Facile Conversion of Toxic Cigarette Butts to N,S-Codoped Carbon Dots and Their Application in Fluorescent Film, Security Ink, Bioimaging, Sensing and Logic Gate Operation.

The present work is emphasized on converting toxic cigarette butts (CBs) into highly fluorescent N,S-codoped carbon dots by a facile hydrothermal approach and exploring their multiple applications. The as-produced carbon dots (CBCDs) exhibited bright and stable fluorescence with a quantum yield of 26% and used as a label-free probe for "on-off-on" sequential detection of Fe3+ and ascorbic acid (AA). The fluorescence of CBCDs can be significantly quenched by Fe3+ ions through static quenching and restored upon the subsequent addition of AA due to the reduction of Fe3+ to Fe2+ by AA. This nanoprobe presented great selectivity and excellent sensitivity to Fe3+ and AA with a detection limit of 0.13 and 0.2 μM, respectively. Furthermore, the nanoprobe was extended to biosystem (intracellular detection) and successfully applied for the detection of Fe3+ in real water (tap, bore, and pond) and AA in biological samples (human urine and serum). In addition, we have constructed an IMPLICATION logic gate based on these unique sensing characteristics. The "visible-invisible" and "UV-visible" property explored their use as invisible ink for security applications. Furthermore, highly photostable fluorescent polymer films were prepared by incorporating CBCDs in poly(vinyl alcohol). It is anticipated that the strong and stable fluorescence emission nature of these films might find direct or indirect applications in various optical/optoelectronic devices, ranging from fluorescent displays to light-emitting diodes.

Introduction

One of the major goals of United Nations Development Programme 2030 is responsible consumption and production of resources. This can be achieved only by efficient management of available resources and proper disposal of toxin class="Gene">c waste and pollutants. According to a recent report given by the World Health Organization (WHO 2017), cigarette butts (CBs) and other tobacco wastes make up the major number of individual pieces of litter in the world accounting to more than 40% of all items collected during urban cleanups.[1] These butts are washed down by drains and eventually make their way to lakes, rivers, and oceans. Ocean Conservancy also reported similar results in this regard.[2] Most of these CBs are made up of cellulose acetate, which possess poor photo- and biodegradable properties, making them persistent in the environment. Furthermore, the toxic leachates of CBs pose a severe threat to aquatic life (both marine and freshwater).[3,4] According to a recent report, CBs are potential sources of nicotine contamination in urban water and can be a major threat to the water quality.[5] Hence, effective management of these wastes is essential. Conventional disposal methods like landfilling or incineration are neither universally sustainable nor economically feasible for this purpose.[6]

Several researchers have made significant exertions in this concern. Mohajerani et al. made notable contributions by incorporating CBs in fired clay bricks[6] and asphalt concrete.[7] These methods offer reduction of large quantities of CBs. However, transforming them into desired products is considered to be a more promising way, as it can offer value-added products besides waste management. But only a few efforts were made in this regard. Yi et al.[8] produced high-performing energy-storage material from used cigarette filters. Wei et al.[9] reported an eco-friendly approach for extracting n class="Chemical">cellulose acetate from CBs to construct a cellulose-based membrane separator for high-performance lithium-ion batteries. Recently, Yilin et al.[10] have reported the synthesis of carbon dots (CDs) from cigarette filters and their application for fluorescence detection of Sudan I. However, sufficient characterization data was not presented (chemical composition of formed CDs is not studied) and the possible formation mechanism of CDs (how the water-insoluble cellulose acetate fibers are converted to water-soluble CDs) was not explained.

Fluorescent CDs are a new member of nanon class="Chemical">carbon family that comprise discrete, quasi-spherical nanoparticles with sizes below 10 nm.[11] After their serendipitous discovery from carbon nanotubes, they have gained ever-increasing attention due to their fascinating properties like unique optical properties, multiple functional groups, excellent biocompatibility, and chemical and photostability.[12,13] They are considered as promising alternatives to fluorescent dyes and quantum dots and are advocated for diversified applications such as sensing,[14,15] bioimaging,[16−18] optoelectronic conversion,[19,20] visible-light-activated bactericide,[21] fingerprint detection,[22] dye degradation,[23−25] solar cells,[26] printing inks,[27] and gene delivery.[28]

Preparation methods of CDs reported so far can broadly be grouped into either top-down or bottom-up. The top-down approach involves the breakdown of bulk n class="Chemical">carbon sources like graphite, carbon nanotubes, and nanodiamonds into fluorescent CDs by employing techniques like arc discharge, laser ablation, chemical oxidation in strong acid, and electrochemical synthesis.[11,29] Conversely, in the bottom-up approach, the CDs are formed from molecular precursors by applying solvothermal/hydrothermal methods,[30−32] ultrasound/microwave[33−35] treatments, or simple thermal combustion.[36,37] Although a large variety of techniques and starting materials were employed for the production of CDs, the demand for the sustainable synthetic routes that adhere to the principles of green chemistry is still high. To fulfill this requirement, researchers have explored the use of waste materials like food waste,[38] agriculture waste,[39] waste paper,[40] waste frying oil,[41] etc. as precursors. However, the fluorescence quantum yields (QYs) of the as-produced CDs are less than 10%, limiting the range of their practical applications. Hence, there is a great need of developing facile methods for the large-scale utilization of waste resources toward the production of highly fluorescent CDs.

