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Synthesis, Characterization, Magnetic Properties, and Applications of Carbon Dots as Diamagnetic Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agents: A Review

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Submitted:

05 March 2025

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05 March 2025

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Abstract
Carbon dots (CDs) are metal-free carbon-based nanoparticles. They possess excellent photoluminescent properties, various physical properties, good chemical stability, high water solubility, high biocompatibility, and tunable surface functionalities, suitable for biomedical applications. Their properties are subject to synthetic conditions such as pH, reaction time, temperature, precursor, and solvent. Until now a large amount of articles on synthesis and biomedical applications of CDs using their photoluminescent properties have been reported. However, their researches on magnetic properties and especially, diamagnetic chemical exchange saturation transfer (diaCEST) in magnetic resonance imaging (MRI) are very poor. The diaCEST MRI contrast agents are based on exchangeable protons of materials with bulk water protons and thus, different from conventional MRI contrast agents which are based on enhancements of proton spin relaxations of bulk water and tissue. In this review, various syntheses, characterizations, magnetic properties, and potential applications of CDs as diaCEST MRI contrast agents are reviewed. Finally, future perspectives of CDs as the next generation diaCEST MRI contrast agents are discussed.
Keywords: 
carbon dot
;  
synthesis
;  
characterization
;  
magnetic properties
;  
diaCEST
;  
MRI contrast agent
Subject: 
Chemistry and Materials Science  -   Nanotechnology

1. Introduction

Carbon dots (CDs) had been noticed ~20 years ago and exhibit emission in the visible region [1]. Sun et al. had prepared bright and colorful photoluminescent CDs by a laser ablation method from graphite powder and cement [2]. CDs are great potential for applications to biomedical and environmental areas [3‒6]. For example, bioimaging and chemo-sensing, cellular imaging, drug delivery, cancer therapy, pollutant removal, waste water treatment, and environmental remediation are the possible application areas [7‒13]. Moreover, surface composition engineering through post-synthetic approaches allows to expand and optimize the range of CD applications. Recently, CDs have received great attention owing to their simple synthesis and surface functionalization, good biocompatibility, and highly stable emission [14]. CDs can contain various surface functional groups such as hydroxyl (‒OH), carboxyl (‒COOH), carbonyl (‒CO), and amine (‒NH2) groups, which are suitable for further surface functionalizations [15‒17]. Surface functional groups enhance the reactivity of CDs towards conjugation reactions and tuning their surface properties. Surface functionalization of CDs with oxygen-containing groups tend to exhibit negatively charged surfaces, while nitrogen-containing functional groups can result in positively charged surfaces, making CDs stable colloids in aqueous media. Since the discovery of CDs, two decades have gone and a lot of CD-based research papers have been published [18]. To highlight the importance of CD-based researches and progress, we used the Scopus database to find out the total number of articles related to CDs published for the last ten years (≥ 2015). Figure 1 displays the number of research papers related to CDs and definitely reveal that the trend keeps on increasing every year.
So far, CDs have been applied to various biomedical areas [7‒11,19]. However, little application studies on contrast agents in magnetic resonance imaging (MRI) exist. MRI is one of the primary techniques used in disease detection, diagnosis, and monitoring. Over the last decades, MRI technology has been continuously improved [20]. These improvements include the enhancement of the image clarity, reduction in the scan times, and development of the high-field scanners. Furthermore, MRI image qualities and diagnostic precisions have been significantly improved with the development of contrast agents. Among them, a new class of metal-free MRI contrast agents based on chemical exchange saturation transfer (CEST) had been introduced by Ward et al. [21]. This CEST works through the proton exchange of contrast agents with bulk water protons to enhance image contrasts at the accumulated region of the contrast agents.
In general, the CEST MRI contrast agents can be divided into two groups based on their compositions [21‒23]; paramagnetic CEST (paraCEST) MRI contrast agents which are paramagnetic metal ion (Eu3+, Dy3+, etc) complexes, and diamagnetic CEST (diaCEST) MRI contrast agents which are made of metal-free materials with exchangeable protons with bulk water protons. In this review, various synthetic approaches and characterization techniques of CDs are briefly reviewed. Then, magnetic properties and applications of CDs as diaCEST MRI contrast agents as a new class of metal-free MRI contrast agents are discussed and highlighted along with their future perspectives.

2. Synthesis of CDs

Until now various synthetic approaches of CDs have been reported. A well-established synthesis method will afford CDs with uniform size, high quantum yield (QY), and scalable and cost-effective production. CDs can be prepared largely by two approaches “top-down” and “bottom-up” as depicted in Figure 2.

2.1. Top-Down Approach

The top-down approach involves cleavage and exfoliation of carbon precursors such as graphite powder, activated carbon, carbon black, carbon nanotubes, and carbon fibers [24]. The top-bottom approach includes chemical oxidation, electrochemical oxidation, laser ablation, and ultrasonication as depicted in Figure 3.

