Supporting Information of

Microwave assisted green synthesis of Zwitterionicphotolumenescent N-doped carbon dots:

an efficient ‘on-off’ chemosensor for tracer Cr(VI) considering the inner filter effect and

nano drug-delivery vector

Sayan Ganguly1#, Poushali Das2#, Subhayan Das3, Uttamkumar Ghorai4, Madhuparna Bose5,

Sabyasachi Ghosh1, Mahitosh Mondal3, Amit Kumar Das5, Susanta Banerjee2,6, Narayan Ch

Das1,2*

1Rubber Technology Centre, Indian Institute of Technology, Kharagpur 721302, India2School of Nanoscience and Technology, Indian Institute of Technology, Kharagpur 721302,

India3School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302,

India4Department of Industrial Chemistry and Applied Chemistry, Swami Vivekananda Research

Center, Ramakrishna Mission Vidyamandira, Belurmath, Howrah-711202, India5Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India6Materials Science Centre, Indian Institute of Technology, Kharagpur 721302, India# Authors have equal contributions.* Corresponding author (ncdas@rtc.iitkgp.ac.in; Ph. No.: 03222-283190)

Experimental

Materials

Melamine, L-arginine and quinine sulphate were purchased from Sigma Aldrich, Germany. The

water for using the whole experiments was purified through a Millipore system (Bedford, MA,

USA). Other chemicals used here were of analytical grades and were used without further

purification.

Synthesis of N-rich melamine-L-arginine carbon dots (MANCDs)

The N-rich carbon dots were prepared by microwave irradiation of melamine and L-arginine.

0.65 g melamine and 2.60 g L-arginine were dissolved in hot water (50-60oC, 20 mL) and sealed

in a Teflon capped borosilicate glass vial. Then the colorless solution was heated in a domestic

microwave oven (750 W) for 8 min. The glass vialwas cooled to room temperature and

centrifuged at 12000 rpm for 15 min to separate out the large particles. The suspension obtained

was further dialyzed against Milli-Q water with a semi-permeable membrane (cellulose ester

membrane bag with average molecular weight of 3500 Da). The MANCDs were then freeze

dried under vacuum to get a brownish powdered mass (yield ca. 37%). The preparation was done

in three different time viz. 5 min, 8 min and 12 min and the specimens obtained from this

different times were designated as MANCDs-5, MANCDs-8 and MANCDs-12 respectively.

Characterization

The FTIR spectrum of MANCDs was carried out in a FTIR spectrophotometer (Perkin Elmer,

model-Spectrum-2, Singapore) in ATR mode with a resolution of 4 cm-1 and 16 consecutive

scans. The morphology was evaluated by high resolution transmission electron microscope,

HRTEM (JEOL, Japan operating voltage 200 kV with filament LaB6). Surface topography of

MANCDs was scanned by Agilent 5500 scanning probe microscope (non-contact mode, scan

area of ̴ 500×500 nm2. The X-ray photoelectron spectra (XPS) have been performed in PHI 5000

Versa Probe II scanning X-ray photoelectron spectrometer, with a monochromatic Al Kα source

(1486 eV). UV-visible spectrum of MANCDs in aqueous suspension was performed in a UV-vis

spectrophotometer (Perkin Elmer, Singapore PTE Ltd., Model lambda 35). The zeta potential

was recorded on a Zetasizer Nano ZS90 (Malvern Instruments, Manchester, U.K.)Steady state

PL spectra and fluorescence lifetime were taken on Edinburgh, FLSP-980

spectrofluorometer whereas for the time resolved PL spectra, Edinburgh, FLSP-

980 luminescence spectrometer was used along with a 375 nm picosecond pulse diode laser as a

source of excitation.

Calculation of fluorescence quantum yield

The quantum yield of MANCDs was calculated at an excitation wavelength of 360 nm by the

following equation,[1]

QY =QR .IMANCDs

I R.

AR

ACD.ηMANCDs

2

ηR2 (1)

Where ‘Q’, ‘I’, ‘A’ and ‘η’ represent quantum yield, intensity of luminescent spectra, absorbance

at particular exited wavelength and refractive index of the solvent, respectively.The subscript ‘R’

and ‘U’ stand for the reference and unknown QY of MANCDs, respectively. Quinine sulfate in

0.1 M H2SO4 was used as standard and its quantum yield (QY) is known to be 54% in 0.1 M

H2SO4 solution.