On the other hand, doping CDs with nonn class="Chemical">metals such as N, S, B, and P received a great scientific attention as it can offer CDs with improved properties like resistance to self-quenching, enhanced fluorescence QY, and sensing selectivity.[42] Recently, codoping with multiple heteroatoms especially N and S has gained extensive consideration because of the improved efficiency resulting from the synergistic effect of the doped N and S atoms.[43]

In the present work, we explored the use of CBs as source material for the production of highly fluorescent n class="Chemical">N and S-codoped CDs. The as-produced CDs (CBCDs) were systematically characterized by various analytical techniques, and their stability toward diverse environmental conditions like high ionic strength, pH, temperature, storage, and continuous irradiation was evaluated. The CBCDs exhibited bright and stable fluorescence with a QY of 26%. Physicochemical characterizations revealed that the CBCDs are spherical with a mean diameter of 3.7 ± 1.4 and composed of graphitic core with amorphous/polar surface functionalities, which impart aqueous solubility. CBCDs were used as “on–off–on” fluorescent probe for the detection of Fe3+ and ascorbic acid (AA). The probe was successfully applied for the analysis of real water (tap, bore, and pond) and biological samples (human urine and serum), and a possible quenching mechanism was proposed. In addition, we have constructed an IMPLICATION logic gate based on these unique sensing characteristics. Excitation-dependent emission behavior coupled with high biocompatibility revealed the multicolor cellular imaging potential of CBCDs. Furthermore, we have prepared fluorescent polymer films by incorporating CBCDs into poly(vinyl alcohol) (PVA). Also these CBCDs were applied as invisible ink for security applications.

Results and Discussion

Synthesis of CBCDs

Here, we report a simple hydrothermal method for the production of n class="Chemical">N, S codoped CDs using cigarette butts as source material. The preparation of CDs from CBs is illustrated in Scheme . Cigarette butts are mainly composed of cellulose acetate fibers, and they also contain minor amount of chemicals like nicotine, polycyclic aromatic hydrocarbons (PAHs), N-nitrosamine, aromatic amines, and other tarlike components trapped during smoking.[6] Conversion of cellulose acetate fibers into water-soluble CDs is the critical step. Concentrated sulfuric acid is selected to achieve this conversion due to (1) its strong acid hydrolyzing property, which produces cellulose from cellulose acetate and further facilitates the conversion of cellulose into hydrophilic cellulose nanoparticles;[44] (2) its strong dehydrating property, which assists the creation of unsaturated C=C bonds from saturated C–C bonds; and (3) its strong oxidizing property, which facilitates the generation of hydrophilic C–O–H and O=C–O–H from hydrophobic C–H.[41] As CDs predominantly comprise PAHs in their carbon matrix,[45] the presence of PAHs in the reaction mixture is expected to facilitate the formation of CDs. Further, the presence of N-containing compounds (nicotine and other aromatic amines) is known to promote the formation of CDs and improve the QY by N-doping and amine passivation. Furthermore, sulfuric acid can also serve as S dopant to produce S and N-codoped CDs with improved QY.[46] The crucial role of sulfuric acid is evident from the blank experiment (double-distilled (DD) water is used instead of sulfuric acid), which resulted in CDs with very low QY (3%) and insignificant production yield. To obtain the best CDs out of waste CBs, process parameters like reaction temperature, time, and concentration of acid were optimized (Tables S1 and S2). Under optimal conditions, CDs with QY as high as 26% were obtained with a production yield of 9.6%. A comparison (Table S3) with several previous reports on the production of CDs from waste materials revealed the advantageous features (mild synthetic conditions and higher QY and production yield) of the present method.

Scheme 1

Schematic Representation of the Synthesis of Fluorescent CDs from Cigarette Butts

Physicochemical Properties

The structure and morphological features of CBCDs were exn class="Chemical">plored by transmission electron microscopy (TEM) analysis. As presented in Figure a, CBCDs possess spherical morphology, are uniform in size, and are well separated from each other. The corresponding size distribution histogram obtained by counting 100 nanoparticles is represented in Figure b. Gaussian fitting of the histogram unveiled the statistical diameter of CBCDs as 3.7 ± 1.4. High-resolution TEM (HRTEM) image (inset of Figure a) revealed that most of the CBCDs have clear lattice structure with a fringe spacing of 0.24 nm, which agrees with the basal spacing of (1120) lattice plane of graphene.[47] Powder X-ray diffraction (XRD) patterns of CBCDs (shown in Figure c) presented a single broad peak centered at 2θ = 24.3 indexed to the (002) lattice spacing of graphitic carbon, indicating the graphite-like structure of CBCDs. The interlayer spacing d (0.37 nm) is larger than that of graphite (0.34 nm), articulating the incomplete graphitization and can be attributed to the introduction of oxygen-containing functional groups or owing to the existence of organic functional groups on the surface.[48] Graphitization is further confirmed by Raman analysis, which exhibited two broad peaks (Figure d) centered at 1359 and 1548 cm–1 corresponding to the D and G bands, respectively. The D band is a measure of disorder in the graphite lattice in the sense that it corresponds to the A1g (zone-edge) breathing vibration phonon that is only activated in the presence of a neighboring sp3 defect.[49] The G band corresponds to the E2g mode of graphite and is related to the vibration of sp2-bonded carbon atoms in a two-dimensional hexagonal lattice.[50] The integrated intensity ratio of D and G bands (ID/IG), which is characteristic of the extent of disorder and functionalization (ratio of sp3/sp2 carbons), is found to be 0.66, signifying the partial disordered graphite-like structure of CBCDs.[41]

Figure 1

(a) TEM image (inset: HRTEM image), (b) particle size distribution, (c) XRD pattern, and (d) Raman spectrum of CBCDs.

