Multifunctional carbon dots in nanomaterial surface modification: a descriptive review

The surface properties of nanomaterials are an important consideration in most scientific and technological applications. Several methodologies can maneuver these properties while surface modification is the most common technique. Carbon Dots (CDs) are viable competitive materials for their pacific environment, chemical inertness, tunable photoluminescence, low cost, eco-friendliness, biocompatibility, schematic surface functionalization, and sophisticated utilization in nanomaterial’s surface modification. The nanoparticle surface attribute is modified for a specific purpose to use in several applications by dint of the tunable properties of CDs. Multifunctional CDs have a great potential to replace traditionally toxic and costly quantum dots through surface modification. This review presents how multifunctional CDs conjugated with other nanoparticles take an active part in medicine and biomedical fields with chemical and physical collaborations. Moreover, the basics of conjugate formation by different chemical and physical interactions of functional molecules are appraised from multiple perspectives. This article also describes different modification mechanisms followed by properties of the modified nano-conjugates. The surface modification affects fluorescence quantum yields, complexation potential, fluorescent coloring, and quenching capabilities. Resultant-modified nanoconjugates are powerful surfaces for drug delivery, biosensing, bioimaging, analysis, and therapeutic methods. Finally, the most fruitful current challenges and further possibilities are discussed in the conclusion section. • Classified the modifications of carbon dots (CDs) surface. • Elaborated on the exceptional and recognized features of CDs. • Summarized the implementations of CDs for emerging applications. • Disclosed ongoing research, gaps, challenges, and potential scope of future work.


Graphical Abstract 1 Introduction
Carbon nanodots commonly known as Carbon Dots (CDs) with sizes in the microscopic region (normally 10 mm or less) possess several optical qualities with handsome biocompatibility and many such properties under development or soon to be revealed (Zhou et al. 2017;Hao et al. 2016;Simões et al. 2016).Impressively, steady photoluminescence at the visible range of wavelength in the aqueous solution is noticed when the light is emitted on the excitation wavelength of the CDs.For purposes like visualization, biosensing, bioimaging, drug delivery studies, analytical purposes, therapeutic methods, and photocatalysis as well as in terms of chemical inertness (Zhang et al. 2012), high solubility (Baker and Baker 2010), easy modification (Ray et al. 2009), and high resistance to photobleaching (Li et al. 2011a), CDs are promising aspirants in comparison to covalent organic dies, regular nanoparticles, and semiconductor quantum dots (Li et al. 2015(Li et al. , 2017a;;Das et al. 2014a;Anjana et al. 2018).Lately, much development has been successfully crowned in the process of preparation, exploration of chemical properties, and application of carbon-based quantum dots in therapeutic medicine (Li et al. 2012).Soon after the invention of CDs, they became the hot topic of discussion for their above-mentioned properties and applications in all possible sectors straight from the beginning.The preparation process is no longer a very tough and expensive one and can be easily dealt with for its very inert nature.Nevertheless, pristine CDs that have drawbacks in the procedure of detecting particles due to the presence of interference out of the absence of a specific recognition group for the analytes on CDs surface and low quantum yields in bioimaging generally cannot be conducted smoothly.They also have drawbacks of showing biological functionality as a biological system is poorly interacted (Xia et al. 2017;Zu et al. 2017).As a result, the CDs distort the nanomaterial surface integrated via covalent modification (amide coupling reactions, silylation, and other reactions including esterification, sulfonylation, and copolymerization), noncovalent modification (interactions, complexation/chelation, and electrostatic interactions), and sol-gel coordination (Mao et al. 2012).As a result, the modified CDs thus formed are significantly used in targeting and extracting several analytes and most importantly in drug release.These surface modifications extraordinarily enhance the internal and external properties such as fluorescence quantum yields, complexation capacity, the color of fluorescence, and their quenching capability in a qualitative manner (Yan et al. 2018).Ultimately, the features of Carbon Quantum Dots (CQDs) must be regularly revised and regulated by the precursors used during synthesis, surface passivation, and functionalization as acknowledged.For the accuracy in luminescence properties and development in novel applications, assumingly the most important aspect to be taken care of is modification/ manipulation of the surface of CDs other than only integrating and partially substituting or synthesizing CDs with new precursors.In this review article, we are going to discuss the different procedures to reorientate the nanomaterial's surface properties and utilize them accordingly for multiple functions in biological systems such as bio-imaging, bio-sensing, optical properties as photocatalysis, detection of metals, and many more.Farshbaf et al. (2018) noticed that the synthesis and analysis of CDs are normally easy and handy processes that employ handy and accessible materials.Moreover, ordinary chemical reactions are required for the modification of CDs.Researchers are attracted by the various size-dependent optical properties of CDs and employ these nanoparticles for extensive application in medicine and biomedical sectors (Farshbaf et al. 2018).
Particularly, this nanomaterial has had no abundancy in medical or technological applications till now with large-scale production facilities due to a miniature understanding of its structure-performance relationship, controllable luminescence (fluoro and photo-luminescence) properties, and characteristic relationships when coincides with a wide variety of chemicals trending now.So, this outlook will provide the functional modification processes that have been used in vivo or in vitro so far, opening a door to future application scaling with further research initiatives.The major surface modification processes that can replace the prior performances are explained with extensions.Endowing to the idiosyncratic features of CDs, the modified surfaces gain potentiality in photoluminescence, catalytic surfaces, cytotoxicity, biotoxicity, surface prevention, and many more properties to be used in medical science.Maturity in the modification and applications of CDs with other nanoconjugates reveal the effect of their vast sneaking properties for technological/medicine/chemical/mechanical/ hardware industries.There has been a plethora of research works on the conjugation of CDs and other nanomaterials making them viable for instrumenting biosensors, bioimaging, drug delivery processes, and anatomical analysis or screening.In summary, we accumulated most of the current research on different modifications of CDs, pathways of modified CD utilizations, and their future potentiality in health, technology, and science sectors from the year 2008 to 2021.This article is designed and compiled to learn how CDs can alter the functionality of any biochemical/chemical/technological determinants when their surface is modified with nanoconjugates.

