Heteroatom-Doped Carbon Quantum Dots and Polymer Composite as Dual-Mode Nanoprobe for Fluorometric and Colorimetric Determination of Picric Acid

Oxygen- and nitrogen-heteroatom-doped, water-dispersible, and bright blue-fluorescent carbon dots (ON–CDs) were prepared for the selective and sensitive determination of 2,4,6-trinitrophenol (picric acid, PA). ON–CDs with 49.7% quantum yield were one-pot manufactured by the reflux method using citric acid, d-glucose, and ethylenediamine precursors. The surface morphology of ON–CDs was determined by scanning transmission electron microscopy, high-resolution transmission electron microscopy, dynamic light scattering, Raman, infrared, and X-ray photoelectron spectroscopy techniques, and their photophysical properties were estimated by fluorescence spectroscopy, UV–vis spectroscopy, fluorescence lifetime measurement, and 3D-fluorescence excitation–emission matrix analysis. ON–CDs at an average particle size of 3.0 nm had excitation/emission wavelengths of 355 and 455 nm, respectively. With the dominant inner-filter effect- and hydrogen-bonding interaction-based static fluorescence quenching phenomena supported by ground-state charge-transfer complexation (CTC), the fluorescence of ON–CDs was selectively quenched with PA in the presence of various types of explosives (i.e., 2,4,6-trinitrotoluene, tetryl, 1,3,5-trinitroperhydro–1,3,5-triazine, 1,3,5,7-tetranitro-1,3,5,7-tetraazacyclooctane, pentaerythritol tetranitrate, 3-nitro-1,2,4-triazole-5-one, and TATP-hydrolyzed H2O2). The analytical results showed that the emission intensity varied linearly with a correlation coefficient of 0.9987 over a PA concentration range from 1.0 × 10–9 to 11.0 × 10–9 M. As a result of ground-state interaction (H-bonding and CTC) of ON–CDs with PA, an orange-colored complex was formed different from the characteristic yellow color of PA in an aqueous medium, allowing naked-eye detection of PA. The detection limits for PA with ON–CDs were 12.5 × 10–12 M (12.5 pM) by emission measurement and 9.0 × 10–10 M (0.9 nM) by absorption measurement. In the presence of synthetic explosive mixtures, common soil cations/anions, and camouflage materials, PA was recovered in the range of 95.2 and 102.5%. The developed method was statistically validated against a reference liquid chromatography coupled to tandem mass spectrometry method applied to PA-contaminated soil. In addition, a poly(vinyl alcohol)-based polymer composite film {PF(ON–CDs)} was prepared by incorporating ON–CDs, enabling the smartphone-assisted fluorometric detection of PA.


Supporting Information Contains
Abbreviations; preparation of solutions; procedure of LC-MS/MS method for validation of PA detection; optimization of experimental conditions; 3D model of STEM images with 30 nm zoom; fluorescence spectra and intensities of ON-CDs prepared at different temperatures and at different reaction times; fluorescence spectra and histogram of ON-CDs obtained at different excitation wavelengths, and ON-CDs prepared in different solvents and solvent mixtures; fluorescence intensity values of ON-CDs and ON-CDs/PA obtained at different buffer solutions and pH values; the relationship between I0-I of ON-CDs and increasing concentrations of PA in solvent mixture medium and aqueous medium; fluorescence spectra and histogram of ON-CDs in the presence of various explosives; fluorescence spectra and histogram of ON-CDs without PA and with PA in the presence of different metal cations, anions, and camouflage materials; digital photographs of PVA-based polymer film and PF(ON-CDs) with different conditions under daylight and UV-lamp; XPS survey spectra of PVA-based polymer film and PF(ON-CD s); stability of PF(ON-CDs) in aqueous-medium; parameters of the quantum yield (QY) calculation of ON-CDs; comparison of the analytical performance parameters of fluorescent probes developed for PA detection; recovery values of PA in admixtures with possible interferents such as metal ions, anions, and camouflage materials; molecular formulas and molecular masses of explosives; references.

Procedure of LC-MS/MS Method for Validation of PA Detection
The working solutions of PA at 0.44×10 -6 M, 0.87×10 -6 M, 1.75×10 -6 M, 3.5×10 -6 M, and 4.4×10 -6 M were prepared from the corresponding stock solutions at 450.0×10 -6 M in acetone.LC-MS analysis was performed on a UPLC-MS/MS equipment (Shimadzu, 20A) employing an injection volume of 15.0 µL.LC was equipped with a Rectek Ultra-AQ column (100×2.1 mm, 3 µm, C18).The column temperature was 40 °C; the injector temperature 4 °C, and the column flow rate 1.0 mL min -1 .Two different ammonium acetate solutions, each at 5 mM concentration, in ultrapure-water (mobile phase A) and in pure methanol (mobile phase B), were used as mobile phases A and B, respectively, at a flow rate of 0.3 mL min −1 .LC−MS/MS analysis was carried out using the negative ion mode electrospray ionization method, and the ionization voltage was 3.5 kV.The product ion and precursor ion were 240.7 m/z for PA (collision energy: 15.0 V).

