On the Molecular Origin of Photoluminescence of Nonblinking Carbon

Apr 11, 2017 - The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b02433. 1H NMR and MAL...
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On the Molecular Origin of Photoluminescence of Nonblinking Carbon Dot Ananya Das, Venkatesh Gude, Debjit Roy, Tanmay Chatterjee, Chayan K. De, and Prasun K. Mandal* Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur, West Bengal 741246, India S Supporting Information *

ABSTRACT: The molecular origin of the photoluminescence (PL) of carbon dots (CDs) is not fully understood. In this article it is shown that CDs are composed of aggregated 2pyridone derivatives employing π−π stacking and H-bonding, etc. The PL quantum yield of this CD is quite high in aqueous medium (∼75%). Unlike literature reports the PL emission maximum of this CD is excitation wavelength independent, and PL decay follows a single-exponential decay equation. These CDs have a long PL lifetime (from ∼10 to 15 ns), so that solvation is complete before emission. The extent of trap states could be reduced quite significantly. A high PLQY and long and single-exponential PL lifetime and it’s polarity dependence would make this CD a suitable probe for FRET and FLIM. It could be shown that unlike literature reports this CD as a single particle does not blink. Unlike literature reports where CDs are bleached within a few seconds these CDs at the single-particle level are alive for about a few minutes. All these aggregation-induced much improved optical properties will make this CD a suitable optical emitter at the ensemble as well as single-particle level toward bioimaging. As the molecular origin is now known several optical properties can now be tuned.

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((F)RET), fluorescence lifetime imaging microscopy (FLIM), etc. It would be easier to calculate dynamical time constants of processes like FRET and FLIM when CDs exhibit singleexponential decay behavior. Moreover, even for ensemble measurements in a cellular environment it is necessary to have CDs with a very high PLQY in aqueous medium. Thus, for fruitful single-particle tracking with CDs, it is necessary to make CD with high PLQY and nonblinking at the single-particle level which does not get bleached within a few seconds. Thus, the quest for understanding the molecular origin or composition of CDs with high PLQY in aqueous medium is highly indispensable. Only when the composition or molecular origin of CDs will be known it will be possible to design CDs with desired optical properties. CDs should possess long singleexponential PL lifetime so that solvation is complete before PL and then CDs can act as a better donor in FRET experiments. If the long PL lifetime is dependent on polarity then CDs could act as a better optical probe for FLIM. If CDs with a long, single-PL lifetime and without trap states could be prepared then CDs will not show excitation wavelength-dependent PL emission and PL decay. Possibly blinking can be stopped under that condition. It is also desirable that CDs should be alive for a longer time at the single-particle level without blinking, so that

INTRODUCTION Carbon dots (CDs) are gaining increasing importance as photoluminescent markers over many other nanomaterials in bioimaging;1−9 however, still the molecular origin for its photoluminescence (PL) is not completely understood. Reported results show that the PL quantum yield of most of the CDs is low10−17 and the PL emission maximum is dependent on excitation wavelength.3−9,12−26 It has been mentioned that incomplete solvation is the cause of the excitation-dependent PL of CDs.26 In most of the literature reports, however, the PL decay of CD has been shown to be multiexponential in nature.5,6,27,28 Single-particle studies have been carried out to show that similar to QDs, CDs exhibit blinking behavior and a single CD gets bleached within a few seconds.29 However, what is intriguing is the fact that a detailed investigation to comprehend what is the molecular origin of PL of CDs is quite rare.1 There is no reason to believe that CDs prepared from common precursors like citric acid would fluoresce as there is no aromatic moiety or extended conjugation in citric acid. Hence, it is necessary to investigate in detail why CDs would emit. As CDs have been shown to blink with a very short on time and mostly off time and get bleached within a few seconds, CDs are not suitable for singleparticle tracking. Moreover, multiexponential PL decay behavior makes ultrafast dynamical analyses quite difficult, especially in experiments like resonance energy transfer © 2017 American Chemical Society

