Co., Ltd, Japan) and a SEM (JEOL, Japan),

respectively. The TEM image and the selected area electron diffraction pattern were obtained on

a JEOL 2100 Field Emission Gun TEM. Photo-luminescent, excitation and emission were

measure by luminescent analyzer FluoroMate FS-2 (Scinco, Korea). The XRD data were

collected on a Rigaku D/Max Ultima II Powder X-ray diffractometer. XPS analyses were carried


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out on a PHI Quantera x-ray photoelectron spectrometer with a chamber pressure of 5×10-9 torr

and an Al cathode as the X-ray source. The source power was set at 100 W, and pass energies of

140.00 eV for survey scans and 26.00 eV for high-resolution scans were used. Band gap values

of near infrared graphene nanoprticles was measured based on absorbance and reflectance the

respective graphene nanoprticles. The calculation methods has been elaborately discussed

elsewhere.

Cytotoxicity evaluation. In vitro cytotoxicty study of near infrared graphene nanoprticles has

been examined in MDA-MB231 cell for 24 and 48 hr. At 37 oC and 5 % CO2 containing

humidified atmosphere, cells were grown in a medium containing MEM with 10 % fetal calf

serum. The cells (5×104 cells/mL) grown as a monolayer were harvested by 0.25 % trypsin-0.03

% EDTA solution. 200 μL of cells containing medium were placed in 96 well plates and

incubated for 24 hr. After 24 hr, the complete medium was suctioned and sample was added in to

the well at different concentrations (10, 25, 50, 100 and 200 µg/ml) with complete medium.

MTT solution aliquots at 5 mg/mL in PBS were prepared followed by culture incubation with

this solution at 5 % in the culture medium for 4 hr in an incubator with a moist atmosphere of 5

% CO2 and 95 % air at 37 oC. After 4 hr, 100 μL of MTT solubilizing solution was added and

made a gentle shake for 15 min. Finally, the absorbance of MTT colorimetric assay was

measured by Varioskan flash (Thermo Scientific, USA) at a wavelength of 570 nm. The viable

quantity of cells was calculated by the following equation:

Cell viability (%) = (absorbance of sample cells/absorbance of control cells)×100

In vitro cellular uptake. For a cellular uptake study of near-IR GNPs, the nanoparticles were

incubated with MDA-MB231 cell line. The MDA-MB231 cells were cultured at 37 oC in a


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humidified atmosphere containing 5 % CO2 in a MEM medium with 10 % fetal bovine serum.

The cells (5×104 cells/mL) grown as a monolayer were harvested by 0.25 % trypsin-0.03 %

EDTA solution. The cells (200 μL) in their respective media were seeded in an 8-well plate and

pre-incubated for 24 hr before the assay. The GNPs (0.1 mg/mL) was added with the 8-well plate

and incubated for 1 hr before observation by a confocal laser scanning microscope (CLMS). The

wells were washed 5 times by PBS to remove the free particles from the outside of the cell

membrane. A 4% formaldehyde solution was added to preserve the cell, and observed by CLMS

to get a noise free clear cellular image.

Animal experiments. Six to seven-week-old SKH1 female nude mice (average body weight of

21-25 g) were purchased from Orient Bio INC., (Seoul, Korea) and maintained under specific

pathogen-free conditions. All experiments were approved by institutional guidelines of the

Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea

College of Medicine in accordance with the NIH Guidelines. For in-vivo imaging studies, SKH1

mice were administered 2.5 mg/kg of near infrared graphene naoparticles through tail vain. Mice

were anesthetized with ketamine (87 mg/kg, Virbac Laboratories, France) and xylazine (13

mg/kg, Kepro B.V., Netherland) via intraperitoneal injection. In vivo mice images were taken by

a time-domain diffuse optical tomography system. In experimental section, mice were placed on

the imaging platform. Images were taken at 0.5, 1, 2, 4, 8, and 16 hr of post injection. The 3D

scanning region of interest was selected using bottom-view CCD. All images were taken by

using the Kodak in vivo imaging system (4000MN PRO, Kodak, USA). The ex-vivo images of

organs were taken after dissection of the mice. The organs were isolated after 16 hr observation.