Covalent modification
Among many chemical reaction-based modifications, the covalent modification of CDs is the result of a reaction between functional molecules and reducing groups on the surface of CDs.The chemistry behind the synthetic process is often used to control the size, passivation, and physical properties of CDs, which can aid in the light generation traps that split radiation when stimulated.On the other hand, non-covalent modification of CDs and modified nano agents that are established on π interaction or the van der Waals force have comparatively less negative impact than that of covalent modification, thus providing easily tunable photoluminescence and interfacial properties.The biocompatibility and optical properties are seen to be increased remarkably after the modification as we can see high quantum yield (QY) and tunable photoluminescence (PL) of CDs with amino groups (-NH 2 ) for π orbital and delocalized molecular orbitals of aromatic rings (Lim et al. 2015).Furthermore, the PL property is also enhanced for the virtue of surface energy traps and electron-hole recombination of the oxygenated functional groups on the CD's outer layer.This feature, however, can be enhanced by controlling the degree of oxidation on the surface of CDs.In addition, this tunable PL of CDs is useful in detecting and targeting a specific molecule, which is possible when drugs are altered in conjunction with the standard CD surface.It can, therefore, specify the sites of disease and release drugs in the drug delivery site which make CDs a significant carrier in drug delivery applications (Yan et al. 2016;Wang et al. 2011a;Fu et al. 2017).Surface modification can be classified into various types based on chemical reactions between distinct functional groups such as amide coupling, sialylation, esterification, sulfonylation, and copolymerization reaction.