2.1.1. Chemical Oxidation

Chemical oxidation method utilizes strong oxidants such as HNO3 and H2SO4 to oxidize carbon precursors to prepare CDs [26]. Chemical oxidation is a facile and convenient method for mass production of CDs.
Qiao et al. prepared multicolor photoluminescent CDs from three different activated carbon precursors by chemical oxidation [27]. Coal-activated carbon (CAC), wood-activated carbon (WAC), and coconut-activated carbon (CAC) were treated with HNO3 to obtain CDs. Further, the CDs were coated with amine-terminated compounds. Figure 4a‒4c exhibit TEM images and size distribution of CDs prepared from CAC, WAC, and CAC precursors; they had average diameters of 4.5±0.6 nm, 4.2±0.8 nm, and 4.2±0.6 nm, respectively. The CDs were water-soluble and displayed multicolor photoluminescent properties with high quantum yields and good biocompatibility.

2.1.2. Electrochemical Oxidation

Electrochemical oxidation is suitable to tune the properties of CDs by controlling electrochemical parameters, solvent, and carbon precursors [28]. The size of CDs can be tuned by changing the applied potential [29].
Liu et al. prepared monodispersed and highly crystalline CDs using electrochemical oxidation of graphite electrode precursor in alkaline solution [30]. The CDs exhibited temperature-dependent color change such that colorless CDs synthesized at 4 oC tinted bright-yellow color at room temperature. This color change was attributed to the oxygenation of CD surfaces. Figure 5a and 5b display the TEM and HRTEM images, size distribution, and photograph of the colorless CDs, and Figure 5c and 5d exhibit the TEM and HRTEM images, size distribution, and photograph of the bright-yellow CDs. TEM images revealed that colorless CDs were monodispersed in size with an average diameter of 4.0±0.2 nm, while bright-yellow CDs afforded two-size distributions; one from monodispersed CDs, similar to the colorless CDs, and the other from aggregated CDs with an average diameter of 8.0±0.3 nm.

2.1.3. Laser Ablation

In laser ablation, a graphite precursor is exposed to laser irradiation and CDs are produced from the precursor [31,32]. Laser ablation is classified into two categories: laser ablation in solution and that of powdered sample [32]. Notably, the double beam laser ablation method provided smaller CDs with a narrower size distribution than the single beam laser ablation method [33].
Hu et al. prepared CDs by laser ablation of graphite flakes in polyethylene glycol (Mn = 1500 amu) solution [34]. The size of CDs were tuned by controlling the laser pulse width. The longer laser pulse width provided a larger particle size of CDs. Figure 6a‒6c exhibit the HRTEM images and Figure 6d‒6f display the size distributions of CDs prepared by 0.3, 0.9, and 1.5 ms laser pulse widths, respectively. Figure 6a exhibits the single crystalline CDs whereas CDs in Figure 6b and 6c are composed of two or more crystalline grains.

2.1.4. Ultrasonic-Assisted Method

This method utilizes ultrasound irradiation to synthesize CDs [35]. The ultrasonic-assisted method has an advantage of low cost and simplicity in operation to synthesize CDs.
Wu et al. prepared water-soluble photoluminescent CDs by an ultrasonic-assisted chemical oxidation method of petroleum coke [36]. The CD surfaces were rich in oxygen-containing functional groups. Then, CDs were further treated hydrothermally in ammonia to prepare N-CDs. Figure 7a and 7b exhibit the TEM and HRTEM images and size distribution of CDs with an average size of 5.0 nm. Figure 7c and 7d display the TEM and HRTEM images and size distribution of N-CDs with an average size of 2.7 nm. The HRTEM images of CDs and N-CDs showed their lattice spaces of 0.332 and 0.334 nm, respectively.

2.2. Bottom-Up Approach

The bottom-up approach involves the carbonization of organic molecules as carbon sources or precursors. The carbonizing molecules are coupled together to form sp2 carbons in CDs [37]. Owing to commercial availability and facile carbonization, organic molecules with hydroxy (‒OH), carboxylic acid (‒COOH), and amine (‒NH2) functional groups are generally used as precursors [38]. Figure 8 exhibits the examples of organic carbon precursors such as melamine, citric acid, and phloroglucinol as well as the synthesis of CDs through ‘‘bottom-up” approaches.

2.2.1. Microwave-Assisted Method

The microwave method involves microwave irradiation to precursors to produce CDs. This method is conveniently applied to various kinds of precursors to prepare CDs in a short reaction time [40,41]. For example, Yu et al. prepared CDs using phthalic acid and triethylenediamine as precursors in a 60 s reaction time [42].
Jiang et al. prepared RNA targeting CDs by microwave thermal decomposition method of neutral red and levofloxacin as precursors for the liver cell imaging [43]. Figure 9a and 9b exhibit a TEM image with size distribution and an HRTEM image of CDs, respectively. The TEM image revealed that CDs were well-dispersed with an average size diameter of 1.60 nm. The HRTEM image exhibited 0.18 and 0.29 nm lattice fringes.