Received: March 15, 2017 Revised: April 7, 2017 Published: April 11, 2017 9634

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Time-Resolved Measurements. Fluorescence lifetime measurement at the picosecond to nanosecond time domain was carried out using a time-correlated single-photon counting (TCSPC) spectrometer (Horiba Jobin Yvon IBH) equipped with a diode laser (λex = 377 nm) that was used as the excitation source and an MCP photomultiplier tube (PMT) (Hamamatsu R3809U-50 series) as the detector. The width (fwhm) of the instrument response function (IRF), which was limited by the fwhm of the exciting pulse, was less than 100 ps for a 377 nm excitation source. IRF was recorded using a scatterer (dilute solution of Ludox in water). Nonlinear leastsquares iterative reconvolution procedure using IBH DAS6 (version 2.2) was employed to fit the fluorescence decay curve using a proper exponential decay equation. The quality of the fit was assessed from the χ2 values and the distribution of the residuals. Single-Particle Measurements. Carbon dots were immobilized on a cleaned glass coverslip by spin coating in poly(methyl methacrylate) (PMMA) matrix. These single carbon dots were probed using home-built total internal reflection fluorescence (TIRF) and confocal microscopy. Excitation of the sample was achieved through an oil-immersion objective (Zeiss, PlanApo, 100×, NA 1.46 for TIRF and Zeiss, PlanApo, 63×, NA 1.00 for confocal) using a 402 nm laser (COHERENT CUBE). To excite the sample laser power was maintained at 0.5 mW (measured before objective) in both cases. The PL was also collected through the same objective. For TIRF microscopy signals were detected with an EMCCD camera (ANDOR iXON3) with 100 ms integration time and a suitable EM gain. The intensity profiles were analyzed within the region of interest of the sample. All data from TIRF microscopy were analyzed using Andor Solis software. (A movie created using TIRF mode is also uploaded as Supporting Information.) For confocal microscopy PL emission spectra from carbon dots were recorded using a Princeton Instruments Acton SP2500 spectrograph (equipped with a grating having groove density 150g/mm and blaze wavelength 500 nm) coupled with an EMCCD camera (ProEM 512). Integration time was chosen to be 1 s with a suitable EM gain. All data from confocal microscopy were analyzed using WinSpec software.

it’s usability for single-particle imaging/tracking will be improved. In this manuscript we could achieve CDs with all these desired superior optical properties. The molecular origin of the CDs could be understood.

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METHODS Materials. Ethanolamine and citric acid monohydrate were procured from Sigma-Aldrich and used as received. Sodium hydroxide pellets were procured from Sisco Research Laboratories. For spectroscopic studies acetonitrile, methanol, and water were of HPLC grade. Synthesis and Purification of Carbon Dot. Citric acid monohydrate and ethanolamine (1:3 molar ratio) are refluxed at 180 °C for 1 h. A dark brown product is formed. The product is then fractionalized by column chromatography using 10:90 (MeOH:DCM). The most fluorescent spot in the TLC plate was separated. Finally, the purified product was dried under high vacuum (10−3 mbar). Thin layer chromatography (TLC) analysis was performed on a Merck Kieselgel 60 F254 plate using 100−200 mesh size silica gel). Characterization Techniques. 1H NMR spectra were recorded in D2O and DMSO-d6 in a JEOL (400 MHz) spectrometer. IR spectra were recorded on a Bruker (model ALPHA) FT-IR spectrometer. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was carried out on a Bruker ultrafleXtreme instrument equipped with a smart beam-II laser in the reflector mode and 22 kV acceleration voltage. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as matrix. The XRD patterns were collected using the Rigaku-smartLab diffractometer attached with D/tex ultradetector and Cu Kα source operating at 70 mA and 35 kV. Scan range was set from 10° to 80° 2θ with a step size of 0.02°, and scan speed was 3°/min. The sample was gel type and spread evenly on a quartz slide. The Raman spectroscopic measurement was carried out using a Horiba Jobin Yvon LabRam HR800 Raman spectrometer. The sample was excited with an Ar+ ion (488 nm) laser of power 10 mW, and Raman signal was collected in back scattering geometry with an 1800 g/mm grating. TEM. Transmission electron microscopy (TEM) was taken on a UHR-FEG_TEM, JEOL, JEM 2100 F model using a 200 kV electron source. Samples were prepared by drop casting a methanolic solution of the nanoparticles on a carbon-coated copper grid, and the grid was dried under air. Steady State Measurements. Steady state absorption and corrected emission spectra were recorded in a Carry 300 Bio UV−Vis spectrophotometer and Fluoromax-4 Horiba Jobin Yvon spectrofluorimeter, respectively. Quantum yield determination was accomplished by comparing the wavelengthintegrated intensity of the carbon dot to that of the standard (quinine sulfate). Fluorescence quantum yields were calculated with solutions having absorbance (OD) less than 0.07 to avoid concentration related discrepancies. Both the compound and the reference were excited at 350 nm. Quantum yield was calculated using the following equation Q = QR