Via amide coupling reaction
Typically, CDs are prepared by using oxidizing agents to treat organic intermediates that have a lot of carboxyl groups on the surface, which gives them exceptional water solubility, selectivity, and the ability to be modified.However, numerous carboxylic groups on the surface of CDs recombine electron-hole pairs, and emissive traps form as a result, affecting the fluorescence properties of CDs dramatically (Wang et al. 2016a;ChandanH et al. 2018;Huifang et al. 2017).
Condensation of carboxyl groups with amino compounds is commonly utilized as a surface modification technique to improve intrinsic fluorescence emission, according to Dong et al. (2015) who used a conventional EDC/NHS (N-ethyl-N-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide)-catalyzed reaction.Using glucose as a carbon source, it generated ethylenediamine (EDA) modified CDs-COOH via an EDC coupling mechanism.Due to the property of well-passivized strong linkage of CDs-COOH improved QY from 1.3 to 3.0% was obtained (Fig. 1).Since free amino groups were bounded with the surfaces of CDs, these modified CDs showed magnificent optical properties along with biological compatibility, hence it is signed in for microscopic imaging and biolabeling of human gastric carcinoma cells (Dong et al. 2015).
D' Angelis do E. S. Barbosa et al. (2015), on the other hand, synthesized CDs-COOH by chemical oxidation from cow manure (Fig. 2).Under nitric acid activation conditions, modified CDs surfaces were yielded through aggregation with EDA.The QY was found to be as high as 65% from the previous study of Dong's group; the modified CDs with EDA have good biocompatibility that can be used to label the nuclei of breast cancer cells MCF-7 with precision.Subsequently, they were used as a subcellular-specific live-cell fluorescence-imaging assay (D' Angelis do E. S. Barbosa et al. 2015).
Dopamine can participate in the amide coupling reaction to manipulate CDs-COOH and the phenolic moiety can be catalyzed to quinone compounds in the presence of the matching oxidant that can suppress the fluorescence of CDs after an alteration of CDs-COOH through the amide coupling process.Taking advantage of it, Chai et al. (2015) have taken activated carbon dust as a raw material to temper CDs-COOH and then changed dopamine via an amide coupling reaction to create new CDs that are used to monitor tyrosinase (TYR) activity (Fig. 3).Hence, under the particular catalysis of TYR, the dopamine molecule in CDs was oxidized to a dopaquinone derivative.Amid CDs and the dopaquinone moiety, there was an intraparticle photo-induced electron transfer (PET) mechanism, followed by the quenching of fluorescence of the newly modified CDs.Later on, for TYR/ pH-associated medical diagnosis and disease monitoring, this fluorescence assay was employed.Moreover, the catalytic activities of CDs can be enhanced by using β-CD as the dopamine receptor unit even after the competitive host-guest interaction between β-CDs conjugated target analytes and stain molecules.
Amino acid combining supramolecular compounds can also be connected covalently with CDs-COOH through an amide coupling mechanism.Cyclodextrin (β-CD) has been used as a host molecule for a unique molecular microstructure comprising a hydrophilic external surface and a hydrophobic internal cavity.Previous study designed fluorescence probes in which they used 6-aminoethyl amino-β-cyclodextrin to modify CDs with CDs-COOH via the EDC/NHS coupling mechanism (Sun et al. 2017).The structure thus formed can generate host-guest inclusion complexes combining with the guest molecules P-nitrophenol to quench the fluorescence of CDs and host molecule cholesterol to remove p-nitrophenol to initiate the fluorescence power output.This technique is very popular in distinguishing cholesterol from the blood serum sample with high efficiency in medical pathology (Fig. 4).
A very popular glycopeptide microbicidal, vancomycin (van) is sometimes used as a wide-ranging antibiotic.CDs can also be associated with the vancomycin group antibiotics to combat dangerous germs like Staphylococcus Aureus and Methicillin-resistant Staphylococcus Aureus (MRSA) for gram-positive staph infection treatment.The amide coupling reaction was employed by Zhong et al. (2015) to modify CDs by the combination of citric acid and carbon from urea along with the modifying agent van (Zhong et al. 2015).It is easy for these nano surface modified CDS to preserve CD properties, i.e., CDs conjugated with a van cannot cause a noticeable change in the fluorescence spectra of CDs.Identification of Staphylococcus aureus is led by the ligand-receptor interaction of van and the cell wall by the application of this modified nanoparticle (Fig. S1).Van can also lead to fluorescence quenching by forming a hydrogen bond with the surface of the gram-positive bacterium and the peptide D-Ala-D-Ala.It has been verified that there is a noticeable linear relation between the fluorescence intensity and the concentrations of S. aureus.Detection of gram-positive bacteria in a practicable initiative following this assay is in practice (Zhong et al. 2015).
A common bio probe that achieved high appreciation and attention in medicine and nanotechnology is fluorescence immunoassay because it possesses the particular benefit of high sensitivity fluorescence technique and Fig. 3 Molecular surface modification with CDs for determination of TYR via amide coupling (Chai et al. 2015) Fig. 4 Synthesis of the modified CDs and method of determining Cholesterol (Sun et al. 2017) high selective immunoassay.The traditional fluorophores such as Fluorescein amidites (FAM) and Texas red on the other hand are not so reliable and reasonable for longterm detection owing to their photobleaching effect.Mohammadi and coworkers showed the development of a selective Förster resonance energy transfer (FRET)immunoassay in order to detect sensitively CA 15-3 tumor marker (Fig. 5) (Mohammadi et al. 2018).Though this assay is a sandwich-type immunoassay, consisting of CDs-COOH and CA 15-3 antibody (by EDC/NHS coupling reaction)-as a fluorescence generator and gold nanoparticles (AuNPs) labeled poly-amidoamine dendrimer (PAMAM) aptamer as the suppressor.The rigid complex interaction of CA 15-3 antigen aptamer can be produced with the conjugation of AuNPs-PAMAMaptamer, which can result in adjacent CDs and AuNPs.The fluorescence intensity of CDs reduces when the FRET across CDs and AuNPs approach closer.However, further advantages of the assay include simple operation, rapid detection, low cost, the requirement of normal and affordable equipment, and be extended to determine biomarkers of various types.
Another study shows that nano-hydroxyapatite (HAP) or its analogs can be prudent multifunctional bio-nanoplatforms or efficient in building biocompatible scaffolds proving its successful biomedical application.Zhao et al. (2015a) using a hydrothermal route, employed monodisperse F-substituted hydroxyapatite (FHAP) nanorods and phosphoethanolamine (PEA) to make amino-terminated fluorine-substituted hydroxyapatite.Taking ethylenediamine carbon source and ClCH 2 COONa as modifying regent, CDs-COOH was prepared with citric acid.Then these modified CD nanomaterial surfaces were acquired through an amide coupling reaction by interfusing CDs-COOH and FHAP.They have been effectively used as bio-nanoplatforms to image MCF-7 breast cancer cells in vitro (Fig. 6).
Semiconductor nanoparticles CQDs with a radius in the range of exciton Bohr radium are mostly CdTe QDs, CdSe QDs, and CdS QDs.The surface modification  (Zhao et al. 2015a) allows them to link other fluorophores or biomolecules, which might be useful in chemical engineering and biomedicine.Xu et al. (2017) developed a silica shell encapsulating amino-passivated cadmium telluride (CdTe) CDs to strengthen the optical and molecular stabilities and minimize the toxic effects of CdTe QD.Through an EDC/NHS coupling process, CDs-COOH were mounted with CdTe QDs-NH 2 to generate nanosurface-modified CDs and these modified CDs were used for the detection of Hg 2+ .A filter paper-based assay was applied, which was non-toxic and made the identification of Hg 2+ quite fast and reliable (Fig. 7).