In vitro cytotoxicity (MTT) assay

The in vitro cytotoxicity assay of MANCDs against dermal fibroblasts (HiFi™ Adult Dermal

Fibroblasts, HADF, HiMedia, Catalog No: CL005). cell line was evaluated by MTT (3-(4,5-

Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay [2]. Fibroblasts cell line was

maintained in Dulbecco's modified Eagles Medium (DMEM) affixed with 10% FBS. The cells

broth was taken of 100 µL in 96-well tissue culture plates at the density of 1 × 104 cells/well in

DMEM and incubated in incubator having 5% CO2 environment and 37 ⁰C for 48 h. The cells

were exposed to different concentrations of MANCDs (10 – 100 µg/mL) for 48 h at 37⁰C. Then,

the 20 µL of MTT (5 mg/mL in 10 mM PBS) solution was added in each well and were

incubated for next 4 h. A volume of 200 µL dimethyl sulfoxide (DMSO) was added in each well

to eradicate the formed unwanted formazan crystals. The absorbance was measured at 570 nm

with microplate reader (Bio-Rad I Mark Microplate reader, USA). Control experiment was done

under same conditions without MANCDs. All data in this study were conveyed as the mean ±

standard deviation (SD). Statistical analysis was performed using one-way ANOVA and tukey

post hoc analysis in Origin Pro 8.5® software. Significance was estimated at p < 0.05.

Diltiazem loading on MANCDs

Being a readily water soluble drug, diltiazem hydrochloride (Dil. HCl) aqueous solution (12

mg/100 mL; w/V) was mixed with MANCDs aqueous dispersion (50 mg/100 mL; w/V). After

that the mixed system was stirred in a 100 mL beaker for overnight (at least 10-14 h). During

stirring the drug-MANCDs mixture was sealed with Parafilm stretch wrapper and covered the

beaker with aluminum foil to protect from direct sunlight. Then the mixture was centrifuged at

10000 rpm for 20 min if there would be any wanted immiscible solid particles. After that the

whole supernatant (drug-MANCDs) was collected and transferred into dialysis bag (MWCO

10kDa) and dialyzed against MQ water for next three days. To quantify the drug loading the

supernatant was collected and estimated as per calibration plot of Dil.HCl at 236 nm (λmax). The

drug loading efficiency was calculated as the following equation;

Drug loading efficiency (%)=amount of drug−MANCDs−amount of free Dil . HClamount of Dil . HCl

× 100

(2)

In vitro drug release study

The drug release study was done at three different pH media i.e. pH 5.3, 7.4 and 9.6. 2.022 mg of

drug loaded MANCDs were homogeneously dispersed into 20 mL PBS buffer solution keeping

the above mentioned pH. Then 10 mL of the dispersion was transferred into a dialysis bag

(MWCO 100-200 Da) and immersed into a 500 mL glass beaker having PBS solution. The

system was kept at constant temperature around 35-37oC. During stirring the system, the whole

beaker was covered with an aluminum sheet. At specific time interval the 1.5 mL aliquot was

taken in a quartz vial and measured by UV-vis spectrophotometer (λmax = 236 nm). Diffusion of

diltiazem drug through dialysis membrane was taken as control. The cumulative drug release

(CDR) was calculated by the following formula,

CDR = [(Total absorbance – Initial absorbance)/Total absorbance] × 100 (3)

Where, total absorbance and Initial absorbance stand for the absorbance intensity at different

time interval (in min.) and absorbance at time = 0 min.

Fig. S1 (a) HRTEM image of MANCDs in aqueous dispersion (b) AFM image of MANCDs

after drop casted in silicon wafer

Fig. S2 UV-visibleabsorption spectra of Cr(+6) in aqueous solution

Fig.S3 Chelating ability of MANCDs.Effect of EDTA on the fluorescence intensity of MANCDs/Cr(+6) complex with different concentrations of Cr(+6)

Table S1 Comparative study of other prepared quantum dots regarding their quantum yield

References

[1] C. Parker, W. Rees, Fluorescence spectrometry. A review, Analyst, 87 (1962) 83-111.

[2] H. Zheng, Q. Wang, Y. Long, H. Zhang, X. Huang, R. Zhu, Enhancing the luminescence of

carbon dots with a reduction pathway, Chemical Communications, 47 (2011) 10650-10652.

[3] S. Liu, R. Liu, X. Xing, C. Yang, Y. Xu, D. Wu, Highly photoluminescent nitrogen-rich

carbon dots from melamine and citric acid for the selective detection of iron (III) ion, RSC

Advances, 6 (2016) 31884-31888.

