silicon nanoparticle based fluorescent biological label via low … · 2013-04-29 · supporting...

Supporting Informations

Silicon Nanoparticle based Fluorescent Biological Label via Low

Temperature Thermal Degradation of Chloroalkylsilane

By Pradip Das,a Arindam Saha,a Amit Ranjan Maity,a Sekhar C. Rayb and Nikhil R. Janaa,*

Experimental Section

Materials: Chloro(dimethyl)octadecylsilane (95%), octadecylamine (ODA, 90%), folic acid (FA, 97%),

triethylamine (Et3N, 99%), poly(maleic anhydride-alt-1-octadecene) (Mn 30,000-50,000), O,O-Bis(2-

aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol (Mr 500),

dimethyl sulfoxide (DMSO), N,N-dicyclohexylcarbodiimide (DCC, 99%), N-hydroxy succinimide

(NHS, 98%) were purchased from Sigma-Aldrich. 1,3,5-trimethylbenzene (TMB, 98%) was purchased

from Spectrochem.

Synthesis of silicon nanoparticles: Silicon nanoparticles were synthesized by low temperature thermal

decomposition of chloro(dimethyl)octadecylsilane as a precursor in presence of octadecylamine as

capping agent at normal atmospheric environment. Briefly, chloro(dimethyl)octadecylsilane (69.4 mg)

and octadecylamine (6.7 mg) were added into 12 mL of TMB taken in three naked round bottom flask.

This solution was then heated to 140 ºC for different times from 6 to 72 hours. Depending on heating

time, the produced silicon nanoparticles were named as Si-6, Si-18, Si-36, Si-48 and Si-72. Silicon

nanoparticles were isolated from free reagents by conventional precipitation-redispersion method.

Typically, TMB solution of silicon nanoparticle is mixed with equal volume of hexane to precipitate the

particles and precipitated particle were then isolated and dissolved in chloroform. This chloroform

solution was then mixed with minimum volume of hexane to precipitate the particles. This precipitate is

again isolated and dissolves in chloroform. This hexane based precipitation and chloroform based

dissolution is repeated 2-3 times and finally particles were dissolved in chloroform.

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

Synthesis of polymer coated Si-72 nanoparticles: Polymer coated Si-72 nanoparticles were prepared

using the previously described method with minor modification.52,53

In brief, 20 mg poly(maleic

anhydride-alt-1-octadecene) polymer was dissolved in 1 mL chloroform and mixed with 500 µL

chloroform solution of Si-72 nanoparticles (2 mg/mL) followed by sonication for 10 minutes.

Subsequently chloroform solution of 250 µL of O, O’-bis(2-aminopropyl) polypropylene glycol-block-

polyethylene glycol-block-polypropylene glycol (PEG-diamine) (prepared by mixing 3:25 volume ratio

of PEG-diamine and chloroform) was added and sonicated for 10 minutes. After that additional 250 µL

PEG-diamine solution was added and sonicated for further 10 minutes. The resultant solution was

allowed to stand overnight at room temperature and then chloroform was evaporated. The residue was

dispersed in 2 mL of aqueous Na2CO3 solution. The polymer coated aqueous Si-72 nanoparticles was

mixed with twice volume of acetone and precipitated particles were isolated from supernatant containing

free polymer by high speed centrifuge and dissolved in water. Finally particle solution was dialyzed to

remove any excess reagents.

Synthesis of folate-NHS: Folate-NHS was prepared following our reported method.58

Briefly, folic acid

(150 mg) and Et3N (75 µL) were added into 10 mL of distilled DMSO and subsequently DCC (70 mg)

was added. The solution was stirred for one hour in absence of light and then NHS (60 mg) was added to

the solution followed by stirring for overnight under nitrogen atmosphere. The resultant folate-NHS was

separated from solution by the addition of diethyl ether and purified by dry THF.

Folate functionalization of Si-72 nanoparticles: Polymer coated Si-72 nanoparticles were reacted with

folate-NHS to prepare the folate functionalized Si-72 nanoparticles. The primary amine groups present on

the surface of polymer coated particle reacted with NHS group with the resultant covalent linkage. In a

typical process, 1.5 mL polymer coated Si-72 nanoparticles solution was prepared in bicarbonate buffer

solution of pH 9. Next, freshly prepared folate-NHS (1 mM) solution was prepared in DMF and 200 µL

of this solution was added to it. The solution was stirred for overnight and then excesses reagents and free

folic acid were removed by dialyzing against basic water and then normal using dialysis membrane

(MWCO ~12000-14000 Da).


