Figure 1 HRTEM images of N-CQDs at different magnification and scale: (one) 20 nm, (do) 10 nm, (si) Gaussian particle size distribution histogram, (Hey) graphic kernel grid. N-CQDs typically have a particle size of 4.60 ± 0.87 nm. To determine the nature of the functionalization, the synthesized N-CQDs were investigated using Fourier transform infrared (FTIR) spectroscopy. The samples were classified into two groups: (i) N-CQDs with lower ammonia concentration (from N-0.25 to N-1) and (ii) with higher ammonia concentration (from N-2.5 to N-10). . FTIR spectra (Fig. 2) showed that all N-CQDs have hydrophilic groups on their surface, such as OH (hydroxyl) corresponding to the peak at 3389 cm-1 and NH (3263 cm-1), thus confirming their good solubility in water. In addition, C–H (2950 cm−1), C=O (1581 cm−1), C–N (1435 cm−1) and C–O (1080 cm−1) bond vibrations were also observed in each sample 13 ,14,15. Comparison of the FTIR spectra (Fig. S4) of the samples showed that increasing N-doping (ammonia concentration, from N-0.25 to N-1) exhibited a decreasing trend in the C–O bond vibration at 1080 cm−1 . While the group of samples with higher ammonia concentration (N-2.5 to N-10) showed a sharp vibration of the C–N bond at 1435 cm−1. Figure 2 FTIR spectra of N-CQD with lower ammonia concentration (from N-0.25) and higher ammonia concentration (N-10). To gain a deeper understanding of the surface characterization of N-CQDs and also to investigate the chemical composition of N-CQDs, X-ray photoelectron spectroscopy (XPS) was used. The resulting XPS spectra shown in Fig. 3 were deconvoluted using Voigt functions (Lorentzian and Gaussian width) with a discrete inelastic background for each element16. A minimum number of components are used to achieve a convenient fit. The binding energy scale was calibrated to the typical C 1s value of 284.6 eV. The atomic composition has been determined using the integral regions provided by the deconvolution process normalized to the atomic sensitivity factor (Table S1). The XPS spectrum of N-CQDs shows three typical peaks C1s (285.0 eV), N1s (399.0 eV) and O1s (531.0 eV). The fitted C1s spectrum was deconvoluted into four components, corresponding to carbon in the form of bonds C=C/C–C (~ 284.4 eV), C–O/C–N (~ 285.8 eV), C=O (~ 287.3) and O=C–OH (~ 288.4 eV)17. Whereas, the N1s band showed three peaks after deconvolution which are 398.8 eV, 399.6 eV and 400.8 eV, representing pyridinate N, NH and amide CN, respectively18. Figure 3 Representative XPS spectra of N-CQDs showing the lowest (N-0.25) and highest (N-10) nitrogen doped samples. The spectra show three typical peaks C1s (285.0 eV), N1s (399.0 eV) and O1s (531.0 eV). The unfolded N1s band showed three peaks representing pyridinate N, NH and amide CN. The content of each kind of nitrogen doping (pyridinic, pyrrolic, and graphitic) is determined and quantified from the XPS spectra of NCQDs in order to understand their influence on the optical and chemical properties (Table S2). As commonly reported, the fluorescent property of CQDs can be improved by using nitrogen doping. However, only carbon-bound nitrogen can improve emission19. Also, a larger N/C ratio was observed for N-CQD samples synthesized with higher ammonia concentration (Table S1). The O1s region contains three peaks at 530.9 eV, 532.2 eV and 533.3 eV for C–OH/C–O–C, C=O, H–O–H, respectively20. In addition, oxygen content is also a key parameter in N-CQDs emissions as it can maintain the balance between sp2 and sp321 carbon atoms. Therefore, Raman spectroscopy was used to investigate the disorder in the carbon bond arrangement of N-CQDs. The Raman spectra (Fig. S5) of N-CQDs showed typical graphical features consisting of the D mode (at 1368 cm−1) related to the symmetry transformation from the defects, and the G band (at 1586 cm−1) , which is assigned to the sp2 bonds of the graphitic core (such as graphite). This is not surprising, as HRTEM images of N-CQDs showed a typical graphite lattice spacing (see Fig. 1d). When comparing Raman spectra between N-CQDs, at first glance these spectra appear similar, a common ID/IG ratio of 0.95 revealed a balance between sp2 and sp3 bonds in the structure of N-CQDs. This is different from g-CQDs where an ID/IG ratio of 0.83 was observed and attributed to the carbon core (sp2 bonds)10. This is probably attributed to the changes introduced by nitrogen doping resulting in the transformation of CC (sp2 bonds) to sp3 bond between N, O and C.

