One-Pot Microwave-Assisted Synthesis of Fluorescent Carbon Dots from Tomato Industry Residues with Antioxidant and Antibacterial Activities

Abstract

Tomato waste (TW) was employed as a sustainable source for the synthesis of fluorescent carbon dots (CDs) via a microwave-assisted hydrothermal carbonization (Mw-HTC) method, aiming at its valorization. Several amines were used as nitrogen additives to enhance the fluorescence quantum yield (QY) of CDs, and a set of reaction conditions, including additive/TW mass ratio (0.04–0.32), dwell time (15–60 min), and temperature (200–230 °C) of the HTC process, were scrutinized. The structural analysis of the tomato waste carbon dots (TWCDs) was undertaken by FTIR and ¹H NMR techniques, revealing their most relevant features. In solid state, transmission electron microscopy (TEM) analysis showed the presence of nearly spherical nanoparticles with an average lateral size of 8.1 nm. Likewise, the topographical assessment by atomic force microscopy (AFM) also indicated particles’ heights between 3 and 10 nm. Their photophysical properties, revealed by UV–Vis, steady-state, and time-resolved fluorescence spectroscopies, are fully discussed. Higher photoluminescent quantum yields (up to 0.08) were attained when the biomass residues were mixed with organic aliphatic amines during the Mw-HTC process. Emission tunability is a characteristic feature of these CDs, which display an intensity average fluorescence lifetime of 8 ns. The new TWCDs demonstrated good antioxidant properties by the ABTS radical cation method (75% inhibition at TWCDs’ concentration of 5 mg/mL), which proved to be related to the dwell time used in the CDs synthesis. Moreover, the synthesized TWCDs suppressed the growth of Escherichia coli and Staphylococcus aureus at concentrations higher than 2000 μg/mL, encouraging future antibacterial applications.

1. Introduction

Tomatoes are fruits of the Solanum lycopersicum plant, which belongs to the Solanaceae family. They are the second most important vegetable crop globally, just behind potatoes. Other fruits of this family, such as eggplants and peppers, are also included in the Mediterranean diet [1,2]. Tomatoes, native to Central and Western South America, were first used as food in Mexico and later spread worldwide following the European colonization of the Americas. Portugal has experienced a significant increase in tomato production over the past 25 years, with an average annual growth rate of 1.8% [1].

Currently, 80% of the world’s tomato production is consumed fresh, while the remaining 20% is industrially processed into canned tomatoes, purees, ketchup, juices, and sauces. In 2023, the global tomato processing market reached 46.9 million tons [3]. Tomato pomace typically represents about 3–5% (w/w) of the total weight of raw tomatoes processed [4]. Based on this estimate, between 1.4 and 2.4 million tons of tomato pomace are generated each year by processing industries around the world. Tomatoes provide numerous health benefits (e.g., anti-cancer properties, protection against neurodegenerative diseases, maintenance of heart health, and regulation of blood glucose levels in diabetic people). These profits are attributed to tomatoes’ high nutritional value, which comes from lycopene, minerals, vitamins, and phenolic compounds [1,5].

Nevertheless, as a result of tomato fruit processing, this industry generates a significant volume of solid and liquid byproducts, including skins, seeds, vascular tissues, and wastewater. These residues have a high organic matter content (e.g., carbohydrates, proteins, lipids, etc.), low biodegradability, and are mainly utilized as animal feed, soil fertilizers, or disposed of as solid waste [6,7,8,9]. However, there is an increasing demand for their conversion into value-added products, with several examples reported (e.g., oil extraction from tomato seeds, and use of various bioactive molecules from the tomato skin in food, pharmaceutical, and nutraceutical industries) [10,11,12,13,14]. Properties of pomace depend on the type of tomato paste produced, which is influenced by factors such as tomato varieties, breaking temperatures, and the size of the finisher screens employed in juice extraction.

In this context, the development of sustainable synthesis of carbon-based nanomaterials from agro-industrial waste has shown a significant impact on the valorization of these residues, and is currently increasing [7,9,14,15]. Their nanosized dimensions proved crucial for many envisioned applications in the biomedical field [16,17,18,19].

Carbon dots (CDs) are promising candidates for several applications (e.g., in (photo)catalysis, (bio)sensing, and (bio)therapy) due to their relevant fluorescence, water solubility, good biocompatibility, lateral sizes typically below 10 nm, low toxicity, versatile surface functionalization, and low-cost production [20,21,22,23,24,25,26,27,28]. The most widespread CDs synthetic strategies are the so-called top-down and bottom-up methods. Top-down methods involve the use of electric arc discharges, laser ablation, chemical and electrochemical oxidations of carbon substrates, while solvothermal methods, ultrasonic synthesis, and pyrolysis largely represent the bottom-up approaches applied to relatively simple organic molecular precursors, biomass wastes, and other residues.

The valorization of cork cooking wastewater [29,30], olive mill waste [31,32,33,34], coffee grounds [35], and microalgae [36] as renewable and sustainable resources for the synthesis of carbon-based nanomaterials was recently reported by us, and their application as sensors and biosensors was demonstrated [29,31,37].

