Carbon-based designer and programmable fluorescent quantum dots for targeted biological and biomedical applications

Abstract

Carbon dots (CDs) are emerging nano-biomaterials owing to their exceptional optical and physico-chemical properties. In recent years, they have gained attention due to some of their distinctive applications in biological science, including bio-imaging, drug delivery, biosensing, and cancer therapy. Depending on the material, desired fluorescence, and expected size, these carbon-based fluorescent nanoparticles can be made using a variety of chemical and natural compounds and varying methodologies. Owing to the unique tunable optoelectronic properties, they exhibit properties including size-tunable emission, excitation-dependent emission, and solvent-dependent emission. We present here a brief perspective on their synthesis, factors regulating their fluorescence behavior, unique characteristics, advantages, limitations, and very focused biomedical applications. We conclude with a brief discussion on future innovations to make the carbon dots ready for real-world biomedical applications.

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1. Introduction

Quantum dots (QDs) are semiconductor nanoparticles with sizes ranging between 1 and 10 nm in diameter¹ and are smaller than the exciton Bohr radius, possess distinctive optoelectronic and physio-chemical characteristics, and can be used to study various biological processes. Alexey Ekimov pioneered the first discovery of quantum dots who discovered QDs in a glass matrix² and Louis Brus discovered (cadmium sulfide) quantum dots in colloidal suspension.³ For the first time in history, carbon nanoparticles (CNPs) were prepared by Xu et al. in 2004 via the purification of single-walled carbon nanotubes.⁴ Later in 2006, Sun et al. termed these CNPs “Carbon Quantum Dots”. In the current era of nanotechnology, where significant discoveries are going on, researchers are concentrating on carbon-based quantum dots having enormous applications because of their distinctive optical properties and surface modifications owing to their unique size and structure, leading to various biomedical applications including biosensing, bioimaging, theranostics, targeted therapeutics, and photodynamic treatment. When sensing, CDs can be employed as fluorescent probes to detect a variety of analytes, such as enzymes, biomolecules, and heavy metals. Furthermore, CDs can be used as contrast agents in imaging to observe biological entities such as cells and tissues. In addition, due to their small size and biocompatibility, they can be used to deliver therapeutics to specific cells or tissues. CDs’ high water solubility is an essential feature when considering entities in biological systems, and is correlated with the surface groups on CDs created by hydrothermal and solvothermal processes; particularly, oxygen-related carboxyl and hydroxyl groups provide them with high water solubility without requiring additional surface modifications.⁵ On the other hand, the optical properties of QDs are based on their band gap energy where these nanoparticles exhibit a particular wavelength of light that matches their band gap energy. The excited electrons momentarily transition from the valence band to the conduction band and return to the relaxed state by emitting photons in the form of energy. The fluorescence intensity of CDs can be used to differentiate between normal and cancerous cells. Compared to normal cells, cancerous cells have a larger distribution of CDs due to greater permeability caused by changes in the cell membrane, resulting in strong fluorescence. It is also shown that the fluorescence lifetime also plays a major role in the absorbance and emittance of light energy.⁶ The lifetime describes the interval of time from the moment the carbon dot absorbs a photon from the light source to the moment the exciton begins to radiate again.⁷ The electron–hole pair can be trapped at more closely spaced energy levels in larger dots. Further, it was discovered that electron–hole couples live longer in larger dots; CDs have a longer fluorescence lifetime (FL) than most of the organic dyes (1–5 ns) and cell autofluorescence (2–3 ns), facilitating fluorescence signals from CDs to be separated from the background fluorescence.⁸ For instance, the background autofluorescence of proteins is around ten times shorter than the fluorescence lifespan of QDs, which is 20–30 ns. As of now, CDs have the longest 19.5 ns fluorescence lifespan.⁹ Semiconductor QDs have limited applications due to their toxic nature in in vivo biological systems; thus, carbon dots are much more promising fluorescent nanoparticles for biomedical applications. The CDs show superior photostability, biocompatibility,¹⁰ low level of toxicity,¹¹ stable chemical inertness,¹² and high optical properties. They are cheap to synthesize and their optical properties are easily tuneable as compared to conventional semiconductor QDs. CDs are generally composed of carbon, hydrogen, and oxygen and if doped contain other elements such as nitrogen (N), phosphorus (P), and sulfur (S). CDs are currently divided into three categories: carbon quantum dots (CQDs), carbonized polymer dots (CPDs), and graphene quantum dots (GQDs) based on their varying micro and nanostructures, properties, and preparation methodologies, as shown in Fig. 1.¹³

Fig. 1 Three different categories of carbon dots are based on the formation mechanism, nano/microstructures, and properties: graphene quantum dots (GQDs), carbon quantum dots (CQDs), and carbonized polymer dots (CPDs). GQDs are made up of small graphene fragments, usually containing single or few sheets of graphene. CQDs are usually spherical and possess chemical groups on their surface. CPDs are also spherical and contain abundant functional/polymer chains on their surface and carbonized core.¹³

Their photoluminescence properties are greatly influenced by the quantum confinement effect, surface functional groups, size, and elemental doping. Numerous studies are being conducted to exploit their remarkable properties of fluorescence, for example, carbon dots generated from biomolecules showing excitation-dependent emission because of the existence of surface energy trap states that could be modulated by modifying surface functionalization.¹⁴ Carbon dots are among the brightest fluorescent probes ever explored, owing to their extremely high fluorescent quantum yield (QY). Such high QY can be obtained by doping heteroatoms into CDs.¹⁵ Similarly, Zheng et al. obtained CDs with a high fluorescence QY of 93.3%.⁸ CDs can be synthesized using either top-down or bottom-up approaches. Although carbon dots were considered as a side product in the synthesis of carbon tubes, once their fluorescence properties had been known, it attracted researchers to work towards the synthesis of carbon dots. In early synthesis procedures, high energy impacts were used to generate fluorescent carbon nanoparticles using top-down “brute-force” experimental schemes. Laser ablation, arc discharge, and electrochemical synthesis are a few types of top-down approaches. The shortcomings of top-down CD preparation techniques are later addressed using various bottom-up strategies (Fig. 2).¹³

Fig. 2 Schematic diagram representing the different properties of carbon dots. Carbon dots consist of unique properties such as ease of surface modification, and they are biocompatible and nontoxic. As they are synthesized from natural sources as well as from organic compounds, they are mostly water-soluble, which eventually aids in penetrating the cells. Their fluorescence can be easily tuned, they show good stability in an aqueous environment and they are resistant to photobleaching.