Via silylation reaction
Silane is a biocompatible non-toxic material that functions as a silica shell that encases two types of fluorescent components to create colorful, harmless fluorescent probes for the two types of functional groups.Silylation is the word used to describe the interaction between silane and functional hydrogen on the exterior of CDs.This strategy aims to enhance the CD solubility in water, selectivity, and lower the level of cytotoxicity while increasing the surface area to ensure excellent dispersion.It is very popular in ion detection and temperature sensing.
CDs-NH 2 was developed by undergoing a hydrothermal method taking acetic acid as the source of carbon by Rao et al. (2016) in the year 2016.Then through the silylation reaction, they managed to encapsulate CDs-NH 2 in a silica shell operating with tetraethyl orthosilicate (TEOS) and 3-aminopropyl triethoxysilane (APTES).A healthy mix of inactive and active functional groups, minimization of conglomeration, and indifferent bindings are the primary advantages of this modification method.Plenty of room for amino groups is noticed on the nanomaterial-modified CDs surface that can undergo EDC/NHS coupling reaction to get covalently associated with thioglycolic acid-modified CdTe QDs and thus associates with the neighboring carboxyl and CdTe QDs surface hydroxyl groups.This is how Cu 2+ ions can be detected (Fig. S2).
CDs@CdTe QDs progressively collaborate with families in the hybrid spheres system, causing the CdTe QDs' fluorescence to fade.CDs' fluorescence is unaltered even by the addition of Cu 2+ in the range of 0.1-1.0M with such a LOD of 0.096 M, but CdTe QDs' fluorescence is quenched.This technology is widely used in the detection of Cu 2+ in several fruits and spiked vegetables.
Amino passivated CDs were developed by Liu et al. (2014) taking raw material N-(β-aminoethyl)-γ aminopropyl methyl-dimethoxy silane (AEAPMS) and through a silylation operation, they were converted into Rhodamine B-doped silica nanoparticles, yielding CDs@SiO 2 .Since the main raw material (AEAPMS) that is used, contained methoxy group, saline groups, and the remaining ethylenediamine groups on CDs exterior, they could serve as Cu 2+ recognization sites, so these were used for the detection of Cu 2+ .Furthermore, in real-life, Cu 2+ in MCF-7 cells and actual tap water may be measured with this fluorescent probe.