Hydrothermal carbonization (HTC) is one of the most widely used methods for preparing nanomaterials. Given that biomass is rich in organic matter and inherently biodegradable, HTC enables the transformation of low-value by-products into functional nanomaterials, such as carbon dots, in a single-step, cost-effective, and environmentally friendly process. As a result, a wide variety of precursors such as papaya [38], sweet potato [39], and cornstalk [40] have been used for the synthesis of CDs using the hydrothermal approach. The microwave-assisted solvothermal method has gained increasing attention due to its advantages as an eco-friendly synthesis approach, at least at a laboratory scale, such as simple equipment, shorter reaction times, and higher efficiency [41,42,43,44]. The present work reports the valorization of tomato waste (TW) pomace from a Portuguese industry through the synthesis of fluorescent carbon dots using a microwave-assisted hydrothermal carbonization (HTC) method, by developing a low-cost, sustainable route for producing functional nanomaterials. Several reaction conditions were explored, and the bioactivity regarding antioxidant and antibacterial properties of the resulting carbon dots was evaluated. This study explores both waste management challenges and the growing demand for eco-friendly nanomaterials for a wide range of applications.

2. Materials and Methods

2.1. General

Tomato waste (TW) was collected from a Portuguese tomato industry, processed by trituration, dried at 60 °C in an oven for one week, and stored in polyethylene boxes until further use at room temperature under a nitrogen atmosphere.

Gallic acid (97.5%, Sigma, Sigma–Aldrich Corp., St. Louis, MO, USA), tannic acid (pure, Carlo Erba, Milan, Italy), salicylic acid (98%, Merck, KGaA, Darmstadt, Germany), β-D-glucose monohydrate (98%, Alfa Aesar, MA, USA), ethylenediamine (EDA, >99.5%, Fluka, Sigma-Aldrich Corp., St. Louis, MO, USA), diethylenetriamine (DETA, 99%, Aldrich, St. Louis, MO, USA), melamine (99%, Acros Organics, Bvba, Belgium), p-phenylenediamine (p-PD, >97%, Fluka, Sigma–Aldrich Corp., St. Louis, MO, USA), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS, ≥98%, Sigma-Aldrich, St. Louis, MO, USA), ascorbic acid (>99.7%, Merck KGaA, Darmstadt, Germany), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; 97%, Acros Organics, Bvba, Belgium), quercetin.2H₂O (crystallized, Merck, Darmstadt, Germany), Mueller-Hinton Broth (MHB, Oxoid, Hampshire, UK), Mueller-Hinton Agar (MHA, Sharlau, Scharlab S.L., Barcelona, Spain), Technical Agar (Oxoid, Hampshire, UK), gentamicin (Sigma-Aldrich, St. Louis, MO, USA), resazurin sodium salt (>85%, Tokyo Chemical Industry Europe, Zwijndrecht, Belgium), BSA (Bovine Serum Albumin; Acros Organics, Bvba, Belgium), Brillant Coomassie Blue G250 (Bio-Rad, Hercules, CA, USA), Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA), quinine hemisulphate monohydrate (>98%, Fluka, St. Louis, MO, USA) were used as received. Urea was recrystallized from ethanol. All other reagents and solvents were of analytical grade and were purified and/or dried by standard methods.

Ultrapure water (Milli-Q, Millipore; Merck KGaA, Darmstadt, Germany) was used in all experiments, except if noticed otherwise.

2.2. Instruments and Methods

FTIR spectra were obtained on a Brüker Vertex 70 spectrophotometer (Bruker Optik GmbH, Ettlingen, Germany) as KBr pellets (transmission mode).

¹H NMR spectra were collected on a Brüker II+ spectrometer (400 MHz; Bruker BioSpin AG, Fällanden, Switzerland) at 25 °C; reported chemical shifts (δ/ppm) are internally referenced to D₂O (4.790 ppm).

Ground-state UV–Vis spectra were recorded on a Jasco UV V-750 spectrophotometer (Jasco Inc., Tokyo, Japan) using 1 cm path quartz cells at 25 °C.

Steady-state fluorescence spectra were acquired on a Perkin Elmer LS45 fluorimeter (PerkinElmer, Waltham, MA, USA) using a 1 cm path quartz cuvette at right angle (RA) at 25 °C under air-equilibrated conditions. The fluorescence quantum yields (QYs) were measured in aqueous solutions using quinine sulfate in 0.01 M H₂SO₄ (QY = 0.54; air equilibrated conditions, RA) as a reference standard at 25 °C. The quantum yields were determined by the slope method [45], keeping the optical density below 0.05 at the excitation wavelength to prevent inner filter effects.

Time-resolved picosecond fluorescence intensity decays were obtained using a HORIBA DeltaFlex (HORIBA UK Ltd., Northampton, UK) Correlated Single Photon Counting (TCSPC) system. The samples were excited by a DeltaDiode LED at 362 nm, with the polarizer set in a vertical position. The emission monochromator was set at 480 nm with the polarizer set at the magic angle (55°). Experimental intensity decays were fitted to the multi-exponential model: $I\left(t\right)={\sum }_i{\mathsf{\alpha }}_{\mathrm{i}}\exp (-t/{\tau }_{\mathrm{i}})$, where α_i are the amplitudes of the component decays at t = 0, and τ_i the respective fluorescence lifetimes. The intensity average lifetime was determined by the expression:

$${\tau }_{ave}=\sum {\alpha }_i{\tau }_i^2/\sum {\alpha }_i{\tau }_i$$

(1)

The transmission electron microscopy (TEM) was carried out on a JEOL JEM1010F microscope, working at 100 kV and equipped with an Orius SC1000 CCD camera from Gatan (Gatan Inc., Weiterstadt, Germany), at the Electron Microscopy Unit of the C.A.C.T.I., University of Vigo, Spain. The sample was sonicated for 10 min, and some drops were deposited on a copper grid (400 mesh) coated with a formvar/carbon film. The Gatan Digital Micrograph (DM) software ver. 3.53.4137.0, 2023, was used to analyze the size distribution of TWCDs.

