S1

Supporting Information

Self-assembled nanoporous graphene quantum dot-Mn3O4

nanocomposites for surface-enhanced Raman scattering based

identification of cancer cells

Chuanqing Lan, Jingjin Zhao, Liangliang Zhang,* Changchun Wen, Yong Huang, Shulin

Zhao*

State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal

Resources, Key Laboratory of Ecology of Rare and Endangered Species and

Environmental Protection of Ministry Education, Guangxi Normal University, Guilin,

541004, China

Experimental CalculationsBand-gap calculation for GQD-Mn3O4

ELUMO, which correlates to the conduction-band energy level, was calculated using

ELUMO=eEred+4.5 V1 (on NHE).However, from SCE to NHE, ELUMO is +0.25 V2, so

ELUMO=eEred+4.75 V. The valence-band energy level was also calculated from the onset

oxidation potential, EHOMO = eEox + 4.75 V, and the energy gap is defined as Eg = ELUMO -

EHOMO. From these calculations, the ECB = -4.39 eV, and EVB = -5.91 eV. For RhB,

ELUMO = -3.50 eV, and EHOMO = -5.50 eV1.

MTT assay.

The cells were grown in a humidified atmosphere containing 5% CO2 at 37 °C in RPMI

1640 medium supplemented with 10% fetal bovine serum (FBS, invitrogen), 100 mg/ mL

streptomycin and 100 units/mL penicillin. The cytotoxicity of the GQD-Mn3O4 was

evaluated using a MTT assay. The cells were seeded and incubated in a 96-well plate

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2017

S2

overnight at a density of 1×104 cells per well for 24 h. The culture medium was then

removed, the GQD-Mn3O4 solution was added into each well with increasing

concentration from 0 to 800 μg mL-1 and incubated for 24 h before replacing the medium

with 200 mL of fresh complete medium containing 20 mL of MTT (5 mg/mL in PBS).

The plate was incubated for another 4 h before all medium was removed and 150

mL/well of DMSO was added, followed by shaking for 15 min. The absorbance of each

well was measured at 570 nm using an Enzyme-linked Immunosorbent Assay (ELISA)

reader with pure DMSO as a blank. Non-treated cells were used as a control and the

relative cell viability (mean%×SD, n=3) was expressed as Abssample/Abscontrol×100%.

Calculation of Raman enhancement factor (EF)

The EF was calculated by comparing the intensity of the surface-enhanced Raman signals

(Isurf) with that of the aqueous RhB solution (INsurf).3, 4

EF = (Isurf / INsurf)(NNsurf/Nsurf)

Nsurf represents the number of RhB molecules that contribute to the SERS obtained from

the active substrate, and NNsurf represents the number of moleculesthat contribute to the

Raman signal obtained in the RhB aqueous solution. The spot size of the laser is 1 µm

and the penetration depth is ∼2 µm. In this calculation, we assume that the surface of the

active substrate is fully covered by a closely packed monolayer of RhB molecules

(corresponding to maximum coverage). Using this coverage assumption, the estimated

EF is the lowest possible value (lower coverage would lead to a higher estimation of the

EF).Each EF is calculated referring to the nearest peak of the Raman spectrum obtained

in RhB aqueous solution for maximum EF.

The calculation of the contribution of photo-induced charge transfer (CT) to the

molecule polarization tensor in the semiconductor-molecule system

The Herzberg-Teller theory indicates that the contribution of photo-induced charge

transfer (CT) to the molecular polarizability tensor in the semiconductor can be

calculated. This is similar to the calculation for a semiconductor-molecule system

reported by Lombardi, Wang and Zhao.4-8

The intensity of a Raman transition may be obtained from the molecular

S3

polarizability tensor by the expression:

(1) 2

4

4

'

9

8

c

I I LII

Raman

IL is the incident laser intensity at ω0. ωIF can be shown to be the proportional to the

electromagnetic enhancement factor L2(ωL)L2(ωS), where ω and ωI’I is a molecular

transition frequency between the states I and I’ (presumably two different vibronic levels

of the ground state Ie), respectively.

