Supporting Information Time Resolved Emission Reveals Ensemble of Emissive States as the Origin of Multicolor Fluorescence in Carbon Dots Syamantak Khan, Abhishek Gupta, Navneet C. Verma and Chayan K. Nandi* School of Basic Sciences, Indian Institute of Technology Mandi, Himachal Pradesh, India Corresponding author *Chayan K. Nandi School of Basic Sciences, Indian Institute of Technology Mandi, Himachal Pradesh-1751, India Email: chayan@iitmandi.ac.in Tel. No. 19526747 Contents Materials and Method, Figures S1-S14 Tables S1-S2 1
Materials and Methods: Nomenclature Starting Material(s a Carbon dots 1 (CND1 Chitosan and PEG b Carbon dots 2 (CND2 Adenine sulphate c Carbon dots 3 (CND3 Sodium citrate and Sodium thiosulphate d Carbon dots 4 (CND4 Potato-dextrose agar Materials: All Glass wares were washed with aqua regia (3 HCl: 1 HNO 3, followed by rinsing several times with double distilled water. Chitosan, polyethylene glycol (PEG, potato dextrose agar, Sodium citrate, sodium thiosulphate were purchased from Sigma Aldrich. Adenine sulphate was purchased from HiMedia chemicals and NaOH purchased from Merck chemicals. Double distilled (18.3 mω deionized (DI water (Elga Purelab Ultra was used throughout the entire process. Synthesis of Carbon dot: 1. Synthesis of CND1: Chitosan (CHS gel (2% was prepared by dissolving 2 gm CHS in 99 ml of water in the presence of 1 ml of acetic acid, under vigorous stirring condition. 25 ml of PEG was dissolved in 75 ml water (25 % PEG solution. For synthesizing CND1, in a beaker 4.5 ml of the prepared CHS gel was mixed with 4.5 ml of the 25% of PEG. In the resulting solution 1 ml of 5 M NaOH was added. The mixture was heated inside a domestic microwave at 6 W powers for 3 minutes. The resulting material was dissolved in 15 ml of distilled water. The solution was filtered by Whatman filter paper. 1.5 ml of the filtered solution was centrifuged twice (Sorvall Lynx 6, thermo scientific at 23 rpm for 3 minutes each. The resulting pellets were removed each time and the final supernatant was collected. This solution was dialyzed for 2 days using 3-4 kd membrane against water. 2. Preparation of CND2: To synthesize CND2,.5gm of Adenine Sulphate (AdS and 1mM NaOH dissolved in 5ml of water. The mixture was heated inside a domestic microwave at 6 W powers for 3 minutes. The resulting material was dissolved in 15 ml of distilled water. The solution was filtered by Whatman filter paper. 1.5 ml of the filtered solution was centrifuged twice (Sorvall Lynx 6, thermo scientific at 23 rpm for 3 minutes each. The resulting 2
pellets were removed each time and the final supernatant was collected. This solution was dialyzed for 2 days using 3-4 kd membrane against water. 3. Synthesis of CND3: CND3 were synthesized by adding 25 ml of sodium citrate solution (.1 M to sodium thiosulfate (.1M solution and the mixture was heated at 6 W for 3 minutes using a normal microwave. The resulting material was dissolved in 15 ml of distilled water. The solution was filtered by Whatman filter paper. 1.5 ml of the filtered solution was centrifuged twice (Sorvall Lynx 6, thermo scientific at 23 rpm for 3 minutes each. The resulting pellets were removed each time and the final supernatant was collected. This solution was dialyzed for 2 days using 3-4 kd membrane against water. 4. Synthesis of CND4: For the synthesis of CND4, 2 gm of PDA broth was dissolved in 2 ml of distilled. The mixture was heated at 6 W for 3 minutes using a normal microwave. The resulting material was dissolved in 15 ml of distilled water. The solution was filtered by Whatman filter paper. 