Via esterification reaction
Synthesizing of CDs requires organic precursors that usually carry an ample amount of the hydroxyl group on their exterior.The esterification reaction can manipulate them in several routes effectively by interacting with different functional components and the nanomaterial's surface is modified accordingly.The modified CD surfaces have qualities such as good selectivity, reversibility towards targets, and good imaging, which derives an implication in the detection and cell imaging fields.Moreover, this strategy helps to change the CD's hydrophobic and oleophilic properties to lipophilic hydrophobicity.
A group of researchers prepared CDs-OH (Algarra et al. 2014) by taking lactose as a carbon source at a relatively low temperature and the quantum yield jumped to 46% directly (Fig. 8).These CDs were prepared using Fig. 7 The modification method of CDs via amide coupling reaction and the detection pathway for Hg 2+ (Xu et al. 2017) mercaptosuccinic acid (MSA) nucleophilic additionelimination process with the help of hydroxyl groups on the CD's surface.The MSA's thiol groups were potentially combined with Ag + to generate a ground-state complex; adding 0-30 μM of Ag + resulted in fluorescence quenching via static quenching.CDs-OH was used for the static quenching technique Ag + detection and further in the dissolution of silver nanoparticles.It could efficiently quantify Ag + ion.
A solid-state synthesis procedure of CD modification was developed by Lai et al. (2016) taking carbon from ammonium citrate along with the identification ligand folic acid (Fig. 9).The modified CD surface was generated by the dry heating method, where the folic acid microstructure was anchored with CD surfaces via a simple dehydration reaction.These CDs were therefore served in the labeling of tumor cell selectivity, because they have the property of high labeling efficiency and bright fluorescence for their selected targets.These modified nanosurfaces with CDs have wide applications in tumor tissue targeting and imaging that are confirmed by the selectivity level of HeLa cells.Soon after that, the research team demonstrated another probe of CD modified with Mannose in terms of general E. coli acknowledgment to mannose.Various nanomaterial component preparations such as carbon nanodiamonds, carbon nanotubes, graphene oxide nanosheets, and fullerene nanoparticles are some inspirations from this modification method as well as other kinds of biolabeling exercise with selective therapies.

Via sulfonylation reaction
Nevertheless, CDs that contain amino groups can be rearranged remarkably by surface modification employing sulfonylation reaction agglomerating with several sulfonyl chloride complexes.Upon adding sulfur atoms on the surface, the energy trap site develops, for photoexcited electron capture, the emissive trap states rise, and the modification range of the electronic composition of CDs is enhanced.Sulphonyl bonds are always  9 The modification mechanism of CDs via esterification reaction and the application of biolabeling (Lai et al. 2016) a water-soluble phenomenon that takes place in buffer condition of alkali solution significantly optimizing the variety in pristine CD's application and performance.Lin et al. (2018) employed the host-guest recognition principle undergoing a two-step sulfonylation reaction to develop the β-CD modified CDs-NH 2 .Generally, contingent on the unique molecular formation CDs-NH 2 is often employed in the determination of analyses of catechol (CC) and hydroquinone (HQ).Depending on the hydrophilic external surface and hydrophobic internal cavity, a rigid host-guest complex generates upon the entry of analytes into cavities of β-CD ensuring intensive fluorescence quenching.Furthermore, the CDs-NH 2 modified surface has an elevated water solubility level and reveals strong fluorescence.Therefore, CC and HQ in real water samples can be detected.
Besides, DN S -modified CDs-NH 2 has been prepared via sulfonylation reaction by Wang et al. (2015) (Fig. S3).In this process a precursor, phenylenediamines was employed to prepare CDs-NH 2 .The newly modified CDs produced afterward exhibited non-fluorescent behavior useful for the identification of selenocysteine (SEC).With the help of the nucleophilic substitution process, the DNs moiety of the modified CDs surface was readily cleaved by selenolate after a period of incubation in an aqueous solution at room temperature using SEC.This procedure increased the fluorescence properties dramatically owing to a modified compound Y-G-CDs.These modified CDs with the surface of nanomaterials are capable of high-level selectivity towards SEC compared with other bio thiols and other bio-species along with enhanced sensitivity.Therefore, this can efficiently help in L929 cells exogenous SEC imaging.

Via copolymerization reaction
CDs normally contain functional groups as active hydrogen and hence are modified by copolymerization reaction in cooperation with cyclic or double bond compounds.Bulky CD nanomaterials can be generated by this method.Li et al. (2017b) managed to prepare CDs-OH by taking carbon from cyclodextrin (Fig. 10).Then the nanomaterial surface modifications of CDs were performed by glycidol anionic ring-opening polymerization taking the hydroxyl group as an initiator.The outstanding solubility, biocompatible property, and extended circulation half-time in vivo made this dedicated hyperbranched polyglycerol (HPG) a highly acceptable compound for copolymerization modification.The HPG layer lessened the CD's energy traps, causing the declined QY of the modified CDs (1.2%) compared to that of pristine (1.5%).The emission peak of modified CDs showed mild cytotoxicity, strong water dispersibility, strong blue fluorescence, and improved hemocompatibility for which it is generally used for A549 cell imaging.Yuan et al. (2014) managed to prepare CDs-NH 2 by pyrolyzing the mixture of 4, 7, 10-trioxa-1, 13 tridecanediamine, and citric acid in glycerol (Fig. 11).To make fluorescence probe CDs, carbon disulfide was added to the amine groups on the surface of the CDs, then Cu 2+ and ammonium N-(dithicarbaxy) sarcosine (DTCS) were coordinated.The application of this modified CD was appropriate for the detection of Hg 2+ because of the strong fluorescence quenching by CuDTC2 via the process of energy transfer and electron transfer.The chelating ability of Hg 2+ was seemingly higher than Cu 2+ to dithiocarbamates, so an extra Hg 2+ (of 0.04-1.0μM) could develop stability in the molecular surface bond Fig. 10 Modification procedure of CDs via co-polymerization reaction (Li et al. 2017b) stiffness of CuDTC2 complex enhancing the fluorescence.Hg 2+ from real samples was identified by a whole complex fluorescence assay which is often reliable.The drawbacks of different covalent modification reactions associated with CD surface modification are enlisted in this study (Table 1).