Because the GQDs have EM enhancement properties, the plasmon resonance in SERS

should be considered. The IL can be expressed as a function of the complex dielectric

function ε(ω) = ε1+ iε2, and the dielectric constant ε0 as follows:

(2)

201

0

2 iI L

Thus the expression for the polarizability tensor by second-order perturbation theory in

the molecule-semiconductor system may be written as:

(3)

',

''

IISIKIS EE

ISSI

EE

ISSI

hh

Where S represents all the other states of the molecule, µ is the dipole moment

operatorand the subscripts ρ and σ represent the three directions in space (X, Y, Z). Using

the zero-order Born-Oppenheimer approximation, all the states (I, I’, S) can be written as

a product of the electronic and vibrational wave functions as follows:

(4)iII e

(5)fII e'

(6)kSS e

Where the subscript e indicates purely electronic states and lowercase letters represent

vibrational functions. The Herzberg-Teller theory states that even small vibrations may

cause mixing of zero-order Born-Oppenheimer states, allowing us to describe the

semiconductor-molecule system as:

S4

(7)S eSRe kRQkSS ,,

(8) 00/ SRSRSR EEh

(9)kRQHkSh eeNeSR ,/,

Where zero refer to zero-order Born-Oppenheimer states, and HeN is the electron nuclear

attraction term in the Hamiltonian, evaluated at the equilibrium nuclear positions (0).Q is

the displacement of the nuclei for a normal mode of the molecule-semiconductor system

and includes molecular normal modes as well as phonon modes of the semiconductor. R

runs over all states of the molecule-semiconductor system. For the purely electronic

transition moment between states, we write:

(10)eeSI IS

(11)eeRI IR

(12)eeSR RS

Substituting this into Eq.(1), the polarizability tensor ασρ in the semiconductor-molecule

system can be written as follows:

(13)CBA

For molecule-semiconductor systems, it should be considered that the most intense SERS

enhancement occurs for the transitions terminating at band edges (EC and EV for the

conduction and valence bands, respectively). So, the charge transfer can borrow intensity

either from molecular transitions (µKI) or from exciton transitions (µVC). Therefore the

exchange of subscripts changes the equations from intensity borrowing in the molecular

states manifold to that in the exciton states manifold. Corresponding changes in the

Herzberg−Teller coupling term (h) follow similarly. In the expressions for A, B and C,

the damping term γ which tends to broaden the resonances and is usually determined

empirically by observation of the homogeneous bandwidth in the optical absorption

spectrum, should be considered.

The A-terms are:

(14) 2222

2

2

01 2 ICIC

ICIC fkki

S5

(15) 2222

2

2

01 2 VKVK

VKVK

VK

fkkiR

For the A-terms, only one other resonance is obtained–either the molecular-HOMO to

conduction-band edge CT (at ω =ωCT) or the valence-band edge tomolecular LUMO CT

(at ω = ωVK).

The B-terms are:

(16) 2222222

2

2

01 2 KIKIICIC

kCKICKI fQih

(17) 2222222

2

2

01 2 VCVCVCIC

kIVICVC

ICV

fQihR

Where, B represents the contribution of photo-induced charge transfer from the molecular

HOMO to semiconductor conduction band edge transitions (at ω = ωIC).

The C-terms are:

(18) 2222222

2

2

01 2 KIKIVKIC

kIVVKVK

IVK

fQihR

(19) 2222222

2

2

01 2 CVCVVKIC

kKCVKVC

KVC

fQiR

C represents the contribution of photo-induced charge transfer of the semiconductor-to-

molecule from the valence-band edge to molecular LUMO transitions (at ω = ωVK). This

is illustrated in Figure S13.

S6

Supplementary figures and tables

Figure S1. TEM image of GQDs extracted from the GQD-Mn3O4 composite after

treatment with 3M HCl.

Figure S2. The size distribution of the GQDs.

S7

Figure S3. HRTEM image of GQDs extracted from the GQD-Mn3O4 composite after

treatment with 3M HCl.

Figure S4. FTIR of GQDs and the GQD-Mn3O4 nanocomposites.

S8

Figure S5. Raman spectra of GQDs and the GQD-Mn3O4 nanocomposites where the

labels D and G indicate the position of the D- and G-bands, as described in the main text.

Figure S6. The UV-Vis spectra of GQDs and the GQD-Mn3O4 nanocomposites.

S9

Figure S7. Raman spectra of RhB on the GQDs after subtracting the baseline.

