Carbon Nanodots: A Novel Carbon Material with Multifacet Applications in Healthcare

Abstract

Carbon nanodots are quasi-spherical carbon materials with dimensions <10 nm (also termed zero-dimensional) and are endowed with several peculiar physiochemical features and attributes such as excellent biocompatibility and dispersibility, low cytotoxicity and cost, high stability, high photoluminescence and fluorescence, and easily tunable through surface modifications by addition of functional groups such as carboxyl (–COOH), hydroxyl (–OH), and amino (–NH₂). This chapter summarizes the synthesis and diverse applications of carbon nanodots in the healthcare sector. The various strategies utilized for the synthesis of carbon nanodots in the recent past include top-down approaches such as arc-discharge, laser ablation, electrochemical approach, chemical exfoliation, and ultrasonic treatment; and bottom-up approaches such as thermal pyrolysis, microwave heating, chemical vapor deposition, and template method. Further, the functionalization of the carbon nanodots for enhanced photochemical properties for varied applications in the clinical context is discussed. The functionalization is achieved through nanodot surface modification using different functional groups from polymers and ions and heteroatom doping using metals and non-metals. The tunable properties of carbon nanodots have brought a revolution in the health sector where these nanomaterials are finding applications in various bioimaging tools functioning on varied optical principles such as fluorescence, photoacoustic (optical luminance in combination with ultrasound detection) effect, and photothermal effect. The carbon nanodots have been used as antioxidants, antimicrobial agents, tumor detection and treatment, drug and gene delivery vehicle, and as biosensors. This is followed by a detailed account of the application of carbon nanodots in medicine and health care where the use of carbon nanodots in the diagnosis and treatment of diseases is emphasized.

1 Introduction

The ongoing advances in medical science have led to the emergence of various new fields, one of which is photomedicine, which involves the use of light and offers the advantages of space–time controllability, and is minimally invasive. The application of such techniques requires the use of photosensitizing agents. These agents should be highly compatible with the human body, stable to light, and have excellent quantum yield.

However, such photosensitizers have several limitations such as less solubility in aqueous solutions (TiO₂) and toxicity issues (CdS). Thus carbon dots have emerged as a potential substitute for conventional photosensitizers. They offer several advantages such as robustness, lesser preparation cost, high water solubility, high photoluminescence (PL efficiency), photostable, and compatibility with human tissues. They have various superficial functions such as –NH_2, –COOH, and –OH which can bind with the biological moieties increasing their accessibility (Fig. 1) [1].

Carbon dots are a subtype of nanoparticles with a size <10 nm, quasi-spherical morphology and are zero-dimensional (all dimensions in the nano range). They are highly fluorescent and their core comprises either sp² carbon constituting turbostratic or graphitic carbon or sp³ hybridized graphene oxide sheets, graphene, and diamond-like carbon which is stabilized by –COOH, –OH, and –NH₂ groups on the surface [2]. These were first discovered in 2004 as a by-product recovered while preparing single-walled carbon and isolated from the arc discharge soot. Their surface passivation was carried out in 2006 to improve their photoluminescence and surface properties. Crystalline Cds were isolated in 2010 and amorphous CDs were synthesized in 2013 [3]. By changing the degree of carbonization, graphitization, and polymerization, several types of carbon dots including carbon quantum dots (CQDs), carbonized polymer dots (CPDs), graphene quantum dots (GQDs), and carbon nanodots (CNDs) have been synthesized [1].

Fig. 1

Unique features and applications of carbon dots

2 Properties of Carbon Dots

2.1 Photoluminescence

The structural diversity of CDs enhances their photoluminescent properties which include phosphorescence, piezo-chromic fluorescence, up-conversion photoluminescence, and solid-state fluorescence which holds a keen interest for researchers [3]. The majority of the CDs have strong emissions in the blue wavelength region with a rapid decay in the red wavelength region. The CDs’ photoluminescence can be divided into fluorescence and phosphorescence. The CDs’ fluorescent properties are determined mainly by quantum yield and wavelength at which emission occurs. In addition, they are also influenced by the size, defects, functional groups, and state of the phases [2]. Three photoluminescence mechanisms that explain the fluorescence in CDs are (1) core-state emission (2) surface-state emission, and (3) molecular fluorescence [4]. According to several studies, the number of bonded oxygen atoms plays a key role in the bandgap regulation between the highest unoccupied molecular orbital (HOMO) and lowest unoccupied energy orbital (LUMO) of CDs, and the bandgap was found to decrease with an increase in the oxygen atoms at the surface. Other studies reported that surface oxidation increases with an increase in reaction time which resulted in a longer emission wavelength. The bandgap narrowed with an increase in C=N content and particle size [2].

2.2 Ultra-violet Visible Absorbance

The majority of the carbon dots share a similar UV–visible spectrum. The absorption band decreases from UV to the visible region. The absorbance occurs due to n-π* transitions from O or heteroatom and π-π* transitions of the conjugated electron. The charged surface functional groups affect this absorbance. CDs are highly resistant to photobleaching. Temperature and reaction time are the factors that affect photobleaching. The presence of larger p-conjugated domains, pyridine-similar structures, and higher carbonization degrees contribute to the enhanced photobleaching property of Carbon dots [2].