ODR I n2 OD IR nR2

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RESULTS AND DISCUSSION Formation of Unsaturated Aromatic Unit from Saturated Precursors. As a first step it was necessary to understand how unsaturation develops from saturated precursors (citric acid and ethanolamine). The mechanism of formation of fluorophore (2-pyridone derivative) has been described in Figure 1. Spectroscopic Characterization. FTIR analysis of purified CDs is shown in Figure 2a, which confirms the presence of functional groups such as −O−H (stretching, in the region of about 3150−3550 cm−1), sp2-C−H (stretching at 3100 cm−1), and sp3-C−H (asymmetric and symmetric stretching at 2940 and 2882 cm−1, respectively). The strong signals at 1635 and 1540 cm−1 could be attributed to >CO stretching of −CONH and −N−H bending mode of secondary amide, respectively. The signal at 1422 cm−1 could be assigned to stretching band of sp2 carbon atoms (−CC−); the peaks at 1355, 1231, and 1055 cm−1 could be assigned to different modes of −C−N− and −C−O− groups present in CDs. The 1H NMR spectrum of purified CDs was recorded in DMSO-d6 solvent (2.5 ppm) and is shown in Figure S1, Supporting Information. The chemical shift (δ) values of triplet

(1)

where Q, OD, I, and n stand for quantum yield, absorbance, integrated luminescence intensity, and refractive index of the solvents, respectively. Subscript R stands for reference (standard dye). 9635