Interaction reaction modification
When new functional groups association is necessary or the provision of new functional groups is evident, π interactions or electrostatic interactions between CDs and modified molecules help in targeting a different nanoparticle or molecule interfacial properties to provide an effective bridge at the junction of nanoparticle and biological systems interaction (Luo et al. 2013).The main advantage of this kind of modification is structural integrity that can be generated depending on the structure characters of different functional groups.

Via π interaction
An extended π system is located at some parts of CDs and when aromatic molecules are encountered, the surface can be modified with CDs through π interaction.These modified nanomaterials have high homogeneous fluorescence probes possessed by extended conjugation domain due to modification through π interaction.They have enhanced QY, photostability, and biocompatibility.
With the help of the microwave-assisted method, Jiang et al. (2016a) prepared Si-doped CDs (Si-CDs) employing glycerol and APTES as the initiating compound (Fig. 12).The π interactions among Si-CDs and dopamine on a one-to-one basis were practiced for the production of these modified surfaces.These modified CDs had a QY of 12.4% that was utilized for the determination of Ag + ion, due to the ultra-bright fluorescence emissive properties.In this media, Ag + can be reduced to nanoparticles of silver in contact with dopamine.Further applications of these modified nano-dots were exposed in assaying intracellular Ag + and HeLa cells for their salt stability, photostability, and lower cytotoxicity.
According to Li et al. (2011b), π interactions of CDs with DNA may be strategically employed as a binding scheme to detect any specific nucleic acid.As they have proposed so far, via π interactions fluorescence from dyelabeled single-stranded DNA probes can be quenched right after their absorption into the outer layer of CD to form double-stranded DNA by the target DNA and dyelabeled DNA accumulation.They highly enhance fluorescence recovery by falling off the CD's surface.This unique strategy is employed for the safe detection of metal ions  ii) Granting conditions.iii) Disproportionation reaction necessary.
i) The reaction is uncontrollable.
ii) The surface of the modified CDs is usually contaminated.

Via electrostatic interaction
Due to the functional groups such as carboxyl, amino, and hydroxyl on the surface of CDs, they usually inherit either a negative or positive charge.The fun fact here is that by the electrostatic interaction method, the charges of CDs can be interchanged.This kind of modified nanodots generate some targeting molecules and fluorophores that result in highly efficient implementation in several therapeutic/theranostic methods and bio-targeting.
By using carbon from natural carrot Jin et al. ( 2017) synthesized negatively charged CDs and then modified them with the help of opposite potential Nile Blue (NB) and polyethyleneimine (PEI) via electrostatic interaction to generate CD@TPF two-photon fluorescence nanocomposite.In the conjugated nanomaterial CD's aqueous solubility was enhanced by PEI.These modified CDs are employed in S 2− detection.In addition, these CDs are found to be highly sensitive and selective towards S 2− (Fig. 13).
In addition, electrostatically modified CD nanomaterials generally have specific targeting and binding properties of molecules, making it a potential candidate to be used in drug delivery systems.Recent findings by Sarkar et al. (2017) prove the control of cell proliferation in cancer cells for the binding capacity of estrogen receptors to 17β-Estradiol.They generated estradiol hemisuccinate (E2) out of 17β-estradiol which is a negatively charged compound.Side by side, using betaine hydrochloride and tris(hydroxymethyl) aminomethane the positively surface-charged blue-emitting CDs had been generated.Ultimately, a modified surface of CDs prepared through electronic interaction of noncovalently coupled blue emitted Cationic Carbon Dots (CCDs) with negatively charged estradiol hemisuccinate (E2).These CDs' functionality has been extended to include precise cell-based marks of estrogen receptor-rich (ER-positive) MCF-7 cells atop estrogen receptor-negative (ER-negative) MDA-MB-231 cells with non-cancerous Chinese Hamster Ovary Cells CHO cells.Indulging these CDs to the mammalian cell in vivo exhibited outstanding stability in the biological milieu.Therefore, minimal biological compatibility issues made it a useful drug carrier in vivo.Taking CDs loaded with the anticancer drug doxorubicin (dox) (modified by Sarkar and follow researchers) and following the delayed apoptotic pathway selective killing of (ER-positive) MCF7 cells is possible.It shows the efficacy of 2 folds more in comparison with (ER-negative) MDA-MB-231 cells and no-carcinogenic Chinese Hamster Ovary Cells or (CHO) cells.Furthermore, for cancertargeting theranostic agents, these surface-modified CDs are treated as research candidates of great potential.

