# Unraveling the synthesis-structure-property relationship in aromatic amino acids-derived carbon dots

Yalei Hu, Alberto Bianco

► To cite this version:

Yalei Hu, Alberto Bianco. Unraveling the synthesis-structure-property relationship in aromatic amino acids-derived carbon dots. Carbon, 2025, 235, pp.120073. 10.1016/j.carbon.2025.120073. hal-05338047

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# Unraveling the synthesis-structure-property relationship in aromatic amino acids-derived carbon dots

Yalei Hu* and Alberto Bianco*

CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University of Strasbourg, ISIS, 67000 Strasbourg, France

* Corresponding authors.

E-mail addresses: yalei.hu@ibmc-cnrs.unistra.fr (Y. Hu); a.bianco@ibmc-cnrs.unistra.fr (A. Bianco)

**ABSTRACT:** Carbon dots (CDs) have attracted increasing attention among various fields due to their unique physicochemical properties. However, the development of CDs remains challenging, particularly because their synthesis-structure-property relationship is still poorly understood. In this work, we systematically investigate this relationship by synthesizing CDs using two aromatic amino acids with distinct reactive sites, phenylalanine (Phe) and tyrosine (Tyr), with citric acid under hydrothermal treatments. The as-prepared Phe-derived CDs (Phe-CDs) and Tyr-derived CDs (Tyr-CDs) display remarkable differences in structural and photophysical properties. Based on a series of characterizations, a possible mechanism is proposed. Phe undergoes amidation and polymerization to form relatively homogeneous sp3/sp2 hybrid Phe-CDs with blue emission, while Tyr undergoes additional phenolic esterification, forming both sp3 and larger crosslinked sp2 regions that are carbonized into a core-shell structure with green dual-emission. These mechanistic insights into the synthesis-structure-property relationship offer a deeper understanding of CDs' formation pathways and provide guidance for their rational design and potential applications.

**KEYWORDS:** carbon materials, crosslinking, molecular state, core state, fluorescence
# 1. Introduction

Carbon dots (CDs), a class of carbon-based nanoparticles with a size of 1-10 nm, are emerging as a new star in the family of carbon nanomaterials [1-3]. CDs first came into the researchers' view by chance, when researchers discovered some fluorescent fragments while purifying single-walled carbon nanotubes [4]. Since then, research in the field of CDs has been growing rapidly. CDs have been found to possess many unique fluorescent properties. Firstly, a wide range of precursor sources, nearly all carbon-containing substances can be converted into CDs [5, 6]. Secondly, CDs possess low toxicity and good biocompatibility compared to traditional cadmium- or lead-based quantum dots [7]. Thirdly, CDs show good chemical stability and photobleaching resistance compared to organic dyes [8]. With these superiorities, CDs have been widely used in a wide range of interdisciplinary fields, such as biosensing, bioimaging, cancer therapy, catalysis, energy conversion, and optoelectronics [9-13].

Extensive efforts have been spent on CDs regarding their synthesis from various precursors and their applications. For example, blue, green, and red emissive CDs were prepared through solvothermal process by using *m*-, *o*-, *p*-phenylenediamines as precursors [14]. Researchers then explored the applications of these CDs in luminescent display and for bioimaging. Similarly, multicolor emissive CDs were prepared *via* the microwave method by using 1,2,4,5-benzenetetracarboxylic anhydride and *o*-phenylenediamine as precursors [15]. The authors then studied the application of these multicolored CDs in light-emitting diodes and fingerprint detection. There are also reports on the investigation of the synthesis and the fluorescent mechanisms of CDs. For instance, *in situ* infrared analysis as a function of temperature has been used to monitor the hydrothermal growth of CDs [16]. In another work, the fluorescence mechanism of CDs was studied at single particle level [17]. The researchers proved that their CDs possessed a core-shell structure, and that the observed fluorescence originates from CDs rather than from the fluorophore impurities. However, the synthesis-structure-property relationship of CDs remain ambiguous, and more efforts are needed to further reveal their mechanisms of formation and their luminescence mechanisms.
Amino acids are also widely used in the synthesis of CDs; however, related synthetic pathways remain unclear. In this work, we will select phenylalanine (Phe) that possesses amino and carboxyl groups, and tyrosine (Tyr) that possesses amino, carboxyl, and phenolic hydroxyl groups as model precursors to prepare CDs with citric acid (CA) through hydrothermal processes. As they possess different functional groups, we assume that the main initial reaction in Phe-CA system will be amidation reaction, while in Tyr-CA system, amidation and esterification reactions occur simultaneously. In addition, other cross-linking reactions involving the hydroxyl groups of citric acid are likely taking place during the preparation of CDs. Different types of reactions may form CDs with different structures and properties, from which we can investigate their synthesis-structure-property relationship. We first synthesized two kinds of CDs *via* hydrothermal processes. As-prepared Phe-derived CDs (Phe-CDs) showed a single blue-emission peak, while Tyr-derived CDs (Tyr-CDs) possessed green dual-emission peaks in aqueous solution. In addition, Phe-CDs and Tyr-CDs showed different solvent-dependent behavior. Phe-CDs displayed slightly different optical properties, while Tyr-CDs exhibited largely different optical properties in different solvents. The structural characterization results, including Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and X-ray photoelectron spectroscopy (XPS), indicated that Phe-CDs and Tyr-CDs owned different structures and surface functional groups. Based on structural and optical properties, the mechanisms for the underlying synthesis-structure-property relationship are proposed involving multi-step reactions, and molecular and core state emissions. In addition, these CDs show multifunctional applications in construction of luminescent solar concentrators (LSCs) and sensing of water in organic solvents. This study compares CDs derived from structurally similar amino acids, Phe and Tyr, to elucidate their synthesis-structure-property relationship and provide insights for the rational design of CDs with tailored properties.