Topographical analysis by atomic force microscopy (AFM) was carried out on a Veeco Multimode 8 Nanoscope V, at the Nanotechnology and Analysis Surface Service of the C.A.C.T.I., University of Vigo, Spain. The measurements were made in tapping mode using a cantilever NCHV-A (Tip Roc < 10 nm, Cantilever Antimony (n) Doped Si, K = 20–80 N/m, Frequency = 339–388 kHz). The images were obtained using a 14 μm scanner. The sample was analyzed in different areas, taking images with different fields of view/magnifications. All images have been processed by Nanoscope Analysis V.1.9 software, applying a plane correction; incorrect lines representing cantilever “jumps” that do not correspond to the topography have also been removed. The sample (ca. 6 mg/mL) was removed from the refrigerator and left for 10 min to stabilize the temperature, and then put in an ultrasonic bath for 10 min to disperse any major aggregates. After this time, 5 μL of each sample was deposited by spin coating (800 rpm for 3 s and 1600 rpm for 10 s) on mica substrates, exfoliated several times before deposition. Once deposited, the sample was air dried at ambient temperature.

Elementary analyses (CHNS) of TW were performed in duplicate using a Carlo Erba EA 1108 analyzer (Carlo Erba, Milan, Italy) at 1013 °C under an oxygen flux of 15 mL/min at C.A.C.T.I., University of Vigo, Spain.

The zeta (ζ) potential determinations of aqueous solutions of CDs (1.0 mg/mL) were carried out on a ZetaSizer Nano ZS (model ZEN 3601, Malvern Instruments Ltd., Worcestershire, UK), at 25 °C within a pH range from 8.2 to 9.5, after the samples were sonicated, using the optimized conditions resulting from the automatic procedure of the equipment. All measurements were repeated five times to verify the reproducibility of the results. Analytical data was processed by Zetasizer software version 7.12.

The microwave-assisted hydrothermal carbonization (Mw-HTC) method was carried out in a monowave reactor from Anton Paar, model Monowave 300 (Anton-Paar, Graz, Austria), using pressure-rated reaction vials of 30 mL with poly(tetrafluoroethylene)-silicon caps.

The pHs of aqueous solutions were determined at approximately 25 °C using a pH VWR pHenomenal^® UM 6100L equipped with a pH electrode phenomenal 221 (VWR International Bvba, Leuven, Belgium).

Colorimetric assays based on the ABTS^•+ radical inhibition method were evaluated on a Bio-Rad 680 (Bio-Rad Laboratories, Hercules, CA, USA) microplate reader.

The antimicrobial activity was assessed against Escherichia coli (E. coli) ATCC^® 25922 and Staphylococcus aureus (S. aureus) ATCC^® 25923, handled in a SafeFAST Classic 209 cabinet. The determination of the minimum inhibitory concentration (MIC) was carried out in a BMG Labtech FLUOstar OPTIMA fluorescence microplate reader.

2.3. Characterization of TW

The TW (25 g) was extracted with water in the Soxhlet apparatus, and the extract evaporated to dryness. After drying under vacuum at 105 °C for ca. 18 h, the total solids in aqueous extracts were determined. The total phenols, flavonoids, carbohydrates, and protein determinations were carried out in triplicate on the aqueous extracts obtained.

The total phenolic compounds were determined by the Folin–Ciocalteu method [46], with gallic and tannic acids as standards (concentrations range 0–250 μg/mL). An aliquot of 1.5 mL of Folin–Ciocalteu reagent (10% v/v) was added to 200 μL of aqueous extract (previously diluted). After 5 min, 1.5 mL of NaHCO₃ (60 g/L) was added and the mixture stood for more than 90 min at 25 °C. After this period, the absorbance was read at 725 nm. The results were expressed in terms of gallic acid equivalent (mg GAE/g extract) and tannic acid equivalent (mg TAE/g extract).

The flavonoids in the aqueous extract of TW were quantified using the aluminum chloride colorimetric method [47] with quercetin as the standard in the range of 0 to 100 μg/mL and expressed in quercetin equivalent (mg QE/g extract). To a 1 mL aliquot of aqueous extract of TW, 0.5 mL of a solution of AlCl₃ (2% w/v) and 0.5 mL of Millipore^® water were added. After stirring and resting for 10 min at 25 °C, the absorbance was read at 427 nm.

The quantification of carbohydrates in the aqueous extract of TW was achieved by adapting the phenol-sulfuric colorimetric method [48]. To a 0.5 mL aliquot of the aqueous extract of the TW, 0.5 mL of phenol solution (80% w/w) was added. Then, 2.5 mL of 96% H₂SO₄ was quickly added to the surface of the solution. The resulting solution was shaken vigorously and left to stand for 25 min at 25 °C. After this period, the absorbance was read at 488 nm. The total carbohydrate content was quantified using β-D-glucose as reference in a concentration range of 0–50 μg/mL.

Protein concentration was quantified by the Bradford assay [49] with bovine serum albumin (BSA) as the protein standard in a concentration range of 0–25 μg/mL. To a 1 mL aliquot of TW aqueous extract was added 1 mL of Coomassie brilliant blue G250 solution (0.06% (w/w) in 0.6 N HCl). The mixture was stirred and left to stand for 2 min. Subsequently, the absorbance was measured at 620 nm.