2.3 Dispersibility

Dispersibility is one of the fundamental properties of CDs which has wide applications in industries and depends upon the surface morphology and functional groups. The majority of the CDs are hydrophilic with high solubility due to O-containing groups either in the precursors or their generation during preparation. Hydrophobic CDs can be obtained through carbonization. In addition, hydrophilic moieties can be converted to hydrophobic CDs by modifying the functional groups of the surface such as through covalent attachment. The microwave pyrolysis of Pluronic F-68 led to the formation of hydrophobic CDs [2].

2.4 Toxicity and Biocompatibility

Due to their small size and low concentrations (<10 μg/ml), the CDs have very low toxicity levels and are eliminated by the excretory system directly. However, the toxicity was found to increase with an increase in concentration, which can have adverse effects on organ development [5]. Graphite hydrothermal treatment yielded CDs that were non-toxic in absence of light but exhibited toxicity in the presence of light due to reactive oxygen species (ROS) generation [2].

3 Strategies for Design, Synthesis, and Functionalization of CNDs

Two strategies are employed for the synthesis of CDs—Top-down and bottom-up approaches categorized based on the carbon material used as the precursor [4]. The bottom-up approach uses organic monomers or polymers such as carbohydrates, and organic acids as the starting material whereas the top-down approach uses graphite, carbon nanotubes, etc. In addition, hetero-atom doping and surface passivation are always conducted during their synthesis to improve the optical properties of the CDs such as quantum yield, photostability, etc. [5] (Fig. 2).

Fig. 2

Various routes of carbon dots synthesis

3.1 Top-Down Approach

3.1.1 Arc-Discharge Method

Arc–discharge refers to the production of plasma from the electrical breakdown of gas with the help of an electric current at a temperature of 4000 K [6]. It was the first process used for CDs preparation [2]. In 2004, the arc-discharged SWCNTs soot was purified by the preparative electrophoresis method by Xu et al. Along with the long nanotubes and short tubular materials, a new type of nanodot was obtained that emitted bright light upon excitation with 365 nm light. Elemental analysis showed that these types of nanodots constituted only C, H, N, and O, and lacked heavy metal constituents [4].

3.1.2 Laser Ablation Method

This synthesis approach was put forward by Sun et al. in 2006 [4]. Preparation of carbon dots via laser ablation involves the following steps-

• a. Absorption of the high energy of the laser pulse by the target.

• b. Knocking of the electrons from the atoms by photoelectric and thermionic emissions.

• c. The electric field leads to the development of repulsive forces between the positively charged ions and the target material which results in the breakdown of CDs [3].

Laser ablation was applied to a carbon target composed of graphite powder and cement mixture at a temperature of 900 °C. However, the researchers observed that these carbon dots generated through laser ablation lacked photoluminescence despite acidic treatment. The photoluminescence was achieved by their surface passivation with polyethylene glycol (PEG). The surface properties played an important role in the photoluminescence of these CDs and the passivation of the surfaces made them soluble in water, which contributed to their applications in bioimaging. Apart from PEG, propionylethylenimine-co-ethylenimine (PPEI-EI) can also be used for surface passivation. Carbon dots doped with semiconductor salts such as zinc sulfide have shown an increased quantum yield. But the production of carbon dots from organic materials results in lesser toxicity, which gives them an advantage over other inorganic nanomaterials (Fig. 3).

Fig. 3

A representation of the laser ablation method for the preparation of CQDs

3.1.3 Electrochemical Approach

This technique was initially used by Ding’s group, who prepared blue CDs from multi-walled carbon nanotubes (MWCNTs). The setup consisted of an electrochemical cell containing a counter electrode of Pt wire and a reference electrode composed of Ag/AgClO₄. The working electrode is comprised of MWCNT synthesized by the chemical vapor deposition technique. The electrolytic solution is comprised of acetonitrile solution. A potential of −2.0 to 2.0 V was applied at a 0.5 V/s rate. The change of the electrolytic solution from colorless to dark brown confirmed the production of CDs [4]. CDs were also synthesized by Lee et al. by utilizing graphite as an anode as well as a cathode and NaOH/Ethanol as the electrolytic solution. This approach of CD synthesis is much simpler and results in higher product recovery, however, the CDs obtained require further purification and separation processes.

3.1.4 Chemical Exfoliation Method

The chemical exfoliation method is widely used for the production of superior quality carbon dots masses. Also, this method does not require complex types of equipment. In this method, the precursors are treated with strong acids such as nitric acid, sulphuric acid, etc. [7]. This approach was first used by Liu and co-workers who prepared CDs from candle soot. The candle soot was treated with 5 M HNO₃, and further centrifuged and neutralized. Electrophoresis and AFM results showed the production of CDs of size 1 nm [4].

Also, N-doped graphene quantum dots (GQDs) from green tea leaf residues subjected to concentrated H₂SO₄ were synthesized by Gunjal et al. which can be used for sensing gefitinib, a medicine used in breast, and lung cancers, thus they can be used as biosensors. Mild oxidants such as hydrogen peroxide (H₂O₂) can also be used for the cleavage of precursor molecules and reduce the further purification steps for the obtained CDs, and are much more environmentally friendly [7].