[4] J. Gong, X. An, X. Yan, A novel rapid and green synthesis of highly luminescent carbon dots

with good biocompatibility for cell imaging, New Journal of Chemistry, 38 (2014) 1376-1379.

Precursor/name Method of preparation

Quantum yield (%)

Area of application

References

Melamine-citric acid Hydrothermal 41.5 Fe(III) detection [3]Ascorbic acid Microwave 15.0 Cell imaging [4]Cellulose-urea Hydrothermal 21.7 Cell imaging [5]

dopamine and cysteine Hydrothermal 5.10 Cell imaging [6]Polyethylene glycol Microwave 16.0 Cell imaging [7]Polyethylene glycol Reflux 45.1 Cell imaging [8]Ammonium citrate Hydrothermal 13.5 - [9]maleic anhydride

and tetraethylenepentaminePyrolysis 21.0 Cu(II) detection

and cell imaging[10]

Sugarcane bagasse pulp Combustion 18.7 - [11]Thioglycolic acid & cadmium chloride

Reflux 47.0 Photovoltaic [12]

Mannose & ammonium citrate

Dry heating 9.80 Bacteria labeling [13]

Anthracite & H2O Ball mill and sonication

20.0 Photovoltaic [14]

Glucosamine & H2O Hydrothermal 16.8 Biosensor [15]L-glutamic acid Hydrothermal; 17.8 Fe(III) detection [16]

Melamine & L-arginine Microwave 58.4 Cr(+6) detection & drug delivery

This work

[5] P. Shen, J. Gao, J. Cong, Z. Liu, C. Li, J. Yao, Synthesis of Cellulose‐Based Carbon Dots for

Bioimaging, ChemistrySelect, 1 (2016) 1314-1317.

[6] J. Jana, M. Ganguly, B. Das, S. Dhara, Y. Negishi, T. Pal, One pot synthesis of intriguing

fluorescent carbon dots for sensing and live cell imaging, Talanta, 150 (2016) 253-264.

[7] A. Jaiswal, S.S. Ghosh, A. Chattopadhyay, One step synthesis of C-dots by microwave

mediated caramelization of poly (ethylene glycol), Chemical Communications, 48 (2012) 407-

409.

[8] W. Kong, R. Liu, H. Li, J. Liu, H. Huang, Y. Liu, Z. Kang, High-bright fluorescent carbon

dots and their application in selective nucleoli staining, Journal of Materials Chemistry B, 2

(2014) 5077-5082.

[9] Z. Yang, M. Xu, Y. Liu, F. He, F. Gao, Y. Su, H. Wei, Y. Zhang, Nitrogen-doped, carbon-

rich, highly photoluminescent carbon dots from ammonium citrate, Nanoscale, 6 (2014) 1890-

1895.

[10] N. Thongsai, Y. Nagae, T. Hirai, A. Takahara, T. Uchiyama, K. Kamitani, P. Paoprasert,

Multifunctional nitrogen-doped carbon dots from maleic anhydride and tetraethylenepentamine

via pyrolysis for sensing, adsorbance, and imaging applications, Sensors and Actuators B:

Chemical, (2017).

[11] S. Thambiraj, R. Shankaran, Green synthesis of highly fluorescent carbon quantum dots

from sugarcane bagasse pulp, Applied Surface Science, 390 (2016) 435-443.

[12] A. Arivarasan, G. Sasikala, R. Jayavel, In situ synthesis of CdTe:CdS quantum dot

nanocomposites for photovoltaic applications, Materials Science in Semiconductor Processing,

25 (2014) 238-243.

[13] C.-I. Weng, H.-T. Chang, C.-H. Lin, Y.-W. Shen, B. Unnikrishnan, Y.-J. Li, C.-C. Huang,

One-step synthesis of biofunctional carbon quantum dots for bacterial labeling, Biosensors and

Bioelectronics, 68 (2015) 1-6.

[14] S. Hu, Z. Wei, Q. Chang, A. Trinchi, J. Yang, A facile and green method towards coal-

based fluorescent carbon dots with photocatalytic activity, Applied Surface Science, 378 (2016)

402-407.

[15] S. Liu, N. Zhao, Z. Cheng, H. Liu, Amino-functionalized green fluorescent carbon dots as

surface energy transfer biosensors for hyaluronidase, Nanoscale, 7 (2015) 6836-6842.

[16] J. Yu, C. Xu, Z. Tian, Y. Lin, Z. Shi, Facilely synthesized N-doped carbon quantum dots

with high fluorescent yield for sensing Fe 3+, New Journal of Chemistry, 40 (2016) 2083-2088.