Optical properties of N-CQDs

The absorption spectra of the as-prepared N-CQDs measured using UV-Vis spectrophotometry are shown in Fig. 4. The N-CQD samples have a strong peak around 265 nm and a shoulder around 295 nm (Fig. 4a). The absorption peak at 265 nm is characteristic of π–π* transitions of the graphitic core (C=C or C–C) of the sp2 domains present in the sp3 environment and 295 nm is attributed to n–π* (C=O ) transitions and C–N/C=N22,23 bonds. For comparison, the absorption spectrum of CQDs without nitrogen doping was also measured. It is noted that the absorption peaks associated with N-CQDs are red-shifted compared to g-CQDs (synthesized from the same glucose source but without nitrogen doping), Fig. 4b. These transitions are observed at 225 nm = π–π* (graphic core), and 280 nm = n–π* transitions (C=O)10. Therefore, the absorption peak observed at 295 nm in the case of N-CQDs is due to the formation of C–N/C=N bonds related to the doping effect caused by the presence of graphitic nitrogen24,25. Figure 4 (one) UV–Vis absorption spectra of (one) N-CQD and (si) g-CQDs without nitrogen doping. The presence of C–N/C=N bonds is observed at 295 nm. The photoluminescence (PL) spectra of the as-prepared N-CQDs were measured using a range of different excitation wavelengths, as shown in Fig. 5. The PL emission of each sample clearly showed the excitation-dependent PL which is beneficial for a variety of applications such as biosensors , bioimages or LED devices26,27. The PL emission peaks shifted when different excitation wavelengths were applied, and each sample exhibited an optimal excitation wavelength. Overall, the PL study revealed interesting optical properties of N-CQDs. First, the PL results are consistent with previous reports where the excitation-dependent CQD emission effect was observed28. Second, the maximum excitation wavelengths varied from 360 to 320 nm with ammonia concentration. Figure 5 Photoluminescence spectra of CQD with and without nitrogen doping measured using excitation wavelengths in the range from 300 to 500 nm, (one) g-CQDs (without nitrogen doping). (si) N-0.25; (do) N-2.5, (Hey) N-10. However, the mechanisms behind the excitation-dependent properties of CQDs are not clear. One of the most comprehensive and widely accepted mechanisms to explain the excitation-dependent PL of CQDs is the quantum confinement effect also known as the size effect14,21,28,29. In general, CQDs possess broad particle size distributions which leads to a range of different energy gaps and is the reason for the variation in emission wavelengths30,31. But here, HRTEM image data analyzes confirmed that the increased amount of nitrogen doping did not contribute to the increase in particle size for the as-prepared samples. Therefore, the observed red-shift character can be attributed to the radiative recombination of the e−h pairs hosted in the sp232 complexes. In addition to the quantum confinement effect, surface state theory is rather widely adopted to explain the excitation-dependent PL behavior of CQDs33,34,35. UV–Vis absorption showed that the peak of N-CQDs at 265 nm is related to the π–π* transition, which indicates the existence of a large number of π-electrons. Surface electronic states can couple with these π-electrons as a result of surface oxidation which results in modification of the electronic structure of N-CQDs34,36. To interpret the mechanism of this effect, the PL lifetime and PLQY of N-CQDs were measured. The obtained results (Table 1) showed an increase in both PL lifetime and PLQY upon nitrogen doping and the highest lifetime and PLQY values ​​were obtained for (\left[N\right]\ge 7.5 M). The obtained PLQY value of 9.6%(\pm) 0.9 for N-10 is a significant improvement compared to g-CQDs that showed PLQY < 1%10. These results are comparable to the literature (shown in Table S3), where CQDs and N-CQDs were synthesized through different methodologies. Table 1 The photoluminescence quantum yield (PLQY), average lifetime, 1/e lifetime, radiative (kr) and non-radiative (knr) rates of N-CQDs. The radiative rate (kr) and non-radiative rate (knr) were calculated using Eq. (2) and (3) 37. $${k}{r}=\frac{\Phi }{{\tau }{1/e}}$$ (2)
$$\Phi =\frac{{k}{r}}{{k}{r}+{k}{nr}}$$ (3)
where (\Phi) is PLQY of N-CQD and ({\tau }{1/e}) corresponds to the lifetime when the fluorescence drops 1/e of its initial value. Table 1 and Figure 6 show that when a higher concentration of ammonia was used, the nonradiation rates were significantly reduced. This is due to surface coating activities…