Apart from different synthesis techniques of CDs, different biomolecules such as antibodies, aptamers, and peptides are also conjugated with CDs, giving them the ability to bind with specific antigens present on specific cells or tissues for designing sensitive and accurate targeted bioimaging and detection in in vitro and in vivo administration.^16,17 CDs have so far been applied to identify cancer markers; for example, CDs conjugated with ZnO were synthesized by treating the pericarp of citrus lemon hydrothermally and then carrying out reduction reaction to obtain blue fluorescent CD-ZnO nanocomposites, which were then used for detection of tumor markers for lung cancer (CYFRA 21-1).¹⁸ For labeling proteins, for instance, taking citric acid and urea, Rao et al. synthesized orange fluorescent CDs and conjugated them with Bovine Serum Albumin (BSA) and demonstrated that N-hydroxy succinimide (NHS) may activate carboxyl functionalized CDs to effectively conjugate with the lysine residues found on the proteins. Thus, any protein having lysine on its surface can be labeled using this method.¹⁹ In a similar way to nucleus labeling, for example, Kumawat et al. synthesized self-assembled graphene QDs (GQDs) for nucleus labeling. They have shown that the synthesized GQDs self localize inside the nucleus of numerous cell lines, including L929, MG-63, HeLa, HT-1080, and MIA PaCa-2 cells, and continue to exhibit the propensity to locate themselves to the cell nucleus, acting as a good nucleus labeling agent as well as a drug delivery carrier inside the nucleus and many more.²⁰

Research on excitable CDs has mainly focused on the region of the visible spectrum from ultraviolet to green, in which absorption, excitation, and emission bands are found. For CDs used in photoluminescence-based applications, when high excitation energy is given, it can induce photobleaching and photodamage and the penetration depth is constrained, resulting in poor performance,^21–23 To get around these constraints, researchers have begun concentrating on red-emitting carbon dots that could be stimulated in the visible region of the electromagnetic spectrum. These red-emissive CDs are the ideal candidate for enhanced targeted and efficient diagnostic and therapeutic applications since they exhibit reduced photobleaching, photostability for a longer duration, minimal photodamage to healthy tissues, good tissue penetration ability, and minimum overlap with biological autofluorescence.^24,25 Through this review, we critically review the progress and development of carbon dots. We discuss the determining parameters which need to be noted while synthesizing CDs and their application in biomedical fields, especially bioimaging, biosensing, and drug delivery.

1.1. Synthesis of carbon dots

Carbon dots can be formed by chemical or physical procedures (Fig. 3). The method used during the formation of the carbon dots has a significant impact on their size as well as subsequent photoluminescence characteristics along with their biocompatibility.²⁶ The properties of CDs and related applications are significantly influenced by the choice of synthesis technique and starting material. Three issues with CD preparation should be noted: (i) during carbonization, there are chances of aggregation, so to avoid carbonaceous aggregation confined pyrolysis, electrochemical synthesis, or solution chemistry approaches can be used; (ii) to retain uniform features and for mechanistic analysis, the uniform size must be maintained as well, which can be achieved by post-treatment procedures including gel electrophoresis, dialysis, and centrifugation; and (iii) surface characteristics, which are essential for solubility and specific functions, may be manipulated throughout the synthesis process.²⁷ Wang and Hu reported achieving uniform CDs after their synthesis by using different treatments such as dialysis, sonication, filtration, column chromatography, centrifugation, and gel-electrophoresis. Various methods are so far discovered for synthesizing CDs; these methods are primarily categorized into top-down and bottom-up approaches.

Fig. 3 Schematic diagram of different methods of synthesis of carbon dots by both top-down and bottom-up approaches.

In the top-down approach, methods like electrochemical oxidation,^28,29 laser ablation,^30,31 and arc discharge methods^32,33 are used to break bulk materials into nano-sized particles. Top-down methods for synthesizing CDs often involve difficult operating procedures, high-cost instruments, and abrasive experimental circumstances, which severely restrict their operable applications. On the other hand, in bottom-up approaches, CDs are formed by combining atom-by-atom or molecule-by-molecule, often entailing self-assembly, by using methods such as microwave irradiation,^20,34 hydrothermal carbonization,^35–37 and solvothermal method.^38,39 The top-down method uses sophisticated instrumentation and harmful chemical processing. The “bottom-up” method relies on more environmentally friendly techniques such as microwave irradiation and hydrothermal/solvothermal treatment, which is why they are preferred over top-down approaches of synthesis. The hydrothermal/solvothermal techniques are more widely used since the CDs synthesized by this method have a high quantum yield as well as being environmentally favorable. Here, a high-temperature hydrothermal reactor is used to enclose the solution as it undergoes hydrothermal or solvothermal carbonization to synthesis.⁴⁰ These techniques are also quicker, more easily scaled up, and less expensive. The only impediment of existing methods is that they have low stability and require complex machinery which is challenging to operate, and sophisticated synthetic conditions are also applied. The microwave method is now replacing the conventional method as it is following the new trend for greener synthesis, low energy consumption with a fast-heating process, and does not require highly complex machinery. Overall, it is a straightforward, inexpensive, and quicker approach to producing carbon dots. It also reduces the reaction time and enhances the quality of nanoparticles.⁴¹ It does, however, also have significant disadvantages, such as inadequate control over the size of synthesized CDs.

1.2. Carbon dots are a better alternative than organic dyes

Carbon dots are robust and highly luminous tools that can be used in fluorescence-based applications. CDs have a longer fluorescence lifetime compared to the majority of organic dyes.⁴² CDs have been shown to exhibit higher photostability than traditional fluorophores. CDs are more convenient, unlike many conventional detection methods which use high-frequency rays for diagnostics. The traditional methods of bioimaging have a huge chance of inducing cell damage, and DNA damage and this may lead to the development of cancer due to mutations in patients. Additionally, carbon dots have several advantages over conventional diagnostic procedures as they emit radiation for days, unlike other organic dyes which work in a smaller time frame.⁴³ They are less toxic and can detect cancer at earlier stages. CDs have exceptional characteristics that make them highly effective for detection, such as size- and composition-tuneable light emission, improved signal transmittance, and tolerance to photo-bleaching, including simultaneous stimulation of fluorescence spectra.⁴⁴

1.3. Factors determining the fluorescence properties of carbon dots

The primary allure of carbon dots can be attributed to their tuneable optical characteristics. Owing to CDs' advantageous optical characteristics, such as their excitation-dependent PL, high QY, and long PL lifetime, CDs have garnered a great deal of interest for their use in biomedical applications. It has been discovered that two or more of the parameters work together to control the luminescence behavior of CDs rather than just one of them.⁴⁵ Even though considerable research has been conducted in an attempt to investigate the basis of CDs’ PL, there are still controversial theories on what causes the diversity of CDs' photoluminescence. Numerous explanations for the complex optical properties of carbon dots have been offered, such as (i) graphitic core-driven optical characteristic in which the quantum confinement effect drives photoluminescence, (ii) the surface-mediated optical characteristics illustrate the photoluminescence of carbon dots caused by surface defect states, (iii) molecular fluorescence resulting due to several π-conjugated islands within CDs, and (iv) while doping of CDs does not directly cause photoluminescence, it does influence the PL behavior to tune optical features with high QY qualities.¹⁴ The properties of CDs are known to vary between different types and can be determined by various factors. These factors can be categorized as intrinsic (size, carbon precursor, surface modification, and band gap) and extrinsic factors (pH, temperature, time, and solvent) that determine the fluorescence property of carbon dots. Changes in the external environment may influence how CDs interact with the environment, modifying emission sites including electrical energy levels. The environment could also have a significant influence on the aggregation of CDs or even alter their structural makeup, which may influence PL mechanisms. While internal components predominate in emission processes, external influences greatly complement these internal factors. Since altering the synthesis parameters is the key means of controlling the characteristics of CDs, the following section will describe the consequences of some of the important parameters which govern the fluorescence features of carbon dots.