Cell and tissue labeling: HeLa cells were cultured in cell culture flask and then subcultured in cell

culture plate with 0.5 mL folate free RPMI-1640 media having 10 % fetal bovine serum (FBS) and 1 %

penicillin/streptomycin. After overnight, cells which were attached to the tissue culture plate were washed

with phosphate buffer solution and then 500 µL fresh media was added. After overnight 10-150 µL of

nanoparticles solution (2 mg/mL) was added and incubated for 4 hours. After that 10 µL of nuclear

straining dye (Hoechst 33342) was added and incubated for 10 minutes. Next, unbound nanoparticles and

excesses dye were removed by repeated washing with PBS buffer solution. The washed cells were fixed

by using of 4 % paraformaldehyde followed by mounting with 50 % glycerol. The cells were then used

for imaging.

Human dissected cervical cancer positive tissue was frozen and sectioned following the standard

procedure.58

The conventional histopathological assay was used to detect cancer of the biopsy tissue

samples. For labeling study, 5 μm thick sectioned tissue sample was incubated with nanoparticle solution

for 4 hours and then extensively washed with PBS buffer solution to remove any unbound nanoparticles.

Next, sample was incubated with solution of hoechst dye for 5 minutes for staining of cell nucleus.

Finally, washed sample was imaged under bright field and fluorescence mode.

MTT assay: HeLa cells were seeded into 24-well plate in 500 µL folate free RPMI-1640 media. After 24

hours, cell were treated with various amounts of nanoparticles having different final concentration (0.15 -

2.5 mg/mL) and incubated for 24 hours. After that cells were washed with PBS buffer solution. Next, 50

µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg /mL in PBS buffer) was

added to every well and incubated for 4 hours. Next, the supernatant was removed carefully, leaving the

violet formazan in the plate. The precipitate was mixed with 500 µL of 1:1 water-DMF mixture and then

absorbance was measured at 570 nm using microplate reader. The relative cell viability was calculated

assuming 100 % cell viability for sample having no nanoparticle.

Instrumentation: UV-Visible absorption spectra were measured using Shimadzu UV-2550 UV-Visible

spectrophotometer and photoluminescence emission, excitation spectra were obtained using BioTek


SynergyMx microplate reader. Transmission electron microscopy (TEM) imaging and energy dispersive

X-ray (EDS) spectroscopy was performed using FEI Technai G2 F20 electron microscope. X-ray

photoelectron spectroscopy (XPS) was performed using a Omicron (Serial No-0571) X-ray photoelectron

spectrometer. Time correlated single photon counting (TCSPC) spectra was obtained through exciting the

sample with picoseconds diode laser (IBH Nanoled) using Horiba Jobin Yvon IBH Fluorocube apparatus.

Temperature dependent photoluminescence (PL) of solid Si-72 nanoparticles was performed by Triax 310

monochromator and a multichannel photomultiplier detector under UV excitation with 325 nm line of He-

Cd laser. Fourier transform infrared (FTIR) spectroscopy was performed on Perkin Elmer Spectrum 100

FTIR spectrometer. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were

performed using TA SDT Q600 and TA DSC Q2000 instrument, respectively. Electron paramagnetic

resonance (EPR) was measured using JEOL JES-FA 200 ESR spectrometer using purified solid samples

at 77 °K. Dynamic light scattering (DLS) and Zeta potential study were performed using a NanoZS

(Malvern) instrument, after dialyzing the samples. Fluorescence images and photostability of different

silicon nanoparticles were performed by drop casting of sample solution on glass slide and images were

taken using Olympus IX 81 microscope attached with digital camera. The fluorescence images of the

cell/tissue were captured by Carl Zeiss Apotome Imager.Z1 fluorescence microscope.


Table S1. Summary of different synthetic methods for fluorescent silicon nanoparticles.

Silicon

precursor

Method Emission (QY) Size Functionalization/

application

Ref.

SiCl4 reduction with sodium

naphthalenide

blue (-) 2-10 nm --- 28

SiCl4 reduction with LiAlH4 blue (10 %) 1.4 nm bioimaging 29

Si(OMe)4 reduction with LiAlH4 blue (11-13 %) 1.6 nm bioimaging 30

SiCl4 reduction with LiAlH4 blue (13-23 %) 1-2 nm --- 31

SiCl4 reduction with NaPh2 blue (20 %) 1-4 nm --- 32

SiCl4,

hexyltrichloros

ilane,

reduction with LiAlH4 blue (25 %) 3 nm --- 33

diphenylsilane thermal degradation at 500°

and 345 bar

blue-green (23 %) 1-4 nm --- 16

Diphenylsilane thermal degradation at 500°

and 83 bar

blue to red

(---)