The elemental composition of CBCDs revealed by scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) (Figure S1a) is found to be n class="Gene">C (64.70%), N (8.37%), O (25.27%), and S (1.66%). The surface functional groups of CBCDs were characterized through Fourier transform infrared (FTIR) spectroscopy (Figure S1b). The broad vibrational band in the range of 3600–3100 cm –1 can be attributed to the stretching vibrations of N–H and O–H. A strong band at 1641 cm–1 is attributed to the stretching modes of C=N/C=O stretching vibration. The peaks at 1568 and 1375 cm–1 can be assigned to C=C bond stretching and C–H vibrations, respectively.[11] The vibrational band at 1047 cm–1 is assigned to the presence of −SO3–, C–O–C, and C–O bonds. The peak at 1313 cm–1 can be due to C(sp2)–N and C–S bonds,[42] while the peak at 1155 cm–1 is ascribed to C–O, C–N, and C–S bonds.[14] Further information regarding the nature of bonds and chemical composition of CBCDs was obtained from X-ray photoelectron spectroscopy (XPS). The XPS survey spectrum (Figure a) of CBCDs presented four peaks at 168, 285, 400, and 532 eV, which are ascribed to S 2p, C 1s, N 1s, and O 1s signals, respectively. The high-resolution C 1s spectrum (Figure b) reveals four types of carbon bonds: sp3 (C–C and C–H) at 284.2 eV, sp2 (C=C) at 285.7 eV, C–S/C–N/C–O at 287.2 eV, and C=O/C=N at 288.7 eV.[47] High-resolution N 1s spectrum (Figure c) designates the existence of pyridinic N s, 1°/2° amino N s (399.0 eV), and pyrrolic N s (400.7 eV). S 2p spectrum (Figure d) can be resolved into three distinct peaks at 167.5, 168.7, and 169.5 eV, which designates the existence of C–SO (x = 2, 3, 4) on the surface of CBCDs.[42] ζ potential of the as-prepared CBCDs suspension was found to be −14.9 mV, demonstrating the negative surface charge of CBCDs (Figure S2). All of these results manifested the occurrence of multiple functional groups like −OH, −C=O, −COOH, and −NH and successful doping of N, S elements in the CB-derived CDs.

Figure 2

(a) XPS survey spectrum of CBCDs. High-resolution spectra of (b) C 1s, (c) N 1s, and (d) S 2p peaks.

(a) XPS survey spectrum of CBCDs. High-resolution spectra of (b) n class="Gene">C 1s, (c) N 1s, and (d) S 2p peaks.

Optical Properties

The UV–vis absorption, excitation, and emission spectra are represented in Figure a. As shown in the figure, the absorption spectrum displays two poorly resolved bands around 270 and 320 nm, which can be ascribed to the π–π* transition of n class="Chemical">carbonic core center and n−π* transition of heteroatomic surface functionalities or molecule center, respectively.[51] Similar to several previous reports, aqueous solution of CBCDs exhibit bright blue emission under 365 nm UV light, and it appeared transparent and pale yellow under daylight (inset of Figure a). The corresponding excitation spectrum shows two peaks, which can be ascribed to core and surface excitations. Like most of the fluorescent CDs, CBCDs also exhibited excitation tunable emission behavior. A steady increase in λex from 300 to 480 nm resulted in a red shift in the emission peak position along with a concurrent first increase and then decrease in the emission intensity (Figure b). This red shift can be clearly observed in the corresponding normalized emission spectra (Figure S3). This tunable emission is considered to be the versatile characteristic of CDs and has been most prominently ascribed to the selective excitation of subsets of CDs within the CD ensemble.[52] The maximum emission peak centered at 430 nm was observed under the excitation of 360 nm with a large stokes shift of 70 nm. As presented in Figure S4, the fluorescence QY of CBCDs at room temperature was calculated to be 26% (using quinine sulfate (QS) as reference), which is greater compared to various previous reports (Table S3). The reason for this higher QY may be due to the synergistic effect of doped N and S atoms.[43]

Figure 3

(a) Absorption (black), excitation (red), and emission (blue) spectra of CBCDs (inset: CBCDs aqueous solution under daylight (left) and UV light (right)). (b) Excitation wavelength-dependent emission spectra of CBCDs.

(a) Absorption (black), excitation (red), and emission (blue) spectra of CBCDs (inset: n class="Chemical">CBCDs aqueous solution under daylight (left) and UV light (right)). (b) Excitation wavelength-dependent emission spectra of CBCDs.

Stability

In view of the fact that fluorescence emission of many fluorescent probes can be disrupted by the complex environmental conditions, we have examined the emission spectra of CBCDs under diverse conditions to verify their practical applicability. The effect of ionic strength was examined by recording the emission spectra under various KCl concentrations. As depicted in Figure S5a, only a slight diminution in the photoluminescence (PL) intensity is observed even at a high KCl concentration of 2 M, which verifies the excellent stability of CBCDs under high-ionic-strength environments and outspreads their usage to identical ion-rich biological conditions. As shown in Figure S5b, change in the solution pH did not alter the peak position, but caused fluctuations in the emission intensity. No significant alteration in the emission intensity is recorded over a pH range of 5–9, which is a beneficial property for the fluorescent probe to be used in complex biological system and practical applications. Photostability studies revealed their excellent property of resistance to photobleaching (Figure S5c). As presented in Figure S5d, the fluorescence intensity of CBCDs was found almost stable in the temperature range of 25–45 °C representing their thermal stability. Further, the storage stability of CBCDs was examined by keeping those under (1) ambient conditions and (2) refrigerator for 60 days. Virtually no fluctuations in their emission intensity and no obvious precipitation were observed in both the cases (Figure S6), demonstrating their great stability and long shelf life.