2. Intrinsic factors

2.1. Band gap

A key characteristic of carbon dots is the quantum confinement effect (QCE), which occurs when the CDs are smaller compared to the exciton Bohr radius. When the particle's size is decreased to a critical threshold where electron mobility is constrained, it leads to the formation of a band gap energy level. In the case of CDs the size and shape, surface structure, and doping state with heteroatoms affect the band gap energy level. Fluorescence is primarily obtained from the size of CDs and the conjugated pi-electron structure of the sp² hybridized carbon percentage present in CDs. A large conjugated π-domain of sp² hybridized carbon at the core of CDs will shift the band gap energy toward a lower energy level. If the sp² is imperfectly formed, it leads to the formation of surface defects and sp³ hybridized carbon bonds. This imperfection forms dangling bonds to which other functional groups are attached and also the defects form many electron–hole recombination sites, changing the electronic state of CDs.^46,47 Hence, when there is an increase in aromatic ring structures, the band gap reduces, suggesting that the conjugated π-domain size may be tuned to control the PL emissions.²⁷ Kim et al. showed size/shape-dependent PL emission dependent on electrical transitions of CDs, which may be controlled by varying their size or shape.⁴⁸ Zhou et al. concluded that the carbon core is what determines the quantum confinement effect or the conjugated-domains, whereas the surface state is determined mostly by the hybridization of the carbon backbone and linked chemical groups, and even the molecule state is solely determined by the fluorescent molecules linked in the interior or on the surface of the CDs.⁴⁹ As the electrons are excited from the valence band to the conduction band part of the energy generated during this short excitation period is lost due to molecular collisions or it is transferred to a surrounding molecule, which results in the emission of light with a larger wavelength and lesser energy.⁵⁰ As the size plays a major role in affecting the band gap energy of CDs, with the increase in the size of CDs there is a decrease in band gap energy and vice versa,⁵¹ which is well demonstrated in Fig. 4.

Fig. 4 Demonstration of how the band gap energy of CDs is affected by the size of CDs. The band gap energy reduces as the size increases, which causes CDs to emit in the near-infrared or red regions of the electromagnetic spectrum, as there is an inverse relationship between the size and the band gap.

2.2. Size

CDs are made up of two domains: a surface domain and a carbon-core domain. The surface domain has a lot of functional groups, whilst the carbon-core domain has an sp² graphene structure. The fluorescence properties of CDs are mainly influenced by these two domains. According to Scrivens and Teng's group, the PL of CDs is dependent on size, whereas Wei et al. showed that the photoluminescence of carbon dots was size-independent.^32,52,53 The materials needed to create CDs influence the inherent energy signature of carbon nanoparticles. To achieve stable characteristics for various applications, optimizing the size of CDs is a crucial step. Numerous studies have been conducted up to this point to produce consistent, homogeneous CDs by synthesis or post-treatment^54,55. Due to the different energy levels within the crystal, different-sized quantum dots alter the color that the crystal emits or absorbs. The size of CDs plays a major role in the absorbance and emittance of light. The electron energy levels in small-size nanoparticles are quite well separated compared to semi-continuous ones in larger-size particles. Due to the tiny, atom-like structure of CDs, the latter characteristic leads to the “quantum confinement” phenomenon. There is a distinct band gap energy level formed because of a smaller number of atoms present in CDs. The frequency and color of the light emitted depend on the carbon dot's size and band gap energy level, which are inversely connected. In most cases, larger size carbon nanoparticles produce emission in red light, while smaller carbon nanoparticles give emission in the blue region of visible spectra. If we try to explain the mechanism of how small size affects the fluorescence properties of CDs at the atomic level it has already been proved that small sp² hybridised carbon domains are dispersed in the amorphous sp³ hybridised core of CDs. More such sp² hybridised domains are present; there is more narrowing down of the energy band gap of CDs because of the delocalization of electrons from the valence band in these domains. Thus we can shift the emission of CDs towards higher wavelength regions of the visible spectrum by increasing the number of such sp² hybridised domains. So, if we increase the number of sp² hybridized carbon atoms in CDs it will shift their fluorescence towards the red region of the visible spectrum.⁵⁶ The shape of the CDs is also being looked at to study the optical properties but the results are yet inconclusive.

3. Environmental factors

3.1. Carbon precursor

The optical characteristics of carbon dots are significantly influenced by the source from which they are produced. Both the source material and the synthesis strategy play a vital role in determining the pl (photoluminescence) property of CDs. The components added for the synthesis and the atomic ratios of the starting material play a significant role. The functional groups present in the precursor material can ultimately become the functional groups present on CDs synthesized. These functional groups form more surface defects on the surface of CDs, lowering their band gap energy and shifting their fluorescence towards the red region of the light spectrum. Therefore precursor materials and heteroatom doping play an important role in controlling the fluorescence properties of CDs.⁵⁷ The functional groups from various solvents also used for synthesis, interacting with the source material, can generate different surface states and generate additional energy levels to its electronic structure, which leads to the alteration of the color-emission of CDs.⁵⁰ Liang Wang et al. reported the full color of highly fluorescent CDs using the solvothermal method. Their experiment showed that using the same source but manipulating different acid reagents in ethanol as a solvent can form different fluorescence carbon dots.⁵⁸ Using the electrochemical fabrication method, Li and colleagues created full-color CDs. They concluded that, rather than an oxidative surface, the quantum-sized graphite architecture controlled the luminous qualities of CDs.⁵⁹ Additionally, it was reported by other researchers also that by using the same source but varying different passivating agents and employing different methods, we can get a diverse range of fluorescent carbon dots.^60–62

3.2. Surface modification

The uncoated surface of CDs is not only susceptible to impurities but also unprotected, as carbon and oxygen have a natural tendency to react among organic molecules. This reaction might cause CDs to lose their optoelectronic properties. A protective layer is therefore essential for the stability and long-term use of CDs. Since CDs possess a significant amount of carbon and oxygen groups, it is easy to functionalize their surfaces with various organic compounds and heteroatoms, which will subsequently change the emission wavelength.⁶³ Surface modification and conjugation play a very important role in CD synthesis as well applications. Li et al. demonstrated that CDs alone cannot penetrate the Blood Brain Barrier (BBB); however, after conjugation to a glycoprotein, human transferrin, the CD conjugate was capable of crossing the BBB via receptor-mediated endocytosis.⁶⁴ Currently, effective tweaking of CDs' intrinsic structure and surface state is made possible by functionalization. According to Sun et al. and other research teams, surface passivation can cause surface defects on CDs, which will entrap the excitons, which eventually impart changes in fluorescence.^65,66 Yang et al. prepared highly amino-functionalized fluorescent CDs by employing hydrothermal carbonization of chitosan at 180 °C, which will cause the dehydration of the chitosan, which leads to carbonization and functionalization, forming fluorescent CDs as bioimaging agents. They concluded that providing different functional groups on the CD surface can induce PL properties and increase its water solubility and lower its potential biotoxicity.⁶⁷

Two functionalization techniques, that is, heteroatom doping and surface modification are used for enhancing the photophysical performance and expanding the area of applications for luminous CDs. Several studies were conducted in recent years to functionalize and modify the surface of CDs, incorporating covalent bonding,⁶⁸ coordination,⁶⁹ sol–gel,⁷⁰ and π–π interactions.⁷¹ The intrinsic structure as well as electron distribution of CDs may be altered by heteroatom doping, involving non-metal or metal doping. Heteroatom doping using N (nitrogen),^15,72 F (fluorine),^73,74 S (sulphur),^75,76 and P (phosphorus),^77,78 as well as certain heavy elements⁷⁹ is a common method of changing the electronic structure of CDs. Nitrogen and sulfur are frequently used dopants, as they have an atomic radius close to that of carbon, while the latter possesses electronegativity similar to carbon. The N and S co-doped CDs prepared by the hydrothermal process of citric acid and glutathione (GSH) demonstrated an excitation-independent PL with an 80.3% quantum yield.⁸⁰ S-doped CDs synthesized by the hydrothermal process using sodium citrate and sodium thiosulfate serve as a fluorescence sensor for detecting Fe³⁺ ions.⁸¹ Nitrogen doping (N-doping) is a particularly effective way to increase the QY of CDs among non-metals because the newly created N-state may trap electrons with a high fluorescence yield.^82,83 It was also discovered that the presence of a significant number of oxygen-containing groups causes non-radiative coupling of localized electron–hole pairs, restricting radiative transition, and hence when doped with high-electronegativity oxygen, will result in very low QY.⁷⁹ Because of its simplicity, heteroatom doping has attracted a lot of research attention as a way to tune CDs' inherent features. Therefore, chemical heteroatom doping is a good way to modify the electronic structure and optical characteristics of CDs; however, it is important to pay attention to the concurrent rise in toxicity.⁸⁴