## 2. Experimental section

### 2.1. Reagents

All chemicals were used as obtained. Citric acid (CA) was purchased from VWR (Belgium). Phenylalanine (Phe, 99+) and tyrosine (Tyr, 99+) were obtained from Iris Biotech (Germany).
Sodium hydroxide (NaOH) was purchased from Fisher Chemical (UK). Polyvinylpyrrolidone (PVP) K30 and PVP K90 were obtained from Sigma-Aldrich (USA). All aqueous solutions were prepared by using ultrapure water obtained from Millipore Milli-Q water purification system.

## 2.2. Synthesis of Phe-CDs and Tyr-CDs

Phe-CDs and Tyr-CDs were synthesized through hydrothermal processes. For Phe-CDs, 0.330 g Phe and 0.768 g CA were first dissolved into 50 mL 0.1 mol/L NaOH aqueous solution, followed by sonication for 10 min. Then the mixture was transferred into a Teflon-lined stainless-steel autoclave and heated at 180 °C for 6 h. After cooling down, the crude product was first filtered through 0.2 µm syringe filter and then the filtrate was dialyzed (dialysis membrane MWCO: 1000 Da) against water for 3 days (the water was changed four times per day, and the volume ratio of filtrate to water is around 1: 100). CDs powders were obtained by freeze-drying. For Tyr-CDs, 0.330 g Phe were replaced with 0.362 g Tyr, and the other steps were the same as above. After five independent experiments, the yields of Phe-CDs and Tyr-CDs were 0.6% and 1%, respectively. Control Phe-CDs and Tyr-CDs were also prepared by hydrothermally reacting the precursors for 2 and 4 h. These CDs are referred as Phe-CDs-2h, Phe-CDs-4h, Tyr-CDs-2h, and Tyr-CDs-4h, respectively.

## 2.3. Material characterization

The morphologies of Phe-CDs and Tyr-CDs were characterized by transmission electron microscopy (TEM) using Hitachi H 7500 and Jeol 2100F instruments. X-ray photoelectron spectroscopy (XPS) was conducted by a multiprobe system (Thermo Scientific) equipped with an Al Kα source (1486 eV). UV-vis absorption and fluorescence emission spectra were recorded by using Cary 5000 UV-Vis-NIR spectrometer (Agilent Technologies) and FP-8300 fluorimeter (JASCO), respectively. Fourier-transform infrared spectroscopy spectra were collected by using FTIR-4100 spectrometer (JASCO).

Kaiser test: 0.2 mg CDs was weight in a tube. Then, 75 µL of phenol solution (10 g phenol in 20 mL ethanol), 100 µL potassium cyanide (in H₂O/pyridine), and 75 µL ninhydrin (1g ninhydrin in 20 mL ethanol) were added to the CDs. The resulting mixture was sonicated then heated 120 °C for 5 min, followed by diluting with 4750 µL of 60% ethanol. The blank was prepared the same way but without CDs. The absorbance at 570 nm was correlated to the amount of free amino groups
on CDs surface.

Relative quantum yield calculation: Quinine sulfate was selected as the reference. The relative quantum yields of Phe-CDs and Tyr-CDs were calculated use the following equation,

$$ \Phi_{CDs} = \Phi_{R} \frac{k_{CDs}}{k_{R}} \frac{\eta_{CDs}^{2}}{\eta_{R}^{2}} $$

where kCDs and kR are the slopes of CDs and reference integral fluorescence intensity versus absorbance curve. ηCDs and ηR are the refractive indices of the solvent.

## 2.4. Luminescent solar concentrator fabrication

Phe-CDs were first dispersed into methanol to prepare a 1 mg/mL stock solution, and then 0.3 mL and 1.5 mL of the stock solution were added into 3 mL of a PVP methanol solution (100 mg/mL PVP K30 and 100 mg/mL PVP K90) under magnetic stirring, respectively. The mixtures were first stirred for 2 h to obtain clear dispersions, followed by 10 min sonication to remove bubbles. The clear dispersions were then drop-casted onto two glass substrates, dried in air for 6 h, to obtain Phe-CDs film LSCs. Tyr-CDs-based LSCs were prepared use the same method with Phe-CDs.

## 2.5. Sensing of water in THF

Typically, Tyr-CDs (1 mg) were first dispersed in THF (1 mL) as stock solution. A series of THF-water mix solutions (water content: 0, 0.1%, 0.2%, 0.5%, 1%, 2%, 4%, 6%, 8%, and 10%) containing 10 µg/mL Tyr-CDs were prepared. All fluorescence emission spectra were recorded after 10 min stabilization at room temperature.

# 3. Results and discussion

## 3.1. Synthesis and morphological properties of Phe-CDs and Tyr-CDs

Phe-CDs and Tyr-CDs were prepared by facile hydrothermal treatment of Phe with CA, and Tyr with CA at 180 °C for 6 h, respectively. After purification by filtration and dialysis, the morphologies of these CDs were first characterized by using transmission electron microscopy (TEM). As shown in Fig. 1a and 1b, Phe-CDs and Tyr-CDs are monodispersed and nearly spherical with average diameters of 5.1 ± 0.6 nm and 5.0 ± 0.4 nm, respectively. From the size distribution histograms, Tyr-CDs are more uniform in size than Phe-CDs. In addition, the high-resolution TEM
(HRTEM) images reveal that the Phe-CDs and Tyr-CDs have a certain crystallinity with lattice planes with a distance of 0.21 nm, which is attributed to the (100) in-plane lattice spacing of graphene [18].