Chemical complexation reaction modification
Complexation here refers to the conjugation of CDs and metal ions on the surface of the nanomaterials that sometimes take place for some definite outcome in a specific condition through a coordination bond.On the surface of CDs, molecules such as hydroxyl, amino, or carboxyl groups are present that take part in complexation with metal ions.When the hydrophobic part increases the stability of CDs in organic solvents, the amphiphilic part/ molecule gets attached to the surface of CDs through complexation and thus modulates the external and internal behavior of the CDs that are most advantageous in practical utilizations.
Unique ratiometric fluorescent nanoprobe CDs were generated by Chen et al. (2015), where they used ethylenediaminetetraacetic acid (EDTA) as the generation source of CDs and carried out complexation reaction of terbium in association with amino and carboxyl groups on the CDs surface (Fig. S4).CDs were successfully organized to work as podiums along with the retaliation unit of Dipicolinic Acid (DPA) as the fluorescence reference unit Tb 3+ .This kind of nanomaterial-modified CD surface is used normally for the detection of DPA.In the presence of two carboxyl groups and a pyridine group, the DPA can maintain very strong interactions with lanthanide ions via ion-pairing interactions and metal-ligand chelate coordination.DPA has a very low energy level in triplet conditions which makes it a suitable candidate to detect Tb 3+ by accurate energy transformation to the central metal ion from the ligand.Hence, the selectivity of this modified CD can be too much inclined to the DPA.Moreover, these modified CD surfaces are known to have quick and functional detection properties of several other bacterial spores and biomolecules.In addition, Wang et al. (2020) used EDTA as the generation source of CDs and carried out a complexation reaction of europium in association with amino and carboxyl groups on the CDs surface for successfully applying it as a biomarker for quantitative and qualitative (visual) assay of DPA in river water (Wang et al. 2020).
Another research shows the activity of nanoprobes generated by CD modified with a lanthanide, which is used for expansion in the application of bioimaging and other biomolecular identification.Shi et al. (2015) managed to modify and form a different kind of CDs-modified surface (CDs-NH 2 ) for magnetic resonance imaging (MRI) nanoprobe or dual model fluorescence imaging in the presence of PEI through low-temperature pyrolysis, Modification was continued through carbodiimide chemistry with cyclic Diethylenetriamine pentaacetate (DTPA) dianhydride and then gradually complexed with harmonious addition of Gd 3+ to obtain dual-modal fluorescence/MR imaging nanoprobe CDs (Fig. 14).As the Gd 3+ ions were only chelated onto the outer surfaces of nanoprobes the modified CDs possessed negligible cytotoxicity and enhanced longitudinal.These CDs were successfully applied in the imaging of HeLa showing improved sensitivity and upgraded MRI resolution with indications of future practical application in bioimaging and medical translation.

Sol-gel modification
A very useful method of modifying the surface of CDs is the sol-gel strategy.In the process, organosilane is employed as a coordinating solvent to create amorphous CDs that are extremely luminous (QY = 47%) in just one minute as illustrated in Fig. 15.The process is preceded for a complete minute at 240 ℃ through pyrolysis of anhydrous citric acid in N-(b-aminoethyl)-c-aminopropyl methyl dimethoxy silane (AEAPMS).The modified nanodot thus generated (with a diameter of 0.9 nm) was marked up with the methoxysilyl group.Moreover, when these CDs are simply heated (80 ℃ for 24 h.), they get converted into pure fluorescent films, popularly known as monoliths.Furthermore, CDs with hydrophilic silica encapsulation (CDs/Silica) can be generated from hydrophobic CDs that are biologically compatible and do not bear any sign of toxicity in the selected cell lines (Wang et al. 2011a).A molecular perception item CDs@MIP (molecularly imprinted polymer) was used in the same study to structure the dopamine (DA) Fluoroluminescence (FL) Opto sensor in the beginning.Then taking AEAPMS as an organosilane precursor undergoing a single-step hydrothermal reaction highly luminescent CDs were shaped.After that, the CDs@MIP composite could be easily separated from the anchored MIP matrix.The obtained combination of CDs and MIP revealed high template selectivity and photo-stability in DA detection via the sample's FL intensity module.