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1:3) is different from the work reported by Yubin et al. (citric acid:ethanolamine = 1:1).31 Hence, it is expected that the fluorophore which is formed in the present case (1b in Figure 1) will be different from the fluorophore reported by Yubin et al. The AB quartet type of 1H NMR signal (2.82−2.60 ppm) and mass spectral peak at 322 (m/z value) (Figure 2C) indicate the presence of condensed product (1a) in the CDs (Figure. 1). Krysmann et al. also has shown formation of the same condensed product from the same starting materials with the same stoichiometric ratio.32 However, no further chemical reactions were shown, which can lead to formation of fluorophore from the condensed product. We have shown that the condensed product further undergoes nucleophilic addition followed by dehydration reaction leading to formation of 6-membered fluorophore (2-pyridone derivative) (Figure 1). Two 1H NMR singlet peaks at 5.94 and 5.86 ppm (Figure. 2b) and a mass spectral peak at 286 m/z (Figure. 2c) indicate the presence of 2-pyridone derivative (fluorophore) in the CDs. We would like to mention here that Krysmann et al. showed 1H NMR only up to 4 ppm, so it was not possible to know whether they obtained two 1H NMR peaks at 5.94 and 5.86 ppm which we obtained. Here it is worth mentioning that although Krysmann et al. and we started from same starting material with the same stoichiometric ratio same temperature, however, the duration of the reaction was different (Krysmann et al. did the reaction for 30 min; we did it for 1 h). We would also like to mention that the spectroscopic properties (absorption maximum, quantum yield, etc.) we obtained are also significantly different from what is reported by Krysmann et al.32 Thus, it is expected that the product Krysmann et al. obtained and what we obtained is different (see later for details). We obtained the molecular weight of chemical species of CDs using the matrix-assisted laser desorption ionization (MALDI) technique with α-cyanohydroxycinnamicacid (CHCA) as matrix. The mass spectrum and m/z values of CHCA matrix are shown in Figure S3 and Table S1, Supporting Information. The magnified portion of the mass spectrum of CDs is shown in Figure 2c. Peaks related to CDs have been extracted (after comparison with the CHCA matrix peaks) and are highlighted in red (Figure 2c, Table S1, Supporting Information). The molecular weight of the formed condensed product (1a) (MW = 321) (Figure 1) exactly matches with the observed mass spectroscopic signal (322, proton adduct). The peaks at 344 [M − Na+] and 360 [M − K+] m/z correspond to its sodium and potassium ion adduct. The peak at 286 m/z corresponds to fluorophore [M − H+] compound (product 1b in Figure 1). Peaks at lower 286 m/z correspond to fragmented species (shown in Figure 1) of fluorophore compound. Thus, from the above spectroscopic analyses it can be concluded that as-prepared CDs are composed of both 2-pyridone derivative and condensed product reported by Krysmann et al. The mass spectra of CDs with CHCA matrix have also been shown in Figure S3, Supporting Information. No prominent signal is observed above 500 m/z, indicating that CDs are not composed of any polymeric structures. The presence of initially formed condensed product (1a in Figure 1) confirmed by NMR and MS perhaps forms the shell surrounding the fluorophore present in the core (Figure S4, Supporting Information). The XRD pattern of purified CDs exhibits a broad peak centered at ∼22° and a weak peak centered at ∼40° (Figure 2d). This XRD pattern matches well with the literature reports of CDs synthesized from various precursors.33,1,18 The

Figure 1. Transformation of aliphatic precursor molecules to aromatic moieties and their possible fragmentation pathways.

peaks in the region from 8.307 to 7.68 ppm correspond to Hbonded amide protons, and those are in a fashion like −CO− NH−CH2−. The magnitudes of coupling constants of all triplet peaks are in the range of 5−7 Hz, indicating spin−spin interaction of amide proton (−NH) with adjacent methylene protons. Peaks at 6.33 and 5.72 ppm correspond to hydroxyl (−OH) protons, and the peaks at 5.87 and 5.63 ppm correspond to protons attached to sp2 (−CC−) carbon. A small peak is observed at 4.7 ppm corresponding to the proton attached to nitrogen. The triplet peaks in the region from 4.90 to 3.57 ppm and multiplet peaks from 3.28 to 3.08 ppm correspond to protons of the methylene group (−CH2) which is attached to oxygen and the nitrogen atom in the ethanolamine moieties. The AB quartet type of signal is from 2.54 to 2.41 ppm corresponding to methylene group protons (diastereotopic protons) of citric acid.30 The 1H NMR spectrum of the same sample was recorded in D2O solvent (4.7 ppm) and is shown Figure 2b. Here, it can be seen that when compared with the previous spectrum (in DMSO-d6) amide and hydroxyl functional group protons signals (9−6 ppm shown in Figure S2 in Supporting Information) disappeared, indicating deuterium exchange occurred. The peaks at 5.94 and 5.86 ppm correspond to protons attached to the sp2 (−C C−) carbon. The triplet peaks and AB quartet type of peaks in the region from 4.19 to 2.60 ppm correspond to the methylene group protons of the ethanolamine moiety and citric acid, respectively. It is worth mentioning that the molar ratio of the precursors used in the present case (citric acid: ethanolamine = 9636