1.5 ml of the filtered solution was centrifuged twice (Sorvall Lynx 6, thermo scientific at 23 rpm for 3 minutes each. The resulting pellets were removed each time and the final supernatant was collected. This solution was dialyzed for 2 days using 3-4 kd membrane against water. UV-Vis Absorption spectroscopy The UV-Vis absorption spectra were recorded using Shimadzu UV-Vis 245 spectrophotometer. The spectra were collected using a 1ml quartz cuvette of 1 mm path length. All the measurements were repeated at least three times. Transmission Electron Microscope (TEM The particle size and dispersity of the synthesized CND were checked using a TECNAI G2 2 kv TEM (FEI, Electron Optics electron microscope with 2 kv input voltage. TEM grids were prepared by placing 5 µl diluted and well sonicated sample solutions on a carbon coated copper grid and evaporated the solution at room temperature completely. Precautions were taken to avoid contamination from various sources like dust particles. 3
Fourier Transform Infrared Spectroscopy (FTIR FTIR spectra were recorded using an Agilent FTIR spectrophotometer equipped with a horizontal attenuated total reflectance (ATR accessories containing a zinc selenide crystal and operating at 4 cm 1 resolution. The use of the spectral subtraction provided reliable and reproducible results. Atomic Force Microscopy (AFM AFM analysis was carried out using a Digital Instruments Bruker AFM. Standard Veeco tapping mode silicon probes were used for scanning the samples. Typically, aqueous suspensions of CND samples were dried on silicon substrate for 3 hours. Once dried, samples were placed on the AFM stage and scanned. Pertinent scanning parameters were as follows: Resonant frequency (probe: 6-8 khz; Tip velocity for all measurements are: (4 m/s for 2 m, (15 m/s for 5 m, (3 m/s for 1 m. Aspect ratio: 1:1; Resolution: 512 samples/line, 256 lines. X-ray photoelectron spectroscopy (XPS X-ray Photo-Electron Spectroscopy (XPS with Auger Electron Spectroscopy (AES module PHI 5 Versa Prob II, FEI Inc. and C6 sputter gun have been used for characterization and scanning the spectra from C 1s, N 1s, O 1s and S 2p region. Al K X-ray radiation was used as the source for excitation (1486.8 ev, 5 mm. Samples were loaded on copper strips, and surface adherence done by double sided adhesive tape. CND samples were prepared by concentrating the solution by centrifugation at 23 rpm, followed by drying. Thus obtained CND samples were used for recording XPS spectra. Steady state and Time Resolved Fluorescence Spectroscopy Steady state fluorescence was measured using Horiba Fluorolog-3 Spectrofluorometer. All the experiments were performed at room temperature. The fluorescence was measured in 1 ml quartz cuvette. The fluorescence lifetime and time resolved emission spectra (TRES were measured using Horiba Scientific Delta Flex TCSPC system with Pulsed LED Sources. Ludox has been used to calculate IRF for de-convolution of the spectral value. The photon decays in different channels were fitted tri-exponentially with a chi-squared value <1.2 in 4