2-6 nm --- 17

tetramethylsila

ne/

tetraethylsilane

thermal degradation at 680 °

C

--- 1-10 nm --- 19

SiBr4 plasma assisted

decomposition

blue-green(24 %) 2-3 nm polymer coating (hydrodynamic

size 50 nm), cell imaging

23-24

silane laser pyrolysis followed by

HF-HNO3 etching

blue to red

(---)

5 nm --- 25


HF-HNO3 etching

red (17 % in

CHCl3, 2 % in

water)

4 nm micelle incorporated (50-120

nm), transferin functionalized

and cell imaging

26


HF-HNO3 etching

blue-NIR (---) 2-8 nm micelle encapsulated, in vivo

imaging

27

trichlorosilane pyrolysis followed by HF

etching

red-NIR (5-40 % in

toluene)

1-5 nm --- 22

octyltrichlorosi

lane

electrochemical reduction blue (6 %) 5 nm --- 34

Si wafer electrochemical etching in

presence of HF, H2O2,

polyoxometalate

blue-NIR (---) 1-4 nm --- 35

silicon wafer electrochemical etching red to NIR (10 %) 150

nm(porou

s)

in vivo application 36

hydrogen

silsesquioxane

thermal degradation at 500-

11000C followed by HF

etching

green-red (4 %) 3.4 nm --- 18

hydrogen

silsesquioxane

thermal degradation at

1100-12000C

NIR (26 %) 4 nm lipid capping, bioimaging 20

hydrogen

silsesquioxane

thermal degradation at

1100-14000C

red-NIR (0.4-8 %) 3-90 nm, -

---

---- 21

NaSi reaction with NH4Br blue (---) 4-5 nm -- 37

Mg2Si reaction with Br2 green (7 %) 2.4 nm functionalized with DNA 40

NaSi heating with NH4Br and

glutaric acid at 200 oC

green (13 %) 4 nm Gluteric acid functionalized,

bioimaging

38

Na4Si4 microwave heating with

NH4Br

blue (23 %) 3.4 nm --- 39

Si nanowire microwave heating with

glutaric acid

red (15 %) 4 nm antibody conjugated, cell

imaging

41

Si nanowire microwave heating with

immunoglobulin

red (18 %) 3.2 nm 40 nm hydrodynamic size,

bioimaging

42

Silicon pieces ball milling blue (60 %) <10 nm --- 43


Table S2. Properties of various silicon nanoparticle prepared by present approach.

Sample Excitation/

Emission

Wavelength (nm)

Fluorescence

quantum yield1

()

Lifetime (ns)

Si-6 370 nm/430 nm 10 %a 0.8

Si-18 430 nm /500 nm 13 %b 0.01, 3.3, 9.2

Si-36 480 nm/570 nm 6 %c 1.4, 4.6

Si-48 480 nm/570 nm 6 %c 0.8, 2.7, 6.8

Si-72 515 nm/600 nm 8 %d 1.5, 4.6

Polymer

coated Si-722

515 nm/600 nm 5 %d 0.5, 1.9, 5.9

1Quantum yield was measured using different standards such as quinine sulfate

a, fluorescein

b, rhodamine

6Gc

and rhodamine Bd. Fluorescence quantum yield for each type of silicon nanoparticle is measured at

the excitation wavelength that produces maximum emission intensity.

2The quantum yield of polymer coated Si-6, Si-18, Si-36 and Si-48 nanoparticles are 10 %, 11 %, 3 %

and 3 %, respectively.


Figure S1. TEM images of different size silicon nanoparticles under low and high magnifications. Size

distributions of these nanoparticles are shown in Figure 1.

Si-6

Si-6Si-48

Polymer coated Si-72Si-72 Polymer coated Si-72Si-72

Si-6

Si-48


Figure S2. Energy dispersive X-ray (EDS) spectrum of Si-48 nanoparticles after complete separation of

free reagents. The spectrum demonstrates the presence of silicon and oxygen suggesting that silicon

nanoparticles surface has some oxides.


4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1

)

Si-72

% T

ran

smit

tan

ce

Polymer

Polymer coated Si-72

4000 3500 3000 2500 2000 1500 1000 500

% T

ran

smit

tan

ce

Wavenumber (cm-1

)

CDOS

ODA

Si-72

a) b)

Figure S3. a) FTIR spectra of chloro(dimethyl)octadecylsilane(CDOS), octadecylamine(ODS) and Si-72

nanoparticle. The strong peaks around 3000 cm-1

is due to C-H stretching of long chain hydrocarbons and

strong peaks around 3400 cm-1

is due to N-H/O-H stretching of octadecylamines/silanols. Si-C stretching

frequency present at 1245 cm-1

for CDOS is substantially decreased in Si-72, suggesting that Si-C bond in

chloro(dimethyl)octadecylsilane precursor is broken in nanoparticle formation condition. (b) FTIR spectra

of Si-72 nanoparticle, polymer and polymer coated Si-72 nanoparticle. The peaks at 1112 cm-1

and 1551

cm-1

in polymer coated samples indicates the presence of C-O and NH2 groups, respectively, which

proves effective polymer coating on the samples.