3.1.5 Ultrasonic Treatment

The ultrasonic approach was first used by Zhou et al. for graphene quantum dots (GQDs) synthesis from graphene [7]. This method involves the use of high and low-pressure ultrasound waves for CD synthesis. These pressure waves generate small vacuum bubbles with high shear forces, which are homogeneously distributed in the solution. The cavitation in the bubbles breaks down the large precursors like graphite and activated carbon into nanosized carbon dots. The CDs obtained have high photoluminescence, photostability, and dispersibility. They exhibit lesser cytotoxicity and crystallinity. Also, this approach does not require complex equipment, hence is much cheaper and easier [8]. Using this approach, Park et al. prepared carbon quantum dots (CQDs) of 2–4 nm diameter from food wastes and ethanol. These CQDs were water-soluble. Adjustment of the reaction time, carbon source to solvent ratio, and power results in the preparation of CDs with different properties [7].

3.2 Bottom-Up Approach

3.2.1 Thermal Pyrolysis Method

Emmanuel P. Giannelis’group first used the thermal pyrolysis method for CD synthesis. In the presence of highly concentrated acid or alkali and at high temperatures, the macroscopic precursors undergo cleavage to give CDs. This method provides the benefits of lower cost, and lesser reaction time, and does not require solvent [7].

It is a four-step technique that includes heating, dehydration, degradation, and carbonization. To date, CDs have been prepared from citric acid salt, EDTA salts, polymers such as PS, and even hair, flour, etc. The citrate in salts serves as the C source whereas the NH₄⁺ act as a surface modifier. Both hydrophobic and hydrophilic CDs can be obtained by proper choice of surface modifiers and temperature regulation. Treatment of citric acid salts at 300 °C with octadecyl ammonium as surface modifier yields hydrophobic CDs with 7.5 nm diameter whereas treatment with 2-(2-aminoethoxy)-ethanol at 65 °C produces hydrophilic moieties.

Further studies have shown that carbonization at a low temperature of 180 °C for 30 min yields CDs precursors with high photoluminescence (termed as CNP 180) whereas a temperature of 230 °C (CNP230) produces CDs with a diameter of 19 nm. Further treatment at 300 °C produces CDs with 8 nm diameter (size reduction due to CO₂ release) and causes sample darkening. Treatment at 400 °C resulted in the accumulation of CDs causing a size increase to about a few hundred nm. But the photoluminescence decreases continuously with the increase in temperature (Fig. 4).

Fig. 4

Representation of thermal pyrolysis method for carbon dots preparation

3.2.2 Hydrothermal Method

This approach for CD production first came to light in 2011 [9]. It is a widely used technique as it is highly cost-effective since even simple materials such as fruit, chocolate, and waste too can be used as starting materials. Thus the approach is easy and environmentally friendly [2]. In this technique, the precursor solution and nitrogen dopants such as EDA are dissolved in ultrapure water and put in an autoclave line with Teflon, followed by heating in an oven at 110 °C for 2 h. After cooling down, methanol is added and then centrifuged for further purification. The pellet is re-dissolved in pure water and lyophilized [10].

3.2.3 Microwave Heating Method

This technique of CD preparation was developed by Yang’s group [4]. This approach requires lesser time and is cheaper as compared to other synthesis techniques and thus is widely preferred. It involves three main processes-polymerization, drying, and carbonization [3]. In this method, polyethylene glycol (PEG) and precursors such as glucose, dissolved in ultrapure water, are heated for 2–10 min at 500 W in a microwave oven. The appearance of dark brown color confirms the formation of CDs. The size of CDs is correlated with the heating time, heating for about 10 min yields CDs with a 3.65 nm diameter whereas that for about 5 min produces 2.57 nm sized particles [4]. Oxidants such as hydrogen peroxide can be used to increase the reaction rate further [7].

3.2.4 Chemical Vapor Deposition

This technique for CD synthesis has been discovered in recent years and was first used by Fan et al. for the preparation of CQDs using methane as a carbon source. The characteristics of the final product are dependent upon the choice of carbon source, time of the reaction, substrate temperature, and the rate of hydrogen gas flow. The substrate is heated to a temperature of 1000 °C in the presence of hydrogen and argon. Then the hydrogen supply is stopped and only argon is continuously fed to remove the hydrogen residues, followed by the pumping of methane gas at a rate of 2 ml per min for a short duration of 3 s. Altering the argon: methane: hydrogen gas ratio results in a change in the number of products obtained. This method offers a high product yield but the high cost and complicated procedure limit its usage [7].

3.2.5 Template Method

This technique was first used by Zhi-Gang Gu and involves the use of metal–organic frameworks (MOFs) as templates for CDs preparation. Since these templates have well-defined pore sizes, the CDs produced are uniform in dimensions [4]. This technique involves two steps-calcination and etching [8]. The MOF powder, synthesized using the solvothermal method, is immersed in carbon sources such as glucose and EtOH/H₂O. The calcination is conducted at 200 °C which results in the decomposition of glucose while the MOF template remains intact. A color change from blue to green indicates CD formation.