2.2.2. Hydrothermal Method

Hydrothermal method is considered as an ecofriendly, nontoxic, and cost-effective method for preparing CDs using diverse carbon sources [44‒47].
Bao et al. prepared dual-function fluorescent CD probe by hydrothermal method using citric acid as a carbon source and o-phenylenediamine as a nitrogen source [48]. Figure 10a displays the TEM image and particle size distribution of the CDs with spherical morphology and good desperation. The particle size of CDs ranged from 1.24 to 6 nm, with the average diameter of 3.23 nm. The HRTEM image exhibited that the CDs had a lattice spacing of 0.21 nm as depicted in Figure 10b. The CDs worked as a dual-function fluorescent probe for the detection of Fe3+ in the brown sugar and sunset yellow dye in beverages.

2.2.3. Pyrolysis Method

This method involves thermal decomposition of carbon precursors at high temperatures to produce CDs. The pyrolysis method is considered an easy operation, low cost, solvent free, and fast reaction method.
Wang et al. successfully prepared CDs using one-pot solid phase pyrolysis in an autoclave (closed environment) and a crucible (open environment) [49]. The CDs prepared in open environment had a longer emission wavelength, a higher crystallinity, and less surface state emission than CDs prepared in closed environment. Figure 11a and 11b exhibit the TEM image with size distribution and the HRTEM image with lattice fringes of CDs-1, respectively, and Figure 11c and 11d display the TEM with size distribution, and the HRTEM image with lattice fringes of CDs-2. Both CDs were well dispersed with average sizes of 2.08 and 2.09 nm, respectively.

2.2.4. Templated Method

The templated method involves a support material (i.e., a template) to produce CDs. The size, morphology, and surface properties of CDs can be controlled using templates.
Yang et. al. prepared monodispersed photoluminescent CDs by soft-hard template approach [39]. Photoluminescent CDs were prepared using copolymer Pluronic P123 as a soft-template, ordered mesoporous silica (OMS) as a hard-template, and 1,3,5-trimethylbenzene (TMB), diaminebenzene (DAB), pyrene (PY) and phenanthroline (PHA) as carbon sources. Figure 12a‒12d exhibit HRTEM images of CDs, which were prepared using TMB, DAB, PY, and PHA, respectively. The copolymers served as micelles (i.e., soft template) to encapsulate carbon precursors and the OMS served as separator (i.e., hard template) of the micelles. The HRTEM image revealed that the soft-hard templated approach prevented the aggregation of CDs during pyrolysis.

3. Characterizations

Several analytical techniques have been used to characterize various physicochemical properties of CDs such as the size, crystal structure, elemental composition, surface charge, hydrodynamic diameter, cytotoxicity, and magnetic properties. The characterization helps to modify and optimize the synthesis. This section will provide overviews of various characterization methods to analyze various physicochemical properties of CDs. TEM, Fourier transform-infrared (FT-IR) absorption spectroscopy, Raman spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential measurement, vibrating sample magnetometry (VSM), electron paramagnetic resonance (EPR) spectroscopy, in vitro and in vivo cytotoxicity measurement are such methods to elucidate properties of CDs.
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Figure 13. Schematic illustration of various characterization methods to elucidate various physicochemical properties of CDs.
Figure 13. Schematic illustration of various characterization methods to elucidate various physicochemical properties of CDs.
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3.1. FT-IR Absorption Spectroscopy

FT-IR absorption spectroscopy is used to identify the functional groups present on the surfaces of CDs and allows to follow the post-surface modification progress [50]. FT-IR absorption spectroscopy is suitable to measure gaseous, liquid, and solid-state samples. Figure 14a exhibits FT-IR absorption spectra of amorphous CDs and dextrose precursor [51], and Table 1 lists the FT-IR absorption wavenumbers of characteristic vibrations of CDs prepared from different carbon sources.

3.2. Raman Spectroscopy

Raman spectroscopy can be used to identify CDs and evaluate crystalline or amorphous nature of CDs [52]. Raman spectrum of CDs displays two distinct peaks, D and G bands at ~1360 and ~1580 cm‒1, respectively, arising from sp2 carbons (i.e., C=C double bonds). D band appears due to the vibrations of defect sp2 graphitic carbons, whereas G band is the primary mode in graphene and graphite which is due to planar sp2 graphitic carbons; therefore, highly crystalline graphene and graphite have a strong G and weak D bands. Figure 14b shows Raman spectrum of the CDs prepared from citric acid and neutral red [56] and Table 1 lists D and G bands of CDs prepared from different carbon sources.