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Figure 2. Spectroscopic characterization of purified CDs: FTIR (a), 1H NMR (b), MALDI-mass (c), XRD (d), and Raman (e).

consonance with the value obtained from XRD. The FFT pattern from the HRTEM image is shown in Figure 3d. Distinct lines (Figure 3c) and bright dots (Figure 3d) clearly demonstrate the crystalline nature of the CDs. Optical Spectroscopic Results. Evidence of Single Type of Fluorophores from Spectroscopic Characterization at the Ensemble Level. It has been shown above that 2-pyridone derivatives form an aggregated structure through π−π stacking as well as H-bonding. Theoretical calculations using the Gaussian 09 package show that the absorption maximum of the 2-pyridone derivative is around 353 nm (Figure S7, Supporting Information). Experimentally it has been observed that the absorption maximum of CD is at 360 nm. It has been shown in the literature that 2-pyridone derivatives exhibit emission maximum at around 380 nm.34−36 Experimentally we observed that the emission maximum of CD is at around 450 nm. Thus, we observed an emission maximum which is 80 nm Stokes shifted. As there is no significant change in the dipole moment (see orbital electron cloud picture (HOMO−LUMO plot) in Figure S7, Supporting Information) from the ground state to the excited state, no significant red shift of the emission

weak peak corresponds to the (100) plane and broad peak corresponds to the (002) plane of graphite. The broad peak corresponds to a d spacing of ∼0.38 nm. This value is larger than that of the (002) planes of bulk graphite (0.33 nm). The recorded Raman spectrum of CDs is shown in Figure 2e. The broad peak in the region 1200−1800 cm−1 with a maximum at 1580 cm−1 corresponds to the −CC− stretching vibration of fluorophores of the CD. Similar broad Raman bands have been reported in the literature when CDs are synthesized using citric acid and ethylenediamine.33,18 The TEM image of the CDs is shown in Figure 3a. The size distribution of CDs is shown in Figure 3b. As can be seen from this figure the size of CDs is less than 8.5 nm, similar to what has been reported in the literature.1 The AFM image and the size distribution from AFM images of the CDs is shown in Figure S5, Supporting Information. The size of the CDs obtained from the AFM image is around 7 nm. Thus, the size obtained from TEM and AFM images matches quite well. The HRTEM image of the CDs is shown in Figure 3c. Distinct planes could be observed in the HRTEM image with an interplanar spacing of 0.38 nm (Figure 3c). This value is in 9637

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Figure 3. TEM image of the CDs (a), size distribution from TEM (b), HRTEM image of the CDs (c), and fast Fourier transform pattern of HRTEM image (d).

Figure 4. Absorption spectra of CD in three different solvents (a), emission spectra in aqueous medium at different excitation wavelength (b), PL decay of CD in aqueous medium (c), photostability of CD (d), emission spectra of CD at different pH (e), and PLQY of CD at different pH (f).

maximum is expected; indeed, we could observe no change of the emission maximum on changing the polarity or proticity of the medium (on moving from ACN to MeOH to water) (Figure S8, Supporting Information). Literature results show that the excited state lifetime of 2-pyridone derivatives is around 5 ns in alcoholic solvents and around 8 ns in aqueous medium.34−36 Experimentally it has been observed that our CDs exhibit single-exponential PL decay with an excited state lifetime of CD around 10 ns in methanol and around 15 ns in aqueous medium (Figure S9 in Supporting Information). Thus, the fluorescence lifetime of our CD (similar to emission

maximum) is higher than literature reports for 2-pyridone derivatives (Figure 4, Table 1). Single-exponential PL decay indicates that only one type of fluorophore is present. Thus, we are convinced from characterization (NMR, IR, MS, etc.) as well as from optical spectroscopic analyses that the 2-pyridone derivative is the fluorophore responsible for PL of CD. In comparison to literature reports of 2-pyridone derivatives, the emission maximum as well as fluorescence lifetime of our CDs are higher. One possible way of explaining this optical behavior is to consider aggregation-induced emission.33 It has been reported in the literature that in comparison to monomer a 9638