After the calcination step, etching is carried out to separate the CDs from the templates. For this purpose, the templates are dissolved in an aqueous KOH solution. The CDs obtained under such conditions are about 1.5 nm in diameter. CDs with different sizes can be prepared by using templates with different pore sizes, such as HKUST-1 (1.35 nm pore size for 1.5 nm CDs), ZIF-8 (1.9 nm pore size for 2 nm CDs), and MIL-101 (3.0 nm pore size for 3.2 nm CDs). Apart from these, mixed soft templates such as copolymer Pluronic P123 and hard templates such as ordered mesoporous silica (OMS) SBA-15) can also be used for CD synthesis [4]. Table 1 provides a quick comparison of various synthetic approaches highlighting their merits and demerits.

Table 1 Different approaches of carbon nanodot synthesis

4 Functionalization of Carbon Dots

Functionalization of carbon dots (CDs) is an effective method to enhance the photochemical and photophysical features of CDs such as photoluminescence and quantum yield. The two main approaches used for the functionalization of the CDs are surface modification and heteroatom doping. Heteroatom doping is usually a one-step bottom-up process where the doped atom can be either a non-metal such as nitrogen (highly efficient), sulphur, a metal such as copper, or different atom combinations, such as in the case of co-doping. Heteroatom doping modifies the internal structure of the CDs and affects the distribution of electrons, thus causing an impact on the energy difference between HOMO and LUMO, which in turn influences fluorescence efficiency. Non-metallic doping helps to increase the quantum yield whereas metallic doping modifies the banding pattern. Surface modification is another strategy for functionalizing the CDs with ions, DNA, or proteins and usually consists of two or more steps and thus is more complex than heteroatom doping. These strategies confer CDs with unique properties, which widens their scope of application [12]. Table 2 provides a brief description of the various functionalization types, the resulting properties changes caused by their doping, and their possible application areas.

Table 2 Various functionalization strategies of CNDs and their applications

5 Carbon Nanodots as Imaging Tools

Nanotechnology has progressed to the point where nanoparticle-based contrast agents, with diameters ranging from 1 nm to several hundred nanometres, have made a significant contribution. Many applications based on CNDs have been developed in the last decade. They have been employed as bioimaging agents to identify cells and tissues utilizing imaging techniques and Imaging-Guided Drug Delivery with CDs, photoacoustic imaging, fluorescence imaging, and multimodal imaging among others [14] (Fig. 5).

Fig. 5

Different applications of Carbon nanodots in bioimaging

Carbon dots made by various methods have some common characteristics, such as a size of less than 10 nm, brilliant fluorescence emission, and a rich hydrophilic surface group that gives carbon dots high water solubility and biocompatibility [4]. Carbon dots have considerable advantages over fluorescent chemical dyes and genetically engineered fluorescent proteins, such as high photo-stability, photoluminescence quantum yield, and metabolic degradation resistance. These properties are responsible for the tremendous potential of CDs-based bio-applications [4]. The need for early disease diagnosis has prompted the development of bioimaging tools. Bioimaging is a technology that uses signaling probes and detectors to directly see biological activities.

5.1 Fluorescence Imaging

Carbon nanodots, a new type of nanomaterials, are getting a lot of attention because of their inherent benefits, which include ease of functionalization, photoluminescence, photobleaching resistance, biocompatibility, strong water solubility, low toxicity, immunogenicity [15]. CDs are commonly used in biological and clinical applications due to their exceptional biocompatibility and luminous fluorescence. CDs’ fluorescence ability in bioimaging has been demonstrated in numerous experiments.

5.2 Photoacoustic Imaging

Photoacoustic imaging (optoacoustic imaging) is a sophisticated biomedical imaging technique that uses optical luminance with ultrasound detection [16]. The application of molecular bioimaging in living tissues to monitor various physiological processes in interior environments has expanded with the advent of photoacoustic imaging [17]. In the last two decades, it has gained popularity and has been explored for biomedical imaging applications. There are various imaging techniques in the biomedical engineering field which are used to carry out biomedical research and diagnosis but photoacoustic imaging is one of its kind which is very useful and very satisfying. In comparison to other optical imaging techniques, imaging living organisms utilizing a photoacoustic imaging approach will provide a greater resolution and allow in-depth viewing of animal tissues [18]. Photoacoustic is a blended modality that combines optical spectroscopic-based specificity and high contrast specificity of optical imaging and high spatial resolution of ultrasonic imaging [19]. Photoacoustic imaging can provide anatomical, physiological, molecular, and dynamic insights by utilizing lipid, hemoglobin, and melanin as endogenous contrast agents or other exogenous contrast agents [18].

5.3 Photothermal Therapy

Phototherapy is the act of utilizing light energy into heat energy for the destruction of tumors or some pathophysiological tissues [20]. It is a safe therapeutic means as it uses a non-ionizing electromagnetic light source. Its non-invasiveness makes it safer and more reliable as it does not involve surgery and does not require insertion into the body. The majority of materials under investigation for photothermal treatment are nanoscale materials. The increased permeability and retention effect found with particles in a given size range is one of the main explanations for this (typically 20–300 nm) [21].