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** TEM images of Phe-CDs (a) and Tyr-CDs (b), with representative HRTEM images and size distribution histograms.</figcaption></figure>

## 3.2. *Structural characterizations of Phe-CDs and Tyr-CDs*

FTIR was first employed to characterize the functional groups on Phe-CDs and Tyr-CDs. As shown in **Fig. 2a** and 2b, the FTIR spectra of Phe-CDs and Tyr-CDs are similar. Stretching vibrations of O-H and N-H groups are observed at around 3376 and 3237 cm⁻¹. The two peaks at around 3012 and 2925 are attributed to the asymmetric stretching vibration of C-H bonds. The vibration bands of the COOH, CONH/COO, and C=C groups appear at around 1698, 1647, and 1570 cm⁻¹, respectively. The two peaks at around 1397 and 1171 cm⁻¹ are assigned to the C-OH/C-N and C-O groups [19]. Meanwhile, the FTIR band positions and intensities also show significant differences. The amino groups of Tyr-CDs appearing at 1516 cm⁻¹ are more intense than those of Phe-CDs at 1497 cm⁻¹ [20]. Kaiser test was then used to evaluate the amount of primary amines. Kaiser test yields 110 µmol g⁻¹ for Phe-CDs and 275 µmol g⁻¹ for Tyr-CDs, confirming the FTIR results that there are more amines on Tyr-CDs than on Phe-CDs. In addition, Tyr-CDs show a
characteristic absorption of Tyr, the phenolic C-OH band at around 1234 cm⁻¹, which is invisible in Phe-CDs [21].

Recently, NMR has been recommended to assess the successful synthesis of CDs [22, 23]. The expected NMR spectra of CDs should possess broad peaks, which are different from the sharp signals of the small molecular precursors. As can be seen from **Fig. 2c**, both Phe-CDs and Tyr-CDs show broad peaks in the aromatic region. Compared with Phe-CDs, the aromatic region peaks of Tyr-CDs are characterized by a shift to high field, which is generally attributed to being surrounded by electron-donating groups [24]. In this work, the shift is caused by the surface amino groups and phenols. NMR results are in good agreement with FTIR results, indicating that there are more amino groups on the surface of Tyr-CDs.

XPS was then utilized to reveal more details of the chemical composition of Phe-CDs and Tyr-CDs. XPS survey spectra indicate that both Phe-CDs and Tyr-CDs contain carbon, nitrogen, and oxygen elements, and there are more nitrogen elements in Tyr-CDs than in Phe-CDs (**Fig. S1**). The high-resolution XPS spectra of C 1s could be deconvoluted into three peaks located at 284.4, 285.8, and 288.2 eV, corresponding to the bonds of C-C, C-N/C-O, and C=O/COOH, respectively (**Fig. 2d** and 2g). The deconvolution of N 1s spectra displays also three species, primary amines (-NH₂), pyridinic N, and pyrrolic N (**Fig. 2e** and 2h) [25]. The high-resolution spectra of O 1s reveal the presence of C=O and C-O (**Fig. 2f** and 2i). These results indicate that there are abundant oxygen- and nitrogen-containing groups on the surface of Phe-CDs and Tyr-CDs. In addition, the differences between Phe-CDs and Tyr-CDs are very clear. There are more -NH₂ and C-O on the surface of Tyr-CDs than on Phe-CDs, in good agreement with the NMR and the FTIR results. These results indicate that during the Tyr hydrothermal process, the phenolic hydroxyl groups in Tyr may undergo condensation with the carboxyl group in CA, and allow the formation of dityrosine fragments through radical reactions. Together, these processes leave a portion of unreacted amino groups, and results in more amino groups and C-O on the surface of Tyr-CDs than on Phe-CDs.
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Structural characterizations of Phe-CDs and Tyr-CDs. (a) and (b) FTIR spectra of the two CDs; (c) ¹H NMR spectra of the two kinds of CDs in DMSO-d6; (d), (e), and (f) high-resolution (HR) XPS spectra of C 1s, N 1s, and O 1s of Phe-CDs; (g), (h), and (i) HR XPS spectra of C 1s, N 1s, and O 1s of Tyr-CDs.</figcaption></figure>

### 3.3. *Optical properties of Phe-CDs and Tyr-CDs*

To assess the optical properties, UV-Vis absorption spectra of Phe-CDs and Tyr-CDs were first collected. Phe-CDs' absorption spectrum shows two absorption bands located at around 250 nm and 340 nm, while Tyr-CDs' absorption spectrum possesses two absorption peaks at around 270 nm and 350 nm, respectively (Fig. 3a). We also recorded the UV-Vis absorption spectra of Phe-
CDs and Tyr-CDs in various solvents. As shown in **Fig. S2**, the positions of UV-Vis absorption bands of Phe-CDs or Tyr-CDs are almost identical in the different solvents, which indicates that the ground state is less sensitive to solvents, and the difference in the UV-Vis absorption values stems from the difference in the molar extinction coefficients. The absorption bands at 250 nm and 270 nm are assigned to π-π* electronic transitions of the aromatic sp² domains, while the absorption peaks at 340 nm and 350 nm are attributed to the n-π* transitions of C=O and N-related groups present on the surface of the dots [26, 27]. The redshift of the π-π* transition in Tyr-CDs suggests that Tyr-CDs possess a larger sp² domain, and the broader and redshift of n-π* transition in Tyr-CDs reflect that these dots possess more abundant surface functional groups [28].

The fluorescence emission spectra of Phe-CDs and Tyr-CDs were then recorded in aqueous solution. Phe-CDs display a single blue-emission peak, centered at 420 nm (Fig. 3b). The single emission peak suggests that the structure of Phe-CDs may be relatively homogeneous. The type of luminescence of the Phe-CDs is attributed to surface molecular state. Tyr-CDs instead exhibit a dual-emission behavior, centered at 490 nm and 530 nm (Fig. 3b). Generally, CDs are considered to possess core-shell structures [29-31]. In some cases, the core and the shell may exhibit different fluorescence behavior [32, 33]. In the Tyr-CDs case, we hypothesize that they possess a core-shell-like structure. The 490 nm emissive species are attributed to the shell, i.e., surface molecular state, while the 530 nm emissive species are attributed to the carbonized sp² core. The long-wavelength core state emission of Tyr-CDs coincides with the larger sp² domain proved by the absorption spectra. As shown in Fig. S3, both Phe-CDs and Tyr-CDs show excitation-independent emission behavior in aqueous solution. Using quinine sulfate as a reference, the relative fluorescence quantum yields of Phe-CDs and Tyr-CDs in aqueous solution were 5.0% and 4.2%, respectively (Fig. S4). The photostability of these CDs was also assessed. Both Phe-CDs and Tyr-CDs retain more than 98% intensity after 1 h of 365 nm UV light irradiation, reflecting a good photostability (Fig. S5).