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The Journal of Physical Chemistry C Table 1. Optical Properties of CD at the Ensemble Level PL decay

a

solvent

polarity parametera ENT

proticity parametera (α)

λabs (nm)

λems (nm)

acetonitrile

0.460

0.19

360

448

methanol

0.762

0.98

360

450

water

1.000

1.17

360

450

λex (nm)

λem (nm)

τ (ns)

χ2

0.50

377

0.50

377

410 450 530 410 450 530 410 450 530

9.45 9.40 9.60 11.50 11.50 11.50 15.90 15.80 15.50

1.1 1.2 1.1 1.1 1.1 1.1 1.0 1.1 1.1

PLQY (Φ)

∼0.75 (pH 4−10)

377

Reichardt, C. Solvents and Solvent Ef fects in Organic Chemistry, 3rd ed.; WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2003.

of our CD is comparable to CdSe/CdS type of core/shell QDs (Figure 4d; for details see the Supporting Information). As saturated alkyl chain bearing −OH groups remain outward and are not conjugated to the fluorophore unit (2-pyridone) and the PL emission is coming from an aggregated structure, the PL behavior of CDs should not depend on pH of the medium. Thus, absorption and emission maxima are expected to be independent of pH. This is what has been observed experimentally (see Figure S11, Supporting Information, and Figure 4e). Moreover, under this condition, the PLQY should also be independent of pH. As can be seen from Figure 4f, the PLQY value remains close to 0.75 in the pH range from 4 to 10. However, in extreme acidic or alkaline condition the PLQY value drops perhaps indicating under harsh condition of pH (say pH ≈ 4 and lower or 10 and higher) protonation or deprotonation of different functional groups is taking place. For example, ring nitrogen or conjugated amine (pKa ≈ 4 or lower) may get protonated at pH 4 or lower. Single-Particle Investigations: Direct Evidence of CDs as Aggregated Same Dye Structure and without Surface Traps. Single-particle spectroscopic measurements are known to depict those photophysical properties which are often masked in ensemble measurements.39,40 Single-particle time trace as well as single-particle PL emission of CD embedded in PMMA could be recorded from these single CD particles (Figure 5).

significantly large Stokes shift of the emission (a maximum of 150−160 nm) has been observed in the case of carbon dots33 and some other nanomaterials.37 Quite interestingly, the fluorescence quantum yield as well as fluorescence lifetime of our CD have been observed to increase with increasing polarity and proticity of the medium (on moving from ACN to MeOH to water). The fluorescence quantum yield has increased from 50% to 75% on changing the solvent from ACN to MeOH to water. Similarly, the single-exponential fluorescence lifetime has increased from 9.5 to 11.5 to 15.9 ns on changing the solvent from ACN to MeOH to water. Exactly the opposite behavior is observed for most of the dyes reported in the literature.38 We have already shown that 2-pyridone derivatives are aggregated using π−π interaction and a H-bond network in CD (Figure S7, Supporting Information). For very few molecules the excited state lifetime has been reported to increase with increasing polarity or proticity of the medium. This kind of interesting optical behavior has been ascribed to increased aggregation or rigidity of the fluorophore. In protic solvents intermolecular Hbonding reduces out of plane vibrations34 and hence decreases the nonradiative pathway (internal conversion). A similar result has been reported in the literature.35 In the present case, 2pyridone derivative has been shown to form aggregates through H-bonding and π−π interaction. When the solvent polarity and proticity increase the extended 3-D H-bonding network becomes more pronounced, thereby increasing the aggregation/rigidity of the system. Hence, both the quantum yield as well as the fluorescence lifetime have been observed to increase. Polarity/proticity-dependent PL lifetime is a very important observation pointing to the fact that this CD could be used for fluorescence lifetime imaging (FLIM) experiments to probe differential polarity/proticity regions of a biological environment, say a live cell. Thus, aggregated 2-pyridone derivatives can be considered to be present in the core surrounded by the initially formed condensed product (1a) shell. We have clearly shown through NMR and mass spectra that although Krysmann et al. started the reaction with the same starting material with the same stoichiometric ratio, because the synthesis was of different time duration the product they obtained and the product we obtained are different. The fluorescence quantum yield of the product that Krysmann et al. obtained is 45%, and what we have obtained is nearly 75%. This clearly demonstrates that the product Krysmann et al. obtained and what we obtained are distinctly different. The photostability of the present CDs is better than bare CdSe QDs. Under the same illumination, after 300 min, the intensity of bare CdSe QDs decays to ∼30% whereas the intensity of our CDs is the range of ∼90%. The photostability