5.4 Multimodal Imaging

Multimodality imaging is typically thought to entail the use of two or more imaging techniques, for example, in the nuclear medicine field, multimodalities are combined for assistive therapy and diagnosis. Some of the types of multimodal imaging are PECT-CT, and PET-SPECT [22]. Because of their small size, nanoparticles have been actively investigated as multimodal platforms capable of merging many imaging methods. Using only a single light source, the multimodal beneficial effects of carbon nanodot-based techniques have recently been studied [23]. The merging of established fields of in vivo imaging technology with molecular and cell biology has resulted in the rapid expansion of in vivo multimodality imaging. Photoacoustic imaging (PAI), also known as optoacoustic imaging, is a hybrid imaging technique that combines optical illumination with ultrasonic detection.

5.5 Two-Photon Induced Imaging

Traditional single-photon FI (for example, confocal microscopy) has flaws such as poor light penetration depth of organisms (100 m), UV-induced photodamage, and autofluorescence. The potential of two-photon excitation imaging to produce superior optical sectioning at longer depths in thick specimens than other imaging technologies is its most potent benefit [4].

5.6 Imaging-Guided Drug Delivery Using Carbon Dots

Most chemotherapy drugs are administered in vivo systems by intravenous infusion of drug-loaded carriers to date. Imaging-guided drug delivery systems combine imaging techniques and drug delivery systems to improve in vivo drug targeting and therapeutic capabilities. The standard technique, on the other hand, is to monitor the release of drugs in physiological systems [4]. Tao Feng et al. experimented with imaging-guided drug delivery using CDs, by preparing a microenvironment-responsive extracellular drug nanocarrier that was based on prodrug-loaded convertible CD-ROMs (CD-Pt(IV)@PEG-(PAH/DMMA)) [6]. Importantly, the positively charged nanocarrier has a strong affinity for the negatively charged membrane of the cancer cell, resulting in better absorption of the primary cisplatin (IV) in the cytoplasm [23]. The in vitro results showed that this prospective switchable nanocarrier had superior therapeutic efficacy in the tumor extracellular milieu than the non-transferable nanocarrier under normal physiological settings. Convertible CDs also showed great cancer suppressive viability and low symptoms in vivo, revealing their actual potential as an efficient drug nanocarrier with worked-on beneficial effects [23]. Sung et al. revealed trackable GQDs paired with small nanosponge vesicles that showed promising drug administration and photolytic and imaging capabilities against deep-seated cancers [1].

6 Carbon Dots in Health Care and Medicine

CDs find enormous applications in drug delivery, bioimaging, phototherapy, as microfluidics markers, LEDs, sensing, logic gates, etc. Owing to their excellent phosphorescent and fluorescent properties, the CDs are utilized for in vitro and in vivo bioimaging and diagnostic studies. The hydrophilic CDs are used as a probe to image and identify tumours and hydrophobic ones are used in organic reactions as catalysts [5]. They also find use in drug delivery as they possess the capability to carry active agents. CDs are used in data security, chirality, optoelectronic devices, etc. Their remarkable properties confer them the advantage to be used for theranostic applications and thus these nanoparticles can help in the diagnosis as well as treatment of diseases. Owing to their lesser cost, photostability, non-toxicity, and ease of preparation, CDs are widely used in photocatalytic and electrocatalytic devices and the conversion of energy (energy storage devices) [3].

6.1 Carbon Dots as Antioxidants and Prooxidants

ROS plays an important part in biological structure damage, biomolecule distortions, chemical product degradation, food spoiling, and other processes. They are chemically reactive and affect most biological activities. One of the most reactive species is hydroxyl radicals, which cause cancer, and significant oxidative stress in pathogenic operations, such as inflammatory illnesses and tumors by interfering with normal body functions and causing the destruction of cellular materials such as proteins, DNA, and lipids. As a result, free-radical antioxidants and scavengers (FRSs) are necessary for utilization in medicine, packaging, food preservation, cosmetics, and corrosion protection. Compounds, in general, with long conjugated C=C chains, are highly resistant to free radicals [24]. CDs can donate as well as accept electrons, so can function as both antioxidants and prooxidants.

In solutions containing chemically generated free radicals, both novel spherical CDs and commercial quantum dots (made of CdSe with a ZnS shell) display antioxidant characteristics. Their antioxidant activity is mostly focused on singlet oxygen scavenging. These nanoparticles, on the other hand, can also generate reactive oxygen species (ROS) and singlet oxygen upon exposure to blue light. Dhr123 (dihydrorhodamine 123) and singlet oxygen sensor green (SOSG) were used to detect free radicals. Dhr123 is an uncharged, nonfluorescent indicator that oxidizes to the fluorescent product rhodamine when exposed to a range of reactive oxygen species (ROS) such as peroxynitrite anion, hydrogen peroxide, superoxide anion, and hypochlorous acid. In contrast, SOSG is very selective for singlet oxygen. A radical cascade reaction frequently causes oxidation. A sequence of redox processes can produce singlet oxygen from superoxide and hydrogen peroxide. Peroxyl radicals can combine with one other during lipid peroxidation, creating singlet oxygen that can generate additional peroxides. In solutions, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) decomposes into two carbon-centered radicals followed by peroxyl radicals (ROO·) over time or under heating. The CDs act as antioxidants after being added to the solutions with either 1M Dhr123 or 2M SOSG and then commence radical production with 1 mM AAPH. Both forms of CDs scavenge singlet oxygen, preventing SOSG from being oxidized.