3.3. XRD

XRD is used to determine the crystal structure of CDs. It also allows us to examine chemical composition, phase purity, and particle size of CDs [60,61]. In XRD patterns, broad peaks indicate the poor degree of crystallinity (or amorphous) of CDs. Figure 14c shows two broad peaks at 2θ = ~19° and ~38° from the graphitic carbon C(002) and C(004) crystal planes in the amorphous CDs [51].

3.4. DLS

Dynamic light scattering (DLS) allows us to examine the hydrodynamic particle diameter distribution or aggregation of CDs in aqueous media [62]. Figure 14d exhibits a DLS pattern of CDs (prepared using citric acid) with an average hydrodynamic diameter below 10 nm [63].

3.5. Zeta Potential

Zeta potential of CDs reflects their surface charge [62,64]. It helps to predict the functional groups, hydrophilicity, and electrostatic stability of the CDs in aqueous solution. For instance, CDs with -COOH groups will have negative zeta potentials, while CDs with NH2 groups will have positive zeta potentials [65]. Figure 14e shows the zeta potential of CDs prepared from citric acid with a value of ‒16 mV [63], indicating that the CDs have negatively charged surfaces owing to -COOH groups.
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Figure 14. Various data of CDs: (a) FT-IR absorption spectra of CDs and dextrose precursor [51]. (b) Raman spectra [56]. (c) XRD pattern [51]. (d) DLS pattern [63]. (e) Zeta potential curve [63].
Figure 14. Various data of CDs: (a) FT-IR absorption spectra of CDs and dextrose precursor [51]. (b) Raman spectra [56]. (c) XRD pattern [51]. (d) DLS pattern [63]. (e) Zeta potential curve [63].
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3.6. Magnetic Properties

The magnetic ordering of CDs makes them magnetic materials [66]. The magnetic ordering of CDs is due to the presence of intrinsic disorder and surface defects, providing unpaired electrons [67]. The unpaired electrons cause magnetic ordering in CDs [68].

3.6.1. VSM

Tripti et al. prepared CDs using the biomass precursor pennisteum glaucum and a simple pyrolysis method [69]. Magnetic properties of the synthesized CDs, i.e., B 1 h, B 2 h, and B 3 h where “B” represents the biomass precursor and “1 h”, “2 h”, and “3 h” represent the pyrolysis times, were investigated. The magnetic ordering was attributed to the interaction of unpaired electrons. Figure 15a shows the saturation magnetization values of 0.02412, 0.02006, and 0.01872 emu/g for B 1 h, B 2 h, and B 3 h CDs, respectively, revealing that as the pyrolysis time increased, the saturation magnetization decreased; this implies that defect structures and unpaired electrons increased with increasing pyrolysis time. Tegafaw et al. prepared amorphous CDs with an average diameter of 2.2 nm using dextrose precursor and a wet-chemical method. The M‒H curve of the CDs revealed that the amorphous CDs had a weak paramagnetic property at room temperature as depicted in Figure 15b [51]. Therefore, magnetic properties of CDs depend on synthesis method and carbon precursor.

3.6.2. EPR

Bhunia et al. prepared fluorescent CDs using carbohydrate derivatives and chemical method [70]. Four kinds of fluorescent CDs with different emission colors were prepared by changing synthesis conditions; they were CDblue, CDgreen, CDyellow, and CDred. Figure 16a displays the EPR spectra of four kinds of CDs with different emission colors at 25 oC, confirming the existence of free electrons in CDs. Zhao et al. prepared CDs with a 2‒4 nm diameter using a microwave approach and glucose and PEG 1500 as the carbon sources [71]. Figure 16b exhibits the EPR spectra of CDs. g-value of 2.00094 revealed that the ground state of CDs was singly occupied in orbital. When NaOH was added into CD solution, the peak intensity and area increased, indicating more singly occupied orbitals by free electrons in CDs.

3.7. In Vitro and In Vivo Cytotoxicity

CDs are potential candidates in biological and biomedical applications owing to their very low or nontoxic performance [72‒74]. Furthermore, their cytotoxicity can be improved through surface modifications [75].
Wang et al. prepared ultrasmall and highly biocompatible CDs using the natural plant Pollen Typhae (PT) and a one-pot pyrolysis method [76]. Figure 17a‒17c exhibit the cell viability of mouse macrophage tumor (RAW 264.7), cervical cancer line HeLa derivative (L02), and human embryonic kidney (293T) cells, respectively, at the concentration range from 19.53 to 2,500 µg CDs/mL, displaying almost no cellular toxicity. Figure 15d presents the in vitro cytotoxicity of amorphous CDs using human prostate cancer (DU145) and normal mouse hepatocyte (NCTC1469) cells, indicating nontoxicity up to the treated carbon concentration of 500 µM in both cells [51]. Wang et al. explored the in vivo toxicity of CDs in various organs (i.e., heart, liver, spleen, lung, and kidneys) 1 and 14 days after injection of CD solution into rats [77]. Figure 17e shows no significant histological changes in the organs after injection as compared with those of the control, confirming nontoxicity of the CDs.