order to calculate fluorescence lifetime. TRES was plotted by taking 5 slices of the 3D plot at 13 channel (~.35ns interval. Separation of two components of CND1: As the two components are formed at different stages of oxidative reaction, it was expected to have certain degree of dissimilarities in terms of their polarity. We used column chromatographic technique using silica gel to separate the mentioned two components with respect to polarity. In one of the four cases (CND1, we achieved enough purity with individual components for their characterization. As confirmed by thin layer chromatography (TLC, the blue (shorter wavelength coloured fraction was less polar and eluted with 5% ethyl acetate in hexane. The other fraction with a high degree of polarity, binds strongly with silica and can only be eluted with > 4% ethyl acetate in hexane. The absorption and fluorescence spectra show the photoluminescence properties of the individual components of CND1. The two components are contrasting in terms of their absorption maxima, emission maxima, excitation dependence and peak broadening (Figure S2. Mass spectroscopy shows the fraction has majorly single component with certain degree of purity. (Figure S13 However the molecular weight of the species was found to be in the lower range (<5D denying the possibility of a large carbon core. It should be noted that, the more polar multicolour fraction, which possibly was formed in a later stage of reaction, shows a lower molecular weight than the other one. FTIR spectra of the multicolour fraction show the presence of carboxylic acid (Figure S3. The broadened peak through 36-25 cm -1 region overlapped with C-H vibrations, a sharp peak at 1699 cm -1 and C-O stretch at 1248 cm -1 is visible. FTIR of blue fraction shows the presence of secondary Amide, characterized by NH stretching and Amide I / Amide II band. Stretching vibration of both methyl (296 & 2875 cm -1 for asymmetric & symmetric respectively and methylene (293 & 2855 cm -1 for asymmetric & symmetric respectively is visible in the spectrum. C-H (CH 2 bending vibration is present at 146 cm -1 along with other bending vibrations. In both of the fractions, CH set of vibration for CH 2 and CH 3 is below 3 cm -1, proving them to be aliphatic Compounds. 5
Counts Counts Counts Counts Supporting Figures: 14M 12M 1M 8M 6M 4M 2M 3 4 5 6 CND 1 14.M 12.M 1.M 8.M 6.M 4.M 2.M 39. 3 35 4 45 5 55 6 CND 2 3.5M 425 6M 3.M 2.5M CND 3 5M 4M CND4 2.M 1.5M 3M 1.M 2M 5.k. 3 35 4 45 5 55 6 65 1M 3 35 4 45 5 55 6 65 7 Figure S1: Multicolour excitation dependent fluorescence spectra of CND (CND1, CND2, CND3 and CND4 synthesized from four different starting materials. The intensity of the red shifted spectra drops with increasing wavelength. Sometimes a shorter wavelength excitation independent component is also observed (CND2 and CND4. 6
Intensity Intensity (a.u. Absorbance Intensity (a.u..4.2. 253nm 27nm 3 4 5 Wavelength nm. Blue Multicolour 3 28 26 24 22 2 18 16 14 12 Multicolour / Longer wavelength Component (Water 8 6 4 2 27nm 419nm 25 3 35 4 45 5 55 448nm 28 29 3 31 32 33 34 35 36 37 38 39 4 41 42 43 44 45 46 47 48 49 5 Absorption Spectra 8 6 4 2 Blue/Shorter wavelength Component (Hexane Ex_34nm Ex_35nm Ex_36nm Ex_37nm Ex_38nm Ex_39nm Ex_4nm 2 15 5 Blue / Shorter wavelength Component (Water 43nm 254nm Ex_28nm Ex_29nm Ex_3nm Ex_31nm Ex_32nm Ex_33nm Ex_34nm Ex_35nm Ex_36nm Ex_37nm Ex_38nm Ex_39nm Ex_4nm Absorption Spectra 35 4 45 5 55 6 25 3 35 4 45 5 55 463nm 6.4ns IRF 33nm 3.5ns 1 1 2 3 4 5 6 Time (ns Figure S2: UV-VIS Absorption and overall-fluorescence spectra of fixed blue colour (shorter wavelength and multicolour (longer wavelength fraction of CND1. The two components are contrasting in terms of their absorption maxima, emission maxima, excitation dependence and peak broadening. The spectrum of blue (shorter wavelength component is dependent on the polarity of the solvent. The fluorescent lifetime decay is clearly different for the two above components. Average lifetime is higher for longer wavelength fraction (~6.4ns compared to the blue fraction (~3.5ns. 7