On the other hand, through surface modification, it is possible to functionalize CDs with ions, chemical compounds, polymers, proteins, and even DNA. The inclusion of functional groups like carboxyl, carbonyl, and amines, which can cause a variety of surface defects on CDs, makes surface functionalization significant. These defects may serve as surface energy traps and alter the way the CDs emit fluorescence.⁸⁵ The findings of Pandit et al. demonstrate that surface charge as well as hydrophobicity may be altered and that the QY could be modified and improved by up to 62% based on functionalization.⁸⁶ Contrary to heteroatom doping, surface modification via functional ligands could be utilized to change the surface states by enhancing the number of active sites.⁸⁷ CDs when modified with L-cysteine can be used for long-term in situ imaging of the Golgi body, as the Golgi body can form cross-links with L-cysteine or even its residue 6. Bhunia et al. demonstrated the use of TAT peptide- or folate-functionalized CDs for biological labeling and imaging.⁸⁸ Fluorescent CDs can be endowed with distinctive features arising from functional ligands by applying surface modification to the various active sites and functional groups on the surface of the CDs. Furthermore, some functional groups demonstrate very selective binding to particular ions. The hydroxyl group, for example, has a specific and strong coordination association with Fe³⁺ ions,⁸⁹ whereas the carboxylic group has a high affinity for Hg²⁺ ions,⁹⁰ while the amino group forms complexes by interacting with Cu²⁺ ions.⁹¹ Bera et al. found that when ruthenium-doped-CDs are amine-functionalized, excitation-dependent emission switches to excitation-independent emission, increasing the QY. They suggested that the excitation independent emission as well as the rise in QY caused by amine modification may be attributed to the transition of many sp² arrays into a single type, which decreases cross conjugates and thus boosts the quantum yield. Thus, judiciously introducing certain functional groups through surface passivation or functionalization might improve the fluorescence response of CDs.⁹² Wu et al. created a multi-functionalized, theranostic nano-agent centered on folate-conjugated reducible polyethyleneimine passivated (fc-rPEI) CDs which can encapsulate several siRNAs (EGFR and cyclin B1) before delivering them in an intracellular reductive condition. Additionally, fc-rPEI-Cdots are a highly biocompatible material and an effective siRNA gene delivery carrier for personalized lung cancer therapy.⁹³ In another experiment, Shoval et al. used surface-functionalized CDs of 8 nm to effectively penetrate the ocular, along with the epithelial layer, cornea, eye cavity, and lens.⁹⁴ Uncoated CDs have a poor luminescence efficiency (quantum yield (QY) <5%) due to the presence of epoxy and carboxylic groups on the surface of uncoated CDs constantly triggering nonradiative recombination of localized electron–hole pairs, inhibiting pristine emission.⁹⁵ Non-functionalized CDs often exhibit blue fluorescence and have a limited number of active sites because of the unicity of active sites, which restricts their applicability. Functionalization of CDs is, therefore, a potent tactic for enhancing the photophysical as well as photochemical properties.

3.3. Temperature

Reaction time has a temperature-dependent impact on the optical characteristics of carbon dots. Two mechanisms that contribute to the temperature-dependent energy gap are (i) renormalization of band energies via interaction of electrons-phonons and (ii) thermal expansion of QDs.⁹⁶ Yu et al. synthesized CDs from glacial acetic acid to check the temperature-dependent fluorescence from cryogenic to room temperatures. They observed that when the temperature increases, the intensity decreases. They explained this phenomenon as temperature-induced nonradiative trapping, whereby excited electrons at relatively low temperatures could only release photons through radiative emission because the nonradiative channel is not thermally stimulated, whereas a temperature rise activates several nonradiative channels, including defect/ionized impurity states, leading to lower PL intensities.⁹⁷ Thus, the temperature-dependent property may be responsible for the temperature-enhanced population of non-radiative surface (trap/defect) state channels. At a higher temperature, more non-radiative channels would be active; thus, more excited electrons would revert back to the ground state through a non-radiative mechanism, resulting in a drop in fluorescence intensity.^98–100 Another theory states that the reaction temperature and period both have an impact on the optical characteristics of carbon dots. Due to the long reaction time, over-carbonization will occur, which will cause the surface structure of CDs to be disrupted. However, if kept for a shorter time, the carbon source won't be sufficiently carbonized, which will yield CDs with subpar fluorescence emission.^98,99 Wang and coworkers proposed that the temperature-dependent PL may be due agglomeration of CDs as temperature changes. They fabricated blue fluorescent CDs by hydrothermally treating glucose and glutathione and discovered that the PL intensity of CDs reduced with the temperature rise, but the UV-vis spectra of CDs were unaffected by variations in the ambient temperature; however, the particle size did change, dropping from 4.4 nm at 80 °C to 2.6 nm at room temperature, which suggests that the CDs may have aggregated during heating, causing PL quenching.¹⁰⁰ Thus, the temperature dependency of CDs' PL behavior still needs more clarity; however, CDs are indeed a good choice for the development of temperature-monitoring devices due to their temperature-sensitive feature. Optimization of temperature and time should be done accordingly, which will give a fruitful result.

3.4. pH

The pH-dependent response is linked with the surface functional groups of the CDs by protonation–deprotonation. Deprotonation can result in electrostatic doping/charging of CDs and a drop in the Fermi level. Choudhury et al. observed that CD emission was red-shifted when the pH was increased due to the progressive formation of new CDs in the environment that absorb and emit at a higher wavelength. Furthermore, CDs showed high-intensity fluorescence under acidic conditions as there was a strong vibrational coupling of OH (hydroxyl) groups caused by hydrogen-bonding effects that promoted energy level broadening, thus enhancing CDs' conformational rigidity, whereas under the basic conditions where hydrogen bonding was eliminated upon deprotonation it may cause lower vibrational coupling and even more discrete energy levels, resulting in a fluorescence drop in CDs.¹⁰¹ It has been well documented that CDs exhibit pH-dependent PL, which can be monitored at different pH to understand the effect of surface groups on optical characteristics.¹⁰² The fluorescence emission intensities of CDs formed from various carbon sources using different synthesis procedures are affected differently by the pH of a solution. Kumar et al. synthesized CDs by a one-step hydrolysis method for Pb2+ ion sensing; they observed the fluorescence behavior of CDs under different pH where they showed that the synthesized CDs were showing strong and stable fluorescence at neutral pH.¹⁰³ Some are stable across a wide range of pH values. Yao et al. prepared CDs that were highly stable at different pH values even after 72 h, with no obvious variation in emission intensity.¹⁰⁴ Similarly, Zhou et al. and Wang et al. groups prepared highly stable water-soluble blue fluorescent CDs which showed good stability over a wide range of pH environments (pH 2.0–11.0) and (pH 4.0–12.0), respectively.^15,105 This behavior was probably due to an alteration in the surface charge during the protonation or deprotonation process. Meanwhile, Anmei et al. synthesized CDs that showed stable and strong fluorescence in an alkaline environment (pH 9.15).¹⁰⁶ Some act well under acidic pH. Hu and coworkers synthesized sulfur-doped CDs with pH-sensitive photoluminescence, where they observed that within the pH range of 3 to 9, PL showed a linear relationship with pH. It is discovered that the PL intensity-pH response is reversible. They deduce that the CD protonation and deprotonation are related to the PL intensity. The degree of deprotonation increases with pH from 3 to 9, resulting in a larger concentration of carboxyl groups that function as fluorophore species on their surface, resulting in increased PL intensity. PL intensity decreases when the carboxyl group concentration reaches its maximum value (pH 9–12).⁹⁹ Interestingly there are findings that state that pH-dependent response is driven by pH-induced aggregation. According to Zhang et al., CDs’ red-shifted emission is caused by the generation of CD aggregates. Under acidic pH, CDs can be a well-dispersed solution, but when pH increases, aggregation occurs due to noncovalent molecular interactions, such as H-bonds formed between carboxyl groups.¹⁰⁷ This optically pH-sensitive feature will be desirable as a function of fluorescent probes for pH monitoring in biological and biomedical applications.