Detection of Fe3+ and AA

The aforementioned optical merits, along with the existence of abundant surface functional groups encouraged us to further investigate the possible sensing applications of n class="Chemical">CBCDs. According to several previous reports, the surface functional groups of CDs can selectively interact with metal ions and produce a fluorescence change; thus, we have studied the fluorescence response of CBCDs toward various biologically and environmentally important metal ions, such as Fe2+, Ca2+, Na+, Pb2+, Cu2+, Mn2+, Cd2+, Sn2+, Cr3+, Al3+, Ni2+, Hg2+, Mg2+, Ba2+, K+, Zn2+, and Fe3+. As shown in Figure S7, among all ions, only Fe3+ caused a severe decline in the PL intensity, which unveiled the applicability of CBCDs as highly selective turn-off fluorescent probes for Fe3+ detection. This discrimination effect for Fe3+ can be credited to the special coordination between electron-deficient Fe3+ ions and electron-rich surface functional groups of CBCDs.[46] In the course of developing an efficient probe, some critical parameters, including usage concentration of CBCDs, incubation time, and solution pH, were optimized. In general, in the presence of quencher of a given concentration, lower concentration of fluorophore will result in greater sensitivity and higher concentration will achieve a broader detection range;[53] hence, through comprehensive consideration of both sensitivity (limit of detection (LOD)) and linear detection range, 0.05 mg mL–1 of CBCDs was selected as optimum. Quenching kinetic investigations (Figure S8a) revealed that 2 min of interaction with Fe3+ is sufficient to produce maximum fluorescence response, which remained stable in the following 20 min of observation, suggesting the complexation between Fe3+ and CBCDs is quick and stable, which is useful in rapid sensing without strict time control.[54] As presented in Figure S8b, Fe3+ ions were able to produce decent response in the pH range of 6–9, which is beneficial as most environmental and biological samples lie in this pH scale and maximum response is achieved at pH 7, which is taken as optimum.

Under optimal circumstances, sensitivity was inspected by computing the fluorescence response of CBCDs to several concentrations of n class="Chemical">Fe3+ in the range of 0–100 μM. As depicted in Figure a, the PL intensity gradually dwindled with increasing concentrations of Fe3+. The plot of quenching efficiency (F0/F) versus Fe3+ concentration (Figure b) displayed a good linearity (R2 = 0.998) in the range of 0–100 μM, where F0 and F are the PL intensities of CBCDs at 430 nm in the absence and presence of Fe3+, respectively. The detection limit (LOD) was estimated to be 0.13 μM based on the equation 3σ/m, where σ is the standard deviation of the blank signal (n = 6) and m is the slope of the linear fit. The LOD presented by our method (0.13 μM) is much lower than the maximum permissible limit (5.36 μM) stipulated by World Health Organization (WHO) and LOD presented by other researchers for Fe3+ in drinking water.[55] Comparison of analytical performance of CBCDs nanoprobe with several chosen probes in the literature (Table S4) revealed the superiority of the present sensor in terms of linear range, LOD, and applicability to real samples.

Figure 4

(a) Fluorescence intensity response of CBCDs with increasing concentration of Fe3+. (b) Relationship between F0/F and the concentration of Fe3+ ions in the range of 0–100 μM (0, 0.5, 1, 2, 4, 6, 10, 15, 20, 40, 60, 100 μM). (c) Fluorescence response (F/F0) of CBCDs toward various metal ions (black) and subsequent addition of Fe3+ ions (red). (d) Fluorescence intensity response of CBCDs/Fe3+ with increasing concentration of AA. (e) Relationship between F/F0 and the concentration of AA in the range of 0–100 μM (0, 0.5, 2, 4, 6, 10, 20, 40, 60, 80, 100 μM). (f) Fluorescence response (F/F0) of CBCDs/Fe3+ system toward various commonly interfering species.

(a) Fluorescence intensity response of CBCDs with increasing concentration of n class="Chemical">Fe3+. (b) Relationship between F0/F and the concentration of Fe3+ ions in the range of 0–100 μM (0, 0.5, 1, 2, 4, 6, 10, 15, 20, 40, 60, 100 μM). (c) Fluorescence response (F/F0) of CBCDs toward various metal ions (black) and subsequent addition of Fe3+ ions (red). (d) Fluorescence intensity response of CBCDs/Fe3+ with increasing concentration of AA. (e) Relationship between F/F0 and the concentration of AA in the range of 0–100 μM (0, 0.5, 2, 4, 6, 10, 20, 40, 60, 80, 100 μM). (f) Fluorescence response (F/F0) of CBCDs/Fe3+ system toward various commonly interfering species.