4. CDs as diaCEST MRI Contrast Agents

4.1. Principle of CEST

Advancement of MRI transformed with the development of contrast agents because they improved the images and enhanced diagnostic precision through contrast enhancements. Until now various kinds of metal-based MRI contrast agents have been developed; these are Gd(III)-chelates, Mn(II)-chelates, and iron oxide nanoparticles [78‒84]. However, these metal-based MRI contrast agents are restricted to low-concentration injection owing to their toxicity. Therefore, metal-fee MRI contrast agents such as CEST MRI contrast agents have been recently introduced [85‒90].
Figure 18a displays the principle of CEST mechanism in which the saturated solute protons are exchanged with bulk water protons at the rate Ksw and the unsaturated bulk water protons return to the solutes at the rate Kws [86,90]. The left spectrum in Figure 18b presents the solute protons which resonate at a different frequency from that of bulk water protons. The saturated solute protons at a specific resonance frequency are transferred to bulk water through exchange with unsaturated water protons, decreasing the water proton resonance signal, as depicted in the right spectrum in Figure 18b. Figure 18c exhibits the normalized proton spectrum; this spectrum is called as the Z-spectrum or CEST spectrum. Figure 18d displays the result of magnetization transfer ratio (MTR) asymmetry analysis in % of the Z-spectrum after removing the effect of water proton signals. Figure 18e displays the chemical shift of various exchangeable proton sources and their MTR efficiencies [91], showing that the higher MTR efficiency and the higher saturation offset from H2O will provide sharper and stronger CEST signals. Therefore, the effective CEST MRI contrast agents should have the high MTR efficiency and the high saturation offset from H2O.

4.2. Applications of CDs as diaCEST MRI Contrast Agents

The CEST MRI contrast agents can be divided into two catagories based on their composition: paramagnetic CEST (paraCEST) agents [22,92,93] and diamagnetic CEST (diaCEST) agents [94‒96]. The metal-free CDs can be used as diaCEST MRI contrast agents to amplify MRI contrast efficiency.
Zhang et al. prepared arginine-modified carbon dots (AC-dots) as a new class of diaCEST MRI contrast agents [94]. Figure 19a shows the synthesis of AC-dots with an average diameter of 4.7 nm using glucose and arginine as precursors and microwave irradiation. The arginine was used to modify the surfaces of CDs. Figure 19b and 19c display the Z-spectra and MTRasym plots, respectively, with an increment of AC-dot concentration in which MTRasym plots exhibited the signal increment with the increase of AC-dot concentration. Figure 19d exhibits that MTRasym plots at pH = 6.1 and 6.5 had maximum signals at ~1 ppm owing to hydroxyl protons of AC-dots, but at pH ≥ 7, maximum signals were observed at ~2 ppm because the CEST signals were replaced into guanidinium protons. Figure 19e and 19f show that liposome (Lipo)-AC-dot-labeled cells had higher CEST contrast enhancements than the control liposome-labeled cells. Importantly, as shown in Figure 19f, the T2 MR images showed similar contrasts at the left and right mouse brains, but the CEST image showed higher contrasts at the left brain (Lipo-AC-dot-labeled cells injected) than the right brain (control Lipo-labeled cells injected). This work clearly demonstrated the effectiveness of the CDs as new class of diaCEST MRI contrast agents in sensitively detecting diseases with minimal artificial defect contrasts.
Pandey et al. prepared water-soluble CDs as diaCEST MRI contrast agents [95]. The amino-thioamide precursor was ineffective as a diaCEST MRI contrast agent owing to its poor water solubility. However, CDs prepared using thermal treatment served as diaCEST contrast agents owing to their improved water-solubility. Figure 20a exhibits the synthesis of CDs using hydrothermal treatment and Figure 20b displays the CEST effect as a function of pH for the amide (pink) and ammonium (green) exchangeable protons in Figure 20a. As shown in Figure 20c, the precursor in PBS at pH = 5.5 exhibited a broad diaCEST spectrum with 9.7% maximum efficiency of MTRasym at Δω = 2.25 ppm. The CEST results of CDs are presented in Figure 20d‒20f. At pH = 9.9, a strong and sharp diaCEST signal with 50.3% MTRasym was observed from amide protons of CDs at Δω = 5.25 ppm (Figure 20d). However, as shown in Figure 20e, the diaCEST efficiency was poor at physiological pH = 7.4. However, improved diaCEST efficiency was obtained at physiological pH by the variation of the reaction time, temperature, and precursor concentration [96]. At pH = 5.5, the CDs exhibited the maximum MTRasym of ~69% from ammonium protons owing to improved water solubility of CDs (Figure 20f). This pH dependent CEST experiment indicated that the best CEST image using CDs prepared using amino-thioamide precursor could be obtained at pH = 9.9.