To verify the above hypotheses, the fluorescence behavior of Phe-CDs and Tyr-CDs among different solvents was investigated. The fluorescence emission maxima of Phe-CDs show slight
shifts in the different solvents (Fig. 3c). With the increase of polarity, the fluorescence intensity of Phe-CDs increases (Fig. S6). Deviations are also observed, which may be attributed to specific interactions. However, in the case of Tyr-CDs, the 490 nm emission peak shows a largely blue-shift in the same solvents used for Phe-CDs, while the peak positions of the maximum emission at around 530 nm remain nearly unchanged (Fig. 3d). And the polarity of the solvent affects the extent of the blue-shift. In addition, the fluorescence intensity of Tyr-CDs decreases as the polarity increases. However, deviations are observed, which may be attributed to specific interactions. These phenomena indicate that the 530 nm emission peaks in Tyr-CDs are minimally affected by the solvents, which fits well with the above hypothesis that the peak originates from sp² core that is not in direct contact with the liquid environment, and thus is more stable. As proposed, the 490 nm emission center in Tyr-CDs belongs to the surface molecular state. Based on the above structural characterizations, there are many N-related groups, such as amino groups on the surface of Tyr-CDs, and these functional groups are easily affected by the surrounding solvent molecules due to the solvation effect, thus leading to a large difference in their optical behavior among the different solvents [34, 35]. Furthermore, nitrogen-containing functional groups such as amino groups can act as electron-donating groups to cause intramolecular charge transfer effects in Tyr-CDs. Moreover, the polarity of the solvent affects the intramolecular charge transfer process, thus making the Tyr-CDs solvent-sensitive. The presence of hydrogen bonds also affects the intramolecular charge transfer process in these CDs, which corresponds to the red-shift of Tyr-CDs in ethanol and water.
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Optical properties of Phe-CDs and Tyr-CDs. (a) UV-Vis absorption spectra of Phe-CDs and Tyr-CDs in aqueous solution; (b) Fluorescence emission spectra of Phe-CDs and Tyr-CDs in aqueous solution; (c) Solvent-dependent fluorescence emission spectra of Phe-CDs; (d) Solvent-dependent fluorescence emission spectra of Tyr-CDs (THF: tetrahydrofuran; DMF: dimethylformamide).</figcaption></figure>

To further study the difference between Phe-CDs and Tyr-CDs, the effect of viscosity on the fluorescence emission of these CDs was then studied (Fig. S7). The ethanol (EtOH)-polyethylene glycol (PEG) system was used as solvent. The higher the PEG content is, the higher viscosity and lower polarity are. With the increase of PEG content, the positions of the fluorescence emission peaks of Phe-CDs keep unchanged, again indicating their weak polarity-sensitive behavior. Instead, the fluorescence emission intensity gradually increases, which is attributed to the large viscosity-
induced restriction of the rotation of CDs [36]. In Tyr-CDs (Fig. S5b), with the increase of PEG content, the fluorescence emission intensity first increases and then the emission peaks gradually blue-shift, which is due to the restriction of rotation and solvent-sensitive nature of Tyr-CDs [37].

### 3.4. Influence of pH

Subsequently, the influence of pH on the fluorescence emission of Phe-CDs and Tyr-CDs was examined. In aqueous solution, the polar functional groups like carboxylic groups (pKa ~ 4.5) and amino groups (pKa ~ 9.0) can be deprotonated or protonated [38]. Here, the fluorescence emission spectra were measured in Britton-Robinson buffer solutions. As shown in Fig. 4a and 4b, the fluorescence emission intensity of Phe-CDs gradually increases when the pH changes from acidic to neutral and remains constant under alkaline conditions. Tyr-CDs exhibit distinct fluorescence behavior under different pH conditions. The fluorescence emission intensity remains relatively constant in acidic and neutral conditions but decreases progressively as the pH transitions from neutral to alkaline. This behavior can be also partially influenced by the phenolic hydroxyl group in Tyr, which is a weak acid and can undergo deprotonation under alkaline conditions. pH may affect the luminescence of the carbon core indirectly by altering the electronic structure or energy alignment of surface states, which modulates the efficiency of electron or energy transfer between core and surface states. These results indicate that the prominent functional groups of the surface molecular states on Phe-CDs and Tyr-CDs are different. In Phe-CDs, the prominent groups are carboxylic acid, while amino groups are protruding groups in Tyr-CDs. These results are consistent with previous structural characterizations and solvent-related fluorescence emission behavior.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 4.** pH-dependent fluorescence emission behavior of Phe-CDs and Tyr-CDs. (a) Fluorescence emission spectra of Phe-CDs at different pH values; (b) Maximum fluorescence emission intensities of Phe-CDs at different pH values; (c) Fluorescence emission spectra of Tyr-CDs at different pH values; (d) Maximum fluorescence emission intensities at 530 nm of Tyr-CDs at different pH values with Boltzmann fitting.</figcaption></figure>

