Fast, eco-friendly synthesis of blue luminescent nitrogen-doped graphene quantum dots in glycerol

Nitrogen-functionalized graphene quantum dots (N-GQDs) were synthesized by hydrothermal transformation of glycine precursors in glycerol. Functionalization of GQDs with nitrogen was confirmed with FTIR spectroscopy. Atomic force microscopy (AFM) analysis revealed that the thickness of the quantum dots is around ~ 1 nm while the lateral dimensions are in the range from 20 to 80 nm. N-GQDs exhibit strong fluorescence in the blue (420 nm) region and they are suitable for bioimaging applications.


Introduction
Graphene quantum dots (GQDs), a relatively novel type of carbon-based materials, emerged as a potential new platform for applications in fluorescent biosensing and bioimaging (Das et al. 2018;Yang et al. 2014;Liu et al. 2018;Tang et al. 2012;Li et al. 2014;Younis et al. 2020). They belong to the class of 0D luminescent nanomaterials with the lattice structure similar to that of graphene. Usually, GQDs are described as small fragments of graphene with a variety of chemical moieties at the edges and basal planes (Ghaffarkhah 2022). Due to their specific structure, graphene quantum dots show specific fluorescence and electrocatalytic properties. The mentioned properties coupled with pronounced photostability, water solubility, biocompatibility and non-toxicity make these nanostructured particles suitable for a wide range of novel biomedical applications. Contrary to graphene, which is the zero band-gap material, GQDs have band-gap strongly dependent on their lateral sizes and edge atoms/groups. For this reason, the optical properties of GQDs can be readily modified by controlling their sizes and the edge-group chemistry (Mansuriya and Altintas 2020).
Doping GQDs with heteroatoms (e.g. nitrogen), provides an attractive route for the effective tuning of their intrinsic properties and for improving luminescence efficiency. It was shown that nitrogen doped graphene quantum dots (N-GQDs) exhibit better electrocatalytic activity and tunable luminescence, which extends the area for possible applications. Generally, the methods for fabrication of GQDs can be classified into "bottom-up" and "top-down (Jovanović 2019). We are focused on bottom-up strategy, which involves the fusion of small precursor molecules (e.g., citric acid, glucose, 1,3,6-trinitropyrene, etc.) into larger structures to form GQDs. The most important bottom-up methods are hydrothermal (Xie et al. 2017), microwave-assisted (Singh et al. 2019), soft-template (Tian et al. 2018), and stepwise organic synthesis routes (Tian et al. 2018). It is relatively easy to introduce heteroatom doping by hydrothermal method. Considering fabrication N-GQDs, above mentioned methods usually include multi-step synthesis, high temperature and high pressure. Thus, there are continuous research efforts for developing a facile one-pot method for the preparation of N-GQDs (Tang et al. 2013). Initial syntheses with citric acid carbonization (Dong et al. 2012) offered fast, cost-effective procedure for obtaining "regular" GQDs. Recently, Li et al. (Li and Wang 2015) modified this method by using a glycine precursor for preparation of N functionalized GQDs in ethylene-glycol. Glycine molecule served as both carbon and nitrogen source, opening a new chapter in facile and green syntheses of N-functionalized GQDs.
In the present paper, we report on green synthesis N-GQD based on glycine precursor, where ethylene-glycol is replaced with an even more affordable medium-glycerol. The obtained N-functionalized GQDs have intensive excitation dependent fluorescence in the blue region. The particles are further investigated by microscopic and optical methods and the results will provide information necessary for the future studies on their applications in fluorescent bioimaging.

Synthesis of nitrogen-doped NGQDs
To prepare N-GQDs (Fig. 1), the solution of glycine (0.5 g) and glycerol (10 ml) was heated at 190 °C for 20 min under magnetic stirring (250 rmin −1 ) and then cooled down to room temperature. The brown-yellow product, diluted 15 times with ultrapure (Milli-Q) water, was put in dialysis membrane (molecular weight: 10,000 Da) for 24 h. After dialysis, remaining N-GQD dispersion was used as a stock solution for further characterizations.