5. Conclusions and Future Perspectives

This review overviewed the progress and advancements of synthesis, characterizations, and MRI application of CDs as diaCEST MRI contrast agents. As reviewed here, only a few studies on diaCEST MRI contrast agents based on CDs exist. Nonetheless, the CDs demonstrated the excellent performance suitable for applications as a new class of nontoxic and next generation MRI, i.e., diaCEST MRI contrast agents.
The CDs have received a great attention owing to their great potential for biomedical applications [97‒99]. Until now CDs have been synthesized using various methods with explanations of their plausible formation mechanisms. Although numerous synthetic methods have been introduced, a standard synthetic methodology producing high-quality CDs with required morphology, size, properties, and surface functional groups has not been developed. Therefore, the future research should address this issue to improve and optimize the performance and applications of CDs. Above all, the synthesis should satisfy the high water-solubility of CDs with many exchangeable protons with bulk water protons to apply them as effective diaCEST MRI contrast agents.
To improve the MR image quality, contrast agents can be used [100]. As reviewed here, the diaCEST MRI contrast agents correspond to a new class of MRI contrast agents. They do not rely on metal ions, but exchangeable protons with bulk water protons. The diaCEST MRI technique can provide resonance frequency selectivity because the resonance comes from exchangeable protons of materials, but not the bulk water protons, providing image contrasts with minimal artificial defects from bulk water proton signals. In addition, compared with conventional Gd-chelates [101] and iron oxide-based superparamagnetic nanoparticles [102], diaCEST MRI contrast agents have considerably lower biotoxicity because they are made of nontoxic elements such as C, H, O, and N.
The CD-based diaCEST MRI contrast agents can provide several advantages over conventional MRI contrast agents. Besides non-toxicity and resonance frequency selectivity, they can be easily synthesized using various carbon precursors and various synthetic methods. They can be made highly hydrophilic with many exchangeable protons with bulk water protons. Furthermore, their surfaces can be easily modified to conjugate with various functional molecules such as targeting ligands and drugs to increase specificity and treat diseases. The present status of CD-based diaCEST MRI contrast agents is just beginning at the research level as can be evidenced from only a few research papers published so far. However, based on previous reports, the future of CD-based diaCEST MRI contrast agents is very promising. The high sensitivity and frequency selectivity of the CD-based diaCEST MRI contrast agents will allow us to detect and monitor diseases at the molecular level. Therefore, metal-free CDs as promising potential diaCEST MRI contrast agents will open a new journey to MRI.