### 3.5. Synthesis-structure-property relationship in Phe-CDs and Tyr-CDs

Generally, the bottom-up hydrothermal synthesis of CDs includes dehydration, condensation, polymerization, and carbonization, but the more specific reaction pathways are unclear. In this work, the hydrothermal temperature was 180 °C. Phe, Tyr, and CA were utilized as precursors, mainly containing amino, carboxyl, and phenolic hydroxyl groups. CDs are most likely formed through the following three stages: 1) CA and amino acid undergo multi-step amidation and/or
esterification to form oligomers; 2) oligomers undergo polymerization to form polymers; 3) polymers undergo further carbonization to form the CDs (Fig. 5) [39]. During the formation of Phe-CDs, the initial reaction is mainly amidation, and the single reaction type leads to a homogeneous structure of Phe-CDs with a low degree of conjugation. For the formation of Tyr-CDs, esterification is also involved. The additional esterification reactions enlarge the sp² domain by undergoing more dehydration condensation and by immobilizing the aromatic region in Tyr. In addition, hydrophobic dityrosine fragments may also be formed through radical reactions [40]. The hydrophobic crosslinked sp² regions are further carbonized to form the carbon core, ultimately leading to the formation of sp² core-sp³/sp² hybrid shell Tyr-CDs. To investigate the proposed pathways, Phe-CDs and Tyr-CDs were synthesized at varying hydrothermal reaction times. The yields of Phe-CDs-2h and Tyr-CDs-2h were extremely low (<0.01%), suggesting that during the first 2 hours, the primary products are oligomers, with negligible condensation to CDs. In the case of Phe-CDs-4h and Tyr-CDs-4h, the yields increased to 0.2% and 0.5%, respectively, but remained lower than those of Phe-CDs and Tyr-CDs obtained after 6 h reaction. The UV-Vis absorption spectra and fluorescence emission spectra of these samples are presented in the Supporting Information (Fig. S8a). These spectra showed similar bands to the samples obtained after 6 h, indicating their comparable structures. The emission spectra of Phe-derived CDs have minimal changes at the different reaction times (Fig. S8b). Instead, for Tyr-derived CDs, the peak at ~530 nm in Tyr-CDs-2h was weaker compared to that in Tyr-CDs after 6 h reaction. This observation supports the hypothesis that the ~530 nm emission originates from the core state, which becomes more prominent as the carbon core grows during extended reaction times. These findings indicate that during the initial stages of the hydrothermal reaction, the amino acids and citric acid primarily form oligomers. After 2 h, some of these oligomers polymerize into larger structures, and by 4 h, the polymers begin to undergo partial carbonization to CDs.

To describe the fluorescence mechanisms of CDs, many possibilities have been reported, including quantum confinement, core state, surface state, molecular state, crosslink-enhanced emission, and/or their synergistic effects [41]. Based on the above structural and optical
characterizations, the molecular state and the core state are involved in Phe-CDs and Tyr-CDs. For Phe-CDs (Fig. 5a), the low degree of conjugated sp²/sp³ hybrid structure results in a short-wavelength emission, i.e., blue-emission. Tyr-CDs exhibit instead a typical core-shell dual-emission behavior (Fig. 5b). The highly carbonized sp² core possesses long-wavelength emission, i.e., green-emission, while the less carbonized sp²/sp³ hybrid shell displays a relatively short-wavelength, i.e., blue- or cyan-emission [42]. The solvent-sensitive behavior of Tyr-CDs is attributed to surface nitrogen-containing functional groups, especially abundant amino groups, which trigger charge transfer and hydrogen bonding and are thus sensitive to the environment (polarity or viscosity). [43, 44].

The additional phenolic hydroxyl group in Tyr acts as a "butterfly effect," significantly influencing the hydrothermal reaction by enabling more extensive dehydration and carbonation. This promotes the formation of a larger core state in Tyr-CDs, whereas Phe-CDs, undergo limited reactions, resulting in properties dominated by surface states. Overall, small variations in the precursors used in the synthesis of CDs lead to distinct synthesis pathways, forming CDs with diverse structures, which in turn give rise to corresponding differences in their properties.

<figure><img src="image_12.png" /><figcaption>**Fig. 5.** Illustration proposing the synthesis-structure-property relationship in (a) Phe-CDs and (b)Tyr-CDs.</figcaption></figure>

3.6. Multifunctional applications of Phe-CDs and Tyr-CDs
As proven above, both Phe-CDs and Tyr-CDs show excellent photostability; thus, they can be used to construct luminescent solar concentrators (LSCs). Phe-CDs and Tyr-CDs-based film LSCs were prepared by drop-casting a mixture of CDs (0.05% or 0.25% of CDs) and polyvinylpyrrolidone (PVP) onto glass substrates. As shown in **Fig. 6a** and 6b, the as-prepared thin-film-structured LSCs show good transparency and light gathering under 365 nm UV light at both low and high concentrations of CDs. The results indicate that these CDs could be useful for the fabrication of LSCs.

As Tyr-CDs show solvent-sensitive behavior, these dots may also be useful to sense water. A series of THF-water mixtures with different contents of water was prepared, and their fluorescence emission spectra were recorded. With the increase in water content, the intensity of 415 nm emission peak first increases, then (from 0.1% water) the emission peak gradually redshifts (**Fig. 6c**). The peak positions at 530 nm remain unchanged, and their intensity gradually increases. The redshift and increased intensity were caused by solvent effects like solvation and hydrogen bonding.[45, 46] As shown in **Fig. 6d**, there is a good linear relationship between the ratio of 530 nm peak intensity ($I_{530nm}$) to 415 nm peak intensity ($I_{415nm}$) and the water content. According to 3σ rule, the limit of detection of water in THF is calculated to be 0.07%. The sensitivity is comparable to other methods (**Table S1**). These results indicate that the Tyr-CDs could be used for the detection of water in organic solvents.
<figure><img src="image_13.png" /><figcaption>**Fig. 6.** Multifunctional applications of Phe-CDs and Tyr-CDs. (a) Photographs of Phe-CDs-based LSCs under daylight (left) and 365 nm UV light (right); (b) Photographs of Tyr-CDs-based LSCs under daylight (left) and 365 nm UV light (right); (c) Fluorescence emission spectra of Tyr-CDs in THF-water system with different contents of water. Inset: photos of the samples under 365 nm irradiation; (d) Plot of dual-emissive Tyr-CDs fluorescence intensity ratio (I<sub>530nm</sub>/I<sub>415nm</sub>) versus water content, with a fitted linear curve.</figcaption></figure>