% Transmittance % Transmittance 11 Blue fraction 9 8 7 296 2927 2872 146 1397 6 1696 1652 5 4 35 3 25 2 15 Wavenumber 11 Multicolour fraction 9 8 3258 1649 7 2963 286 2875 1457 1122 1248 6 2929 1699 138 5 4 35 3 25 2 15 Wavenumber Figure S3: FTIR spectra of shorter and longer wavelength fraction respectively. FTIR of blue (shorter wavelength fraction shows the presence of secondary amide, characterized by NH stretching and amide I /amide II band. Stretching vibration of both methyl (296 & 2875 cm -1 for asymmetric & symmetric respectively and methylene (293 & 2855 cm -1 for asymmetric & symmetric respectively is visible in the spectrum. C-H (CH 2 bending vibration is present at 146 cm -1 along with other bending vibrations. FTIR spectra of the longer wavelength fraction show the presence of carboxylic acid. The broadened peak through 36-25 cm -1 region overlapped with C-H vibrations, a sharp peak at 1699 cm -1 and C-O stretch at 1248 cm -1 is visible. In both of the fractions, CH set of vibration for CH 2 and CH 3 is below 3 cm -1, proving them to be aliphatic compounds. 8
Intensity (a.u. Intensity (a.u. Intensity (a.u. Intensity (a.u. 5 4 3 37nm 38nm 39nm CND1 3 25 2 15 39nm 38nm 37nm CND2 2 5 35 4 45 5 55 6 4 45 5 55 6 5 4 3 2 37nm 38nm 39nm CND3 3 25 2 15 5 37nm 39nm 38nm CND 4 4 45 5 55 6 35 4 45 5 55 6 Figure S4: The cooling effect on multicolour emission of CND. A new structured spectrum with many vibrational structures appears when cooled at19k. A little excitation dependent emission is observed in the spectrum. At low temperature and high viscosity, the solvent dynamics slows down further and majority of the photons completes their decay before the onset of the relaxation process. 9
Intensity Intensity Intensity Intensity 3 25 2 15 5 46nm 377nm ph 3 ph 7 ph 12 Excitation : 3nm CND1 35 3 25 2 15 5 373nm 47nm ph 12 ph 7 ph 3 Excitation : 3nm CND2 3 35 4 45 5 55 3 35 4 45 5 55 Wavelength(nm 4 35 3 25 2 15 377nm 46nm ph 3 ph 7 ph 12 Excitation : 3nm CND3 6 5 4 3 2 397nm 419nm ph3 ph7 ph12 Excitation : 3nm CND4 5 3 35 4 45 5 55 3 35 4 45 5 55 6 Wavelength(nm Figure S5: Effect of ph on excitation independent blue emission (excitation wavelength of 3nm CND. A blue shift was observed in reducing environment using NaBH 4 and at higher ph. 1
Intensity (a.u. Intensity (a.u. Intensity (a.u. Intensity (CPS / MicroAmps Intensity (CPS / MicroAmps Intensity (AU a c 1.M 5.M ex:4nm ex:35nm b. 7M 6M 5M 4M 3M 2M 1M 4 45 5 55 6 4 45 5 55 6 65 7 5 1 15 2 d e f 5 521 nm 5 nm 538 nm 3 4 5 6 7 Concentrated (1X 2 15 5 5 463 nm 3 4 5 6 7 Concentration (mg/ml 479 nm 521 nm Dituted (5X 35 Diluted 46 nm (1X 3 25 56 nm 2 15 5 3 4 5 6 7 Figure S6: Concentration dependent fluorescence of CND. (a-c The linear increase in intensity was observed below ~2mg/ml concentration with two different excitation wavelengths. The spectrum shifts towards lower wavelength with increasing brightness from (d-f where the sample was diluted serially (1X, 5X, 1X.. 11
kcounts / 1ms Intensity Intensity Intensity Intensity 7 6 5 4 3 2 379nm 46nm Original Ascorbic Acid Methyl Viologen Sodium Borohydride Nitric Acid Excitation: 3nm CND1 7 6 5 4 3 2 379nm 48nm NaBH4 original HNO3 Methyl Viologen Ascorbic Acid Excitation: 3nm CND2 9 8 7 6 5 4 3 2 3 35 4 45 5 55 6 386nm 44nm Ascorbic Acid Original Methyl Viologen NaBH4 HNO3 Excitation: 3nm 3 35 4 45 5 55 6 CND3 8 7 6 5 4 3 2 3 35 4 45 5 55 6 39nm 46nm Original NaBH4 HNO3 Ascorbic Acid Methyl Viologen Excitation: 3nm 3 35 4 45 5 55 Wavelength(nm CND4 Figure S7: Effect of chemical environment on excitation independent blue emission (excitation wavelength of 3nm CND. Quenching of fluorescence using methyl viologen is common in all CND. 3k 2k 1k 5 1 15 2 25 3 counts/ms 4k time (s 2k 1k counts/ms3k 5 1 15 2 25 3 time (s.3.2.1 1. 1.5 2. 2.5 3. Time (s Figure S8: Single molecule transients of CND shows one step bleaching. 12