Author Contributions

Conceptualization, E.M. and T.T.; methodology, E.M.; validation, Y.L., D.Z. and A.B.; writing—original draft preparation, E.M.; writing—review and editing, Y.C. and G.H.L.; funding acquisition, J.K., Y.C. and G.H.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Basic Science Research Program of the National Research Foundation (NRF) funded by the Korea government (Ministry of Science, and Information and Communications Technology: MSIT) (Basic Research Laboratory, No. RS-2024-00406209) and NRF funded by the Ministry of Education (Post-Doc. Growth Type Cooperational Research, No. RS-2024-00459895).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The annual number of research articles related to CDs. (The statistical data is from the Scopus database up to February 2025).
Figure 1. The annual number of research articles related to CDs. (The statistical data is from the Scopus database up to February 2025).
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Figure 2. Schematic representation of ‘‘top-down” and ‘‘bottom-up” approaches in CD syntheses.
Figure 2. Schematic representation of ‘‘top-down” and ‘‘bottom-up” approaches in CD syntheses.
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Figure 3. Schematic representation of top-down approaches of CD synthesis: (a) chemical oxidation, (b) electrochemical oxidation, (c), laser ablation, and (d) ultrasonication. Reproduced with permission [25].
Figure 3. Schematic representation of top-down approaches of CD synthesis: (a) chemical oxidation, (b) electrochemical oxidation, (c), laser ablation, and (d) ultrasonication. Reproduced with permission [25].
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Figure 4. TEM images and size distributions of CDs prepared from (a) CAC, (b) WAC, and (c) CAC precursors. Reproduced with permission [27].
Figure 4. TEM images and size distributions of CDs prepared from (a) CAC, (b) WAC, and (c) CAC precursors. Reproduced with permission [27].
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Figure 5. (a) TEM and HRTEM images and (b) size distribution and photograph of colorless CDs. (c) HRTEM and TEM images and (d) size distribution and photograph of bright-yellow CDs. Reproduced with permission [30].
Figure 5. (a) TEM and HRTEM images and (b) size distribution and photograph of colorless CDs. (c) HRTEM and TEM images and (d) size distribution and photograph of bright-yellow CDs. Reproduced with permission [30].
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Figure 6. HRTEM images of CDs prepared using (a) 0.3, (b) 0.9, and (c) 1.5 ms laser pulse widths, respectively. (d)‒(f) The corresponding size distributions. Reproduced with permission [34]. “Interface” (as labeled with arrows) indicates boundaries between CDs.
Figure 6. HRTEM images of CDs prepared using (a) 0.3, (b) 0.9, and (c) 1.5 ms laser pulse widths, respectively. (d)‒(f) The corresponding size distributions. Reproduced with permission [34]. “Interface” (as labeled with arrows) indicates boundaries between CDs.
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Figure 7. (a) TEM and HRTEM images and (b) size distribution of CDs. (c) TEM and HRTEM images and (d) size distribution of N-CDs. Reproduced with permission [36].
Figure 7. (a) TEM and HRTEM images and (b) size distribution of CDs. (c) TEM and HRTEM images and (d) size distribution of N-CDs. Reproduced with permission [36].
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Figure 8. Schematic of ‘‘bottom-up” approaches to prepare CDs: (a) microwave [25], (b) hydrothermal [25], (c) pyrolysis [25], and (d) templated methods: TMB = 1,3,5-trimethylbenzene; TEOS = tetraethoxysilane; P123 = copolymer Pluronic P123; OMS = ordered mesoporous silica [39].
Figure 8. Schematic of ‘‘bottom-up” approaches to prepare CDs: (a) microwave [25], (b) hydrothermal [25], (c) pyrolysis [25], and (d) templated methods: TMB = 1,3,5-trimethylbenzene; TEOS = tetraethoxysilane; P123 = copolymer Pluronic P123; OMS = ordered mesoporous silica [39].
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Figure 9. (a) TEM image and size distribution, and (b) HRTEM image and lattice fringes of CDs. Reproduced with permission [43].
Figure 9. (a) TEM image and size distribution, and (b) HRTEM image and lattice fringes of CDs. Reproduced with permission [43].
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Figure 10. (a) TEM image and particle size distribution of CDs. (b) HRTEM image and lattice fringes of CDs. Reproduced with permission [48].
Figure 10. (a) TEM image and particle size distribution of CDs. (b) HRTEM image and lattice fringes of CDs. Reproduced with permission [48].
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Figure 11. CDs-1: (a) TEM image with size distribution histogram (inset) and (b) HRTEM image with lattice fringes. CDs-2: (c) TEM image with size distribution histogram (inset) and (d) HRTEM image with lattice fringes. Reproduced with permission [49].
Figure 11. CDs-1: (a) TEM image with size distribution histogram (inset) and (b) HRTEM image with lattice fringes. CDs-2: (c) TEM image with size distribution histogram (inset) and (d) HRTEM image with lattice fringes. Reproduced with permission [49].
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Figure 12. HRTEM images of CDs: (a) CDTMB, (b) CDDAB, (c) CDPY, and (d) CDPHA. Reproduced with permission [39].
Figure 12. HRTEM images of CDs: (a) CDTMB, (b) CDDAB, (c) CDPY, and (d) CDPHA. Reproduced with permission [39].
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Figure 15. (a) Magnetization curves of B 1 h, B 2 h and B 3 h CDs at room temperature (pennisteum glaucum as the precursor) [69]. (b) Magnetization curve of amorphous CDs at room temperature (dextrose as the carbon precursor) [51].
Figure 15. (a) Magnetization curves of B 1 h, B 2 h and B 3 h CDs at room temperature (pennisteum glaucum as the precursor) [69]. (b) Magnetization curve of amorphous CDs at room temperature (dextrose as the carbon precursor) [51].
