Utility of eco-friendly microwave-assisted nitrogen-doped carbon dots as a luminescent nano-sensor for the ultra-sensitive analysis of tigecycline in dosage form and biological samples

In the presented work, simple, green, sensitive, and selective nitrogen-doped carbon quantum dots (N-CQDs) were developed as nano-sensor for quantification of tigecycline (TIG) in different matrices. The proposed method is based on microwave synthesis of green nitrogen-doped carbon quantum dots with a high quantum yield (41.39%) and size diameter equal to 2.0 nm from the green juice of Eruca sativa leaves. The relative fluorescence intensity (RFI) of the green synthesized quantum dots (N-CQDs) was quenched at emission 512 nm (excitation 445 nm) after the addition of TIG drug. A good linear range between TIG concentration and quenched fluorescence intensity of N-CQDs in the range 20–300 ng mL−1, with the lower limit of quantitation (LOQ) equal to 8.51 ng mL−1. The proposed method was validated using the international conference of harmonization (ICH) recommendation and bio-analytical validation using U.S. food and drug administration (US-FDA) guidelines. The N-CQDs have been fully characterized using a transmission electron microscope (TEM), dynamic light scattering (DLS), X-Ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectrometer (FTIR). The suggested technique is a straightforward analytical procedure that can be used in clinical laboratories. Under the optimum condition, TIG was estimated in human plasma with a high percentage of recovery ranging from 96.95 to 98.54%. In addition, the proposed method was applied effectively in milk samples with percentage of recovery equal to 98.90 ± 1.55.


Introduction
Tigecycline (TIG) is a broad-spectrum antibiotic drug. TIG was prescribed for the treatment of various infectious diseases such as community-acquired pneumonia and skin disease. Community-acquired pneumonia is one of the most common infectious diseases with high mortality and morbidity worldwide. Spreading infectious diseases is the most reason for the need for innovative new antibiotics for bacterial resistance like Streptococcus pneumoniae and Hemophilus influenzae to beta-lactams, macrolides, and earlier generations of tetracyclines . (Honeyman et al. 2015;McKeage et al., 2008;Villano et al. 2016).
Major limitations were observed in previously published techniques that evaluate TIG as suffering low sensitivity and lack of bio-analytical proofs (Ge et al. 2015;Magalhães da Silva et al. 2012;Molina-García et al. 2011), the demand for sophisticated and expensive equipment's, availability for the utilization and disposal of solvents and individuals experienced in these technologies (Ji et al. 2008;Ji Ji et al. 2010). Due to their simplicity, low cost, specificity, and widespread availability in most quality control laboratories, spectrofluorimetric approaches are among the most common and sensitive techniques (Hammad et al. 2017;Salman et al. 2023;Salman et al. 2022a, b, c). The last ten years have seen a strong development of quantum dots (QDs), which have distinct optical features, excellent water solubility, biocompatibility, non-toxicity, and ease of functionalization.
Novel quantum dots are currently seen as a great alternative to fluorescent dyes and fluorescence derivatizing agents because of their exceptional and adaptable fluorescence properties. Compared to metal-based nanoparticles, they also are less toxic and hazardous to the environment (M. F. B. Ali et al. 2023;Salman et al. 2022a, b, c;Salman et al. 2022a, b, c). Additionally, diverse processes such as dry heat, hydrothermal, and solvothermal approaches were used to create carbon quantum dots (CQDs). These techniques have several shortcomings, however, as they go against the notion of "green chemistry," taking up to 24 h, requiring temperatures of 300 degrees, and utilizing harsh chemicals and organic solvents (Wang et al. 2019). Microwave synthesis is a revolutionary technique that has recently been applied to create high-yield quantum dots while reducing the synthesis period from hours to minutes. (Choi et al. 2017;Eskalen et al. 2020).
Eruca sativa plants are frequently grown in Egypt and have very affordable leaves. It is the first leaf vegetable that humans have ever eaten. The leaves of the Eruca sativa plant are a rich source of several nutrients and vitamins, including sugar, fiber, carbs, vitamin A, vitamin B1, riboflavin, glycosides and folic acid (Hussein 2013;Miyazawa et al. 2002;Neriman et al. 2011).
We subsequently created an integrated strategy for greening both the synthesis processes and the source of carbon to restore the greenness and sustainability of the synthesis of N-CQDs. The suggested technique only employs low-power microwave-assisted synthesis for 4 min at 700 W. We also have access to affordable, abundant, and practical flora. The study's environmental friendliness is also in line with broad claims about green chemistry and security.

