Facile Hydrothermal and Solvothermal Synthesis and Characterization of Nitrogen-Doped Carbon Dots from Palm Kernel Shell Precursor

: Carbon dots (CDs), a nanomaterial synthesized from organic precursors rich in carbon content with excellent ﬂuorescent property, are in high demand for many purposes, including sensing and biosensing applications. This research focused on preparing CDs from natural and abundant waste, palm kernel shells (PKS) obtained from palm oil biomass, aiming for sensing and biosensing applications. Ethylenediamine and L-phenylalanine doped CDs were produced via the hydrothermal and solvothermal methods using one-pot synthesis techniques in an autoclave batch reactor. The as-prepared N-CDs shows excellent photoluminescence (PL) property and a quantum yield (QY) of 13.7% for ethylenediamine (EDA) doped N-CDs (CDs-EDA) and 8.6% for L-phenylalanine (L-Ph) doped N-CDs (CDs-LPh) with an excitation/emission wavelength of 360 nm/450 nm. The transmission electron microscopy (TEM) images show the N-CDs have an average particle size of 2 nm for both CDs. UV-Visible spectrophotometric results showed C=C and C=O transition. FTIR results show and conﬁrm the presence of functional groups, such as -OH, -C=O, -NH 2 on the N-CDs, and the X-ray diffraction pattern showed that the N-CDs were crystalline, depicted with sharp peaks. This research work demonstrated that palm kernel shell biomass often thrown away as waste can produce CDs with excellent physicochemical properties. characterization of the as-synthesized CDs-EDA and CDs-LPh. The average particle size of CDs-EDA and CDs-LPh was estimated to be 2.10 ± 0.06 nm for CDs-EDA and 2.00 ± 0.06 nm for CDs-LPh, respectively. The shapes are all approximately spherical


Introduction
Carbon dots (CDs) are a nanomaterial synthesized from organic precursors discovered by the Xu research group in 2004 when they were purifying single-wall carbon nanotubes (SWNTs) [1][2][3]. CDs are in high demand and have caught researchers' keen interest because of their peculiarly low toxicity, suitable photoluminescence property, excellent water-solubility, dispersibility, broad range emission spectrum, biocompatibility, and high resistance to photobleaching. These features are responsible for their photocatalysis, electrocatalysis, chemical/biosensing, and bioimaging applications [4,5]. Unlike semiconductor-based quantum dots (QDs) and some conventional organic dyes with similar properties, they are characterized by an inherent low water solubility and toxicity issues [6].

Characterization Instruments
Vecstar Muffle furnaces (Power: 3 KW, Max Tem.: 1600 • C, Max Cont.: 1500 • C) were used for the carbonization of the powdered palm Kernel Shell while, Protech Oven: Model FAC-50 Malaysia, was used for the heating of the Autoclave and Autoclave batch reactor for the synthesis of the CDs. Optical properties measurements were performed at room temperature using the Spark multimode microplate reader, Ultraviolet-visible spectrophotometer (UV-Visible Spectrophotometer: Infinites F500, TECAN, Ltd., Mannedorf, Switzerland) for photoluminescence (PL) emission spectra measurements at 360 nm and Perkin Elmer Lambda 35 UV-Visible Spectrophotometer, USA, was used to record the absorbance of the CDs, between 200 nm to 700 nm. A Fourier-transform infra-red spectrophotometer (FTIR) was run to determine the functional groups on the CDs, Transmission Electron Microscopy (TEM), X-ray Diffraction study and Dynamic light scattering (DLS) measurement with a Zetasizer Nano ZS instrument (Malvern Instruments, Malvern, UK) were carried out for the surface morphology and surface charge determination of the CDs [59-63].

Preparation of Palm Kernel Shell
The palm kernel shell (PKS) biomass used as precursors in this research work was bio-agricultural waste collected from Perusahaan Minyak Sawit Bintang (Johor, Malaysia). The KPS samples were washed thoroughly several times with distilled-deionized water and air-dried. It was then dried in an oven at 70 • C and crushed into powder [57,64].