For the nitrate analysis, an aliquot of 100 μL of a previously diluted TW aqueous extract was taken and mixed with 0.4 mL of salicylic acid solution in H₂SO₄ (5% (w/v)). The resulting mixture was stirred and left at room temperature for 20 min. After this period, 9.5 mL of 2 N NaOH was added, and the solution was stirred before measuring the absorbance at 415 nm. The NaNO₃ standards were prepared in a concentration range of 0–250 μg/mL, and absorbance readings for the TW aqueous extract were performed in triplicate, using Millipore^® water as a negative control [50].

The lipid content in the TW was quantified using a Soxhlet extraction method with n-hexane as solvent. The resulting extract was dried over anhydrous magnesium sulfate, filtrated, evaporated, and dried under vacuum at room temperature.

The ash content in TW was determined following a previously described method [51]. For that, 100 mg of TW was added to porcelain crucibles previously dried at 105 °C. The samples were first ignited with a flame and then ashed in a muffle furnace at 600 °C for approximately 24 h. Following ashing, the crucibles were cooled in a desiccator to room temperature and weighed repeatedly until a constant mass was achieved. The residual ash was then quantified. All ash content determinations for TW were performed in triplicate to ensure accuracy and reproducibility.

Microanalysis was used to evaluate the CHNS contents of TW.

2.4. General Procedure for the Synthesis of Carbon Dots from Tomato Waste

In a typical setup, the TW (450 mg) was placed into the microwave reactor with Millipore^® water (15 mL), a certain amount of additive (diverse organic amines) was added, and the mixture heated for 15–60 min with stirring (600 rpm) at a given temperature (200–230 °C). For example, using EDA as an additive (2.4 mmol EDA/g TW; EDA/TW mass ratio = 0.32) at 200 °C for 60 min., an aqueous brown solution and a dark brown residue were obtained after filtration of the reaction mixture through a cellulose membrane syringe with a pore size of 0.2 μm. The aqueous solution was then extracted with CH₂Cl₂ (ca. 2 × 15 mL) and AcOEt (ca. 2 × 15 mL) to remove the low to medium-polarity molecular species, resulting in the tomato waste carbon dots (TWCDs) obtained in 36.4% yield (w/w, based on TW and EDA), quantified after drying an aliquot of the aqueous solution at 105 °C. Reaction yields were obtained as mass yields (η_mass = (mass of TWCDs/(mass of TW + mass of additive)) × 100.

2.5. Evaluation of the Bioactivity of TWCDs

2.5.1. Antioxidant Activity Analysis

Antioxidant activity was assessed using the ABTS radical cation inhibition method [52,53], based on the reduction of the blue-colored ABTS radical cation (ABTS^•+) to its non-radical form, which is confirmed by color decrease.

In a 96-well microplate (VWR), 290 μL of ABTS^•+ solution, obtained by reacting a 7 mM ABTS stock solution with 2.5 mM K₂S₂O₈, and previously diluted to absorbance 1.0 at 655 nm, was mixed with 10 μL of TWCDs (5 mg/mL) using ascorbic acid or Trolox as positive controls. After a 5 min period protected from light, absorbance was measured at 655 nm. Antioxidant activity was quantified using Trolox standards [0–1 mg/mL]. Assays were performed in triplicate, with deionized water as negative control, and results were expressed as % inhibition of ABTS^•+ and as μg Trolox equivalents (TE) per gram of TWCDs.

2.5.2. Antimicrobial Activity Analysis

The antimicrobial activity of TWCDs was evaluated using a modified Kirby-Bauer disk diffusion method [54,55]. Bacterial species, E. coli and S. aureus, were inoculated according to the CLSI (Clinical & Laboratory Standards Institute) M100 guidelines [56]. Pre-inoculum was grown overnight in MHB at 37 °C and 180 rpm, with turbidity adjusted to McFarland standard 0.5 (1.5 × 10⁸ CFU/mL; Liofilchem). The diluted suspension was transferred to MHS medium (MHB supplemented with 0.5% (w/v) Technical Agar) and poured over MHA.

Filter paper disks of 9 mm diameter were loaded with 50 μL of TWCDs (variable mass), and inhibition zones were measured after 18 h incubation at 37 °C. Gentamicin (10 μg/disc) and sterilized deionized water were used as positive and negative controls, respectively. Additionally, the MICs of TWCDs were determined against E. coli and S. aureus by a colorimetric microdilution assay [57] based on the reduction of resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide, sodium salt), a poorly fluorescent N-oxide phenoxazine dye, to resorufin, a strongly fluorescent reduced phenoxazine, by viable cells [58]. In sterile 96-well microplates (VWR), 100 μL of cell suspensions adjusted to 5 × 10⁵ CFU/mL were mixed with 100 μL of TWCDs (variable mass). After incubation for approximately 18 h at 37 °C, 20 μL 0.01% (w/v) solution of resazurin in phosphate-buffered saline (PBS) was added to each well, and mixtures were re-incubated at 37 °C for an additional 1–2 h before visual data interpretation and fluorescence intensity measurements (excitation at 550 nm, and monitoring at 650 nm). The possible direct chemical reduction of resazurin by TWCDs was assessed by performing a control test in the absence of bacterial cells, in which the TWCDs were used at concentrations of 500 and 2000 μg/mL.

3. Results and Discussion

3.1. Tomato Waste Characterization

The physicochemical and organic composition of TW and water extract (TW-WE) used in this work were assessed by standard methods and are displayed in

Table 1. Physico-chemical parameters and organic composition of TW and TW-WE.