**4. Conclusion**

In summary, this work studied the synthesis-structure-property relationship of CDs derived from Phe and Tyr, synthesized via hydrothermal treatments. The Phe-CDs displayed a single blue emission, while Tyr-CDs exhibited green dual-emission. The structural evolution of these CDs was closely linked to the functionality of precursors. Phe underwent amidation, polymerization, and partial carbonization to yield homogeneous Phe-CDs with low conjugation degree. In contrast, the
additional phenolic hydroxyl group in Tyr facilitated esterification and formation of larger sp2
domains, which were further carbonized into an extended carbon core, producing core-shell Tyr-
CDs with dual-emission from core and surface molecular states. These insights revealed how, even
little, functional group variations influence CD formation and resulting properties. In addition, this
work demonstrated the potential of these CDs in applications such as photovoltaics and sensing.
The proposed synthesis-structure-property relationship facilitates the design of CDs with tailored
properties, paving the way for their practical uses.

CRediT authorship contribution statement

Yalei Hu: Writing – original draft, Methodology, Investigation, Data curation. Alberto Bianco: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

**Declaration of competing interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique (CNRS), by the
Interdisciplinary Thematic Institute SysChem, through the IdEx Unistra (ANR-10-IDEX-0002).
The authors thank C. Royer and D. Ihiawakrim for TEM and HRTEM observations. We also thank
Y. He for her help on XPS. Y. Hu acknowledges the support of Horizon Europe under Marie
Skłodowska-Curie Actions European Fellowship program (no. 101150852).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at xx.

**Data availability**

Data will be made available on request.
References

[1] B. Wang, G.I.N. Waterhouse, S. Lu, Carbon dots: mysterious past, vibrant present, and expansive future, Trends Chem. 5 (2023) 76-87, https://doi.org/10.1016/j.trechm.2022.10.005.

[2] L. Đorđević, F. Arcudi, M. Cacioppo, M. Prato, A multifunctional chemical toolbox to engineer carbon dots for biomedical and energy applications, Nat. Nanotechnol. 17 (2022) 112-130, https://doi.org/10.1038/s41565-021-01051-7.

[3] C. Xia, S. Zhu, T. Feng, M. Yang, B. Yang, Evolution and Synthesis of Carbon Dots: From Carbon Dots to Carbonized Polymer Dots, Adv. Sci. 6 (2019) 1901316, https://doi.org/10.1002/advs.201901316.

[4] X. Xu, R. Ray, Y. Gu, H.J. Ploehn, L. Gearheart, K. Raker, W.A. Scrivens, Electrophoretic Analysis and Purification of Fluorescent Single-Walled Carbon Nanotube Fragments, J. Am. Chem. Soc. 126 (2004) 12736-12737, https://doi.org/10.1021/ja040082h.

[5] H. Liu, X. Zhong, Q. Pan, Y. Zhang, W. Deng, G. Zou, H. Hou, X. Ji, A review of carbon dots in synthesis strategy, Coord. Chem. Rev. 498 (2024) 215468, https://doi.org/10.1016/j.ccr.2023.215468.

[6] A.U. Khan, Y. Liu, S. Wang, M.W. Ullah, Q. Chen, D. Zhang, Z. Kang, B. Mao, Advancements in the green synthesis of carbon dots for sustainable development, Sustain. Mater. Technol. 41 (2024) e01004, https://doi.org/10.1016/j.susmat.2024.e01004.

[7] Y. Wang, T. Lv, K. Yin, N. Feng, X. Sun, J. Zhou, H. Li, Carbon Dot-Based Hydrogels: Preparations, Properties, and Applications, Small 19 (2023) 2207048, https://doi.org/10.1002/smll.202207048.

[8] Z. Chen, Y. Liu, Z. Kang, Diversity and Tailorability of Photoelectrochemical Properties of Carbon Dots, Acc. Chem. Res. 55 (2022) 3110-3124, https://doi.org/10.1021/acs.accounts.2c00570.

[9] Y. Hu, X. Geng, L. Zhang, Z. Huang, J. Ge, Z. Li, Nitrogen-doped Carbon Dots Mediated Fluorescent on-off Assay for Rapid and Highly Sensitive Pyrophosphate and Alkaline Phosphatase Detection, Sci. Rep. 7 (2017) 5849, https://doi.org/10.1038/s41598-017-06356-z.

[10] D.-K. Ji, G. Reina, S. Guo, M. Eredia, P. Samorì, C. Ménard-Moyon, A. Bianco, Controlled functionalization of carbon nanodots for targeted intracellular production of reactive oxygen species, Nanoscale Horiz. 5 (2020) 1240-1249, https://doi.org/10.1039/D0NH00300J.

[11] H. Song, M. Wu, Z. Tang, J.S. Tse, B. Yang, S. Lu, Single Atom Ruthenium-Doped CoP/CDs Nanosheets via Splicing of Carbon-Dots for Robust Hydrogen Production, Angew. Chem. Int. Ed. 60 (2021)
7234-7244, https://doi.org/10.1002/anie.202017102.

[12] J. Liu, Y. Liu, N. Liu, Y. Han, X. Zhang, H. Huang, Y. Lifshitz, S.-T. Lee, J. Zhong, Z. Kang, Metal-free efficient photocatalyst for stable visible water splitting via a two-electron pathway, Science 347 (2015) 970-974, https://doi.org/10.1126/science.aaa3145.