Characterization of nitrogen-doped NGQDs
Fourier transformed infra-red (FTIR) spectra were recorded in the region from 400 to 4000 cm −1 (with resolution of 0.5 cm −1 ) using a Nicolet iS50 FT-IR, Thermo Fisher Scientific spectrophotometer equipped by Smart iTR attenuated total reflectance (ATR) sampling accessory by placing powder samples on diamond plate and fixing them with pressure tower.
Atomic force microscopy (AFM) measurements were carried out on a Quesant atomic force microscope. 20 µl of NGQD dispersion was deposited on freshly cleaved mica surface using a spin coating method (3500 rpm, 1 min). Images were recorded in a tapping mode at room temperature, using premounted standard silicon probes Q-WM300 with force constant 40 Nm -1 (Nano and More, Germany).
The photoluminescence (PL) measurements of the samples were conducted on Horiba Jobin Yvon Fluoromax-4 spectrometer (Horiba, Kyoto, Japan), equipped with Peltier element and magnetic stirrer for cuvette, using quartz cell with 1 cm path length and 4 ml volume. Excitation wavelengths were in the 300-400 nm range. The spectra were collected in the air environment at a temperature of 25 °C. Figure 2 shows the FTIR spectra of glycine and the obtained N-GQDs. The quantum dots show typical bands of glycine, but slightly red-shifted, which possibly originates from hydroxyl bending vibrations (Dong et al. 2012). A pronounced peak at 1666 cm −1 and multiple weaker bands at 1466 cm −1 in N-GQD spectrum correspond to C = C stretching vibrations, confirming formation of skeletal polycyclic aromatic hydrocarbons (Tucureanu et al. 2016;Pavia et al. 2014). Peaks at 3183 cm −1 (N-GQDs) and 3147 cm −1 (glycine) originate from N-H stretching vibrations, while peaks at 1538 cm −1 (N-GQDs) and 1574 cm −1 (glycine) correspond to NH 3 + symmetric bending. The broad  (Li and Wang 2015). Absorption peaks at 1000-1300 cm −1 (C-O stretching), 1400 cm −1 (COO − stretching) and 1337 cm −1 (C-C stretching) in N-GDQs spectrum are matching glycine peaks in the same spectral range (Tucureanu et al. 2016;Fischer et al. 2005). Surface morphology of N-GQDs was investigated by atomic force microscopy (AFM). Figure 3a shows typical graphene quantum dots. From the height profiles of multiple N-GQDs (inset of Fig. 3a,) it was found that their average height and diameter are ~ 0.72 nm and 68 nm, respectively (Lee and Park 2019). Figure 3b shows the AFM image of a single N-GQD particle with corresponding height profile. Based on AFM measurements, it can be concluded that the size distribution of N-GQDs is relatively uniform and that the particles consist of one or few layers of graphene. Figure 4 shows photoluminescence (PL) emission spectra of dialyzed NGQD sample recorded at various excitation wavelengths in the range from 300 to 400 nm and photoluminescence excitation (PLE) spectrum that corresponds to emission λ em = 420 nm.

Results and discussion
It can be seen that the position of the emission band initially at 415 nm red-shifts with increasing in excitation wavelength. Typically, blue emission of GQD at ~ 420 nm is related to both the size and the surface state of sp 2 domains (Qu 2013). In N-GQD, it is suggested that two type of transitions contribute to PL spectrum: π → π* transitions of carbon sp 2 and n → π* transition of N state (Qu 2015). The excitation-dependent photoluminescence in Fig. 4 suggests that the distribution of sp 2 domains is not uniform probably due to variation in size distribution and chemical composition of the dots (Qu 2015). This also opens a possibility for tuning PL emission by controlling the nature of sp 2 sites via changing size, shape, and fraction of sp 2 domains.

Conclusion
We suggested fast, green, and cost-effective synthesis of luminescent nitrogen functionalized GQD in glycerol. This study successfully continues a new trend in GQD synthesis, employing cheap and non-toxic materials (glycine and glycerol) as a unique procedure compared to usual complicated and toxic synthesis methods. FTIR spectral analysis showed successful transformation of glycine molecules into N-functionalized GQDs. Although it has higher viscosity then previously used ethylene glycol, the glycerol proved to be a suitable medium for fabrication of N-GQDs. The stock solution of photoluminescent N-GQDs in glycerol can be easily diluted with water and adjusted for possible biomedical Fig. 4 Photoluminescence (PL) emission spectra of N-GQDs recorded at different excitation wavelengths. Photoluminescence excitation (PLE) spectrum was recorded at λ em = 420 nm applications. Due to their strong blue fluorescence, they can be employed as an bioimaging agent for monitoring drug delivery in targeted cells.