Parameter	Values
Total solids-WE (%)	47.92 ± 2.11
pH (WE) (25 °C)	4.40
Ashes (TW) (%)	2.89 ± 0.07
Microanalysis (TW)	C, 56.21%; H, 7.47%; N, 3.69% O (calc.), 29.69%; S < 0.30%
Total phenols (mg GAE/g extract) 1	6.52 ± 0.14
Total phenols (mg TAE/g extract) 1	9.35 ± 0.20
Flavonoids (mg QE/g extract) 1	4.79 ± 0.01
Total carbohydrates (mg/g extract) 1	16.41 ± 0.07
Proteins (mg/g extract) 1	6.28 ± 1.04
Nitrates (mg/g) 1	0.70 ± 0.02
Lipids (%) 2	11.46 ± 0.37

¹ Values determined from the water extracts (see Section 2); ² Values obtained from the extraction of TW with n-hexane (see Section 2); Data represent mean ± SD, n = 3.

.

3.2. Synthesis of TWCDs

Microwave-assisted hydrothermal carbonization (Mw-HTC) method was employed to synthesize fluorescent CDs from TW in a mono-mode microwave reactor, using a variety of conditions (e.g., reaction temperature, dwell time, type of organic amine additive), as will be detailed later.

The structural and morphologic analysis that follows used the CDs obtained with EDA as an additive (2.4 mmol EDA/g TW; EDA/TW mass ratio = 0.32) at 200 °C for 60 min as the representative material of TWCDs, except if noted otherwise.

3.3. Structural and Morphologic Characterization of TWCDs and Their Surface Charge

The analysis of surface functional groups of TWCDs was performed by FTIR spectroscopy, being the assignments made according to the general literature [59]. Its spectrum (Figure 1a) shows a strong stretching (str) broad band centered at 3422 cm⁻¹ (O-H), along with an N-H shoulder at ca. 3275 cm⁻¹, both H-bonded [33]. The band (as a shoulder) appearing at 3088 cm⁻¹ may be due to aryl-H stretching vibrations. Aliphatic methyl and methylene groups exhibit bands at 2970, 2931, and 2877 cm⁻¹ (C_sp³-H; str), while the corresponding bending (ben) vibrations appear near 1441, 1404 (CH₂), and 1378 cm⁻¹(CH₃). The strongest band, peaking at 1656 cm⁻¹, is attributed to several C=O (str) contributions, likely those typical of N-substituted amides/primary amides, carbamates, and H-bonded/conjugated carboxylic acids, C=N, and C=C (olefin) groups [33]. The band centered at 1552 cm⁻¹ may stem from contributions of in-plane skeletal vibrations of C=C of aromatic rings, asymmetric carboxylate stretching vibrations, and N-H bending (amides/carbamates/ureas) [33]. Additional bands of lower intensity appear at 1296 cm⁻¹, 1110 cm⁻¹, and 1050 cm⁻¹. The former may be assigned to C-O stretching in carboxylic acids, the second to bending vibrations of C-H mixed with C=C in aryl rings and C-O stretching in H-bonded phenols, and that at 1050 cm⁻¹ to C-O stretching in aliphatic alcohols (primary and secondary), aryl ethers, and carboxylates [33]. The spectrum of TWCDs obtained without any additive (see below) presents a somewhat similar profile (Figure S1).

Nonetheless, some differences are evident. Of major note are the broader band related to O-H (str), with a concomitant higher intensity of vibrations assigned to C-O (str) of alcohols (1125–1025 cm⁻¹), the presence of clear shoulders at higher energy than the main C=O band at 1658 cm⁻¹, and a less pronounced band corresponding to C=C (str) and N-H (ben). The reduced reaction time and the absence of EDA in the synthesis of these CDs may have conditioned this outcome.

The ¹H NMR analysis of TWCDs does not allow any deep insights regarding the structure of the CDs. However, three main regions of resonances may be identified (Figure 1b), which point to the presence of aliphatic C-H (0.65–2.8 ppm), CH-O and CH-N (3.1–4.5 ppm), and aromatic (6.5–8.25 ppm) protons.

The TWCDs were morphologically evaluated through transmission electron microscopy (TEM) and atomic force microscopy (AFM). Both techniques revealed that diverse levels of aggregation occur upon drying the solution of TWCDs on TEM/AFM substrates. That such aggregation occurs at a molecular level, involving small to medium size molecular species existing in solution, leading to the formation of supramolecular structures which are assembled upon removal of the solvent to achieve the solid form in various lateral sizes and heights, has been demonstrated in a recent study [34]. Under our conditions of observation (see Section 2), TEM images of TWCDs show nanoparticles ranging from 3.5 nm to 12.5 nm in their lateral size (Figure 2a), with an average size of 8.1 ± 1.7 nm by statistical analysis (Figure 2b).

The TWCDs synthesized with no additive display a similar range of nanoparticle sizes (4.5–10.5 nm), with only a slightly lower average size (7.5 ± 1.5 nm), as shown in Figure S2.

The topographical analysis of TWCDs performed by AFM furnished, again, clear evidence for the high propensity of aggregation of the species present in solution on going to the solid state upon drying on the mica substrate. Figure 3 depicts a representative topographic image of TWCDs nanoparticles. For this sample, the nanoparticles’ height range varies between 3 and 10 nm. Performing the Mw-HTC of TW in the absence of any additive leads also to the formation of nanoparticles (Figure S3) having an analogous height range (3.2–11 nm). As will be shown below, the fluorescence quantum yields of the samples analyzed above are, however, substantially different.