When exposed to light, nanoparticles generate radicals by transferring electrons and/or energy to adjacent molecules, acting as prooxidants. In photochemistry, energy from the dye molecule is transmitted to oxygen through a dye-oxygen collision complex, resulting in highly reactive singlet oxygen. Photogeneration of exciton pairs (electron–hole (e–h pair)), which undergo redox reactions with biomolecules and oxygen adsorbed on quantum dot surface) is the most likely mechanism of the photosensitized action of quantum dots. Taking oxygen out of the solution protects against photosensitization [25].

CDs’ antioxidant capacity in polymer composites has also been investigated. Surface modification can be accomplished by adding chemical functionalities to the surface, such as polar functional groups, or by covering the surface with thin polar films. Photografting, plasma polymerization, ceric ion-induced grafting, and layer-by-layer ionic grafting are all effective surface grafting procedures. Photografting is an appealing way to add a variety of functional groups to the surface of the polymer, among other methods. It entails the utilization of energy to generate free radicals on the polymer surface, followed by a polymerization step that produces polymers with an active surface. The benefits include facile and controlled chain introduction, as well as covalent chain bonding, which leads to chemical stability and prevents delamination of the chains introduced, as opposed to physically linked layers. The photochemical grafting approach was used to immobilize N, P, and S co-doped CDs (NPSC-dots) onto nonpolar plastic films (PP). This approach relied on the synthesis of the non-radical form of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical in alcohol and a hydrogen-donating antioxidant. NPSC-dots’ antioxidant potential effectively removed superoxide anion radicals [24].

6.2 Tumor Detection and Treatment

When compared to conventional cancer therapies, photodynamic therapy has increased researchers’ interest because of its non-invasive property and rational approach to eliminating tumor cells with fewer side effects. This therapy exhibits minuscule phototoxic effects on the skin and little tissue loss. Furthermore, it shows relatively little medication resistance. The build-up of photosensitizing chemicals in malignant cells, followed by irradiation, produces reactive oxygen species (ROS) derived from intracellular oxygen in this therapy. This causes necrosis and cell death in nearby cancer cells.

Due to their great two-photon absorption, inertness and robustness, efficient resistance to photobleaching, and high aqueous solubility, CDs are used in photodynamic therapy as imaging-oriented therapeutic agents. When mixed with carbon-based nanomaterials like CDs, photosensitizers like methylene blue (MB) could have minimal cytotoxicity and great biocompatibility, allowing for innovative photodynamic therapy. They also show strong resistance to DNA contact, aggregation, and reduction interventions.

Cu-CDs have a quantum yield (QY) of about 36% and demonstrate significant photoinduced cytotoxicity. Furthermore, they effectively stop cervical tumor cells and human neuroblastoma from growing. Wu and his colleagues created nanohybrids with CDs-decorated platinum porphyrin (CDs@PtPor) for use in photodynamic treatment. Xu et al. synthesized Se and N co-doped CDs (Se/N-CDs) for an enhanced and better photodynamic therapy, considering the in vivo limitations of photodynamic therapy which arise due to the restricted diffusion distance of damaging reactive oxygen species (ROS) in the cell and short lifespan.

Photothermal therapy is also another efficient method for tumor treatment. The application of photothermal agents for heat generation through energy absorption is used in this therapy to kill cells. A NIR absorptive substance is utilized for inducing hyperthermia to achieve cell death or irreversible cell damage. The NIR light is less phototoxic and has a high cell penetration rate. Photothermal therapeutic drugs in combination with bioimaging, aid in the precise recognition of the location and size of tumors and monitor the treatment response in real time. CDs are important photothermal agents because they (i) are made up of a lot of electrons and act similar to free electrons in metallic nanomaterials, (ii) can produce large temperature variations when exposed to light, and (iii) they have a high photothermal conversion efficiency [8]. Previous studies have reported the use of folate-functionalized CDs in detecting cancer cells that overexpressed surface folic receptors. CDs also have exhibited complex formation with anticancer drugs through intramolecular or chemical interactions to accomplish efficient and high-loading drug delivery for better and improved therapy [5]. Zhao et al. developed cisplatin(IV) prodrug-loaded charge convertible CDs (CDs-Pt(IV)@PEG-(PAH/DMMA)) for ovarian cancer thermonastics. The anionic polymer containing dimethyl maleic acid moieties (PEG-(PAH/DMMA)) through electrostatic interactions, developed a complex with CDs-Pt(IV) [26].

6.3 In Drug and Gene Delivery

Cationic CDs tend to interact electrostatically with negatively charged nucleic acids and positively charged functionalized CDs, hence can act as gene carriers and delivery vehicles [2]. Multifunctional FRET-based CDs for two-photon imaging and real-time drug administration determination were reported by Tang et al. They demonstrated efficient delivery and high drug release to tumor cells. The polymer of PEG-(PAH/DMMA) was modified from an anionic form to a cationic form in the slightly acidic tumor cell microenvironment, leading to the release of positive CDs-Pt (IV). The positively charged nanocarriers exhibited high affinity to the negative charge on the tumor cell surface resulting in a strong interaction between the tumor cell and the carrier [3].