With inherent complexity, n class="Chemical">metal-ion detection in real samples poses a great challenge to the analytical methods not only in terms of sensitivity but more importantly in selectivity. Hence, to further explore the applicability of the proposed nanoprobe to real samples, selectivity and competition experiments were carried out. The black bars shown in Figure c depict the PL response (F/F0) of CBCDs to various metal ions each at a concentration of 200 μM. It is evident that none of these metal ions caused a considerable decline in the PL intensity. Competition experiments were conducted by subsequently adding 100 μM of Fe3+ to the above solutions. As represented by the red bars in Figure c, no substantial change in the PL response (F/F0) appeared in the co-presence of Fe3+ and other metal ions in comparison to that of Fe3+ alone. All of these results clearly demonstrated the excellent selectivity of CBCD-based nanoprobe and motivated us to investigate their practical applicability in real samples.

It is observed that the quenched fluorescence of CBCDs/n class="Chemical">Fe3+ can be recovered by the addition of AA. This can be attributed to the (i) antioxidant nature of AA, which can reduce Fe3+ to Fe2+ and (ii) the high selectivity of CBCDs toward Fe3+ over Fe2+. Moreover, AA did not exhibit any significant effect on the fluorescence of CBCDs alone. Hence, the CBCDs/Fe3+ system can be employed as a turn on fluorescent probe for the detection of AA (Scheme ). Kinetic investigations disclosed that, 10 min of interaction with AA recovered the emission of CBCDs/Fe3+ system to a stable value (Figure S9), and this 10 min is taken as the incubation time for the assay. Sensitivity of CBCDs/Fe3+ system toward AA is evaluated by recording its fluorescence response to various concentrations of AA. As presented in Figure d, the emission intensity of CBCDs/Fe3+ nicely recovered with increase in the concentration of AA, and the plot of F/F0 versus AA concentration (Figure e) demonstrated a good linearity (R2 = 0.996) in the range of 0.5–100 μM, where F0 and F are the emission intensities of CBCDs/Fe3+ at 430 nm in the absence and presence of AA, respectively. The detection limit (LOD) was estimated to be 0.2 μM based on the equation 3σ/m, where σ is the standard deviation of the blank signal (n = 6) and m is the slope of the linear fit. Selectivity of the probe is evaluated by studying the interference of various amino acids, anions, and sugars. As depicted in Figure f, the probe presented greater selectivity, which opened their further applicability to real samples. The sensing performance (linear range and limit of detection) of the present probe is compared to that of several reported methods (Table S5), which revealed that the presented method is among the best methods reported so far.

Scheme 2

Schematic Illustration of On–Off–On Detection of Fe3+ and Ascorbic Acid

Analysis of Real Samples

Practical applicability of n class="Chemical">CBCDs nanoprobe was assessed by detecting Fe3+ in real water (tap, bore, and pond) and AA in complex biological (human urine and serum) samples. To avoid the particulate matter, real water samples are subjected to centrifugation and filtration (0.45 μm membrane filter). Serum and urine samples are subjected to a 100-fold dilution with PBS before analysis. Then, the water samples were spiked with various concentrations of standard Fe3+ solution, and the serum and urine samples were spiked with AA and analyzed by the proposed method. As shown in Tables S6 and S7, good recoveries (98–102) and high analytical precision with RDS 3.2 (n = 6) were obtained. Furthermore, the nanoprobe is validated by comparing the results of Fe3+ and AA detection in a real sample to those of a standard method (atomic absorption spectrometry for Fe3+ and high-performance liquid chromatography–UV for AA). These results confirm the reliability and feasibility of the proposed nanosensor for monitoring Fe3+ in environmental and AA in biological samples.

Possible Mechanism of Fluorescence Quenching

In general, several sorts of molecular interactions between the fluorophore and quencher such as electron or energy transfer, excited-state reaction, collisional quenching, and ground-state complex formation can lead to fluorescence quenching.[56] The quenching mechanisms are typically classified into (1) dynamin class="Gene">c quenching, involving the transfer of electron from excited-state fluorophore to ground-state quencher by means of collision and (2) static quenching resulting from the formation of a nonfluorescent ground-state complex between the fluorophore and quencher.

To explore the possible mechanism of quenching of n class="Chemical">CBCDs fluorescence by Fe3+ ions, the standard Stern–Volmer equation is applied.where F0 and F refer to the fluorescence intensities of CBCDs in the absence and presence of quencher (Fe3+), respectively; Ksv and kq represent the Stern–Volmer quenching constant and bimolecular quenching constant, respectively; [Q] is the concentration of quencher (Fe3+); and τ0 is the average lifetime of CBCDs in the absence of quencher.

As depicted in Figure b, the plot of F0/F versus [Q] exhibited a good linear correlation (correlation coefficient, R2 = 0.998) and the Stern–Volmer quenching constant (n class="Chemical">Ksv) was found to be 2.2 × 104 M–1. The excellent linear correlation infers that the observed quenching is either purely dynamic or purely static and rules out the possibility of static–dynamic combination.[51] In principle, fluorescence lifetime measurement is the most definitive method to distinguish dynamic and static quenching processes.[57] In light of this, fluorescence lifetime quenching analysis is carried out to confirm the nature of quenching. Figure a reveals that the PL lifetime of CBCDs is not quenched by Fe3+ ions, which rule out the possibility of dynamic quenching. The bimolecular quenching constant (kq) calculated from Ksv and average lifetime τ0 (5.26 ns) is found to be 4.18 × 1013 M–1 s–1, which is higher than the diffusion-controlled limit (1010 M–1 s–1) and further supports the static quenching mechanism involving the ground-state complex formation. Decline in the quenching constant with rise in the temperature (Figure b) further supported the static quenching process. All of these findings designate that the quenching caused by Fe3+ ions is a result of nonfluorescent complex formed between the surface functional groups of CBCDs and Fe3+ ions. FTIR spectral analysis was further used to verify this assumption. As shown in Figure S10, evident changes (especially at the characteristic peaks of C–O, C–N, and SO–) were observed in the FTIR spectrum of CBCDs after the addition of Fe3+, which further confirmed that Fe3+ indeed coordinated with these surface functional groups of CBCDs.