Concerning the TWCDs’ antimicrobial applications, it is important to evaluate the surface charge of the nanomaterials, since their potential toxic effect is highly dependent on TWCDs–cell interactions [60]. The zeta (ζ) potential of TWCDs aqueous solutions synthetized using EDA/TW mass ratio of 0.16 during 15, 30 and 60 min are, respectively, −11.0 ± 4.8 mV (at pH = 9.25), −9.7 ± 2.5 mV (at pH = 9.06), and −19.5 ± 4.7 mV (at pH = 8.83). Increasing the EDA/TW mass ratio to 0.32, but keeping the dwell time at 60 min, a deep reduction in the zeta potential (ζ = −3.9 ± 0.5 mV; at pH = 9.52) was observed. The TWCDs obtained without an additive during a 15 min reaction time also showed a negative potential (ζ = −7.9 ± 1.4 mV; at pH = 8.17). No clear-cut trend could be observed between the samples prepared under the diverse conditions used, viz., dwell time and amount of added amine or even in its absence.

3.4. Photophysical Properties of TWCDs

The photophysical properties of CDs were studied by UV–Vis, steady-state, and time-resolved fluorescence spectroscopies. The ground-state absorption, excitation, and emission spectra of aqueous solutions of TWCDs are shown in Figure 4a. The absorption profile exhibits bands around 275 and 325 nm, with an absorption cut-off near 600 nm. The band at higher energy is assignable to π-π* transitions of sp²-hybridized carbon structures, and the latter to n-π* and π-π* mixed transitions of conjugated systems (e.g., carbonyl, carboxyl, and imine functions conjugated to unsaturated carbon) [33]. The emission of TWCDs is dependent on the excitation energy, with the maxima emission wavelength shifting to the red on its decrease (Figure S4), with a concomitant lowering of the intensity of emission, which may be due to the decrease in absorption. The observed emission tunability may likewise be a result of the spectral heterogeneity of the single emitters, possessing different dimensions and structural features [61]. When excited at 380 nm, the emission spectrum revealed the most prominent band (FWHM = 0.49 eV) peaking in the blue region at 458 nm, with a QY of 0.08. The excitation spectrum, when monitored at 460 nm, showed two main transitions peaking at ca. 245 and 371 nm, with a much larger contribution from the latter chromophore. A significant photostability of TWCDs was found upon continuous irradiation of a CDs’ sample for ca. 60 min (Figure S5).

The time-resolved fluorescence (TRF) intensity profile of TWCDs is displayed in Figure 4b, which was obtained from a time-correlated single photon counting (TCSPC) method. The lifetimes were calculated by iteratively fitting the exponential functions to the multi-exponential model (see Section 2 for details). A sum of three exponentials (τ₁ = 4.55 ns, f₁ = 47.1%; τ₂ = 13.2 ns, f₂ = 41.8%; τ₃ = 0.75 ns, f₃ = 11.1%, where the fi are the fractional contributions of each component) was found to best fit the experimental decays, yielding an intensity average lifetime (τ_ave) of 7.8 ns (χ² = 1.14).

The corresponding set of spectra obtained for TWCDs synthesized without additives is shown in Figure S6. Clear differences may be observed in the absorption, excitation, and emission spectra between the two types of CDs. In the latter CDs, the band at 325 nm is much less intense. The main chromophore responsible for the emission features is now centered at 342 nm instead of 371 nm for the former CDs. Owing to that, a band at 380 nm is clearly evident. At last, the emission spectrum is broader (FWHM = 0.49 eV). Comparing the TRF intensity profile of the above CDs with that of TWCDs prepared without additive (Figure S6b), the decay of the latter is likewise well-fitted with a sum of three exponentials with comparable amplitudes for each component decay and lifetime (τ₁ = 4.0 ns, f₁ = 52.8%; τ₂ = 14.3 ns, f₂ = 32.1%; τ₃ = 0.56 ns, f₃ = 15.1%, with an τ_ave of 6.8 ns; χ² = 1.28). Nonetheless, the longest lifetime component has a lower contribution to the average lifetime, which may be related to the lower QY obtained for the latter CDs.

3.5. Influence of Reaction Parameters on TWCDs’ Optical Properties

To evaluate the influence of reaction conditions on the nanomaterials’ characteristics, namely their photophysical properties, different reaction parameters were tested. The influence of reaction temperature on the photoluminescence and reaction yields of TWCDs was first explored (

Table 2. Temperature effect on the TWCDs’ luminescence and reaction yield ¹.

T (°C)	QY 2	Emissionmax (nm) 2	ηmass (%)
200	0.04	460	23.5
230	0.08	451	22.6

¹ Typical reaction conditions: [TW] = 30 mg/mL, EDA/TW mass ratio = 0.16, 15 min. ² Excitation at 380 nm.

).

Increasing the temperature from 200 to 230 °C has a positive effect on the fluorescence QY (0.04 to 0.08), accompanied by a 9 nm blue shift in the emission, with no impact on the TWCDs mass yield (ca. 23%). From this result, experiments conducted at 230 °C would be advised. However, due to operational limitations of the microwave reactor, specifically its pressure threshold, further experiments at this temperature were not carried out.

The influence of irradiation time was next evaluated, keeping the reaction temperature at 200 °C. Two EDA/TW mass ratios were tested (0.16 and 0.32), being the results obtained collected in

Table 3. Residence time effect on the TWCDs’ luminescence and reaction yield ¹.

EDA/TW Mass Ratio	t (min)	QY 2	Emissionmax (nm) 2	ηmass (%)
0.16	15	0.04	460	23.5
	30	0.04	460	26.3
	60	0.08	456	24.3
0.32	15	0.05	460	30.4
	30	0.05	456	35.6
	60	0.08	458	36.4

¹ Typical reaction conditions: [TW] = 30 mg/mL, 200 °C. ² Excitation at 380 nm.