0 100 200 300 400 500 600 700 8000

20

40

60

80

100

Wei

gh

t lo

ss (

%)

Temperature (0C)

CDOS

ODA

CDOS & ODA

Si-72

50 100 150 200 250-0.3

-0.2

-0.1

0.0

Heat

Flo

w (

W/g

)

Temperature (0C)

50 100 150 200 250

-0.6

-0.4

-0.2

0.0

Heat

Flo

w (

W/g

)

Temperature (0C)

a) b) c)

Figure S4. XPS spectra of (a) C 1s and (b) O 1s of purified Si-72 nanoparticles showing that silicon

nanoparticles consist of oxidized SiOxHy layer and passivating long chain octadecyl groups.

Deconvoluted C 1s has been fitted with two components and assigned as C-C/C-H bond (284.7 eV) and

carbon atoms bonded to electronegative element such as nitrogen or oxygen (286.8). Deconvoluted O 1s

spectrum fitted with three components with peaks at 530.7 eV, 531.7 eV and 533.3 eV. The first two

peaks are due to presence of Si-O and Si=O groups and third component comes from the surface hydroxyl

groups.

Figure S5. a) Thermogravimetric analysis of chloro(dimethyl)octadecylsilane(CDOS),

octadecylamine(ODA), mixture of CDOS and ODA and Si-72 nanoparticle, b) differential scanning

calorimetry of CDOS and c) differential scanning calorimetry of mixture of CDOS and ODA.

538 536 534 532 530 528 526

O 1s

Inte

nsi

ty (

CP

S)

Binding Energy (eV)290 288 286 284 282 280

Inte

nsi

ty (

CP

S)

Binding Energy (eV)

C 1sa b


Figure S6. a) Absorption (—), photoluminescence excitation (…..) and emission spectra of chloroform

solution of silicon nanoparticle produced in the reaction condition after heating for 6 hours (Si-6), 18

hours (Si-18), 36 hours (Si-36), 48 hours (Si-48) and 72 hours (Si-72). Emission has been measured by

exciting at 370 nm for Si-6, 430 nm for Si-18, 480 nm and 515 nm for Si-36, Si-48 and Si-72. (Yellow

and red line indicates the emission spectra at 480 nm and 515 nm excitation, respectively.)

b) Solid Si-72 sample prepared after purification, showing that method can be adapted for milligram to

gram scale synthesis.

300 400 500 600 7000

1

0

10

1

0

10

1

0

10

1

0

10

1

0

1

Em

issi

on

In

ten

sity

(a

.u.)

Ab

sorb

an

ce

Wavelength (nm)

Si-6

Si-18

Si-36

Si-48

Si-72

300 400 500 600 7000

1

0

10

1

0

10

1

0

10

1

0

10

1

0

1

Em

issi

on

In

ten

sity

(a

.u.)

Ab

sorb

an

ce

Wavelength (nm)

Si-6

Si-18

Si-36

Si-48

Si-72

300 400 500 600 7000

1

0

10

1

0

10

1

0

10

1

0

10

1

0

1

Em

issi

on

In

ten

sity

(a

.u.)

Ab

sorb

an

ce

Wavelength (nm)

Si-6

Si-18

Si-36

Si-48

Si-72

300 400 500 600 7000

1

0

10

1

0

10

1

0

10

1

0

10

1

0

1

300 400 500 600 7000

1

0

10

1

0

10

1

0

10

1

0

10

1

0

1

Em

issi

on

In

ten

sity

(a

.u.)

Ab

sorb

an

ce

Wavelength (nm)

Si-6

Si-18

Si-36

Si-48

Si-72

a) b)


Figure S7. Microscopic image of films of Si-6 (a, b, c), Si-18 (d, e, f) and Si-72 (g, h, i) which are

captured by depositing their respective chloroform solutions on the glass slide. The images are acquired

under bright field (a, d, g) or fluorescence modes under UV excitation (b), blue excitation (c, e, h) and

green excitation (f, i).