Figure S8. The Raman spectrum of 50 mM RhB molecules in aqueous solution (black

line) and 10 μM RhB aqueous solution on the GQD-Mn3O4 nanocomposite substrate (red

line).

S10

Figure S9. 3D fluorescence of the GQDs.

Figure S10. Fluorescence spectra of the GQDs and GQD-Mn3O4 nanocomposites.

S11

Figure S11. The fluorescence lifetime of RhB+ GQD-Mn3O4 nanocomposites with excitation at 460 nm.

Figure S12. Fluorescence lifetime of RhB with excitation at 460 nm.

S12

Figure S13. HepG-2 cell viability after add GQD-Mn3O4 by MTT assay

Figure S14. HeLa cell viability after add GQD-Mn3O4 by MTT assay

S13

Figure S15. 7702 cell viability after add GQD-Mn3O4 by MTT assay

S14

Table S1

Comparison of Raman peaks of RhB adsorbed on GQDs and on GQD-Mn3O4

nanocomposites.

RhB in solution

Peak position (cm-1)

GQDsPeak

position (cm-1)

EF GQD-Mn3O4Peak

position (cm-1)

EF Assignments3, 9-11

357 351 410 348 11827 XR stretching

425 421 674 419 20625 XR deformation+ N-H oscillation

585 592 12983 –NH wagging

622 632 60 631 1713 XR + PHR stretching

661 C–H o.p. bending of XR

732 766 164 763 3924 C–H o.p. bending of XR

790 C–H o.p. bending of XR

826 C–H o.p. bending of XR

936 943 5000 XR + PHR stretching

1020 C–H bending of XR

1083 PHR stretching

1147 C–H bending of XR

1204 1190 183 1190 2500 C–H bending of XR

1277 1286 70 1277 1004 C–H bending of XR

1358 1362 131 1361 2770 XR stretching

1388 XR stretching

1437 XR stretching

1516 1508 126 1499 1749 XR stretching

1532 XR stretching

1559 1569 150 1566 5000 PHR stretching

1649 1644 110 1641 795 XR stretching

S15

Table S2

SERS substrate comparisons (data from references 3,4,8,12-19).

Author Substrate Analyte EF Excited wavelength

(nm)X. Ling et.al. Graphene R6G,

PPP, Pc, CV

2-17 457, 514, 632

X. Yu et.al. Chemical-reduced graphene oxide

RhB 61-684 514

H. Cheng et.al. GQDnanotubes R6G 40-74 632 W. Xu et.al. GERS-Au, GERS-Ag R6G,

CuPc85

(GERS-Au)755

(GERS-Ag)

632

X. Wang et.al. H-Si nanowire R6G 8-28 532 L.G.Quagliano InAs/GaAs quantum

dotsPyridine 103 514

L. Jiang et.al. Cu2O nanosphere 4-MBA 105 488D. Qi et.al. TiO2 photonic

microarrayMB 2×104 532

S. Hayashi et.al.

GaP CuPc 700 514

S. Cong et.al. W18O49 R6G 3.4×105 532

This workGQD,

GQD-Mn3O4

nanocomposite

RhB 674 (GQD),2×104

(GQD-Mn3O4)

514

S16

References

1. Hashimoto, K.; Hiramoto, M.; Sakata, T., Photo-induced electron transfer from

adsorbed rhodamine B to oxide semiconductor substrates in vacuo: Semiconductor

dependence. Chemical Physics Letters 1988, 148, 215-220.

2. Pavlishchuk, V. V.; Addison, A. W., Conversion constants for redox potentials

measured versus different reference electrodes in acetonitrile solutions at 25°C.

Inorganica Chimica Acta 2000, 298, 97-102.

3. Yu, X.; Cai, H.; Zhang, W.; Li, X.; Pan, N.; Luo, Y.; Wang, X.; Hou, J. G., Tuning

chemical enhancement of SERS by controlling the chemical reduction of graphene

oxide nanosheets. ACS Nano 2011, 5, 952-8.

4. Cong, S.; Yuan, Y.; Chen, Z.; Hou, J.; Yang, M.; Su, Y.; Zhang, Y.; Li, L.; Li, Q.;

Geng, F.; Zhao, Z., Noble metal-comparable SERS enhancement from

semiconducting metal oxides by making oxygen vacancies. Nature Communications

2015, 6, 7800.