Materials and reagents
All the chemicals and materials used in this work were of high purity and analytical grade. Tigecycline (TIG) 99.9% and Tigacyl® vial (50 mg) were provided from Pfizer, Cairo, Egypt. Egyptian Blood Bank provided human plasma samples, which were then kept in storage at − 24 °C until processing. The standard solution of TIG (100 µg mL −1 ) was prepared by dissolving 10 mg TIG powder in 100 mL distilled water. The working standard solution was prepared by serial dilution with distilled water to obtain a concentration range (0.2-3.0 µg mL −1 ).

Instrumentation of the N-CQDs approach
An FS5 spectrofluorometer was used to collect the data and a 150 W xenon lamp. Moreover, it is coupled to the Fluora-cle® software and has a 1-cm quartz cell, and the slit widths were 2 nm (Edinburgh, UK). The dynamic light scattering measurements (DLS) were scanned by the Zetasizer Red badge instrument of ZEN 3600 Nano ZS model (Malvern, UK). High-resolution transmission electron microscope (TEM) JEOL JEM-100CX II with tungsten EM filament 120 (USA).
The MFMI-100A microwave device was created to facilitate solvent extraction and chemical synthesis (MED Future). IR Temperature Sensor with magnetic and mechanical stirring from 100 to 1500 rpm (Australia). A Fouriertransform infrared (FTIR) spectrometer was utilized to determine the functional groups of the quantum dots (Germany). A Philips X-ray diffractometer was used to scan the powder X-ray diffraction (PXRD). Benchtop pH meter (100 B, China). Unicen 21 centrifuges with a speed 10.000 rpm (Spain).

Synthesis of nitrogen green quantum dots
The green nitrogen-doped carbon quantum dots were synthesized using thermolysis of Eruca Sativa leaves. The leaves were crushed well and filtrated, then 40 mL of the filtrate was transferred into the reaction vessel and then placed in the microwave. Microwave source: 2450 MHz, 700 W for 4 min until a brown solution was formed. The residue was dispersed in 50 mL ultra-pure water and then sonicated for 30 min to remove large particles. The solution was filtered and centrifuged at 4000 rpm for 10 min. the supernatant was filtrated via 0.45 μm cellulose membrane. The obtained yellow filtrate color solution was utilized for the experiment.

The fluorimetric procedure of TIG
In a 10-mL volumetric flask, one milliliter of N-CQDs (0.17 mg mL −1 ) was added together with one milliliter of Britton-Robinson (BR) buffer (pH 6.6). Next, 1.0 ml of the different concentrations of the standard working solution of TIG was added in the range (0.2 -3.0 µg mL −1 ). The solution was completed to the mark via ultrapure water to obtain the final concentration range (20 -300 ng mL −1 ). After 8 min, the relative fluorescence intensity (RFI) was scanned at 512 nm (excitation 445 nm).

Estimation in pharmaceutical product
Tigacyl® vial (50 mg TIG) was analyzed by an equivalent amount equal to (10.0 mg) TIG put into a volumetric flask and dissolved in 100 mL of ultrapure water, to obtain a concentration of 100 µg mL −1 .

Preparation of spiked plasma
A sufficient amount of TIG solution was added to 0.5 mL of human plasma before being spiked into a centrifuge tube. the protein precipitating agent acetonitrile was then added for a total of 1 mL (Omar et al. 2016;Salman et al. 2019). After 30 s of vertexing, the fluid was diluted to 10 mL. The product was centrifuged for 30 min. at 3500 rpm, and 1.0 mL of the supernatant was then employed in the analysis.

Preparation of milk sample
The milk sample (0.5 mL) was combined with 1.0 mL of acetonitrile, TIG was added, and the mixture was shaken for 30 s. The mixture was centrifuged for 10 min at 3500 rpm after being sonicated for 5 min. The supernatant was then removed for further examination (H. R. H. Ali et al. 2021). The Drug-free milk samples were prepared using the same steps without the addition of TIG.

