The citric acid and urea were obtained from HiMedia Laboratories Pvt. Ltd., India. The DMF (N, N-Dimethylformamide) was purchased from Merck Millipore. Hydrogen peroxide was obtained from SRL Pvt. Ltd., India. A Milli-Q device (Millipore, Burlington, MA, USA) provided the deionized water. Human origin myeloperoxidase (MPO) was purchased from Merck, Rahway, NJ, USA.

The functionality and photoluminescence characteristics of carbon quantum dots can be precisely controlled by adjusting the surface functional groups and chemical configurations (27). A comprehensive review by (7) have explored into the synthesis of CQDs highlighting the potential for functionalization in accordance with their application requirements. Carbon quantum dots synthesis may be achieved through two distinct strategies: one is a top-down approach, where larger carbon-based materials are systematically reduced in size to form quantum dots. The second approach, called bottom-up, takes the opposite route by assembling quantum dots from smaller carbon-containing molecules or compounds (28, 29).

Here, the microwave-assisted synthesis method was utilized to prepare CQDs using urea and citric acid as the starting materials. Equal amounts of citric acid and urea, 5.0 grams each, were combined to create a 1:1 mixture for the synthesis of CQDs (30). Then this mixture was supplemented with 2 ml of N, N-dimethylformamide (DMF) solution. The prepared mixture was then microwaved at full power for two minutes using a standard domestic microwave. This process resulted in a charred substance with a chestnut brown color. After allowing this mixture to cool to room temperature, it was diluted with 50 ml of highly purified (Milli Q) water (31). The mixture was thoroughly blended using a high-speed homogenizer at 12,000 rpm for 15 min. The homogenized solution was centrifuged at 10,000 rpm for 40 min to remove larger particles, lumps, and residues from the CQDs. Following centrifugation, the pellet was discarded, and the supernatant was collected for subsequent processing (Figure 1). The supernatant was then dialyzed using 1 KDa dialyzer overnight at room temperature under continuous stirring. The dialyzed CQDs were then dried using a lyophilizer and were kept for long-term storage at 4°C.

For the physicochemical characterization of CQDs, 100 µg/ml of CQDs were suspended in Milli Q water to disperse the CQDs. The suspension was sonicated thoroughly in a bath sonicator (Ikon Industries) for 15 min with ice-cold water. Malvern Zetasizer Advance Series (Malvern, UK) instrument was used for the analysis of the particle size distribution and zeta potential at room temperature to maintain the stability of CQDs. The resulting graph and data were analyzed using zetasizer associated software.

UV absorption scans (200–400 nm) and fluorescence spectrum scans (300–750 nm) of carbon quantum dots were conducted using an Envision device (PerkinElmer, Waltham, MO, USA). The resulting spectra were then analyzed with SpectraGryph software.

During this research, The KBr disk approach was specially used to generate Fourier-transform infrared spectra utilizing a Mattson 5,000 spectrometer (Unicam, United Kingdom).

For surface topology, scanning electron microscope is used through the utilization of an accelerating voltage ranging from 10–15 kV.

When carrying out the enzymatic biodegradation of CQDs, we implemented the methodology outlined by (32). An aliquot of 1 ml of Milli-Q water (autoclaved), containing a concentration of 140 mM NaCl, was employed in the suspension of 5 μg/ml of human-derived MPO, which was subsequently combined with 50 μg of CQDs (resulting in a final concentration of 100 μg/ml). Subsequently, H₂O₂ was then added to the mixture at a rate of 100 μM h⁻¹ for 24 h (32). Following an interval of six hours, the MPO enzyme was reintroduced, and the reaction mixture was sustained at a controlled temperature of 37°C (33). Subsequently, equivalent volumes of CQDs were dispersed in 1 ml of autoclaved Milli-Q H₂O and subjected to a treatment duration of 24 h, with or without the addition of H₂O₂, at a rate of 100 M h⁻¹. Upon the completion of reaction period, the samples were subjected to an analytical procedure to evaluate their absorbance within the wavelength range of 300–500 nm utilizing a UV-1800 spectrometer (Shimadzu, Kyoto, Japan). The final spectra were then processed using SpectraGryph software.