3.5. Solvent

The synthesis and characterization of CDs depend heavily on solvents since they have an impact on both particle dissolution and synthesis. CDs' fluorescence can be hampered by solvents in several ways. The energy of the released fluorescence can be absorbed by the solvent molecules in a process known as quenching. Aggregation of the CDs is an additional factor that can also minimize fluorescence. Moreover, the solvent may have an impact on the CDs' stability, causing them to degrade and exhibit further drops in fluorescence. To avoid interference with their fluorescence, it is essential to select the solvent for CD applications carefully.^108,109 According to Zhang et al., the optical characteristics of CDs can be regulated by solvent interactions including H bonding and internal CD penetration. To function as both electron acceptors and donors, amino groups in N-containing fluorophores create H-bonds with solvent molecules, where H-bonding has more impact in protic than aprotic solvents. In addition to promoting internal conversion, these interactions also shorten PL lifetimes, enhance spectral shifts, and decrease QY values. Additionally, solvent molecules may enter the interior of CDs, modifying their optical characteristics. Fuyou Du et al. showed that fluorescence and emission intensity change with a change in solvent; that is, carbon dots exhibit solvent-dependent photoluminescence.¹¹⁰

4. Advantages of red carbon dots

Red Carbon Dots (RCDs) are in trend due to their low light absorption, minimal auto-fluorescence, and deep tissue penetrating ability; red fluorescence emission from RCDs particularly that generated near-infrared excitation (NIR, 800–1200 nm) demonstrates several benefits in biological applications. Blue and green fluorescent carbon dots are unfavorable for detection in bioimaging as they require a high excitation energy source that can damage the cell, which may lead to cancer. Also, chances of autofluorescence are there, as under the ultraviolet (UV) spectrum certain plant and animal tissues tend to show fluorescence. Furthermore, chances of autofluorescence are there in blue-green fluorescent carbon dots when excited and they interfere with the signal of other CDs and cause damage to cells and tissues, leading to hampering the bioimaging process.¹¹¹ To date, numerous green and blue CDs have been synthesized using chemical sources such as ascorbic acid,^112,113 acetic acid,¹¹² and gelatin¹⁰ as well as natural sources like eggshell,^113,114 orange juice,⁵¹ lotus root,¹¹⁵ carrot,¹¹⁶ watermelon,¹⁰⁵etc. However, it's still challenging to synthesize red fluorescent CDs with high QY as the sp2 π-conjugation domains are much larger, which makes them more prone to deformities and at risk of environmental disruption.¹¹⁷ In the case of red fluorescent carbon dots, there is low light scattering because of biological tissues and fluids. A high signal-to-noise ratio helps in improving the bioimaging of cells and tissues using red fluorescent CDs. For combating the difficulties of tissue damage and scattering by biological cells and fluids, various red fluorescent carbon dots have been synthesized using various carbonaceous sources such as p-phenylenediamine,¹¹⁸ citric acid,³⁸L-glutamic acid,¹¹⁹ polythiophene,³⁵etc.

4.1. Biological applications of carbon dots

The general characteristics of CDs, such as their small particle size, customizable chemical composition and characteristics, strong quantum yield, and high fluorescent property, have attracted a variety of applications. Because of their distinctive size- and shape-dependent optoelectronic characteristics, CDs have invited researchers to explore their uses in different fields like biomedicine and therapeutics, and diagnostics. These nanoparticles have given promising results in single molecule probes, real-time tissue imaging, and drug delivery to name some of their many applications.¹²⁰ Immunofluorescent labeling of tissues and cells that have been fixed and immunostaining of proteins associated with the cell membranes of living cells have both served as examples of the first effective utilization of CDs in medical diagnostics.¹²¹ For the quick detection of disease-relevant biomolecules, the carbon dots are also extensively employed as DNA-functionalized probes.¹²² The properties of CDs, like their broad excitation spectra, make them an asset in studying the imaging of tissues. Their increased intensity develops better imaging with less to no background noise.¹²³ In addition, CDs also have excellent fluorescence lifetime and photostability performance. These nanodots photobleach very low relative to other molecular markers, so we can monitor prolonged actions because their color intensity lasts longer. CDs when synthesized through microfluidics can prove to be an effective platform for the diagnostics of trace disease markers.¹²⁴ In the following section, some of the important biomedical applications of CDs are discussed in detail. Fig. 5 summarizes biomedical applications in bioimaging, photodynamic therapy, biosensing, fluorescence-assisted cell sorting, and drug delivery.

Fig. 5 Depiction of multiple arenas of biomedical applications using carbon dots. Carbon dots have been explored for their applications in bioimaging, targeted drug delivery, photodynamic therapy, fluorescence-assisted cell sorting, and biosensing. The tunable fluorescence and easy surface modification of CDs give them an advantage over other organic and semiconductor fluorophores, thereby interfacing them ideally to probe and program various biological systems as shown above.