Figure 5

(a) Fluorescence decay of CBCDs in the absence (green) and presence (red) of Fe3+ ions. (b) Stern–Volmer plots (F0/F vs Fe3+ concentration) at different temperatures.

(a) Fluorescence decay of CBCDs in the absence (green) and presence (red) of n class="Chemical">Fe3+ ions. (b) Stern–Volmer plots (F0/F vs Fe3+ concentration) at different temperatures.

Cytotoxicity and Cellular Imaging

With the grander optical properties like bright fluorescence (QY = 26%), high ionic strength, and tolerance to photobleaching and complex environmental conditions, the n class="Chemical">CBCDs showcase a great potential to serve as bioimaging agents. However, like various biological applications, low cytotoxicity is the key requirement for a material to be used as bioimaging agent also. Hence, we have evaluated the inherent cytotoxicity of CBCDs toward human normal (HEK-293) and human cancerous (HeLa) cells. Standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay is employed for this purpose. As shown in Figure S11, CBCDs did not exhibit substantial toxicity on both cancerous and noncancerous cells and the cell viabilities remain 85% even at a concentration of 1000 μg mL–1, denoting their excellent biocompatibility. An important observation is that, at the dosage used for bioimaging (200 μg mL–1), virtually no toxicity is witnessed (cell viability is over 95%), which indicated that the CBCDs have great potential for biomedical applications such as in vivo imaging, cellular labeling, and medical imaging.[58] These results are in agreement with similar studies carried out earlier.[59] Further, it is interesting to note that CDs derived from toxic CBs did not exhibit any toxicity toward human normal and cancerous cells demonstrating their versatility.

Cellular imaging potential of CBCDs was evaluated by performing the in vitro cellular uptake experiments with n class="CellLine">HeLa cells. After incubating with 200 μg mL–1 CBCDs for 6 h, the cells were imaged under a confocal laser scanning microscope. As shown in Figure , the cells were brightly illuminated with blue, green, and red colors under the excitation of 408, 488, and 543 nm, respectively. Nevertheless, the control cells did not display any detectable fluorescence under the same exposure conditions. A closer observation of the images revealed that the fluorescence glow is mainly confined to cell membrane and cytoplasmic area while the nucleus had only a weak glow. These results indicate that, similar to some previous reports,[60] CBCDs were also having difficulties in labeling the nucleus, while the cytoplasm and cell membrane are easily stained.

Figure 6

Confocal microscopic images of HeLa cells: cells treated with 200 μg mL–1 CBCDs for 6 h; cells (CBCDs treated) further treated with 200 μM Fe3+ for 1 h; and cells (CBCDs/Fe3+ treated) further treated with 500 μM AA for 1 h, and images taken under three excitation wavelengths of 405 nm (blue), 488 nm (green), and 561 nm (red).

Confocal microscopic images of n class="CellLine">HeLa cells: cells treated with 200 μg mL–1 CBCDs for 6 h; cells (CBCDs treated) further treated with 200 μM Fe3+ for 1 h; and cells (CBCDs/Fe3+ treated) further treated with 500 μM AA for 1 h, and images taken under three excitation wavelengths of 405 nm (blue), 488 nm (green), and 561 nm (red).

Intracellular Detection of Fe3+ and AA

It has been reported that overload and deficiency of Fe3+ can disturb the cellular homeostasis, resulting in various diseases.[61] Hence, developing a simple and sensitive probe for monitoring intracellular Fe3+ is of great importance. Impressed by the favorable biocompatibility, cell imaging ability, and high selectivity, CBCDs were further applied for monitoring Fe3+ in live cells by introducing exogenous Fe3+ into the CBCD-pretreated HeLa cells. As expected, the confocal microscopic images taken after supplementing cells with 200 μM Fe3+ in the growth medium exhibited very weak intracellular fluorescence (Figure ). Further treatment of cells with AA resulted in the nicely recovered intracellular fluorescence indicating that emission of CBCDs/Fe3+ in the cells can be recovered by AA. It can be observed that the fluorescence emission of the cells is in the order of CBCDs > CBCDs/Fe3+/AA > CBCDs/Fe3+ treated. All of these results elucidate that CBCDs could serve as efficient fluorescent probe for “on–off–on” detection of Fe3+ and AA in living cells.

Logic Operations of CBCDs

The fluorescence switching behavior of CBCDs in the presence of n class="Chemical">Fe3+ and AA can be employed as multiple molecular logic gates, performing the Boolean algebraic logic operations.[62] The simple single input molecular logic gate NOT can be constructed using Fe3+ as single input signal (Figure a). The multiple input logic gate IMPLICATION can be realized by taking Fe3+ and AA as input 1 and input 2, respectively. The presence of Fe3+ or AA is taken as 1 and their absence as 0. For output, the maximum fluorescence was taken as 1 and the corresponding quenched fluorescence as 0. As shown in Figure b, only in the presence of Fe3+ and absence of AA, i.e., input (1, 0), significant fluorescent quenching is observed and provided output as 0, while in the case of other inputs (0/0, 0/1, 1/1), the output remains 1. The logic symbol, truth table, and output fluorescence intensity at 430 nm are shown in Figure .