Carbonization of Palm Kernel Shell
Three different PKS biomass material masses were weighed in an aluminum boat, namely 17.97 g, 20.14 g, and 18.58 g, as shown in Table 1, and placed in an inert atmosphere of nitrogen gas in a quartz tube, in the midpoint of the furnace heating chamber, as shown in Figure 2. The nitrogen gas was purged at a flow rate of 50 ccs/min through the quartz tube during the carbonization period at a temperature of 600 • C for 3 h at a 10 • C/min rise in temperature. Nitrogen gas was kept running for 3 h until the furnace chamber temperature was brought to 40 • C [42,57,65,66]. The percentage yield of the carbonized palm kernel shell (CPKS) materials was determined after the sample temperature reached room temperature by again weighing the carbonized materials using Equation (1) below: where MCPKS is the mass of carbonized palm kernel shells, and MPKS is the mass of palm kernel shells, respectively [57,64,67]. of the carbonized palm kernel shell (CPKS) materials was determined after the sample temperature reached room temperature by again weighing the carbonized materials using Equation (1) below: Percentage Yield of CPKS = × 100 (1) where MCPKS is the mass of carbonized palm kernel shells, and MPKS is the mass of palm kernel shells, respectively [57,64,67].

Synthesis of Carbon Dots (CDs) Using Hydrothermal Method
N-doped CDs were synthesized via hydrothermal synthesis from CPKS. A 0.3 g of powdered CPKS was dissolved into the mixture of milli-Q water (15 mL), 1 M NaOH (3 mL), and ethylenediamine (EDA) (0.2 mL), then stirred for 30 min. The mixture transferred to an airtight Teflon-lined stainless-steel autoclave batch reactor for hydrothermal treatment at temperatures of 120-200 • C for 3 h and then brought to 40 • C after the reaction completed. The mixture was filtered using filter paper, and then it was again filtered using a filter syringe disc attached to a 20 mL syringe to obtain the N-CDs. Different ethylenediamine amounts (0.2-1.4 mL) and temperatures (120-200 • C) were investigated for the optimization study.
The CDs doped with L-phenylalanine (CDs-L-Ph) were prepared as the procedure above except that 15 mL ethanol replaced Milli-Q water as the solvent and a different amount of L-phenylalanine (60-140 mg) replaced EDA as the dopant. The as-synthesized CDs, CDs-EDA and CDs-L-Ph, were then adjusted to pH 7.5 and 7.8 using 2 M hydrochloric acid (HCl). The furnace set up for the CDs' preparation illustrated in Figure 2 [15,[68][69][70][71].

Measurement of Quantum Yields
Quantum yield (ΦY) of the synthesized CDs was estimated using a general refractive index of 1.384 from literature (1) by comparing the CDs with quinine sulfate as a reference compound (fluorophores). Quinine quantum yield reported as 0.53 when dissolved in 0.1 M H 2 SO 4 has a refractive index η = 1.346. The quinine sulfate solution was prepared and diluted to have an optical density (OD) = 0.00, 0.02, 0.04, 0.06, 0.08, 0.1, and the synthesized CDs treated in the same manner. The ΦY of CDs in water having refractive index η = 1.384 was estimated using Equation (3) obtained from Equation (2) because the absorbance values were components of the gradients: where ΦY = the quantum yield, IFI is the integrated fluorescence intensity, Grad is the gradient from the plot of integrated fluorescence intensity versus absorbance, A = absorbance, η the refractive index, and the subscripts ST and S denote standard and test sample, respectively [1,26,46,72]. Figure 2 shows the instrumentals for the pyrolytic carbonization of palm kernel shell (PKS) in an inert tubular nitrogen gas chamber of a furnace, which produced a black carbonaceous char with an average yield of 30.96% as tabulated in Table 1. The result agrees with other research groups [42,56]. Their analysis shows that the obtained char was about 30%, the char results from the thermal decomposition of cellulose, lignin, and hemicellulose in the PKS precursor. The average of 30.96% of char obtained in this work produced from the PKS consists of 20.8-27.7% cellulose, 21.6-22.3% hemicelluloses, and 44.0-50.7% lignin [56,73]. Previous studies affirmed that cellulose, hemicellulose, and lignin are the most abundant components of crude biomass materials, including PKS [7,22,55,58]. The studies postulated that the composition of biomass influences the derived carbonaceous material properties, and hence show the possible transformation of cellulose, hemicelluloses, and lignin to graphitic materials by the dehydration condensation process. The dehydration condensation process is one way to form carbonaceous materials or CDs when biomass, precisely, cellulose, hemicellulose, and lignin subjected to pyrolysis. The authors showed that the individual percentage conversion of the constituents of biomass was as follows: cellulose (9%), hemicellulose (0%), and lignin (39%) at 900 • C and 21%, 4.6% and 52% at 400 • C calcination temperature, respectively [7,22,55,58]. Jiang et al. and Nasir et al. have characterized the graphitic material obtained from the char of carbonized PKS to graphene oxide and reduced graphene oxide. The study showed that the highest percentage conversion of PKS to carbonaceous material was 32% at 400 • C [57,58]. The type of graphitic material produced after the carbonization depends on the material source, heating rate, and temperature condition of pyrolysis [56]. This study showed that the average percentage conversion of PKS to carbonaceous material was approximately 30.96% at 600 • C.