4.2. Bioimaging: in vitro and in vivo biological systems

Bioimaging is among the most effective technologies for live cell imaging research using fluorescence microscopy. Cell biology has benefited greatly from the use of live cell imaging, which has shed light on a variety of complex processes such as cell division, morphogenesis, endocytosis, and exocytosis; thus, it enables the direct viewing of biological processes. The use of organic and inorganic nanomaterials as fluorophores for bioimaging has been extensively documented. For bioimaging, organic fluorophores were used, but there were drawbacks including their photobleaching susceptibility and chances of dripping out from the cells into the media, lack of photostability (i.e., degradation of fluorophores while being exposed to light while performing the imaging procedure), and narrow excitation and emission wavelength because of which we cannot regulate the optical properties, and organic fluorophores may interact with the cell during the imaging procedure, which leads to cytotoxicity.⁸⁵ Hence, here CDs come into the picture to overcome those limitations. Conventional organic fluorophores and contemporary inorganic semiconductor QDs lack in several aspects when compared with CDs, including photobleaching resistance, chemical inertness, and ease of surface functionalization. Additionally, they have good water solubility, increased sensitivity, absence of radioactivity threat, minimal cytotoxicity, and improved spatial resolution, and fluorescence imaging has developed into an effective way for observing biological activities within biological systems.¹²⁵ CDs are sometimes coated with certain targeting moieties to visualize intracellular structures via fluorescence microscopy or confocal laser scanning microscopy. CDs may be designed depending on tissue-specific targeting properties, which can be utilized to monitor certain organs, using in vivo imaging techniques. Yang and colleagues for the first time demonstrated optical imaging in vivo by employing CDs.¹²⁶ Yanqiu Chen et al. showed that fungal cells stained by the CDs would be vividly lit when viewed using a confocal microscope triggered by a green laser pulse; the CDs provide an effective tag for fungal cell imaging in the red emission zone.¹²⁶ Yu et al. prepared blue fluorescent gadolinium-doped CDs, using gadopentetic acid as the precursor and glycine functioning as the surface passivating agent, which were used for MRI imaging.¹²⁷ Chen et al. developed 1.7 nm-sized nitrogen-sulfur-doped CDs with a remarkable fluorescence emission yield of about 39.7%. When incubated with HeLa cells, they exhibited minimal cytotoxicity and good biocompatibility. By quenching their fluorescence, these doped CDs can detect Cr(IV) inside the cells.¹²⁸ Han et al. showed the ability of antibody-conjugated linked CDs to penetrate inside the bone marrow and mark individual cells inside, even exceptional groups of progenitor and hematopoietic cells. This technique eliminates the drawbacks of fluorescently tagged antibodies, including their limited photo-stability, low multiphoton action cross-section, and asymmetric emission.¹²⁹ Cao et al. employed PEGylated CDs to recognize the fluorescence in MCF-7 human breast cancer cells.¹³⁰ For cellular imaging of cells, Zhang et al. created CDs from polydopamine. Yellow and green fluorescence was detected in the cytoplasm but not inside the nucleus of the cell when excited at wavelengths of 458 and 405 nm, respectively.¹³¹ In another example to create carbon dots for bioimaging of the human bronchial epithelial cell line (16HBE cells), Chen et al. carbonized sucrose with oleic acid. There was green fluorescence seen in the cell membrane along with the cell cytoplasm and very weak fluorescence was also seen inside the nucleus of 16HBE cells, which shows that the prepared CDs were not able to go inside the nucleus.¹³² CDs can also be employed for nucleus imaging. The nucleolus is the cell's ribosomal RNA (rRNA) manufacturing center and ribosome factory. CDs could be applied for fixed cell nucleolus imaging as well as tracking of live cell nucleolus-related events. The basis of nucleolus-targeting is the selective binding of CDs to RNA (rather than DNA) upon nucleolus invasion.¹³³ Han et al. prepared CDs of 2–3 nm which were able to pass through the germ-cell membrane, intestinal wall, and gonad vesicle wall.¹³⁴ Singh et al. synthesized red fluorescent nanoparticles from para-phenylenediamine by employing the reflux method. These highly fluorescent red nanoparticles showed promising applications as bioimaging agents (Fig. 6).¹³⁵

Fig. 6 (a) Schematic representation of the synthesis of red fluorescent carbon nanoparticles using the reflux method of synthesis and (b) bioimaging of concentration-dependent cellular uptake studies of CNPs in cancer cells like SUM159A at different time intervals; the concentrations used were 10, 25, 50 and 100 μg mL⁻¹. Synthesized fluorescent CNPs were employed in confocal microscopy research for biomedical applications because of their strong and persistent red fluorescence emission.¹³⁵

CDs’ bioimaging applications can be broadened to disease diagnostics. CDs with a size of less than 3 nm are capable of penetrating the blood–brain barrier for selectively targeting glioma cells, serving as an efficient bioimaging and targeting tool. Zheng et al. demonstrated that self-targeted CDs derived from D-glucose and L-aspartic acid pyrolysis can be applied for fluorescence imaging as well as detecting and targeting non-invasive glioma.¹³⁶ Subsequently, Qiao et al. enhanced the CD-targeting capacity for brain glioma cells by optimizing the glucose to aspartic acid ratio, which was found to be 7 : 3.¹³⁷ Bioimaging is a vital method used in both research and therapeutics as it allows observing biological processes such as the delivery of drugs, cellular uptake, and biodistribution of therapeutics in a well-detailed manner with the use of various electromagnetic spectra. Imaging plays a crucial role in cancer diagnosis because sensitive imaging enables early tumor detection and identification of metastasis and the return of the disease.^138–140

4.3. Biosensing

Biosensors are systems that can generate a quantifiable signal in retaliation to any biological event.¹⁴¹ The advantages of good sensitivity, rapid response, and relative inexpensiveness have made fluorescence-based CD sensing a hot topic in recent years. The optical characteristics of carbon dots can be applied to biosensing in addition to bioimaging (Fig. 7). The monitoring of specific cells and tissues of interest using CDs in bioimaging has improved contrast in magnetic resonance (MR) pictures, even though both bioimaging and biosensing applications use CDs and call for the detection of emitted photons². Contrarily, CDs’ function in biosensing systems is to find and signal the presence of biomolecules in the system. The premise for carbon dot-based biosensors is the affinity between certain functional groups located on the surface of CDs and the analyte biomolecule.¹⁴² Carbon dots can be employed as a fluorescent probe for pH measurement^143,144 as well as sensing metal ions such as Hg⁺,^90,144 Cu⁺,¹⁴⁵ Fe³⁺,146 phosphate (Pi),¹⁴⁷ iodide¹⁴⁸ and quercetin.¹⁴⁹ Khose and colleagues synthesized red fluorescence-emitting carbon dots from guava leaves which they used for Hg⁺ metal sensing.³⁹ Fuyou Du et al. reported that alachlor, a herbicide found in soil samples, can be sensed using red-emissive nitrogen-doped carbon dots (NCDs). The developed NCD probe demonstrated exceptional stability, sensitivity and selectivity, and outstanding repeatability in the sensing of alachlor, and it can measure alachlor over a broad concentration range (0.005–150 M).¹¹⁰ To veterinary medicines, red emissive CDs demonstrate a rapid, sensitive, and targeted fluorescence response. Animals that are slaughtered for food are treated with veterinary medicines to prevent or cure illness; food made from these treated animals may include traces of these drugs which are harmful for human consumption. To identify veterinary drug residues in foodstuffs, He Li and co-workers developed red emissive CDs from sorbitol and o-phenylenediamine for ratiometric sensing of these trace compounds.¹⁵⁰ A more advanced and versatile fluorescence resonance energy transfer (FRET) probe based on CDs was shown to be capable of both imaging and detecting mitochondrial hydrogen peroxide (H₂O₂). The CDs act as the sensing system's carrier and energy transfer donor. The CDs were covalently bonded to boronate-protected fluorescein, a hydrogen peroxide recognition component. It could be used to monitor the amounts of exogenous and endogenous hydrogen peroxide in L929 cells (mouse fibrosarcoma cell line) as well as macrophage cells, respectively.¹⁵¹

Fig. 7 Fluorescent carbon dots are being used for biosensing applications. The affinity between particular functional groups present on the surface of CDs and the analyte biomolecule is the basis for CD-based biosensors.

4.4. Drug delivery

As a system for drug delivery, CDs offer several benefits, including ease of synthesis, versatility in drug conjugation, tunable physicochemical features, and intriguing optical characteristics that make them traceable and simple to monitor once injected.¹⁵² In addition, CDs' non-toxic nature and biocompatibility make them a more reliable drug delivery system.⁴⁰ Due to their small size—between 1 and 10 nm, carbon dots have considerable potential as delivery vectors that can penetrate the tumour.¹⁵³ Cells typically uptake functionalized CDs by receptor-mediated endocytosis (Fig. 8). The majority of the research is driven toward anticancer drugs.^154–156 In the case of cancer, early detection and drug delivery are very crucial. All the detection processes are invasive and require tissue autopsy and it has been impossible to detect cancer and deliver the drug simultaneously. Researchers are currently trying to use the CDs as bioimaging probes as well as to perform targeted drug delivery at the same time. This would potentially make the detection non-invasive and drug delivery traceable.¹⁵⁷

Fig. 8 Schematic illustration of the Carbon dot – Aptamer (Drug) Bi-FRET system. Fluorescent CDs are surface functionalized with an aptamer, showing fluorescence even after functionalization. The desired drug individually shows fluorescence. The intercalation of the drug with the aptamer on the surface of CDs forms the CD-Aptamer (Drug) complex, which results in the quenching of both CD and drug fluorescence through the Bi-FRET mechanism. Further the uptake of the CD-Aptamer (Drug) complex into the targeted cell. The release of drugs from the complex results in the regaining of fluorescence properties of both drugs and CDs, thereby CDs acting as targeted drug carriers and also sensing the intracellular drug delivery.