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Supporting figures:

•Nitric acid

•Sulfuric acid

Heating and

stirring

•Carbon fiber

•Sulfuric acid

•DI water

•Tem. regulated by

heating mantle

•Stir for 12-18 h

Ultra-sonication : 10 min

Bath sonication :1 hr

Slowly injected by syringe

Add DW (2X of

reaction

solution)

Add

NaOH

until pH 1

Add Na2CO3

until pH 8 (PPT

appear)

Decantation

the GNPs

solvent

Freeze

drying (48 h)

Add citric

acid and

stirring for

1 h

60 5 oC

60 5 oC

Form

precipitation

0-4 oC,

slowly stirring

Scheme S1. The schematic diagram represents the synthesis process of photoluminoscent

graphene nanoparticles.


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Table S1. Controlling of reaction parameters to synthesis of different graphene nanoparticles

based on excitation.

Carbon

fiber (mg)

H2SO4

(99%)(mL)

HNO3

(60%)(mL)

DI water

(ml)

Citric

acid (mg)

Rxn. Temp.

(C)

Excitati

on (nm)

100 70 20 X 100 105±5 450

100 80 20 X 100 95±5 550

100 90 25 5 100 85±5 655

100 100 25 10 100 75±5 705

100 100 30 10 100 65±5 805

65 C,12 hr

Mono-layered Graphene

Citric acid

Sulfuric acid+ Nitric acid Sonication

CF

Figure S1. Synthesis process of highly photoluminoscent graphene nanoparticles from carbon

fiber.


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450 nm 550 nm 705nm 805 nm

A

B

1 0.5 0.25 0.12 0.06

mg/mL

Figure S2. Optical properties of graphene nanoparticles. The as synthesized graphene

nanoparticles shows different excitation properties with different colors (a) and optical emission

of near infrared graphene nanoprticles at different concentrations (b).

200 300 400 500 600 700 800

Inte

nsity (

a.u

.)

Wavelength (nm)

200 300 400 500 600 700 800

Inte

nsity (

a.u

.)

Wavelength (nm)

200 300 400 500 600 700 800

Inte

nsity (

a.u

.)

Wavelenth (nm)

200 300 400 500 600 700 800

Inte

nsity (

a.u

.)

Wavelength (nm)

200 300 400 500 600 700 800

Inte

nsity (

a.u

.)

Wavelength (nm)

a b c

d e


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Figure S3. Fluorescence excitation spectra of GQDs with emission at (a) 455 nm, (b) 555 nm,

(c) 650 nm, (d) 705 nm and (e) 805 nm.

500 600 700 800

PL inte

nsity (

a.u

.)

Wavelength (nm)

405

395

415

385

Figure S4. Fluorescence spectra of near-IR GQDs, registered at different excitation wavelength.


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b

c

0 3 6 9 12 15

Inte

nsity (

%)

Particle size (nm)

a

50 nm

30 nm

Figure S5. Size and morphologies of near infrared graphene nanoparticles. (a) Field

emission scanning electron microscopy, (b) size distribution from dynamic light scattering, (c)

transmission electron microscopy image of graphene nanoparticles (emission 705 nm). The

magnified image confirms the zig-zag shape of graphene nanoparticles.

A B C


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Figure S6. Morphology of CF and its’ derivatives. TEM images of carbon fiber (a), after

sonication of CF for 10 min (b) , and nano-sized graphene particles (c). The carbon fiber shows rod

type with around 0.5µm in width.

pH 5 pH 7 pH 9 pH 11

10000

20000

30000

40000

50000

60000

70000

80000

Flu

ore

scen

ce I

nte

nsi

ty (

AU

) 705 nm

805 nm

0 1 2 3 4 5 6 7

10000

20000

30000

40000

50000

60000

70000

80000

PL

inte

nsi

ty (

a.u.)

Time (Days)

705 nm

805 nm

0 1 2 3 4 5 6 7

10000

20000

30000

40000

50000

60000

70000

80000P

L i

nte

nsi

ty (

a.u.)

Time (Days)

705 nm

805 nm

A

C D

Day 1 Day 7

B

Figure S7. PL stability. Stability of near infrared graphene nanoparticles was investigated in

different conditions; pH buffer (a), optical stability measured by kodak molecula imaging station

(b) in PBS (c), and 10% FBS solution (d).