Figure 5. Gradual decrease of intensity from single particle, and ultimately the particle gets bleached (a); distribution of bleaching time of single CD particle (b); time-dependent single-particle emission spectra from single CD particle (c). 9639

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Three possibilities may arise: (i) If CDs are made up of only one dye we should observe single-step bleaching as observed in most of the literature reports.29 (ii) If CDs are similar to semiconductor QDs with trap states we should observe blinking behavior.41 Similar results have been reported in the literature for CDs.29,41,42 (iii) If CDs are made up of several aggregated dyes without any trap state then we should observe gradual decay of single-particle time trace intensity without blinking. From Figure 5a (see also a movie provided in the Supporting Information) it is noted that there is a gradual decrease of intensity with respect to time, and finally, a single CD particle bleaches (intensity becomes similar to background). This means the third possibility mentioned above is actually true and neither of the first two possibilities is feasible. Thus, singleparticle investigations provide direct evidence that CDs are made up of aggregated dyes and without the existence of trap states. From about 100 single CD particles we could obtain a distribution of bleaching times (Figure 5b) with a maximum of around 120 s. Thus, our CDs are much more stable in comparison to normal dyes at the single-molecule/particle level.29,39−44 This is expected as single CDs are made up of aggregated dyes. We will also like to mention here that our CDs are nonblinking and much more photostable at the single-particle level than the CDs reported in the literature where CDs have been shown to blink and getting bleached within a few seconds.29 For our CDs at the single-particle level it has been observed that PL emission intensity gradually decreases with time and no new band appears Figure 5c. This means the emission maximum remains the same. Thus, from Figure 5a and 5c we could provide direct evidence to conclude that these CDs are composed of aggregated dyes (2pyridone derivative in this case) and without any surface traps. Unlike bare CdSe QDs45,46 or other CDs, our CDs do not exhibit blinking. Therefore, CDs are better candidates than bare CdSe core QDs for single-particle tracking. However, there is a scope of further improvement of the photostability of CDs at single-particle level.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b02433. 1 H NMR and MALDI mass spectra, AFM details, computational calculation details, optical behavior of CD, photostability and pH-dependent measurement details (PDF) Movie showing the gradual decrease of PL intensity of CD at the single-particle level (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Prasun K. Mandal: 0000-0002-5543-5090 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.K.M. thanks IISER-Kolkata for financial help and instrumental facilities. Support from the Fast-Track Project (SR/FT/CS52/2011) of DST-India is gratefully acknowledged. A.D., D.R, and T.C. thank CSIR; V.G. and C.K.D. thank IISER-Kolkata for their fellowships.