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Figure 16. (a) EPR spectra of four different CDs at 25 oC: control corresponds to CDs with poor fluorescence [70]. (b) EPR spectra of CDs before (black) and after (green) NaOH addition to solution [71].
Figure 16. (a) EPR spectra of four different CDs at 25 oC: control corresponds to CDs with poor fluorescence [70]. (b) EPR spectra of CDs before (black) and after (green) NaOH addition to solution [71].
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Figure 17. In vitro cytotoxicity of CDs: (a) RAW 264.7, (b) L02, (c) 293T [76], and (d) DU145 and NCTC1469 cells [51]. (e) Hematoxylin and eosin stained tissue slices (liver, spleen, kidney, heart, and lung) of mice at 1 and 14 days after injection (dose = 23 mg CDs/kg) [77].
Figure 17. In vitro cytotoxicity of CDs: (a) RAW 264.7, (b) L02, (c) 293T [76], and (d) DU145 and NCTC1469 cells [51]. (e) Hematoxylin and eosin stained tissue slices (liver, spleen, kidney, heart, and lung) of mice at 1 and 14 days after injection (dose = 23 mg CDs/kg) [77].
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Figure 18. (a) Principle of CEST mechanism: the saturated solute protons are exchanged with bulk water protons at the rate Ksw and the unsaturated bulk water protons return to the solutes at the rate Kws. Measurement of CEST: (b) solute protons are saturated at their specific resonance frequency at 8.25 ppm and bulk water protons at 4.75 ppm (left spectrum) and the proton exchange leads to the bulk water proton signal reduction after a period (tsat) (right spectrum), (c) normalized proton signal spectrum, called the Z-spectrum or CEST spectrum, and (d) MTR asymmetry (MTRasym) plot of the Z-spectrum after removing the effect of bulk water proton signal [86]. (e) MTRasym plots for the three agents: salicylic acid (1), barbituric acid (2), and D-glucose (3) [91].
Figure 18. (a) Principle of CEST mechanism: the saturated solute protons are exchanged with bulk water protons at the rate Ksw and the unsaturated bulk water protons return to the solutes at the rate Kws. Measurement of CEST: (b) solute protons are saturated at their specific resonance frequency at 8.25 ppm and bulk water protons at 4.75 ppm (left spectrum) and the proton exchange leads to the bulk water proton signal reduction after a period (tsat) (right spectrum), (c) normalized proton signal spectrum, called the Z-spectrum or CEST spectrum, and (d) MTR asymmetry (MTRasym) plot of the Z-spectrum after removing the effect of bulk water proton signal [86]. (e) MTRasym plots for the three agents: salicylic acid (1), barbituric acid (2), and D-glucose (3) [91].
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Figure 19. (a) Synthesis of AC-dots. (b) Ac-dot concentration dependent Z-spectra. (c) Ac-dot concentration dependent MTRasym plots at pH = 7.4. (d) pH-dependent MTRasym plots of AC-dots (10 mg/ml) in PBS. (e) MTRasym plots of Lipo-AC-dots-labeled cells and Lipo-labeled cells as control. (f) T2-weighted MR image (left) and corresponding CEST image (right) at 2 ppm of a mouse brain at 24 h after implantation [94].
Figure 19. (a) Synthesis of AC-dots. (b) Ac-dot concentration dependent Z-spectra. (c) Ac-dot concentration dependent MTRasym plots at pH = 7.4. (d) pH-dependent MTRasym plots of AC-dots (10 mg/ml) in PBS. (e) MTRasym plots of Lipo-AC-dots-labeled cells and Lipo-labeled cells as control. (f) T2-weighted MR image (left) and corresponding CEST image (right) at 2 ppm of a mouse brain at 24 h after implantation [94].
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Figure 20. (a) Synthesis of CDs showing amide (pink) and ammonium (green) protons exchangeable with bulk water protons in diaCEST MRI, (b) CEST effects (%) of the precursor estimated from Z-spectra for the two types of protons at different pH values. (c) Z-spectrum and MTRasym of the precursor at pH = 5.5. Z-spectra and MTRasym of the CDs at pH = (d) 9.9, (e) 7.4, and (f) 5.5 [95].
Figure 20. (a) Synthesis of CDs showing amide (pink) and ammonium (green) protons exchangeable with bulk water protons in diaCEST MRI, (b) CEST effects (%) of the precursor estimated from Z-spectra for the two types of protons at different pH values. (c) Z-spectrum and MTRasym of the precursor at pH = 5.5. Z-spectra and MTRasym of the CDs at pH = (d) 9.9, (e) 7.4, and (f) 5.5 [95].
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Table 1. FT-IR absorption and Raman shift (cm‒1) of CDs.
Table 1. FT-IR absorption and Raman shift (cm‒1) of CDs.
Carbon precursor FT-IR (cm‒1) Raman shift (cm‒1) Ref
Wavenumber Vibration mode D band G band
Sodium citrate and polyacrylamide 3436 and 1410 N-H/O-H stretching and O-H bending, respectively 1363 1582 [53]
1590 N-H bending or asymmetric stretching of carboxylate anions
1648 and 1059 C=O stretching and C-N stretching, respectively
L-ascorbic acid and β-alanine 1720 C=O stretching 1365 1595 [54]
1370 O-H bending
1214 C-O stretching
1050 C-N stretching
Glucose and m-phenylenediamine 3400 N-H/O-H stretching 1357 1565 [55]
1605 C=N or C=O stretching
1137 Benzene C-H stretching
Mandelic acid and ethylenediamine 3352 to 3031 O-H and N-H stretching 1358 1574 [56]
2926 and 1367 C-H stretching and bending, respectively
1570 C=O stretching
1059 C-O stretching
692 N-H deformation
Oatmeal 3432 O-H/N-H stretching 1359 1584 [57]
2921 C-H stretching
1625 and 1382 C=O asymmetric and symmetric stretching, respectively
1241 and 1151 C-N and C-OH stretching, respectively
1091 C-O stretching
Lychee seeds 3443 O-H or N-H stretching 1387 1585 [58]
2981 C-H stretching
1633 C=O stretching
1055 C-O stretching
Citric acid and neutral red 3496 O-H stretching 1340 1596 [59]
1720 C=O stretching
1210 C-O-C stretching
3296 N-H stretching
1551 and 1412 C=C and C-N stretching, respectively
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