Synthesis and Characterization of CDs
The hydrothermal/solvothermal synthesis of N-doped fluorescent CDs was prepared in a Teflon-lined autoclave batch reactor by a one-step synthesis procedure. The results show that subjecting CPKS to hydrothermal/solvothermal synthesis caused the material to undergo pyrolysis, nucleation, condensation, polymerization carbonization, and surface functionalization [1,22,46]. The aforementioned synthetic route or process is the approach for the fabrication of CDs, which changed the CPKS and resulted in photoluminescent crystalline CDs obtained in this research work [1,46,48]. The produced CDs emit a blueviolet colour at a wavelength between 430 and 450 nm when excited at a wavelength range of 350-400 nm in an alkaline medium, as shown in Figure 4a show that subjecting CPKS to hydrothermal/solvothermal synthesis caused the material to undergo pyrolysis, nucleation, condensation, polymerization carbonization, and surface functionalization [1,22,46]. The aforementioned synthetic route or process is the approach for the fabrication of CDs, which changed the CPKS and resulted in photoluminescent crystalline CDs obtained in this research work [1,46,48]. The produced CDs emit a blue-violet colour at a wavelength between 430 and 450 nm when excited at a wavelength range of 350-400 nm in an alkaline medium, as shown in Figure 4a

Optimization Parameter for the Synthesis of Carbon Dots (CDs)
The best reaction time for synthesis of the CDs determined at a fixed ratio of CPKS/H 2 O (20 mg/1 mL) treated hydrothermally from 1-5 h at 160 • C, without adding any dopants (Figure 3a). The results showed that the CDs synthesized for 3 h and 160 • C temperature fluoresced under UV light, as depicted in Figure 3b. Therefore, the best synthesis time of 3 h selected for this study and application study of CDs.
For surface passivation, ethylenediamine (EDA) and L-phenylalanine (L-Ph) were used as dopants to introduce N-atom possibly into the carbon nuclear lattice of the graphitic carbon of the CPKS materials and as well as to introduce amino (-NH 2 ) functional group on the surface of the CDs [74] (Figure 3c). The synthesized CDs-EDA and CDs-L-Ph have shown fluorescence property when viewed under UV light indicating the CDs passivation process with the dopants still retain the fluorescence property of the CDs (Figure 3d).

Effect of Amount of Dopant
The impact of EDA's volume on the as-prepared CDs-EDA's fluorescence properties was studied by varying the amount of EDA from 0.2-1.0 mL, and the amount of CPKS, NaOH, and H 2 O was fixed at 0.3 mg, 3 mL (1 M) and 15 mL, respectively. As shown in Figure 4c, the fluorescence of CDs-EDA increases with an increase in EDA volume. A volume of 1 mL of EDA produced the highest fluorescence intensity with slight narrower peaks. The peaks become unimportantly small for optical consideration when the amount of EDA is above 1 mL. This observation in agreement with the work reported by [75]. The fluorescence peaks showed a slight redshift with an increase in the volume of EDA. Figure 4d shows L-Ph's effect as a doping agent on the fluorescence properties of CD-LPh. The produced CDs-LPh show varied fluorescence intensities with different amounts of L-Ph used. The increasing quantity of L-Ph from 80-120 mg increases the fluorescence peaks, and maximum intensity observed at 120 mg of L-Ph. Above 1 mL of EDA for CDs-EDA and 120 mg of L-Ph for CDs-LPh, the fluorescence intensity decreases because the concentration of the functional groups functionalizing the CDs at the surface of the CDs increased to a point and overcrowded the surface of the CDs [3,75].