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0 100 200 300 400 50070

75

80

85

90

95

100

CF

GNPs

We

igh

t (%

)

Temperature (oC)

a

1.0 1.5 2.0 2.5 3.00

102030405060

Energy band gap (eV)

Oxyg

en

(%

)

20406080100120 R

ea

ctio

n T

em

. (oC

)

b

0 100 200 300 400 500-0.24-0.18-0.12-0.060.000.060.120.180.24

Tem

p. d

iffe

rence (

oC

/mg)

Temperature (oC)

c

Figure S8. Differential thermal analysis. (a) Characterization of thermal properties measured

by TGA shows variation between CF and NIR graphene nanoparticles. Thermal properties of CF

and near infrared graphene nanoprticles measured by DTA (b). (c) Variation of energy bad gap

of the as synthesized nanoparticles regarding reaction temperature and presence of oxygen atom

with graphene.


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520 525 530 535 540

Cou

nts

/S

Binding energy (eV)275 280 285 290 295

Cou

nts

/S

Binding energy (eV)

0 300 600 900 1200

Cou

nts

/S

Binding energy (eV)

A

B C

Figure S9. XPS survey scan of CF. (a) XPS scanning spectrum shows two major peaks of

carbon and oxygen. High resolution XPS survey scan show (b) C1s (C=95.96%), and (c) O1s

(O=4.04%). The spectrum demonstrate that the starting martials carbon fiber is composed carbon

with partial amount of oxygen. The carbon fiber does not shows any band gap, exciation and

emission properties.


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200 300 400 500 600 700 800

Inte

nsity

(a.u

)

Binding energy (eV)

520 525 530 535 540

Cou

nts/

s

Binding energy (eV)

270 275 280 285 290 295

Cou

nts/

S

Binding energy (eV)

C1s

O1s

385 390 395 400 405 410

Cou

nts/

SBinding energy (eV)

N1s

a b

c d

Figure S10. XPS survey scan of near infrared graphene nanoparticles. (a) XPS scanning

spectrum shows two major peaks of carbon and oxygen. High resolution XPS survey scan show

(b) C1s (C=56.04%), (c) O1s (O=43.16%) and (d) N1s (0.80 %). The composition of GNPs are

different than that of CF as observed from the survey. The as synthesized GNPs contains around

43% oxygen which is responsible for band gap, excitation and emission properties.


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Table S2. XPS scanning survey of carbon fiber (CF) and photolumunoscence graphene

nanopartiles (GNPs) and their energy band gaps.

Element CF GNPs-450 GNPs-

550

GNPs-

655

GNPs-

705

GNPs-

805

Carbon 95.9 71.5 67.3 62.1 56.04 51.2

Oxygen 4.04 27.9 32.4 37.6 43.1 48.4

Nitrogen X 0.51 0.21 0.23 0.8 0.3

Band gap

(eV)

X 1.02 1.34 1.82 2.01 2.63

0 1 2 3 4 5 6 7 80

5

10

15

20

25

30

35

40

Siz

e in

dia

mete

r (n

m)

Time (day)

705 nm

805 nm

Figure S11. Size stabilitty. Size stability of near infrared graphene nanoparticles in 10% FBS

for 7 days shows no mentionable changes in size measured by DLS.


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10 25 50 100 200

24 h 48 h

Concentration (g/ml)

Ce

ll v

iab

ility (%

)IC50

Figure S12. In vitro cytotoxicity. In vitro cytotoxicity assay of near infrared graphene

nanoparticles in MDA-MB231 cancer cell line for 24 hr and 48 hr represents no mentionable

toxicity.

Figure S13. Cellular uptake of GNPs. MDA-MB 231 cancer cells were incubated with

0.1mg/mL of GNPs at 37C for 2 hr. The cell images were analyzed for determining cell

permeability of GNPs using confocal laser microscopy.


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Figure S14. FT-IR spectra of CF and GQDs. FTIR spectrum of carbon fiber shows a straight

line as it does not contains any functional groups. Oxygen containing functional groups such as

carbonyl, carboxyl and hydroxyl groups were introduced to the edges of the monolayer graphene

as shown in the FT-IR spectrum.


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