Fluorescent CDs with a diameter <10 nm, high biocompatibility and quantum yield, little to no cytotoxicity, and inexpensive cost are regarded as emerging stars in nanomedicine. Carboxyl-rich green-emitting CDs are a nontoxic bioimaging agent for drug biodistribution studies and can be utilized as a stable cancer drug delivery system that selectively kills cancer cells through localized therapy owing to their finite size and rich surface chemistry that leads to the formation of non-covalent bonds with drug payloads [27]. Anti-cancer nanocarriers derived from solvothermally produced red emissive CDs were used to carry the anticancer medication doxorubicin. Doxorubicin-loaded CDs were used to reach the nuclei of cancer stem cells in this investigation. The viability of HeLa cells reduced to about 21% after exposure to doxorubicin-loaded CDs, which was significant in comparison to the viability of HeLa cells (about 50%) upon exposure to doxorubicin in absence of CDs.

The biocompatibility and non-toxicity of CDs make them effective as nanocarriers or gene carriers in gene therapy or gene delivery. Yang et al. established a gene delivery technique for micro-RNAs (miRNAs)-combo (MC) delivery through electrostatic interaction using a non-viral vector (branched polyethyleneimine-functionalized nitrogen-doped CDs (BP-NCDs)). Hydrothermally produced polymeric CDs were used in a work by He and colleagues to improve the efficiency of transfection in gene transfer methods [15]. The luciferase assay in HeLa cells suggested that the as-prepared polymeric CDs might function as multifunctional gene vectors with high biocompatibility and transfection efficiency with minimal cytotoxicity. The as-prepared polymeric CDs showed efficient condensation of DNA (positively charged) and prevented DNA degradation. In comparison to the polyethyleneimine polymeric subunits, CDs were found to have higher cell viability, enhanced serum tolerance, and about 2000 times higher transfection effectiveness, implying a significant role in gene transfer [8].

6.4 Biosensors

Fluorescence CDs can identify a wide range of analytes, including macromolecules, anions, cations, medicines, and small molecules, so can be employed as sensors due to their excellent sensitivity and selectivity, and ease of use as biocompatible and inexpensive devices. Designing CDs as a sensor material can be done in three ways: (1) Fluorescence signal intensity varies as the prepared CDs and analyte interact with each other (2) CDs can be modified by conjugating special functional groups to produce sensing ability and (3) CD substrate quenchers and fluorophore combinations could be used as sensory materials [3].

CDs should (i) identify analytes via particular linkages via targeting moieties (e.g., aptamers, antibodies, and enzymes) and (ii) modify their optical characteristics through contact with analytes for biosensing applications. Changes in the photoluminescence intensity of CDs have been readily applied to determine the presence of analytes such as metal ions, cholesterol, glucose, etc. in ratiometric, turn-on, and colorimetric sensors.

The label-free form is the most basic CD-based optical biosensor that requires no additives or targeting ligands and is used to detect metal ions. The interaction between CDs and metal ions leads to the non-radiative electron transfer to empty d orbitals of metal ions from excited CDs, metal ions often produce photoluminescence quenching of CDs in “turn-off” or “on-to-off” sensors.

Certain functional groups on the surface of the CD scan combine with metal ions in the biological system, such as Cu²⁺ and Fe³⁺. Amine-containing PEI has been used as a CD precursor for Cu²⁺ capturing as the amine-containing PEI tends to collect Cu²⁺ and generate cupric amine. The inner filter effect on label-free CDs enhances their sensitivity by detecting small molecules. IFE uses spectrum overlap between energy absorber absorption and fluorophore emission and/or excitation. CDs use specific targeting compounds such as antibodies and enzymes to find and detect biologicals in complex physiological solutions, for example, glucose oxidase (GOx/GOD) is an oxidoreductase that can specifically recognize glucose, which is responsible for >80% of biosensing applications. Gluconic acid and H₂O₂ are produced when glucose is oxidized by GOx. This H₂O₂ is degraded ultimately to hydroxyl radical (OH) through reactions with peroxidase-like CDs and horseradish peroxidase (HRP), or by the iron (Fe²⁺)-mediated Fenton reaction where iron (Fe²⁺) reacts with H₂O₂ (Fe²⁺  + H₂O₂) to generate Fe³⁺, hydroxyl (OH⁻) ion, and hydroxyl (HO^•) free radical. The photoluminescence absorption level of CDs relates to the quantity of glucose as OH induces the breakdown (or aggregation) of CDs’ emission centers.

“Off-to-on” or “turn-on” fluorescence sensing is the polar opposite of quenching-based sensing. A quencher after coming in the contact with fluorescent CDs via electrostatic interaction or coordination leads to quenching of the fluorescence of the CDs via electron/energy transfer. The analyte concentration may be determined in this method by restoring the quenched CDs to an emissive state. Fluorescent CDs have good photoactivity and energy transfer efficiency with minuscule size and these properties aid in improving the optical sensitivity and reduction in the signal-to-noise ratio in a “turn-on” sensing system (for better interactions) [1].