Effect of Temperature of Synthesis on CDs
The effect of different temperatures on the synthesized N-doped CDs and the fluorescence properties shows that the produced CDs' intensity increases with increasing temperature of the synthesis [75]. Figure 4e shows the fluorescence intensity profile of the effect of temperature on the synthesized CDs-EDA. The fluorescence intensities showed incremental gradation with an increase in temperature from 120-200 • C with the highest synthesis temperature of 200 • C, delivering the highest fluorescence intensity. In Figure 4f, the effect of temperature on CDs-L-Ph's fluorescence intensity was displayed. By varying temperature from 120-200 • C, the CDs showed increasing fluorescence intensity with a peak centred in the range of 420-450 nm. The maximum fluorescence peak obtained at 200 • C using an excitation wavelength of 360 nm. However, the fluorescence peak produced at 160 • C and 360 nm excitation wavelength appeared more stable for the two CDs. The fluorescence peaks become unstable above 160 • C, which may be due more to the high degree of carbonization than the degree of surface functionalization. The process leads to an irregular and narrow fluorescence peak [75]. Thus, the temperature of 160 • C was chosen for the CDs synthesis for stable and appreciably better fluorescence peaks produced at that temperature.

Determination of Photoluminescence Quantum Yield
The quantum yields of the prepared CDs were determined by comparing the photoluminescence properties of the CDs with that of quinine sulfate using a UV-Visible spectrophotometer for absorbance measurement and a Tecan Multimode reader for the fluorescence measurement [1,26,76]. The quantum yield obtained for the CDs-EDA was 13.5% with a concentration of 50.1 mg/mL, and CDs-LPh was 8.6% with a concentration of 10.0 mg/mL. The gradient of the plot of integrated fluorescence intensities of quinine sulfate, CDs-EDA, and CDs-LPh against their absorbances is shown in Figure 5. The quantum yield (QY) of CDs-EDA and CDs-L-Ph obtained in this study was also compared with other synthesized CDs. The QY of CDs got from other natural sources are Ginko fruit (3.33%) [47], mangosteen pulp (1.09%) [77], prawn shell (9%) [78], Pyrus pyrifolia or apple juice (4.27%) [79]. glucose (2.4%) [80], sugar cane bagasse (12.4%) [81], banana juice (8.95%) [43], microcrystalline cellulose (51%) [74], and Acacia concinna seed (10.2%) [82]. The obtained QY of 8.5% and 13.7% for the CDs shows that PKS biomass is a source for producing good quality and high QY CDs for optical imaging and other applications.   Figure 6 shows the results of transmission electron microscopy (TEM) for the morphological characterization of the as-synthesized CDs-EDA and CDs-LPh. The average particle size of CDs-EDA and CDs-LPh was estimated to be 2.10 ± 0.06 nm for CDs-EDA and 2.00 ± 0.06 nm for CDs-LPh, respectively. The shapes are all approximately spherical and are monodispersed with both CDs having visible lattice fringes with a lattice space of 0.23 nm according to the TEM results, which indicates crystallinity in the CDs [83]. The similar physical properties observed for the two types of CDs may be due to the precursors' subjection to the same time and temperature of carbonization. The nanomaterials synthesized here in this research have similar shapes, particle size, and excitation/emission wavelengths at 360 nm/430 nm despite utilizing different dopants, and the  Figure 6 shows the results of transmission electron microscopy (TEM) for the morphological characterization of the as-synthesized CDs-EDA and CDs-LPh. The average particle size of CDs-EDA and CDs-LPh was estimated to be 2.10 ± 0.06 nm for CDs-EDA and 2.00 ± 0.06 nm for CDs-LPh, respectively. The shapes are all approximately spherical and are monodispersed with both CDs having visible lattice fringes with a lattice space of 0.23 nm according to the TEM results, which indicates crystallinity in the CDs [83]. The similar physical properties observed for the two types of CDs may be due to the precursors' subjection to the same time and temperature of carbonization. The nanomaterials synthesized here in this research have similar shapes, particle size, and excitation/emission wavelengths at 360 nm/430 nm despite utilizing different dopants, and the different dopants may be responsible for the different levels of crystallinity and peak intensities of the CDs [1,65,75,83,84].