Optimization of Experimental Conditions
Oxygen-and nitrogen-doped ON-CDs were prepared by a one-step reflux method using CA, Glu, and EDA as molecular precursors containing abundant hydroxyl (-OH) and amino bright fluorescent ON-CDs can be obtained with increasing reaction time.However, if the reaction temperature and time continue to increase, the formation rate and the particle size of the carbon cores will increase, which will reduce the fluorescence intensity of the ON-CDs.Considering not only the optimal fluorescence property of the ON-CDs [2], but also cost and energy savings, the final reaction temperature and time were determined as 180 °C and 3 h, respectively.Under these determined conditions, ON-CDs were prepared with a quantum yield of 49.7%.
Optimal wavelength and solvent system were investigated for ON-CDs prepared under optimized synthesis conditions.Emission spectra of ON-CDs excited at different wavelengths (310 nm to 420 nm) were recorded as shown in Figure S4a.When the recorded spectra were examined, the emission band with the highest fluorescence intensity was recorded at 355 nm excitation wavelength (Figure S4b).Also, in experimental studies, it is necessary to know the behavior of ON-CDs in different organic solvents (ethanol, methanol, dimethyl sulfoxide, and acetone) and mixtures with H2O (1:1, v/v).When the spectra in Figure S5a and the fluorescence intensity histogram in Figure S5b are examined, it is seen that the best fluorescence intensity of ON-CDs is obtained in H2O.However, since ON-CDs are used in the determination of explosives, the use of organic solvents is extremely important for the solubility of explosives.For this reason, EtOH was chosen as a suitable solvent, which was prepared as a mixture with water, and the EtOH-H2O (1:1, v/v) solvent mixture was preferred throughout the experimental studies.
Another parameter that is extremely important for ON-CDs is the pH of the medium, because the characteristic structure of ON-CDs and the way it responds in the presence of the target analyte may vary according to the ambient pH.For this reason, emission spectra of ON-CDs at 455 nm wavelength were recorded in the absence and presence of PA with HEPES-Tris buffer solution within a pH range of 4 to 9. As seen in the fluorescence intensity histogram given in Figure S6a, ON-CDs maintains fluorescence stability in the pH range of 4 to 9.However, in the presence of PA, the best signal was recorded at pH 7.After determining pH 7 as the most suitable working medium for ON-CDs, experiments were carried out with different buffers (HEPES-Tris, NH4Ac, PBS, and acetic acid-acetate) that would fix the working medium to pH 7. Fluorescence intensities were recorded in pH 7 medium with different buffer systems of ON-CDs in the absence (Figure S6b) and presence (Figure S6c) of PA.As a result of the results obtained in the histograms, it was determined that the best analytical signals were obtained with HEPES-Tris (pH 7) buffer, which was used throughout the experimental studies.

This work
Table S2 shows probes based on carbon dots using citric acid as the carbon-source with the use of different molecular precursors as heteroatom sources.The synthesized carbon dots are mostly N-doped and contain a high amount of amine groups (-NH2) on the surface.On the other hand, ON-CDs have a high amount of hydroxyl groups (-OH) in addition to the amine group (-NH2) on its surface.The methods of synthesis mostly include thermal methods (i.e., hydrothermal).Unlike these synthesis methods, ON-CDs were synthesized by the reflux method and may be an alternative to the literature.
Looking at the particle sizes, the average smallest particle size belongs to ON-CDs.
Considering the reported quantum yields, ON-CDs have a very high quantum yield.
Although there are carbon dots with higher quantum efficiency in the literature, ON-CDs has a simple synthesis method, more than one heteroatom doped on its surface, shows high photostability, and allows practical application by forming composite material with support material (polymer film).In addition, carbon dot-based probes are mostly used as fluorescent probes, but our selective ON-CDs can determine the analyte both fluorometrically and colorimetrically, owing to their high hydroxyl group content and to the balance between surface hydroxyls and amines.To the best of our knowledge, this is the first time use of an oxygen-rich CDs-sensor owing to the use of glucose as a precursor [8].

Table S3
Comparison of sensing performance of different fluorescent probes for PA detection.

Abbreviations
Abbreviations

(
-NH2) groups, respectively.The molecular precursors were put into the hightemperature reaction flask through a condensation polymerization step forming a polymer-like graphite sheet which was further carbonized to form ON-CDs.The hydroxyland amino-groups functionalized on this graphite layer surface are responsible for the fluorescence of ON-CDs.Extensive research has shown that the fluorescent properties of ON-CDs are strongly influenced by the synthesis technique.Therefore, various reaction conditions such as reaction temperature and time were investigated to optimize the fluorescent performance of ON-CDs.As shown in Figure S2a and S2b, the fluorescence intensity of the prepared ON-CDs first increased (160 °C to 180 °C) and then decreased (180 °C to 190 °C) with increasing reaction temperature.Similarly, as shown in Figure S3a and S3b, the fluorescence intensity of ON-CDs first increased (2 h to 3 h) and then decreased again (3 h to 5 h) with increasing reaction time.With increasing temperature, the reaction is more complete and results in an increased yield of ON-CDs [1].In addition,

Table S2
Comparison of properties of different fluorescent probes.