6.5 Antimicrobial Agents

Photoexcited CDs can produce ROS, which is known for their efficiency in killing or inhibiting the growth of bacteria. CDs’ antimicrobial properties are most likely linked to ROS generation. CDs adhere to the bacterial surface and cause the photoinduced production of ROS. This causes the disintegration and penetration of the bacterial cell wall and membrane and the induction of oxidative stress which significantly damages DNA and RNA. The damage in the nucleic acids causes alterations or modifications in the gene expressions. Oxidative stress also affects other biomolecules such as proteins through their oxidation. CDs generate hydroxyl free radicals (OH^•) or singlet oxygen by activating oxygen in presence of air or water, after coming in contact with the bacterial cells and ultimately generating ROS. As mentioned earlier, ROS leads to oxidative stress and oxidation of biomolecules in the cell via protein inactivation, lipid peroxidation, mitochondrial malfunction, and the slow disintegration of the cell membrane, which causes apoptosis or necrosis of the cell and leads to cell death [28].

Wu and colleagues manufactured CDs with high labeling selectivity toward Gram-positive bacteria and antibacterial efficacy in vivo [29]. By varied reagent ratios, the nanoparticles were synthesized using solvothermal synthesis using glycerol and the quaternary ammonium salt dimethyl octadecyl [3-(trimethoxysilyl)propyl]-ammonium chloride (Si-QAC). The CDs exhibited high selectivity for Gram-positive Staphylococcus aureus cells and reported significant mortality at doses as low as 5–10 g/mL and no impact on Gram-negative Escherichia coli. SEM imaging revealed evident traces of membrane breakdown and cytoplasmic material leaking, demonstrating the death action in the case of S. aureus cells following CD treatment, however E. coli cells sustained the treatment and were found to be intact.

Chekini et al. used a different method other than ROS production and cell death and created a CD/cellulose nanocrystal-based hydrogel capable of sequestering Fe³⁺ from its surroundings. Due to the sequestering of the ions, bacterial cells were depleted of Fe³⁺, which is essentially required for the growth and reproduction of bacteria. The study reported the hindrance in the growth of both Gram-positive and negative bacteria. The CDs were created by hydrothermally preparing cellulose nanocrystals and then cross-linking them with aldehyde-modified cellulose to prepare the hydrogel. When the material was coordinated with the metal, it exhibited a fluorescence quench that was complexation-derived, which could be used to determine the material's effectiveness through the naked eye [29].

The prevalence and treatment of multi-drug resistant (MDR) bacterial infections is a significant problem for hospitalized patients, particularly in those suffering from chronic infections, which happen to be a threat to the patient's survival and significantly increase sanitation and hospitalization expenses, as well as patient recovery time. Because of CDs’ exceptional biocompatibility and bactericidal properties, their incorporation into innovative biomedical materials represents a viable strategy to suppress MDR bacterial infections, as well as utilization as components in wound dressing materials and pharmaceuticals. These new materials have considerable promise for topological infection therapy, limiting bacteria and biofilm formation while protecting against the pathogens in long term. Li et al. used a muffle furnace for the creation of ammonium citrate-based CDs by thermal degradation at 180 °C [2]. Following that, the surface of the CDs was functionalized using gentamicin through the calcination procedure, again at 180 °C. For efficient drug delivery application, the nanomaterials were combined with carboxymethyl chitosan. The chitosan's free amine functionalities were then complexed with aldehydes on a dextran polymer derivative to produce the final hydrogel through imine production. The CD-containing hydrogel demonstrated high physical flexibility which is a requirement for the treatment of wounds with uneven shapes. Furthermore, the hydrogel demonstrated pH-dependent CD release due to its capacity to break imine bonds at acidic pH. Such pH-responsive property is beneficial in biofilm infection treatments, which are typically associated with an acidic microenvironment [29].

7 Conclusions and Future Aspects

Carbon nanodots emerged as novel nanomaterial with unique optical-physical properties. It shows excellent biocompatibility, tissue penetration, and absorption near the IR region. This opens the scope of its wide range of applications in bio-imaging and medical applications. It has a multivariant functionalization ability and can be conjugated with a variety of drugs. This helps in increasing half-life, enhancing bioavailability, reducing non-specific uptake, and assisting in the targeted delivery of drugs. The fluorescence property makes its usefulness in optoelectronic applications. However, challenges like low luminescence and external quantum efficiency are still unaddressed. The role of bond length, bond angle, bond geometry configuration, and local potential distribution in carbon dot emission needs to be explored. These nanomaterials also have the potential for the development of biosensors, microfluidic-based bioassays, and biomimetic systems. The discovery of chiral CDs paves the path for futuristic applications in the field of sensing, catalysis, optical electronics, and other biological applications. But these chiral carbon dots only hold UV range CD signal and there is the limitation of circular polarization emission. The researchers are working to overcome these challenges to put forward this fluorescent carbon nanomaterial for bio-sensing, drug delivery, nanomedicine, and electronics.