Measurement of the accuracy and replication
The accuracy of the N-CQDs technique was studied using various concentrations from TIG (30, 50, 100.0, 200, and 300.0 ng mL-1) within the calibration range was used.
Moreover, the precision of the N-CQDs method was further tested using three replicates of each concentration (100.0, 150.0, and 250.0 ng mL −1 ).

Characterization of N-CQDs
The N-CQDs have been fully characterized using a highresolution transmission electron microscope (TEM), X-Ray photoelectron spectroscopy (XPS), Zeta potential analysis, fluorescence, UV-VIS, and FT-IR spectroscopy. A high-resolution transmission electron microscope (TEM) was used to analyze the surface morphology of N-CQDs. The size was found to be 2.0 ± 0.1 nm (Fig. 1b). The size of the N-CQDs was confirmed using dynamic light scattering (DLS). It was observed that the size was found to be 2.2 ± 0.1 nm, which is compatible with the TEM image (Fig. 1c). The functional groups on the exterior surface of N-CQDs were investigated using FTIR spectroscopy, as shown in Fig. 1d.
The FTIR bands at 3400 cm −1 and 2930 cm −1 , respectively, indicate the function groups (-NH, -OH), and (-CH). The bands at 1600 and 1450 cm −1 demonstrate the -C = O and -C-N groups, respectively. Moreover, the peaks at 1100 cm −1 and 680 cm −1 , respectively, reflect N-O and C-O. (Fig. 1d) The research focuses on the synthesis of multifunctional nitrogen-doped quantum dots.
For the elemental analysis, X-Ray photoelectron spectroscopy (XPS) was also used. Three distinctive significant peaks at 284.9, 395.6, and 538.5 eV, which correspond to C 1 s, N 1 s, and O 1 s, were detected as the XPS peaks of N-CQDs. It means that the main components of C-dots are O (43.00%), C (37.89%), and N (19.11%), as shown in Fig. S1a (supplementary materials). There are 4 peaks in the C1s spectra (Fig. S1b), which correspond to the existence of the C = C, C-N, C-O, and C = O groups, respectively. For N1s spectrum, 2 peaks are observed at 399.2 eV and 400.8 eV, produced due to the presence of C-N and N-H as shown in Fig. S1c (Ghosh et al. 2021). For O1s spectrum has two peaks for C-OH, C-O-C, and C = O at 531.4 eV and 532.6 eV ( Fig. S1d) (Jothi et al. 2021;Salman et al. 2022a, b, c). The quantum dot morphological characteristics show that the structure of N-CQDs contains multiple functional groups (-NH2, -OH, COO-), which interpret the interaction between TIG and N-CQDs via electrostatic interaction and hydrogen bonding. The energy-dispersive X-ray spectrometer (EDX), as depicted in Fig. S2, was used to check for the existence of the elements (C, N, and O). The spectrum reveals the presence of the elements C, N, and O. Additionally, the peak shown at 24.60 is a diagnostic peak of carbon dots (Salman et al. 2022a, b, c;Salman et al. 2022a, b, c), which was observed in Fig. S3 of the powder X-ray diffraction (PXRD) picture used to analyze the generation of N-CQDs.

The quantum yield of N-CQDs
The proposed method provides high quantum yield due to reducing the sizes of N-CQDs (2.0 nm) would increase the quantum yields by creating more optical effects via an increasing number of functional groups on the surface of quantum dots as described in Fig. 1d (Bae et al. 2008;Yang et al. 2016). Additionally, Eruca Sativa leaves are abundant in several elements and vitamins, such as carbohydrates, sugar, fibers, vitamin A, vitamin B1, riboflavin, and folic acid, which resulted in a wide range of functional groups during the production of quantum dots.
The quantum yield (QY) of N-CQDs was studied via single point method using the following equation (Salman et al. 2022a, b, c;Salman et al. 2022a, b, c;Salman et al. 2022a, b, c): Q is the quantum yield, while F is integrated fluorescence. The quantum yield of nitrogen quantum dots was found to be = 41.39%.