Since the blood–brain barrier (BBB) is extremely semipermeable, it prevents the majority of therapeutic agents from entering the central nervous system (CNS), and hence drug delivery across the BBB is one of the largest problems in contemporary medicine. Various kinds of CDs as well as CD-ligand conjugates have been discovered to successfully cross the BBB, indicating a hopeful breakthrough in the use of CD-based drug delivery systems for treating diseases related to the CNS.¹⁵⁸ According to Liyanage et al. findings, non-toxic carbon nitride dots (CNDs) can specifically target cells of pediatric glioblastoma, but a high concentration of the CND-gemcitabine combination was required for GM to efficiently destroy CNS malignant cells.¹⁵⁹ In another study, curcumin-loaded CDs were used for diagnosing Alzheimer's disease (AD).¹⁶⁰ Similarly, Chung and coworkers prepared branched polyethyleneimine (bPEI) conjugated CDs using ammonium citrate, to treat AD by acting as an anti-Alzheimer's β-amyloid (Aβ) neurotoxin agent.¹⁶¹

Mehta et al. demonstrated that due to the multifunctional groups on CD surfaces, CDs can be employed as multifunctional vehicles for drug loading and release. They loaded the drug lisinopril onto CDs, and it was discovered that the HeLa cells effectively uptake Lis-loaded-CDs. This suggests that the fluorescent CDs can serve as drug carriers for loading and releasing the drug due to their ultrafine size, multifunctional groups, and high PL.¹⁶² A broad-spectrum antibiotic ciprofloxacin conjugated to carbon dots with vivid green fluorescence has already been used for bioimaging and providing a possible nanocarrier for regulated drug release with significant antibacterial action in physiological surroundings.¹⁶³ The solubility of a drug poses a significant obstacle when administering it. However, one solution to this issue is utilizing a copolymer of chitosan and polymethyl methacrylate as a theranostic nanocarrier, which is both biocompatible and powerful. By doing so, poorly water-soluble drugs can be embedded in the hydrophobic core of the nanocarrier, while CDs can be conjugated to the hydrophilic portion to aid in bioimaging.¹⁶⁴

4.5. Cancer diagnosis and therapy

Numerous developments have been made to better understand the chemistry of cancer by creating smart nanomaterials that could target cancer cells with specificity, react to the microenvironment in which they are found, and perhaps even aid in non-invasive diagnostics. Quantum dots, especially CDs, can generate detectable acoustic waves after laser irradiation. This makes them a very good contrast agent in photoacoustic imaging. This feature of the CDs helps them to extract valuable information from the smallest of places. These carbon dots are sufficiently taken up at the tumor sites, which makes them very efficient in delivering the drugs to minor tumors, serving as a good nanocarrier. These nanocarriers can be conjugated with different anticancer compounds for active targeting. The carbon dots made it feasible to monitor the release and uptake of the drug by the cells with the help of techniques like FRET. These molecules are easily removed from the body by the kidneys after the successful delivery of the drug; hence, they are also non-toxic and biocompatible.¹⁶⁵ Sui et al. showed that cisplatin's anticancer efficacy can be improved by graphene quantum dots (GQDs) by enhancing its cellular and nuclear uptake and improving its association with DNA.¹⁶⁶ As per the findings by Xiao S. et al., the neuroprotective peptide glycine–proline–glutamate (GQDGs) when conjugated with GQDs could potentially cure neurodegenerative diseases like Alzheimer's. The amyloid-fibril aggregation was inhibited by the GQDGs, and both the quantity and quality of newly formed neurons and neural precursor cells increased.¹⁶⁷ The microenvironment pH can also help in cancer therapeutics for delivering drugs to specific sites. This was demonstrated by Feng and coworkers, in which when cisplatin-loaded CDs were used with a pH-dependent charge reversal polymer, the drug-CD complex was released at the tumor site that promoted their effective delivery into cancerous cells. The conjugated anionic polymer undergoes cationic conversion in the tumor environment, which releases the positive moiety. As a result, drug-loaded CDs can infiltrate tumor cells and get activated under cytosolic reductive conditions. Since cisplatin (as a prodrug) is only active in a reductive tumor environment, using cisplatin in the prodrug form eliminates its non-tumor targeting.¹⁶⁸ Similarly, CDs synthesized from citric acid and urea have carboxyl groups on their surface that may conjugate with the anticancer agent doxorubicin (DOX) via an amino group, generating a pH-sensitive association.¹⁵⁵ Tumor-specific release of drugs happens as a result of CDs-drug breaking down in the tumor environment. To kill cancer cells, Zhou et al. demonstrated the use of a CD encapsulated mesoporous silica complex allowing the pH-triggered intracellular sustained release of the anticancer doxorubicin (DOX) drug. The mesoporous silica NPs which were positively charged were conjugated using CDs’ carboxyl group. They discovered that the dissociation of the CDs-mesoporous silica NPs and subsequent release of a significant amount of DOX molecules occurred at physiological temperature and in a mildly acidic state. Both in vitro and in vivo, the nano complex has a high level of efficiency in killing cancer cells.¹⁶⁹ Utilizing bio-conjugated CDs, it is now feasible to find appropriate molecular biomarkers for cancer diagnosis, treatment, and prognosis. They might enable the doctor to undertake a full surgical resection and map sentinel lymph nodes. Owing to their unique optical characteristics, they are perfect donors for experiments on photodynamic therapy and fluorescence resonance energy transfer. Multifunctional carbon dots are effective materials that have the potential to diagnose, target, and treat cancer simultaneously (Fig. 9).¹³⁶

Fig. 9 Targeting fluorescent carbon dots for recognition of cancerous cells in the mouse brain.¹³⁶