.

From these results, it may be inferred that extended dwell times (60 min) enhance the fluorescence QY, whatever the amount of added amine, pointing to the use of residence times of 60 min for better luminescence performance of CDs.

The impact of the nature of the additive on QY was also investigated, carrying out experiments with various aliphatic and aromatic amines, and in their absence. The sift assays were conducted for 15 min at 200 °C (

Table 4. Additive effect on the TWCDs’ luminescence and reaction yield ¹.

Additive	QY 2	Emissionmax (nm) 2	ηmass (%)
_	0.02	457	16.4
EDA	0.04	460	23.5
DETA	0.07	448	30.8
Urea	0.02	461	27.5
Melamine	0.03	458	19.8
p-PhD	0.01	494	18.6

¹ Typical reaction conditions: [TW] = 30 mg/mL, additive/TW mass ratio = 0.16, 200 °C, 15 min. ² Excitation at 380 nm.

).

From this set of experiments, the aliphatic amines EDA and DETA appear as the most useful additives to enhance the QY of these CDs.

Taking EDA as the additive, the influence of its amount on TWCDs’ QYs was next assessed (

Table 5. Effect of EDA amount on the TWCDs luminescence and reaction yield ¹.

EDA/TW Mass Ratio	QY 2	Emissionmax (nm) 2	ηmass (%)
_	0.02	457	16.4
0.04	0.04	469	19.1
0.08	0.04	461	20.0
0.16	0.04	460	23.5
0.32	0.05	460	30.4

¹ Typical reaction conditions: [TW] = 30 mg/mL, 200 °C, 15 min. ² Excitation at 380 nm.

).

From this data, one might conclude that increased amounts of EDA beyond a certain EDA/TW mass ratio (0.04) do not bring any relevant enhancements in the QY of CDs, which is good news since fewer exogenous synthetic compounds need to be used for attaining reasonable TWCDs’ QY. Nevertheless, the use of EDA clearly enhances the QY of CDs as compared to the assay without it.

At last, the impact of the TW initial concentration on QY and mass yield was evaluated (

Table 6. Effect of TW initial concentration on the TWCDs luminescence and reaction yield ¹.

TW Concentration (mg/mL)	QY 2	Emissionmax (nm) 2	ηmass (%)
15	0.05	455	22.8
30	0.04	460	23.5
70	0.08	455	22.5
100	0.04	457	21.7

¹ Typical reaction conditions: EDA/TW mass ratio = 0.16, 200 °C, 15 min. ² Excitation at 380 nm.

).

The results seem to indicate an increase in fluorescence QY when TW was used at a concentration of 70 mg/mL, with no impact on the mass yield.

Taking all the foregoing results, and having the highest fluorescence QY as the target, suitable experimental conditions to perform the Mw-HTC of TW have been found, which encompass the use of temperatures of 200 °C (and above), residence times of 60 min, and the use of additives (EDA/DETA).

As a complementary note to the above studies, a selected set of reaction conditions (TW concentration = 30 mg/mL, EDA/TW mass ratio = 0.16) was replicated in a domestic microwave oven using reaction times up to 15 min, resulting in TWCDs with lower luminescence (ca. 0.02).

3.6. Antioxidant Activity of TWCDs

The antioxidant properties of aqueous solutions of TWCDs were evaluated using the ABTS radical cation inhibition method [52,53]. The results are expressed in Trolox equivalents per mass unit of TWCDs (Table S1) and percentage of inhibition (Figure 5).

All the tested TWCDs reveal a good antioxidant capacity, very slightly higher for the CDs obtained under longer residence times. On the other hand, the increase in the EDA/TW mass ratio has no effect on the antioxidant activity. The high antioxidant activity observed for TWCDs may stem from the huge presence of hydroxyl and carboxyl groups in their structure [62]. Likewise, the evidence that reactive oxygen species are more efficiently scavenged by N-doped nanomaterials suggests that the nitrogen-rich domains in TWCDs also account for their pronounced antioxidant capacity [63]. Furthermore, the antioxidant properties displayed may also be related to the size of CDs. Large CDs (average size 7.8 nm) have been associated with a high inhibition of radicals due to the presence of more surface antioxidant functionalities [64].

The antioxidant capacity of TWCDs compares well with other CDs synthesized from other natural, renewable sources using microwave-assisted hydrothermal carbonization (

Table 7. Antioxidant capacity of various CDs obtained from different natural sources using Mw-HTC method.

CDs Source	Antioxidant Assay Method	Inhibition (%)	Concentration (mg/mL)	Reference
Showy Asian grapes	DPPH 1	73.8	5.0	[65]
Red Korean ginseng	DPPH 1	85.4	0.6	[66]
Edible swiftlet nest	DPPH 1	47.5	5.0	[67]
Centella asiatica leaves	DPPH 1	79.0	0.25	[62]
Solanum lycopersicum waste	ABTS•+	75.0	5.0	This work

¹ DPPH (2,2-diphenyl-1-picrylhydrazyl) radical inhibition method.

).

3.7. Antimicrobial Activity of TWCDs

Antimicrobial properties of TWCDs were assessed against pathogenic bacteria E. coli (Gram-negative) and S. aureus (Gram-positive), by disc diffusion method [54,55]. Four TWCDs batches, prepared over different reaction times and two EDA/TW mass ratios, were tested, using several amounts of CDs (100, 200, and 400 μg/disc). From the diameters of the inhibition zones obtained around each TWCDs-impregnated disc (Figure S7), after incubation of the cultures (

Table 8. Antibacterial activity of TWCDs by disc diffusion assay.