Figure 7

Logic operations using CBCDs: (a) logic symbol and truth table of NOT logic gate; (b) logic symbol and truth table of IMPLICATION logic gate; and (c) fluorescence response of CBCDs under different inputs.

Logic operations using n class="Chemical">CBCDs: (a) logic symbol and truth table of NOT logic gate; (b) logic symbol and truth table of IMPLICATION logic gate; and (c) fluorescence response of CBCDs under different inputs.

Fluorescent Ink and Polymer

Finding novel applications to emerging materials like n class="Chemical">CDs is always important. Here, CBCDs possess unique properties, including strong and stable fluorescence and good transparency in the visible region, which makes them suitable candidates for fluorescent ink applications.[63] To explore this, a sketch pen is filled with CBCDs aqueous solution (Figure S12) and employed for writing some text on commercial filter paper. As shown in Figure , the words “Osmania University” are clearly visible (showing blue fluorescence) under UV light, whereas the filter paper appeared as blank under daylight. It is worth mentioning that the information on the filter paper remained consistent and can be reproducible even after 30 days (when stored under ambient conditions) (Figure S13). These observations suggest that the CBCDs can be used as invisible ink for loading important information for secret communications and have a great potential for anticounterfeiting applications. Furthermore, with their high biocompatibility (low/nontoxic nature), CBCDs can be safely used as ink pads to form human fingerprints that do not contaminate the fingers.[27]Figure c characterizes the CBCDs-formed fluorescent fingerprint on commercial filter paper, which could reflect human fingerprint clearly. Thus, the water-soluble CBCDs-based fluorescent ink could serve as an alternative to the traditional inks to form a clear, adelomorphic, long-lasting, and blue fluorescent fingerprint that can be easily cleaned with water and is pollution free.[64]

Figure 8

Information loaded on commercial filter paper using CBCDs invisible ink under (a) daylight and (b) 365 nm UV light. (c) CBCDs-formed fingerprints under 365 nm UV light.

Information loaded on commercial filter paper using CBCDs invisible ink under (a) daylight and (b) 365 nm UV light. (c) n class="Chemical">CBCDs-formed fingerprints under 365 nm UV light.

Further extending their applicability to solid-state optic-related fields is interesting and challenging. Efficient solid-state n class="Chemical">PL emission of CDs is extremely important for light-emitting diode (LED) applications. However, CDs in the solid state generally suffer from self-quenching because of the aggregation-caused quenching effect, which might be a result of excessive Forster resonance energy transfer among adjacent CD particles in the aggregated state or direct π–π interaction.[65] Dispersing CDs in the polymer matrix is considered to be an effective strategy to avoid this. Here, we have prepared solid-state films by dispersing CBCDs in poly(vinyl alcohol) (PVA). PVA was selected as polymer host because of its known high optical quality and desirable film properties.[66] The as-fabricated films are highly transparent (about 90% transmittance in the entire visible region) under daylight, while exhibited bright blue fluorescence under UV illumination (Figure ). The absorption spectra (Figure S14a) of CBCDs in the polymer film is similar to those in solution, and the emission spectra (Figure S14b) also exhibited a similar excitation-dependent behavior.

Figure 9

CBCDs/PVA film (a) under daylight, (b) under 365 nm UV light, and (c) corresponding transmittance spectra and fluorescence spectra under optimal excitation of 357 nm.

CBCDs/n class="Chemical">PVA film (a) under daylight, (b) under 365 nm UV light, and (c) corresponding transmittance spectra and fluorescence spectra under optimal excitation of 357 nm.

In comparison to CBCDs aqueous solution, the optimal emission spectra of n class="Chemical">CBCDs/PVA film exhibited a blue shift of 6 nm and a slight red shift (3 nm) in the optimal excitation wavelength (Figure S15), which might be due to the fact that PVA environment is different from aqueous solution.[67] The fluorescence QY of CBCDs in PVA matrix determined using QS as reference was found to be 33% (Table S8), which is higher than that in aqueous solution. This enhancement in the QY can be ascribed to the formation of hydrogen bonds between PVA and surface functional groups of CDs, which provide a stabilization effect on the electrons and holes for more efficient radiative recombination.[63] A similar enhancement was observed in some previous reports also.[63,66] Furthermore, the optical properties of CBCDs/PVA films are quite stable and no obvious decrease in PL emission is observed even after 24 h of continuous exposure to UV light (365 nm) (Figure S16). With improved and stable optical properties, CBCDs/PVA films have a great potential to serve as light conversion film/phosphor material in solid-state lighting systems.

Conclusions

In summary, we have successfully demonstrated a simple one-step hydrothermal method for the production of highly fluorescent (QY = 26%) n class="Chemical">N,S-codoped CDs from toxic CBs for the first time. Moderate reaction conditions, using waste as starting material, good production yield (9.6%), and multifunctional nature of the formed products (CBCDs) make our method economical and scalable. The as-produced CBCDs were well characterized and explored for multiple applications. Bright and stable fluorescence coupled with high biocompatibility made CBCDs a potential candidate for bioimaging applications. They were successfully applied as “on–off–on” fluorescent probe for the sequential detection of Fe3+ and AA in real water and biological samples. CBCDs were further employed as biocompatible and invisible ink for loading data and forming fingerprints. The “visible–invisible” and “UV–visible” properties of CBCDs promise their applicability in anticounterfeiting. Furthermore, the CBCDs/PVA films with strong and stable emission might find direct or indirect applications in various optical/optoelectronic devices, ranging from fluorescent displays to LEDs.