Figure S8. Emission spectra of Si-6 (a), Si-18 (b), Si-36 (c), Si-48 (d), Si-72 (e) and polymer coated Si-

72 (f) under different excitation wavelengths.


Figure S9. Photostability studies of different silicon nanoparticles. Films of Si-6, Si-18, Si-72 and

polymer coated Si-72 were prepared by depositing respective chloroforms solutions on the glass slides

and then imaged under bright field (BF) or under fluorescence mode (F) with UV, blue and green

excitation.

S8

50 m50 m50 m

0 min

50 m

0 min

50 m50 m

10 min

50 m

10 min

50 m50 m

50 m50 m50 m

0 min

50 m

0 min

50 m50 m

10 min

50 m

10 min

50 m50 m

10 min

50 m

10 min

50 m50 m

0 min

50 m

0 min

50 m50 m50 m50 m50 m

50 m50 m50 m 50 m

0 min

50 m50 m

0 min

50 m

10 min

50 m50 m

10 min

50 m50 m50 m 50 m

0 min

50 m50 m

0 min

50 m

10 min

50 m50 m

10 min

50 m50 m50 m 50 m

0 min

50 m50 m

0 min

50 m

10 min

50 m50 m

10 min

Si-6

Si-18

Si-72

Si-72

Si-72-polymer

Si-72-polymer

BF

BF

BF

BF

BF

BF

F (UV)

F (blue)

F (blue)

F (green)

F (blue)

F (green)

Si-6 Si-6F (UV)

F (blue)Si-18 Si-18

F (blue)Si-72 Si-72

Si-72 Si-72F (green)

Si-72-polymer Si-72-polymerF (blue)

Si-72-polymerF (green)Si-72-polymer


Figure S10. Emission spectra of solid Si-72 nanoparticle at different temperatures, showing that emission

decreases with the increasing temperature.


Figure S11. Fluorescence lifetime decay spectra of solutions of Si-6 (a), Si-18 (b), Si-36 (c), Si-48 (d),

Si-72 (e) and polymer coated Si-72 (f). Blue and red lines correspond to experimental and fitted data,

respectively.


a) b) c)

500 550 600 650 700 7500

3500

7000

Em

issi

on

In

ten

sity

(a.u

.)

Wavelength (nm)

control

Si-folate

500 550 600 650 700 7500

3500

7000

Em

issi

on

In

ten

sity

(a.u

.)

Wavelength (nm)

control

Si-folate

500 550 600 650 700 7500

4000

8000

12000

Em

issi

on

In

ten

sity

(a.u

.)

Wavelength (nm)

control

Si-folate

500 550 600 650 700 7500

4000

8000

12000

Em

issi

on

In

ten

sity

(a.u

.)

Wavelength (nm)

control

Si-folate

300 400 500 6000.0

0.5

1.0

1.5

Ab

sorb

an

ce

Wavelength (nm)

control

Si-folate

300 400 500 6000.0

0.5

1.0

1.5

Ab

sorb

an

ce

Wavelength (nm)

control

Si-folate

Figure S12. Photoluminescence (PL) spectra of polymer coated Si-72 nanoparticles before (blue) and

after reaction with fluorescamine (green), indicating the presence of primary amine groups on the surface

of polymer coated Si-72 nanoparticles. Emission of silicon is masked by high emission of fluorescamine-

amine complex.

Figure S13. (a) Absorption spectra of polymer coated Si-72 before and after conjugation with folic acid,

showing the appearance of 375 nm band due to folate.

(b, c) Fluorescence spectra of polymer coated Si-72 under 480 nm excitation (b) and 515 nm excitation

(c), before and after conjugated with folate.


0

25

50

75

100

2.331.621.160.640.14

Cel

l V

iab

ilit

y (

%)

Concentration (mg/mL)

control

Si-folate

0

25

50

75

100

2.331.621.160.640.14

Cel

l V

iab

ilit

y (

%)

Concentration (mg/mL)

control

Si-folate

Figure S14. Fluorescence images of HeLa cells after labeling with polymer coated Si-72 nanoparticle

showing that it does not label cells as nanoparticle is not functionalized with folic acid. Cells were labeled

with nanoparticle and hoechst dye and imaged under differential interference contrast (DIC) mode (a) or

fluorescence mode with UV excitation (b), blue excitation (c) and green excitation (d). Blue emission

under UV excitation is due to nucleus staining by hoechst dye.

Figure S15. Viability of HeLa cells after 24 hours incubation with different concentrations of polymer

coated Si-72 (control) and folic acid conjugated functionalized Si-72 nanoparticle (Si-folate).


silicon nanoparticle based fluorescent biological label via low … · 2013-04-29 · supporting...

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