Bacteria	EDA/TW Mass Ratio 1	Dwell Time (min) 1	Inhibition Zone (mm) 2
			100 μg/disc	200 μg/disc	400 μg/disc
Escherichia coli (E. coli)	0.16	15	-	22 ± 0.5	25 ± 1.0
	0.16	30	-	21 ± 0.5	24 ± 1.0
	0.16	60	14 ± 1.0	23 ± 0.5	27 ± 0.5
	0.32	60	12 ± 0.5	26 ± 1.0	33 ± 0.5
Staphylococcus aureus (S. aureus)	0.16	15	-	20 ± 0.5	23 ± 1.0
	0.16	30	-	22 ± 0.5	24 ± 0.5
	0.16	60	-	22 ± 0.5	26 ± 0.5
	0.32	60	13 ± 0.5	25 ± 0.5	31 ± 1.0

¹ Typical reaction conditions for TWCDs synthesis: [TW] = 30 mg/mL, 200 °C. ² Data represent mean ± SD, n = 3; gentamicin (G) used as control (10 μg/disc): 27 ± 0.5 mm (E. coli) and 23 ± 0.5 mm (S. aureus); - no inhibition detected.

), it was found that TWCDs exhibited activity against both bacteria in a non-selective way, though, in a carbon dots mass-dependent mode (from 100 to 400 μg/disc). The highest inhibitory capacity was observed for TWCDs prepared with an EDA/TW mass ratio of 0.32. It is worth noting that these CDs have the least negatively charged zeta potential. Taking into consideration that the inhibition of bacterial activity may be related to the development of strong electrostatic interactions between the CDs and the bacterial cell walls [60], these are the CDs that are more likely to offer that possibility. Further studies are needed to clarify this issue.

To quantify the antibacterial effect of TWCDs, another susceptibility test was conducted to determine the minimum inhibitory concentration (MIC) and minimal lethal concentration (MLC) of TWCDs [57]. To this end, a microdilution test was carried out by inoculating E. coli and S. aureus in MHB and using resazurin as a cell viability marker [58]. In the presence of metabolically active cells, this poorly fluorescent dye is reduced to the highly fluorescent resorufin. For this purpose, the TWCDs batch (EDA/TW mass ratio = 0.32, 60 min) that stood out for its antimicrobial activity in disc diffusion assays was selected for the study, using gentamicin as a control (1 μg/mL for E. coli and 0.5 μg/mL for S. aureus). The data in Figure 6 show no significant effect on cell viability up to 1000 μg/mL of TWCDs, decreasing more noticeably after that. A sudden reduction in cell viability was clearly observed at a concentration of 4000 μg/mL, pointing to a MIC of TWCDs between 2000 and 4000 μg/mL for both bacteria. To discard any possibility of a direct chemical reduction of resazurin by TWCDs, which would have implications for the cell viability data, an experiment was undertaken in the absence of the cells. No reduction of resazurin was observed whatsoever (Figure S8).

Even though recent studies have reported nanomaterials synthesized from several natural sources with antibacterial activities, including plants, fruits, and microorganisms [68,69], there are no reports in the literature of CDs obtained from tomato or its wastes with such properties. The antimicrobial activity observed by the disc diffusion assays for the TWCDs (Table 8) is comparable with that of the henna plant-derived CDs [70]. Nevertheless, previous findings have revealed higher inhibitory effects on E. coli and S. aureus for nanomaterials derived from osmanthus (1000 μg/mL) [71] and turmeric leaves (250 μg/mL) [68].

As a corollary of the above results, one may also say that TWCDs exhibit a remarkable biocompatibility at concentrations under 1000 μg/mL, prompting their use in bioimaging applications.

4. Conclusions

As a significant step for its valorization, tomato waste from industrial plants was used for the synthesis of fluorescent carbon-based nanomaterials through microwave-assisted hydrothermal carbonization. On going from aqueous solutions to the solid state, under TEM experiments, nanoparticles having average lateral sizes of 8 nm were found. AFM topographical analysis furnished additional evidence for the nanoscale nature of the observed particles in the solid state, with height sizes between 3 and 10 nm. The structural characterization of TWCDs was undertaken by FTIR and NMR, allowing the identification of the main functionalities present in the materials. The TWCDs’ photophysics were studied in detail by absorption, steady-state, and time-resolved fluorescence techniques, permitting their full characterization.

To optimize the photophysical properties of TWCDs, a wide range of reaction parameters, within the Mw-HTC method, was tested, having the best conditions being found. The synthesized CDs exhibit higher photoluminescent quantum yields (up to 0.08) when organic aliphatic amines (EDA/DETA) were used as additives. TWCDs display considerable photostability, which is meaningful for several of their envisioned applications.

The ABTS^•+ assay demonstrated a strong radical scavenging efficiency, indicating promising antioxidant properties for the synthesized TWCDs. Additionally, antimicrobial activity against pathogenic bacteria E. coli and S. aureus was found, revealing a marked impact on bacterial growth by these nanomaterials at concentrations higher than 2000 μg/mL. On the other hand, no cytotoxic effects were found up to TWCDs concentrations of 1000 μg/mL, revealing their pronounced biocompatibility and potential for bioimaging applications.

Studies are currently in progress to further highlight the mechanism underlying their antimicrobial activity toward bacterial cells.