5. Lombardi, J. R.; Birke, R. L., Time-dependent picture of the charge-transfer

contributions to surface enhanced Raman spectroscopy. The Journal of Chemical

Physics 2007, 126, 244709.

6. Lombardi, J. R.; Birke, R. L., A Unified Approach to Surface-Enhanced Raman

Spectroscopy. The Journal of Physical Chemistry C 2008, 112, 5605-5617.

7. Lombardi, J. R.; Birke, R. L., Theory of Surface-Enhanced Raman Scattering in

Semiconductors. The Journal of Physical Chemistry C 2014, 118, 11120-11130.

8. Wang, X.; Shi, W.; She, G.; Mu, L., Using Si and Ge nanostructures as substrates for

surface-enhanced Raman scattering based on photoinduced charge transfer

mechanism. Journal of the American Chemical Society 2011, 133, 16518-23.

9. Shim, S.; Stuart, C. M.; Mathies, R. A., Resonance Raman cross-sections and vibronic

analysis of rhodamine 6G from broadband stimulated Raman spectroscopy.

Chemphyschem: European Journal of Chemical Physics and Physical Chemistry

2008, 9, 697-9.

10. Sarkar, J.; Chowdhury, J.; Pal, P.; Talapatra, G. B., Ab initio, DFT vibrational

calculations and SERRS study of Rhodamine 123 adsorbed on colloidal silver

S17

particles. Vibrational Spectroscopy 2006, 41, 90-96.

11. Alvarez-Puebla, R. A.; Agarwal, A.; Manna, P.; Khanal, B. P.; Aldeanueva-Potel, P.;

Carbo-Argibay, E.; Pazos-Perez, N.; Vigderman, L.; Zubarev, E. R.; Kotov, N. A.;

Liz-Marzan, L. M., Gold nanorods 3D-supercrystals as surface enhanced Raman

scattering spectroscopy substrates for the rapid detection of scrambled prions.

Proceedings of the National Academy of Sciences of the United States of America

2011, 108, 8157-61.

12. Cheng, H.; Zhao, Y.; Fan, Y.; Xie, X.; Qu, L.; Shi, G., Graphene-quantum-dot

assembled nanotubes: a new platform for efficient Raman enhancement. ACS Nano

2012, 6, 2237-44.

13. Qi, D.; Lu, L.; Wang, L.; Zhang, J., Improved SERS sensitivity on plasmon-free

TiO2 photonic microarray by enhancing light-matter coupling. Journal of the

American Chemical Society 2014, 136, 9886-9.

14. Quagliano, L. G., Observation of molecules adsorbed on III-V semiconductor

quantum dots by surface-enhanced Raman scattering. Journal of the American

Chemical Society 2004, 126, 7393-8.

15. Ling, X.; Xie, L.; Fang, Y.; Xu, H.; Zhang, H.; Kong, J.; Dresselhaus, M. S.; Zhang,

J.; Liu, Z., Can graphene be used as a substrate for Raman enhancement? Nano

Letters 2010, 10, 553-61.

16. Jiang, L.; You, T.; Yin, P.; Shang, Y.; Zhang, D.; Guo, L.; Yang, S., Surface-

enhanced Raman scattering spectra of adsorbates on Cu(2)O nanospheres: charge-

transfer and electromagnetic enhancement. Nanoscale 2013, 5, 2784-9.

17. Kim, K.; Kim, K. L.; Shin, K. S., Raman spectral characteristics of 4-

aminobenzenethiol adsorbed on ZnO nanorod arrays. Physical Chemistry Chemical

Physics: PCCP 2013, 15, 9288-94.

18. Hayashi, S.; Koh, R.; Ichiyama, Y.; Yamamoto, K., Evidence for surface-enhanced

Raman scattering on nonmetallic surfaces: Copper phthalocyanine molecules on

GaP small particles. Physical Review Letters1988, 60, 1085-1088.

19. Xu, W.; Ling, X.; Xiao, J.; Dresselhaus, M. S.; Kong, J.; Xu, H.; Liu, Z.; Zhang, J.,

Surface enhanced Raman spectroscopy on a flat graphene surface. Proceedings of

the National Academy of Sciences of the United States of America 2012, 109, 9281-6.

S18