C/s Absorbance a 3. 2.5 b 2. 1.5 1. 257 33 %T 165 137 3331 294 1574.5. 3 4 5 6 7 4 35 3 25 2 15 Wavenumber (cm-1 171 c 3 529.8 (O 1s 25 283.2 (C 1s 2 15 41(N 1s 5 8 6 4 2 Binding Energy (ev d Figure S9: Characterization of synthesized CND1 (a UV-Vis absorbance spectrum (b FTIR spectrum (c X-ray photoelectron spectrum (d AFM image with height profile 13
C/s Absorbance % T a.8.7.6 27nm b 12.5.4.3 261nm 8 6 33 2793 1416 936.2 4 312.1. 2 167 167 c 2 3 4 5 6 5 4 Wavelength nm. 532.1 (O 1s, Na 494 (Na 4 35 3 25 2 15 5 Wavenumber (cm-1 3 2 4.1 (N 1s 284.3 (C 1s 196 167 S 2s,2p Si 8 6 4 2 Binding Energy (ev d Figure S1: Characterization of synthesized CND2 (a UV-Vis absorbance spectrum (b FTIR spectrum (c X-ray photoelectron spectrum (d AFM image with height profile 14
Height Profile C/s % T Only CD (after Baseline correction a b 95 9 85 8 75 3229 165 658 994 535 1115 118 7 65 34 1594 6 4 35 3 25 2 15 5 Wavenumber (cm-1 139 c XPS of Cit -thiosulphate 5 4 53.1 (O 1s 494 (Na 3 2 4.3 (N 1s 283.7 (C 1s 162,166 S 2p Si 8 6 4 2 Binding Energy (ev d 5 4 3 2.8nm 4.3nm 3.4nm 2 1-1 2 4 6 8 Scale Figure S11: Characterization of synthesized CND3 (a UV-Vis absorbance spectrum spectrum (c X-ray photoelectric spectrum (d AFM image with height profile (b FTIR 15
Counts Absorbance a.7.6.5 b 12 8 243 164 831.4.3 256 29 %T 6 2954.2.1 366 4 2 1638. 3465 1372 2 3 4 5 6 7 8 4 35 3 25 2 15 Wavenumber (cm-1 c 531.5 (O 1s 283 (C 1s 397 (N 1s d 8 6 4 2 Binding Energy (ev Figure S12: Characterization of synthesized CND4 (a UV-Vis absorbance spectrum (b FTIR spectrum (c X-ray photoelectron 16 spectrum (d AFM image with height profile
Figure S13: Mass-spectra of shorter wavelength and longer wavelength fraction of CND1 respectively. The molecular weight of the species was found to be in the lower range (<5D denying the possibility of a large carbon core. It should be noted that, the more polar multicolour fraction, which possibly was formed in a later stage of reaction, shows a lower molecular weight than the other one. 17
Counts Counts Counts Counts a 357nm b 397nm 383nm 414nm 1 3 35 4 45 5 1 3 35 4 45 5 55 6 c 337 nm d 42nm 346 nm 3 35 4 45 5 46nm 3 35 4 45 5 55 6 Figure S14: Time resolved emission spectra (TRES show the spectral migration of fluorescence irrespective of different CND materials. (a CND1 (Purified Fraction 1 in water, (b CND2 (in water, (c CND3 (in water and (d CND4 (in water 18
Table S1: Life time components of two purified fraction of CND1 Blue Fraction (shorter wavelength Multicolour Fraction (Longer wavelength λ Em T1(ns T2(ns T3(ns 3 1.68 3.37 6.75 31 1.46 2.93 5.86 32 1.58 3.17 6.35 33 1.48(2% 2.97 (5% 5.94 (3% 34 1.65 3.31 6.62 36 1.63 3.26 6.53 37 1.61 3.23 6.45 38 1.75 3.51 7.2 39 1.77 3.55 7.11 4 1.78 3.57 7.15 λ Em T1(ns T2(ns T3(ns 49 1.96 3.91 7.83 415 2.19 4.38 8.77 421 2.19 4.38 8.77 427 2.29 4.59 9.19 433 2.34 4.68 9.37 439 2.54 5.8 1.18 445 2.63 5.27 1.53 451 2.75 5.5 11.1 457 2.79 5.58 11.16 463 2.81(19% 5.62 (54% 11.23 (27% 469 2.96 5.92 11.85 475 3.14 6.28 12.57 481 3.13 6.26 12.53 487 3.27 6.54 13.1 493 3.3 6.61 13.2 499 3.35 6.7 13.39 Table S2: Rotational correlation time of CND1 (purified at different ph Rotational Correlation Time (ns ph3 ph7 ph12 32nm 1.6 1.51 1.8 345nm.95.9.81 4nm.73.65.64 45nm.6.61.7 19