Optical characterization of N-CQDs
As shown in Fig. 2a, the UV spectrum of quantum dots displays a characteristic absorption peak at 225 nm, which is principally derived from the π-π* transition of carbonaceous skeleton, and at 295 nm for n-π* electronic transition of C = O and C = C which represented in Fig. 1d (Ali et al. 2019).
When the excitation wavelengths used to scan the relative fluorescence spectra of N-CQDs were increased, the Excitation dependent emission spectra of N-CQDs emission spectra of the N-CQDs shifted toward the red, from 380 to 470 nm, and the RFI decreased, demonstrating the excitation-dependent emission of carbon dots (Salman 2023). Figure 2b The reaction mechanism of TIG with N-CQDs According to Fig. 3a, the fluorescence of nitrogen-doped carbon quantum dots gradually decreased as the TIG concentration increased at 512 nm. The Stern-Volmer equation was used to evaluate the reaction mechanism between TIG and N-CQDs and distinguish between dynamic and static quenching using the following expression: Ksv is the Stern-Volmer constant, Q is TIG concentration, and F0 and F are fluorescence in the absence and presence of TIG.
The linearity of the Stern-Volmer curve, as seen in (Fig. 3b), clearly indicates that the quenching process is dynamic. Excited N-CQDs interact with TIG, resulting in an energy/electron transfer and a reduction in the fluorescence of the quantum dots. This mechanism is properly described by the Stern-Volmer model (Fig. 3b).
Stern-Volmer equations at various temperatures were used to confirm dynamic quenching. According to the Stern-Volmer plots (Fig. 3c), dynamic quenching rises as temperature rises (Larsson et al., 2022;Magdy et al. 2022).
Moreover, the presence of multiple functional groups in TIG permits the formation of hydrogen bonds and electrostatic attraction among TIG and nitrogen-doped carbon dots that have various functional groups as in Fig. 1d, in addition to allowing for these other phenomena. (Salman 2023;Salman et al. 2022a, b, c).

Optimization of the quantum dots method with TIG
In the presence of TIG, investigations into the effect of pH on N-CQD fluorescence were carried out. N-CQDs were shown Stern-Volmer curve between N-CQDs using different concentrations of TIG at room temperature F0 in absence and F in the presence of TIG, c Stern-Volmer curve between N-CQDs using different concen-trations of TIG at different temperatures, and d Effect of pH on the reaction of TIG (100 ng mL −1 ) with N-CQDs. *F0 is the relative fluorescence intensity of N-CQDs in absence of TIG. *F is the relative fluorescence intensity of N-CQDs in the presence of TIG to quench continuously in the pH range of 6.4 to 7.2 due to the presence of several function groups confirmed via FTIR curve (Fig. 1d), however, raising the pH to 7.2 caused an unstable drop in RFI. Thus, it was determined that 6.6 was the ideal pH. Figure 3d Additionally, multiple N-CQD concentrations were investigated during the reaction with TIG (100 ng mL −1 ), and it was shown that the quenching produced at the concentration with the greatest stability was 0.17 mg mL −1 (1.0 mL) (Fig. 4a).
At various time intervals spanning from 0 to 30 min, the reactivity of TIG with N-CQDs in pure form was observed. Within 8 min, the greatest stable quenching was recorded (Fig. 4b).
The response of TIG with N-CQDs in pure form was noticed at various intervals ranging from 0 to 30 min. The maximum steady quenching was observed within 8 min (Fig. 4b).