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REFERENCES

(1) Baker, S. N.; Baker, G. A. Luminescent Carbon Nanodots: Emergent Nanolights. Angew. Chem., Int. Ed. 2010, 49, 6726−6744. (2) Jin, H.; Heller, D. A.; Sharma, R.; Strano, M. S. Size-Dependent Cellular Uptake and Expulsion of Single-Walled Carbon Nanotubes: Single Particle Tracking and a Generic Uptake Model for Nanoparticles. ACS Nano 2009, 3, 149−158. (3) Fan, R. J.; Sun, Q.; Zhang, L.; Zhang, Y.; Lu, A. H. Photoluminescent carbon dots directly derived from polyethylene glycol and their application for cellular imaging. Carbon 2014, 71, 87− 93. (4) Karthik, S.; Saha, B.; Ghosh, S. K.; Pradeep Singh, N. D. Photoresponsive quinoline tethered fluorescent carbon dots for regulated anticancer drug delivery. Chem. Commun. 2013, 49, 10471−10473. (5) Xu, Y.; Wu, M.; Liu, Y.; Feng, X.-Z.; Yin, X.-B.; He, X.-W.; Zhang, Y.-K. Nitrogen-Doped Carbon Dots: A Facile and General Preparation Method, Photoluminescence Investigation, and Imaging Applications. Chem. - Eur. J. 2013, 19, 2276−2283. (6) Sun, D.; Ban, R.; Zhang, P. H.; Wu, G. H.; Zhang, J. R.; Zhu, J. J. Hair fiber as a precursor for synthesizing of sulfur- and nitrogen-codoped carbon dots with tunable luminescence properties. Carbon 2013, 64, 424−434. (7) Xu, Z. Q.; Yang, L. Y.; Fan, X. Y.; Jin, J. C.; Mei, J.; Peng, W. Low temperature synthesis of highly stable phosphate functionalized two color carbon nanodots and their application in cell imaging. Carbon 2014, 66, 351−360. (8) Ruan, S.; Qian, J.; Shen, S.; Zhu, J.; Jiang, X.; He, Q.; Gao, H. A simple one-step method to prepare fluorescent carbon dots and their potential application in noninvasive glioma imaging. Nanoscale 2014, 6, 10040−10047. (9) Nandi, S.; Malishev, R.; Parambath Kootery, K.; Mirsky, Y.; Kolusheva, S.; Jelinek, R. Membrane analysis with amphiphilic carbon dots. Chem. Commun. 2014, 50, 10299−10302. (10) Meiling, T. T.; Cywinski, P. J.; Bald, J. White Carbon: Fluorescent carbon nanoparticles with tunable quantum yield in a reproducible green synthesis. Sci. Rep. 2016, 6, 28557. (11) Kwon, W.; Do, S.; Kim, J.-H.; Seok Jeong, M.; Rhee, S.-W. Control of Photoluminescence of Carbon Nanodots via Surface

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CONCLUSIONS In conclusion, we show that CDs are made up of aggregated 2pyridone derivatives employing π−π stacking and H-bonding. It could be shown that the PL emission maximum of CDs is excitation wavelength independent. These CDs are of very high PLQY (∼75%) in aqueous medium. It could also be shown that unlike literature reports the PL decays of CDs in all three solvents, viz, acetonitrile, methanol, and water, are single exponential. These CDs possess a long PL lifetime (from ∼10 to 15 ns). The extent of trap states could be reduced quite significantly. The single-exponential lifetime of CD has been shown to be dependent on the polarity of the medium, and the PL lifetime increases from 9.5 in acetonitrile to 11.5 ns in methanol to 15.8 ns in aqueous medium. The long and singleexponential PL lifetime and its polarity dependence would make this CD a suitable probe for FRET and FLIM, respectively. It could be shown that as single emitters CDs do not blink and are alive for a few minutes. All these much improved and highly desired optical properties will make this CD a better optical emitter in comparison to other CDs at the ensemble as well as single-particle level toward bioimaging. As the molecular origin is now known several optical properties can be tuned. 9640

DOI: 10.1021/acs.jpcc.7b02433 J. Phys. Chem. C 2017, 121, 9634−9641

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DOI: 10.1021/acs.jpcc.7b02433 J. Phys. Chem. C 2017, 121, 9634−9641