Experimental Section

Materials

Cigarette butts were collected from the ash trays of a local bar and restaurant. All of the chemicals were of analytical grade and used without further purification. All solutions were prepared by using double-distilled (DD) n class="Chemical">water.

Synthesis of Carbon Dots

CBs (0.5 g) were cut into small pieces (paper wrap was removed to avoid dust particles) and placed in a 100 mL of Erlenmeyer flask containing 20 mL of 40% n class="Chemical">H2SO4 and sonicated for 1 h. Then, the formed dispersion was transferred to a 25 mL Teflon-lined stainless steel autoclave and heated at 180 °C for 6 h in an electric oven. After cooling down to room temperature, the obtained solution was centrifuged to remove large particles. Then, the solution was neutralized with Na2CO3, centrifuged, and filtered through a 0.22 μm syringe filter to remove the formed precipitate. Afterward, the solution was purified by dialyzing against DD water for 24 h using a dialysis tubing (molecular weight cut-off 3500 Da). Finally, CBCDs powder was obtained by freeze drying and used for the preparation of stock solution (1 mg mL–1).

Characterization

Fluorescence spectral measurements were carried out using a JASCO spectrofluorometer (FP-8500) set with excitation and emission slit widths at 2.5 nm. UV–vis spectral studies were conducted on a Shimadzu UV–vis–NIR 3600 spectrophotometer. Fluorescence decay analysis was done on a Fluoro Cube, Lifetime System (Horiba Jobin Yvon) with a 370 nm Nano LED excitation source. Transmission electron microscopy (TEM) images were acquired on a JEOL 3010 microscope operating at an accelerating voltage of 200 kV by drop-casting a proper dilution of CBCDs aqueous solution onto the carbon-coated copper grids. X-ray photoelectron spectroscopy (XPS) measurements were carried out by Kratos AXIS Ultra spectrometer with Al Kα X-ray as the excitation source (1486.71 eV). Fourier transform infrared (FTIR) spectrum was recorded on a Shimadzu IR Prestige-21 spectrophotometer. ζ potential measurements were conducted using a Malvern, Nano ZS90 Zetasizer. Raman spectra were measured with a Horiba Jobin Yvon LabRAM HR with a focal length of 800 mm and equipped with a He–Ne 633 nm Laser. X-ray diffraction (XRD) patterns of CBCDs were obtained using an X’pert Pro powder X-ray diffractometer (the Netherlands) with Cu Kα radiation, λ = 1.5406 Å.

Calculation of QY

QYs of all of the CBCD samples were calculated by using the slope method in which quinine sulfate (QS) is chosen as standard (Φ = 54%). This particular method first involves the preparation of several concentrations of CBCDs aqueous solutions and QS 0.1 M H2SO4 solutions by maintaining the absorbance values less than 0.1 at their excitation wavelengths. Subsequently, the integrated emission intensities of all of the samples were recorded by exciting at 360 nm. Afterward, the integrated emission intensities were plotted against corresponding absorbance values and the slope values of the obtained linear plots were computed. Finally, quantum yields were calculated by using the following equationwhere Φ is the quantum yield, Grad is the gradient from the plot of integrated emission intensity versus absorbance, and η is the refractive index of the solvent (1.33 for both solvents). The subscripts st and x denote standard (QS) and CBCDs, respectively.

Detection of Fe3+ and Ascorbic Acid

Solutions of all metal ions used in this experiment are prepared from corresponding salts in n class="Chemical">DD water. At room temperature, 1 mL of DD water is added to 1 mL of CBCDs aqueous solution (0.1 mg mL–1), the emission spectra of this sample are recorded at an excitation wavelength of 360 nm, and the emission intensity at 430 nm is denoted as F0. In a similar manner, 1 mL of different metal ion solutions of 200 μM concentration were added to 1 mL of CBCD solution, shaken well, and incubated for 2 min and thereafter the emission intensity is recorded as F. A similar procedure was followed for the quantitative determination of Fe3+. In a typical assay, 1 mL of CBCDs solution was mixed with 1 mL of Fe3+ solution of varying concentrations. For the detection of ascorbic acid (AA), first, 0.5 mL of Fe3+ is added to 0.5 mL of CBCDs solution and then 1 mL of AA with varying concentrations was added. The final concentrations of Fe3+ and CBCDs are 100 μM and 0.05 mg mL–1, respectively. After 10 min of incubation, fluorescence response in recorded.

Cytotoxicity and Cellular Imaging

Standard MTT assay was emn class="Chemical">ployed for evaluating the in vitro cytotoxicity of cigarette butt-derived CDs. Experimental studies were performed on both normal (HEK-293) and cancerous (HeLa) cell lines. Complete details are provided in the Supporting Information.

Preparation of Fluorescent Films (CBCDs/PVA Composite Films)

Poly(vinyl alcohol) (PVA) (1 g) is added to 10 mL of DD water under constant stirring, and the mixture was heated to dissolve PVA. After that, 1 mL of 1 mg mL–1 CBCDs aqueous solution was added to 9 mL of PVA solution and stirred gently for 10 min. Finally, the mixture was drop-casted onto a clean glass slide and dried in an oven. After drying, the film was peeled off from the glass substrate to get a freestanding film.