The validation of TIG with the N-CQDs method
The proposed method for the reaction of TIG in the presence of N-CQDs under ideal conditions was validated using ICH and US-FDA standards (Branch 2005;Zimmer 2014). The green fluorescence of the created N-CQDs was quenched at 512 nm as the TIG concentration increased (excitation at 445 nm) (Fig. 3a).
The regression equation was identified as Y = 0.0047x + 1.154 using the Stern-Volmer equation; adequate linearity was found within the range of 20-300 ng mL −1 ; and the correlation coefficient was 0.9996 (Fig. 3b).
The lower limit of detection (LOD) and limit of quantitation (LOQ) were calculated using the following equations: LOQ and LOD were found to be 8.51 and 2.80 ng mL −1 , respectively, the results refer to ultra-sensitivity of the N-CQDs method (Table 1).
To test the accuracy of the N-CQDs technique, various TIG drug concentrations (30, 50, 100.0, 200, and 300.0 ng mL-1) within the calibration range were used. The relative standard deviation (RSD) scores were obtained from 0.22 to 0.90 in Table 2, and recovery percentages ranged from 100.28 to 101.82%.
The precision of the N-CQDs method was further tested using three replicates of each concentration (100.0, 150.0, and 250.0 ng mL −1 ). According to Table 2, the relative standard deviation ranged from 0.30 to 0.74%, showing the high reproducibility of the N-CQDs method.
Furthermore, by making minor changes to the analytical procedure's parameters, the robustness of the green synthesized N-CQDS for TIG estimation was used. A small adjustment to the procedure variables had no appreciable impact, as shown in Table S1 (Supplementary materials).
Three concentrations were used to conduct a bio-analytical validation of the reaction of TIG and N-CQDs in human plasma in compliance with US-FDA regulations (50, 100, and 250 ng mL −1 ). The RSD value ranged from 1.55 to 2.60 in Table 3, and the results demonstrate that the N-CQDs approach in human plasma produces excellent accuracy.
As demonstrated in Table S2, the N-CQDs approach was also employed to research the stability of TIG in human plasma. The stability was examined under various conditions Fig. 4 a Effect of N-CQDs concentration on the reaction of TIG (100 ng mL −1 ), and b Effect of the reaction time for the reaction of N-CQDs with of TIG (100 ng mL −1 ). *F0 is the relative fluorescence intensity of N-CQDs in absence of TIG. *F is the relative fluorescence intensity of N-CQDs in the presence of TIG using three levels of low-quality controls (LQC), mediumquality controls (MQC), and high-quality samples (HQC). According to the results, TIG is stable in human plasma at diverse conditions. Table S2.

Quantification of TIG using the N-CQDs method in human plasma and milk samples
Due to the ultra-sensitivity of the N-CQDs approach, tigecycline (TIG) was successfully estimated in spiked human plasma. When using six different concentration levels, the recovery percentage for the studied procedure ranged from 96.95 to 98.54%. The analytical method's variability caused by various matrix effects was measured as the relative standard deviation (RSD), which ranged from 1.21 to 2.18 and is given in Table 4. In addition, the proposed method was applied effectively for the estimation of TIG in milk samples. The results refer to the high sensitivity of the proposed method as in Table 4.

Quantification of TIG in commercial form
For quantifying TIG in market dosage form (Tigacyl® 50 mg vial), the suggested method (N-CQDs) was employed successfully. The percentage of recovery RSD discovered was 102.50 ± 1.11 when compared to the reported approach (Salman et al. 2019) (100.34 ± 1.11). The t-value was 1.34 and the F-value was 2.40, respectively.

Assessment of the greenness of the proposed method
Various methods have been recently observed for the evaluation of the greenness of the analytical methods. These methods are useful for the reduction of environmental pollution generated by analytical techniques such as chromatographic methods. AGREE and GAPI methods were utilized for the greenness assessment of the proposed method (Saraya et al. 2022). AGREE depicts a clockshaped symbol with a perimeter divided into 12 pieces, each representing a GAC concept. The pictogram's center displays a number value estimating the ecological impact, with the closer to one, the greater the damage as shown in Fig. 5. Moreover 2 red zones appear while using GAPI method (third and fourth) due to sample transportation and using acetonitrile as a protein precipitating agent (Fig. 5).
The results refer to the high greenness effect of the proposed method (N-CQDs) which agrees with the US climate change conference.

Conclusion
This work aims to establish a green fluorimetric approach using nitrogen-doped carbon quantum dots with high quantum yield. The N-CQDs method is a simple, environmentally friendly, highly sensitive, affordable, and rapid strategy used as a fluorescent probe for analysis of TIG. To quickly generate the N-CQDs, a one-pot, low-energy, chemical-free carbonization microwave assisted was used for the synthesis of quantum dots. The proposed technique was evaluated and bioanalytically validated following ICH and US-FDA guidelines. These N-CQDs were successfully used to determine TIG pharmaceutical dosage form, human plasma, and milk samples. As a result, this simple and label-free sensing technology was used for fluorescence-based analysis of the target analyte. Data availability All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Conflict of interest
The authors have no competing interests to declare that are relevant to the content of this article.
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