Carbon quantum dots were synthesized utilizing a 1:1 mixture of urea and citric acid through a simple single-step microwave-assisted approach using a 900-watt domestic microwave. The resulting CQDs were characterized using dynamic light scattering (DLS) to reveal a narrow size distribution with an average hydrodynamic diameter of 10.73 ± 0.18 nm (Figure 2), indicating the formation of uniform, nano-sized particles. The zeta potential was determined to be −11.9 ± 2.7 mV, suggesting colloidal stability of the CQDs in aqueous suspension (Figure 3). Furthermore, Carbon Quantum Dots (CQDs) were stimulated within the ultraviolet B (UVB) spectrum ranging from 280–320 nm. The resultant CQDs exhibited a photoluminescence (PL) emitting a blue color, as depicted in Figure 4 which is absent in standard white light sources. Neither chemical precursors nor dimethyl formamide shows any blue photoluminescence. This blue photoluminescence exhibited by CQDs arises from a combination of factors, including emissive traps, aromatic conjugate structures, triplet carbenes at zigzag edges, and surface functional groups such as carboxyl and oxygen-rich hydroxyl moieties (34).

The maximum photoluminescence emission observed from the citric acid derived carbon quantum dots was identified at an excitation wavelength of 320 nm, displaying an emission peak at 430 nm (Figure 5). The photoluminescence characterized by blue color observed in carbon dots (CDs) is significantly modulated by the existence of emissive trapping sites, the presence of triplet carbenes situated at the zigzag boundaries, the configuration of aromatic conjugated frameworks, and the incorporation of functional moieties such as carboxyl groups and hydroxyl groups that are abundant in oxygen, all of which contribute to this intricate phenomenon (31).

The FTIR analysis conducted on the citric acid-derived carbon quantum dots (CQDs) elucidated the existence of N–H and O–H functional groups within the spectral range of 3,463–3,186 cm⁻¹, along with a distinctive C=C bond, observed at 1,662 cm⁻¹, while C–H stretching revealed at 2,920 cm⁻¹. The spectrum also exhibited C–O–H vibration bands at 1,412 cm⁻¹, C=O stretching at 1,600 cm⁻¹, and C–O–C linkages at 1,069 cm⁻¹ (Figure 6).

SEM images illustrated the citric acid derived carbon quantum dots exhibited crumpled structures formed by precipitation, devoid of any visible dots due to their minute particle size and the tendency to agglomerate because of prolonged sample storage (Figure 7).

In addition to demonstrating their biocompatibility, it is necessary to investigate the biodegradability of carbon quantum dots. Human myeloperoxidase, along with various other peroxidases, possesses the enzymatic capability to decompose the graphitic matrix of carbon quantum dots (CQDs) and other carbon-based nanomaterials via an oxidative mechanism (35). Therefore, an MPO-based peroxide system was used to investigate the oxidative biodegradation of CQDs. When CQDs were incubated for 24 h with 140 mM NaCl, 5 μg/ml MPO, and 100 μM H₂O₂. In addition, we also performed control experiments where we incubated the CQDs exclusively with H₂O₂ to investigate any potential structural changes. Now the aliquots of the various samples were taken at 0, 4, 12, and 24 h intervals during incubation. Using UV-Vis spectroscopy, the absorbance of these aliquots was measured at various time intervals, and it was discovered that absorption decreased with time (Figures 8, 9).

The oxidative degradation of the CQDs caused by H₂O₂ alone with NaCl was comparatively less than that caused by the H₂O₂-driven peroxide system, MPO, and NaCl (Figure 8). To facilitate the synthesis of sodium hypochlorous acid (NaHOCl), which subsequently disintegrates the graphitic lattice, myeloperoxidase (MPO) interacts with the hydroxyl (OH) groups located on the surface of CQDs situated in proximity to defect sites. The OH group that present on the surface of the CQDs had come from citric acid verified by FTIR spectra (Figure 6). This observation is particularly relevant in the context of the body's innate immune defence mechanism. The primary barrier against invading pathogens and foreign materials consists of resident macrophages and neutrophils, which produce a myeloperoxidase-mediated peroxide system (22).

Additionally, the relative fluorescence intensity of CQDs was assessed over time, revealing a significant reduction in each MPO enzyme-treated sample's fluorescent intensity compared to its respective control (Figure 10). The most significant decrease in fluorescence intensity occurred during the first 4 h of incubation, in contrast to the subsequent time intervals. This phenomenon can likely be attributed to the high concentration of oxygenated functional groups present on the surface of the CQDs, which enhances their water dispersibility and facilitates interaction with the enzyme (35). We propose that after degrading the defective regions of the CQDs, the enzyme began to break down the more graphitic areas, potentially explaining the decreased rate of degradation after 4 h.