Transmission Electron Microscopy (TEM) and X-ray Diffraction Spectroscopy (XRD)
Appl. Sci. 2020, 10, x FOR PEER REVIEW 10 of 18 X-ray diffraction (XRD) technique is a fundamental analytical process that helps in studying, determining, and identifying powdered heteroatoms dopped CDs structure, purity, and crystallinity [85,86]. The XRD was based on diffraction angles of 0°-100° at a scan rate of 2.00 (°/min) at room temperature. The XRD data in Figure 7a,b were obtained to ascertain the CDs' crystallinity, nature, and purity. CDs-EDA shows very sharp absorption peaks at 32.4° and 46.12°. In comparison, CDs-L-Ph peaks ere at 32.62°, 46.26°, and 66.78° at two theta angle measurements for diffraction angles between 32° and 47° for CDs-EDA and 30° to 70° for CDs-L-Ph, which is an indication of high crystallinity of the CDs samples [86]. Though the two CDs have similar optical properties and size, this difference may be due to the different dopants used in their fabrication. X-ray diffraction (XRD) technique is a fundamental analytical process that helps in studying, determining, and identifying powdered heteroatoms dopped CDs structure, purity, and crystallinity [85,86]. The XRD was based on diffraction angles of 0 • -100 • at a scan rate of 2.00 ( • /min) at room temperature. The XRD data in Figure 7a,b were obtained to ascertain the CDs' crystallinity, nature, and purity. CDs-EDA shows very sharp absorption peaks at 32.4 • and 46.12 • . In comparison, CDs-L-Ph peaks ere at 32.62 • , 46.26 • , and 66.78 • at two theta angle measurements for diffraction angles between 32 • and 47 • for CDs-EDA and 30 • to 70 • for CDs-L-Ph, which is an indication of high crystallinity of the CDs samples [86]. Though the two CDs have similar optical properties and size, this difference may be due to the different dopants used in their fabrication.

UV-Visible Spectroscopy
The UV-visible spectroscopic study measures the absorptivity of CDs, and it is a fundamental property of materials and nanomaterials. The CDs showed colour between indigo to blue under UVlight and emission wavelength between 430-450 nm. The specific emission wavelength is related to the colour absorbed by the substance producing the emission. Therefore, the measure of the absorptivity of CDs using a UV-Visible spectrophotometer, among other parameters, can be a quantitative and qualitative characterization technique for the CDs [1]. The UV-Visible spectroscopic measurement of the samples was obtained by scanning the wavelength between 200-700 nm ( Figure  8). It shows prominent absorption peaks at 260-280 nm and 320-340 nm. The peak at 260-280 nm corresponds to π-π* transition of aromatic C=C bonds from the core sp 2 hybridized carbon fragments and the conjugated π nanodomain within the carbon dots structure. The peak at 320-340 nm, were attributed to the n-π* transition of non-bonding electrons in C=C and C=O, an attribute from the molecular state of the carbon dots [1,87,88].