4.6. Photodynamic Therapy (PDT)

In this approach, light irradiation activates a chemical molecule known as a photosensitizer to transfer electrons to the intracellular molecular oxygen, resulting in in situ generation of reactive oxygen species (ROS), which leads to apoptosis in the target tumor cells.¹⁷⁰ Carbon dots have revealed interesting achievements in PDT-based cancer therapies. As a result, currently, there is a significant amount of research being performed on the development of non-toxic, bio-compatible carbon dot-based photosensitizers.¹⁷¹ The first-generation photosensitizers proved effective towards colorectal, lung, neuronal, oral, and breast malignancies and were developed based on a hematoporphyrin backbone. However, they experienced significant impediments, including difficult preparation and sophisticated structures, limited tissue absorption due to a shorter wavelength range, truncated quantum yields, slower pharmacokinetics, and hydrophobicity.¹⁷² For the first time, CDs were discovered to induce ROS when exposed to blue light irradiation in vitro to be employed in PDT in 2011.¹⁷³ PDT is oxygen-dependent; a high amount of oxygen is consumed in this process, which results in hypoxia, which limits its therapeutic effects, as in a tumor environment there is insufficient oxygen supply; thus, PDT is unable to reach its full therapeutic potential.^174,175 To surpass this problem, Mn-CDs were created by using manganese(II) phthalocyanine as a precursor, which enhanced the ROS generation and stimulated the production of oxygen.¹⁷⁶ Carbon dots have been experimentally proved to show antibacterial-photodynamic therapy; that is, E. coli and S. aureus were photodynamically inactivated by CDs. A nanocomposite made of carbon nitride doped CDs and a polymer segment having a photosensitizer and a targeting agent can induce the generation of ROS under light irradiation; hence, the nanocomposite reverses tumor hypoxia, boosting its anticancer effects.¹⁷⁷ Li et al. established that CDs based on triphenyl porphyrin (TPP) are photodynamically active that efficiently decrease the size of the tumor without any side effects when exposed to irradiation.¹⁷⁸ Nie et al. and Xu et al. have demonstrated that selenium-doped carbon dots specifically attach to RNA, which results in the generation of ROS that harms the nuclear membrane.^179,180 Additionally, CDs were proven to be efficient for concurrent improved fluorescence imaging and PDT of gastric malignancy in vivo.¹⁸¹ Because of their low cytotoxicity, CDs can be conjugated with conventional photosensitizers (PS) to increase the organic PS water dispersibility. Additionally, the surface of CDs could be functionalized to aid in the attachment of certain proteins and antibodies, which produces site-specific localization of the photosensitizer and thwart the phototoxicity that is frequently associated with traditional photosensitizers. Hence, the majority of the problems with present modalities may be solved by incorporating carbon dots into PDT.¹⁷¹

5. Drawbacks and limitations

To meet the needs of good biocompatibility, long-life stability, and tissue-specific targetting ability, it is crucial to effectively regulate and optimize the size, structure, morphological features, and surface functionalization of CDs. But synthesizing carbon dots is itself a challenging task as several factors hinder the synthesis process and also affect the PL property of carbon dots, a few of which were mentioned above in this review, such as changes in time and temperature conditions, the solvent and compound (precursors) used, and the synthesis method. Also, they still experience reduced photoluminescence and quantum yields (QY) in the red portion of the spectrum, advancements have been made, and this disadvantage is now being overcome significantly.¹⁸² Even though CDs have been extensively employed in several sensing systems and biomedical applications, there are still numerous issues that must be taken into account: (1) the first problem is maintaining selectivity with the simultaneous increase in sensitivity. It is important to use proper precursors that enhance CDs' chemical characteristics to increase the sensitivity and selectivity. An appealing strategy for creating functional nanoprobes for specific targets is by modifying the surface of CDs using unique functional ligands. (2) Another limiting factor while synthesizing carbon dots is their size and agglomeration. Certain CDs begin to aggregate after a specific duration of time, which causes an increase in their size. Also, it was found that even the solution in which nanodots are dissolved is also responsible for aggregation. When Kumawat et al. dissolved the graphene QDs synthesized from grape seed extract in ethanol the size was 1–8 nm, but when dissolved in an aqueous medium, it formed a cluster of size 50–60 nm approx.²⁰ In an experiment, CDs were prepared from spinach agglomerates after a certain period. It was discovered that the charge of PEI is responsible for CD agglomeration. Additionally, when incubation time extends, color transitions from red to green are observed. While CDs made with ethanol don't change color over time as CDs made with water do, they do display a color change over time. It has been seen that as time passes on, nitrogen concentration reduces because CDs react with oxygen to generate agglomerates, which eliminate excess nitrogen.¹⁸³ The solvent is now known to have a significant function in the aggregation of nanoparticles.¹⁸⁴ It was even found that, in line with earlier results, the size of the graphene QDs increases dramatically below pH 3.^185,186 Thus, there is still a need to concentrate on optimizing the size and preventing the aggregation of these particles. (3) Additionally, separating our desired size of nanodots from the mixture of varying sizes of particles is itself a challenging task. Advanced protocols for purification and separation techniques still need to be ameliorated. (4) The applicability of CDs is constrained since they frequently have poor QY; to overcome this limitation we can perform reduction^187,188 or heteroatom doping^189,190 of carbon dots to raise the quantum yield of CDs. (5) While preparing CDs use of toxic chemicals should be avoided so that the synthesized CDs are harmless and dissipate and stay in the person's body without causing harm to them to be successful. (6) The internalization structure, surface property and surface architectural make-up of carbon dots are still not clear due to the lack of suitable characterization tools to investigate the architecture of CDs. (7). We still need to completely understand the process underlying CDs' fluorescence. The majority of the proposed mechanisms are debatable or inconclusive, although some of them have helped to clarify the photoluminescence process and served as guidelines for controlling the PL features. The task of increasing sensitivity while retaining selectivity in biosensing is daunting. This means that appropriate precursors and synthesis techniques must be carefully chosen to identify the chemical characteristics of quantum dots. For high-quality findings in biological imaging, CDs with long-wavelength emission and high quantum yield are required. Furthermore, their intricate and expensive preparation procedures, in addition to possible cytotoxicity arising during surface modification and unfavorable environmental conditions, could be barriers to their use in real-time applications. It is, therefore, highly desirable to have chemosensors that are precise, biocompatible, safe, inexpensive, and simple to use.

6. Future directions and outlook

Carbon dots are gaining much attention in the field of nanoscience due to their anomalous optoelectronic properties, semi-conductivity, photostability, and size variable emission. The majority of the carboxyl moieties on CDs' surfaces contribute significantly to their water solubility and biocompatibility.^189,190 Before CDs can be used efficiently in clinical applications, there are still some obstacles to be overcome, including overall toxicity, body clearance, scalability of the synthesis technique, environmental impact, manufacturing costs, and others. The preparation of CDs should focus on making them non-hazardous, biocompatible, water-soluble, and customizable in a way that makes them suitable for various applications in the biomedical sector as well as safe to use in vivo. Improved fluorescent materials with enhanced optical and biological properties must be developed to overcome biodegradability and toxicity for the bioimaging of live cells. One of the major obstacles when targeting drug delivery with CDs or bioconjugate CDs is the BBB. For instance, less than 1% of antibody medications used in clinical trials for treating CNS-related diseases can pass across the BBB, requiring large doses and high prices. Taking this into account, drug conjugated CDs with improved therapeutic and penetrating features to cross the BBB should be developed. Nonetheless, the pathways for overcoming the BBB employing CDs are still not properly understood, and they are essential topics of research for future CDs or CD conjugates to breach the BBB for drug delivery. CDs have great potential as novel luminescent thermometers and pH sensors due to their pronounced PL dependency on temperature and pH-responsive behavior due to temperature-induced and pH-induced aggregation of CDs, which causes fluorescence quenching. Notably, the PL intensity is sensitive to temperature but stable in the physiological temperature range, which makes CDs a great choice for cellular temperature sensing.¹⁰⁷ Therapeutic and diagnostic platforms are continuously being developed by combining CDs with different kinds of nanoparticles and/or biologically active compounds. Until now, there have been a lot of studies on the uptake of CDs by cells and using them for biomedical purposes, but the light should be drawn towards the commercialization of CDs as well as the influence and applications of CDs in clinical settings so that CDs can be used extensively in the human body. Exploring the mysteries of CDs will require more study. The future of quantum dots offers a promising outlook for core research and real-world applications.

Conflicts of interest

We do not have any conflict of interest to declare.

Acknowledgements

KB thanks DY Patil Institute, Pune, for permission for her MTech internship at IITGN. US and PY thank IITGN MoE for PhD fellowships. We sincerely thank all the members of DB lab for their critical input on the review. DB thanks SERB-DST GoI for Ramanujan Fellowship, INYAS-INSA for the Young Scientist membership, and IIT Gandhinagar for startup funds.

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