[13] H. Zhao, G. Liu, S. You, F.V.A. Camargo, M. Zavelani-Rossi, X. Wang, C. Sun, B. Liu, Y. Zhang, G. Han, A. Vomiero, X. Gong, Gram-scale synthesis of carbon quantum dots with a large Stokes shift for the fabrication of eco-friendly and high-efficiency luminescent solar concentrators, Energy Environ. Sci. 14 (2021) 396-406, https://doi.org/10.1039/D0EE02235G.

[14] K. Jiang, S. Sun, L. Zhang, Y. Lu, A. Wu, C. Cai, H. Lin, Red, Green, and Blue Luminescence by Carbon Dots: Full-Color Emission Tuning and Multicolor Cellular Imaging, Angew. Chem. Int. Ed. 54 (2015) 5360-5363, https://doi.org/10.1002/anie.201501193.

[15] H. Ding, R. Zhao, Z.-H. Zhang, J.-J. Yang, Z. Wang, L.-L. Xiao, X.-H. Li, X.-J. He, H.-M. Xiong, Kilogram-scale synthesis of carbon dots with high-efficiency full-color solid-state fluorescence using an aggregation-induced emission strategy, Chem. Eng. J. 476 (2023) 146405, https://doi.org/10.1016/j.cej.2023.146405.

[16] L. Fois, L. Stagi, D. Carboni, M. Alboushi, A. Khaleel, R. Anedda, P. Innocenzi, The Formation of Carbon Dots from D-Glucose Studied by Infrared Spectroscopy, Chem. Eur. J. 30 (2024) e202400158, https://doi.org/10.1002/chem.202400158.

[17] B. Wang, Z. Wei, L. Sui, J. Yu, B. Zhang, X. Wang, S. Feng, H. Song, X. Yong, Y. Tian, B. Yang, S. Lu, Electron-phonon coupling-assisted universal red luminescence of o-phenylenediamine-based carbon dots, Light Sci. Appl. 11 (2022) 172, https://doi.org/10.1038/s41377-022-00865-x.

[18] Y. Ru, L. Sui, H. Song, X. Liu, Z. Tang, S.-Q. Zang, B. Yang, S. Lu, Rational Design of Multicolor-Emitting Chiral Carbonized Polymer Dots for Full-Color and White Circularly Polarized Luminescence, Angew. Chem. Int. Ed. 60 (2021) 14091-14099, https://doi.org/10.1002/anie.202103336.

[19] M. Zhang, Y. Ma, H. Wang, B. Wang, Y. Zhou, Y. Liu, M. Shao, H. Huang, F. Lu, Z. Kang, Chiral Control of Carbon Dots via Surface Modification for Tuning the Enzymatic Activity of Glucose Oxidase, ACS Appl. Mater. Interfaces 13 (2021) 5877-5886, https://doi.org/10.1021/acsami.0c21949.
[20] M. Wolpert, P. Hellwig, Infrared spectra and molar absorption coefficients of the 20 alpha amino acids in aqueous solutions in the spectral range from 1800 to 500cm⁻¹, Spectrochim. Acta A 64 (2006) 987-1001, https://doi.org/10.1016/j.saa.2005.08.025.

[21] B. Hernández, F. Pflüger, A. Adenier, S.G. Kruglik, M. Ghomi, Vibrational Analysis of Amino Acids and Short Peptides in Hydrated Media. VIII. Amino Acids with Aromatic Side Chains: l-Phenylalanine, l-Tyrosine, and l-Tryptophan, J. Phys. Chem. B 114 (2010) 15319-15330, https://doi.org/10.1021/jp106786j.

[22] B. Bartolomei, A. Bogo, F. Amato, G. Ragazzon, M. Prato, Nuclear Magnetic Resonance Reveals Molecular Species in Carbon Nanodot Samples Disclosing Flaws, Angew. Chem. Int. Ed. 61 (2022) e202200038, https://doi.org/10.1002/anie.202200038.

[23] B. Bartolomei, M. Prato, The Importance of the Purification Step and the Characterization of the Products in the Synthesis of Carbon Nanodots, Small 19 (2023) 2206714, https://doi.org/10.1002/smll.202206714.

[24] M. Baranac-Stojanović, Can Variations of ¹H NMR Chemical Shifts in Benzene Substituted with an Electron-Accepting (NO₂)/Donating (NH₂) Group be Explained in Terms of Resonance Effects of Substituents?, Chem. Asian J. 13 (2018) 877-881, https://doi.org/10.1002/asia.201800137.

[25] X. Sun, C. Brückner, Y. Lei, One-pot and ultrafast synthesis of nitrogen and phosphorus co-doped carbon dots possessing bright dual wavelength fluorescence emission, Nanoscale 7 (2015) 17278-17282, https://doi.org/10.1039/C5NR05549K.

[26] K. Jiang, Y. Wang, X. Gao, C. Cai, H. Lin, Facile, Quick, and Gram-Scale Synthesis of Ultralong-Lifetime Room-Temperature-Phosphorescent Carbon Dots by Microwave Irradiation, Angew. Chem. Int. Ed. 57 (2018) 6216-6220, https://doi.org/10.1002/anie.201802441.

[27] S. Zhu, L. Wang, N. Zhou, X. Zhao, Y. Song, S. Maharjan, J. Zhang, L. Lu, H. Wang, B. Yang, The crosslink enhanced emission (CEE) in non-conjugated polymer dots: from the photoluminescence mechanism to the cellular uptake mechanism and internalization, Chem. Commun. 50 (2014) 13845-13848, https://doi.org/10.1039/C4CC05806B.

[28] G. Yang, C. Wu, X. Luo, X. Liu, Y. Gao, P. Wu, C. Cai, S.S. Saavedra, Exploring the Emissive States of Heteroatom-Doped Graphene Quantum Dots, J. Phys. Chem. C 122 (2018) 6483-6492,
https://doi.org/10.1021/acs.jpcc.8b01385.

[29] J. Liu, R. Li, B. Yang, Carbon Dots: A New Type of Carbon-Based Nanomaterial with Wide Applications, ACS Cent. Sci. 6 (2020) 2179-2195, https://doi.org/10.1021/acscentsci.0c01306.