UV-Visible Spectroscopy
The UV-visible spectroscopic study measures the absorptivity of CDs, and it is a fundamental property of materials and nanomaterials. The CDs showed colour between indigo to blue under UV-light and emission wavelength between 430-450 nm. The specific emission wavelength is related to the colour absorbed by the substance producing the emission. Therefore, the measure of the absorptivity of CDs using a UV-Visible spectrophotometer, among other parameters, can be a quantitative and qualitative characterization technique for the CDs [1]. The UV-Visible spectroscopic measurement of the samples was obtained by scanning the wavelength between 200-700 nm (Figure 8). It shows prominent absorption peaks at 260-280 nm and 320-340 nm. The peak at 260-280 nm corresponds to π-π* transition of aromatic C=C bonds from the core sp 2 hybridized carbon fragments and the conjugated π nanodomain within the carbon dots structure. The peak at 320-340 nm, were attributed to the n-π* transition of non-bonding electrons in C=C and C=O, an attribute from the molecular state of the carbon dots [1,87,88].   [39,89,90]. FTIR with medium or small and broad absorption bands around 3500-3200 cm −1 could correspond to N-H aliphatic amine [91] but, the peaks were absent in L-Ph maybe because there was only one molecule of -NH2 in L-Ph, making NH2 less abundant to be detected, the broad and medium absorption band at 2964.1 cm −1 in L-Ph is due to -OH of carboxylic acid stretching. A functional group appeared around 1650-1590 cm −1 for all the bands, which corresponds to the -NH2 scissoring vibration of a primary amine. The aminofunctional group appeared in all the four bands, signifying the presence of amine in the EDA, CDs-EDA, L-Ph, and CDs-LPh. By extension, this confirmed the functionalization of amine on the surface of the carbon dots (CDs-EDA and CDs-Lph), respectively. The CDs show similar broad and intense peaks at 3280.4 cm −1 (CDs-EDA) and 3322.8 cm −1 (CDs-LPh), corresponding to normal polymeric hydroxyl (O-H) stretching vibration, an indication that the CDs has -OH functional group. The other peaks at the fingerprint region at 1360.0 cm −1 (CDs-LPh), 1405.9 cm −1 , 1085.7 cm −1 , 1041.4 cm −1 , and 879.4 cm −1 could be due to sp 2 and sp 3 stretching vibrations and out-of-plane bending modes of C-H groups [1,92].  [39,89,90]. FTIR with medium or small and broad absorption bands around 3500-3200 cm −1 could correspond to N-H aliphatic amine [91] but, the peaks were absent in L-Ph maybe because there was only one molecule of -NH 2 in L-Ph, making NH 2 less abundant to be detected, the broad and medium absorption band at 2964.1 cm −1 in L-Ph is due to -OH of carboxylic acid stretching. A functional group appeared around 1650-1590 cm −1 for all the bands, which corresponds to the -NH 2 scissoring vibration of a primary amine. The amino-functional group appeared in all the four bands, signifying the presence of amine in the EDA, CDs-EDA, L-Ph, and CDs-LPh. By extension, this confirmed the functionalization of amine on the surface of the carbon dots (CDs-EDA and CDs-Lph), respectively. The CDs show similar broad and intense peaks at 3280.4 cm −1 (CDs-EDA) and 3322.8 cm −1 (CDs-LPh), corresponding to normal polymeric hydroxyl (O-H) stretching vibration, an indication that the CDs has -OH functional group. The other peaks at the fingerprint region at 1360.0 cm −1 (CDs-LPh), 1405.9 cm −1 , 1085.7 cm −1 , 1041.4 cm −1 , and 879.4 cm −1 could be due to sp 2 and sp 3 stretching vibrations and out-of-plane bending modes of C-H groups [1,92].

Conclusions
This research successfully produced hydrothermal/solvothermal CPKS derived N-doped CDs. The fabricated CDs characterization showed that the manufactured CDs possessed high-intensity fluorescence with good quantum yield value. The as-prepared CDs have shown excellent water solubility, dispersibility, with a stable excitation/emission wavelength of 360 nm/430 nm at neutral pH, which could have a suitable application in biosensing. Two CDs with two different doping agents (EDA and L-Ph) having unique fluorescence properties that increase with an increasing volume of the dopants fabricated. This research work demonstrated that palm kernel shell biomass often thrown away as a waste can be used to produce CDs with sterling physicochemical properties.

Conclusions
This research successfully produced hydrothermal/solvothermal CPKS derived Ndoped CDs. The fabricated CDs characterization showed that the manufactured CDs possessed high-intensity fluorescence with good quantum yield value. The as-prepared CDs have shown excellent water solubility, dispersibility, with a stable excitation/emission wavelength of 360 nm/430 nm at neutral pH, which could have a suitable application in biosensing. Two CDs with two different doping agents (EDA and L-Ph) having unique fluorescence properties that increase with an increasing volume of the dopants fabricated. This research work demonstrated that palm kernel shell biomass often thrown away as a waste can be used to produce CDs with sterling physicochemical properties.