[30] M. Alafeef, I. Srivastava, T. Aditya, D. Pan, Carbon Dots: From Synthesis to Unraveling the Fluorescence Mechanism, Small 20 (2024) 2303937, https://doi.org/10.1002/smll.202303937.

[31] S. Maniappan, K.L. Reddy, J. Kumar, Transmitting biomolecular chirality into carbon nanodots: a facile approach to acquire chiral light emission at the nanoscale, Chem. Sci. 14 (2023) 491-498, https://doi.org/10.1039/D2SC05794H.

[32] T. Zhang, F. Zhao, L. Li, B. Qi, D. Zhu, J. Lü, C. Lü, Tricolor White-Light-Emitting Carbon Dots with Multiple-Cores@Shell Structure for WLED Application, ACS Appl. Mater. Interfaces 10 (2018) 19796-19805, https://doi.org/10.1021/acsami.8b03529.

[33] F. Rigodanza, M. Burian, F. Arcudi, L. Đorđević, H. Amenitsch, M. Prato, Snapshots into carbon dots formation through a combined spectroscopic approach, Nat. Commun. 12 (2021) 2640, https://doi.org/10.1038/s41467-021-22902-w.

[34] L. Ai, Y. Yang, B. Wang, J. Chang, Z. Tang, B. Yang, S. Lu, Insights into photoluminescence mechanisms of carbon dots: advances and perspectives, Sci. Bull. 66 (2021) 839-856, https://doi.org/10.1016/j.scib.2020.12.015.

[35] Y. Hu, C. Neumann, L. Scholtz, A. Turchanin, U. Resch-Genger, S. Eigler, Polarity, intramolecular charge transfer, and hydrogen bond co-mediated solvent effects on the optical properties of graphene quantum dots, Nano Res. 16 (2023) 45-52, https://doi.org/10.1007/s12274-022-4752-1.

[36] F. Yan, X. Sun, T. Ma, Y. Zhang, Y. Jiang, R. Wang, C. Ma, J. Wei, L. Chen, Y. Cui, A viscosity-dependent carbon dots with anti-VEGF properties for monitoring and promoting apoptosis in cancerous cell, Chem. Eng. J. 407 (2021) 127801, https://doi.org/10.1016/j.cej.2020.127801.

[37] Z. He, Y. Sun, C. Zhang, J. Zhang, S. Liu, K. Zhang, M. Lan, Recent advances of solvent-engineered carbon dots: A review, Carbon 204 (2023) 76-93, https://doi.org/10.1016/j.carbon.2022.12.052.

[38] F. Meierhofer, F. Dissinger, F. Weigert, J. Jungclaus, K. Müller-Caspary, S.R. Waldvogel, U. Resch-Genger, T. Voss, Citric Acid Based Carbon Dots with Amine Type Stabilizers: pH-Specific Luminescence
and Quantum Yield Characteristics, J. Phys. Chem. C 124 (2020) 8894-8904,
https://doi.org/10.1021/acs.jpcc.9b11732.

[39] C. Xia, J. Zhong, X. Han, S. Zhu, Y. Li, H. Liu, B. Yang, The Formation Mechanism of Carbonized Polymer Dots: Crosslinking-Induced Nucleation and Carbonization, Angew. Chem. Int. Ed. 63 (2024) e202410519, https://doi.org/10.1002/anie.202410519.

[40] B.P. Partlow, M.B. Applegate, F.G. Omenetto, D.L. Kaplan, Dityrosine Cross-Linking in Designing Biomaterials, ACS Biomater. Sci. Eng. 2 (2016) 2108-2121, https://doi.org/10.1021/acsbiomaterials.6b00454.

[41] B. Wang, S. Lu, The light of carbon dots: From mechanism to applications, Matter 5 (2022) 110-149,
https://doi.org/10.1016/j.matt.2021.10.016.

[42] Y. Liu, D. Cheng, B. Wang, J. Yang, Y. Hao, J. Tan, Q. Li, S. Qu, Carbon Dots-Inked Paper with Single/Two-Photon Excited Dual-Mode Thermochromic Afterglow for Advanced Dynamic Information Encryption, Adv. Mater. 36 (2024) 2403775, https://doi.org/10.1002/adma.202403775.

[43] X. Xu, L. Mo, Y. Li, X. Pan, G. Hu, B. Lei, X. Zhang, M. Zheng, J. Zhuang, Y. Liu, C. Hu, Construction of Carbon Dots with Color-Tunable Aggregation-Induced Emission by Nitrogen-Induced Intramolecular Charge Transfer, Adv. Mater. 33 (2021) 2104872, https://doi.org/10.1002/adma.202104872.

[44] E.V. Kundelev, N.V. Tepliakov, M.Y. Leonov, V.G. Maslov, A.V. Baranov, A.V. Fedorov, I.D. Rukhlenko, A.L. Rogach, Amino Functionalization of Carbon Dots Leads to Red Emission Enhancement, J. Phys. Chem. Lett. 10 (2019) 5111-5116, https://doi.org/10.1021/acs.jpclett.9b01724.

[45] H. Liu, J. Yang, Z. Li, L. Xiao, A.A. Aryee, Y. Sun, R. Yang, H. Meng, L. Qu, Y. Lin, X. Zhang, Hydrogen-Bond-Induced Emission of Carbon Dots for Wash-Free Nucleus Imaging, Anal. Chem. 91 (2019) 9259-9265, https://doi.org/10.1021/acs.analchem.9b02147.

[46] G. Cui, Z. Lan, W. Thiel, Intramolecular Hydrogen Bonding Plays a Crucial Role in the Photophysics and Photochemistry of the GFP Chromophore, J. Am. Chem. Soc. 134 (2012) 1662-1672, https://doi.org/10.1021/ja208496s.