Photoluminescence
A fascinating property of CDs is photoluminescence (PL), also termed size-dependent optical absorption bearing a classic sign of quantum confinement.Further analysis and elucidation about the exact modus operandi of PL are necessary owing to the inconsistent and controversial findings in the existing literature (Zhao et al. 2008).Emission wavelength and intensity is the key feature for the behavior of the PL to be taken for granted on account of diverse emissive traps in CDs outer layer and most importantly nanoparticle size.It was asserted by Zhao et al. (2008) that compared with nanoparticles having a similar size and particle characteristics, the influence of CDs PL on the wavelength of excitation range was tailoring more emissive trap sites.Moreover, the optical behavior of these CDs/CQDs is determined by the dispersion of the diverse emissive sites.As Liu et al. (2009) stated that the emission of surface energy passivation due to the presence of energy traps on the surface, is an attribution to enhancing the CD's photoluminescence.They further assumed that emissive energy traps have a quantum confinement effect, which is present on the surface and is accountable for a powerful exhibition of PL when the surface is passivized.To achieve strong PL emission on modification of the nanosurface, the CDs generated by the supported approach required surface passivation (Liu et al. 2009).This report showed that colorful PL was emitted from CDs due to surface energy traps which eventually emitted strongly in action for the dispersion of emissive trap sites (Fig. S5).
Again, Zhu et al. (2018) structured CD/PVP (polyvinyl pyrrolidone) composite films that made white lights quite visible with photoluminescence Quantum Yields of 15.3%.Then it was confirmed that the blue/green/red emissions originally came from three different emitting states namely the intrinsic state, C = O-and C = N-related surface states, respectively.

Up-conversion photoluminescence (UCPL)
Until the last couple of years, the UCPL of CDs has not yet been yet a much noticeable discovery but recently due to the application of up-conversion fluorescence matter in bioimaging, it became a frontline research topic.The property of CDs that is considered the main reason for UPCL is simply the multi-photon activation process that is at its best level after being conjugated with other nanodots.When two or more photons are absorbed at the same time, they emit light with a wavelength shorter than the excitation light, allowing two-photon luminescence microscopy to be used for cell imaging.Excitation by a femtosecond pulsed laser for two-photon excitation within the N-IR region (800-840nm) or by an argon-ion laser results in significant emission in the visible area (Cao et al. 2007).The representative two-photon luminescence spectrum in such CDs has been used to demonstrate their UCPL features (Fig. S6).
A recent investigation shows that the electrochemical module aided by alkali excels in exhibiting UCPL features.However, on differently modified CDs and raw CDs prepared from different processes, it is confirmed from the Fluoroluminescence (FL) spectrophotometer that CDs did not exhibit observable UCPL.According to these findings, normal FL is the derivative of UCPL, since it is triggered by a leaky component from the second diffraction in the monochromator of the FL spectrophotometer.By installing a long-press filter in the excitation lane, the leaking component and subsequent UCPL can be eliminated from the FL spectrophotometer.Coupled with some other findings, it is assumed that rather than many photon pathways, UCPL is pure fluorescence with a linear response.A very critical step in this detection of up-conversion FL is to eliminate the normal fluorescence (Wen et al. 2014).

Cytotoxicity
In the last few years, a wide range of studies have been conducted place regarding the production of CDs-based bio-probes with high stability, owing to the deep necessity of their application and functionalization in live tissues, cells, and animals in biocompatibility.Recently, both functionalized CDs and pristine CDs have been assessed systematically for their cytotoxic effects, and the results often disgruntled researchers.Yang et al. (2009) prepared CDs by arc discharge of graphite rods that afterward were outflowed with HNO 3 for half a day.Evidence of safe unmodified dose of CDs was found to be only up to a level of 0.4 mg/mL.In another output, it came out that CDs did not show any effective mark on cell viability when a human kidney cell line was employed for the evaluation of electrochemically prepared CDs.
A very improved soot-based method was employed by Ray et al. (2009) for the preparation of CQDs with a diameter of 26nm that had insufficient cytotoxicity at desired concentrations of Fluoroluminescence bioimaging.The CDs were examined after being modified with functional groups such as PAA (polyacrylic acid), BPEI (branched poly ethylenimine), PEI, and Polyethylene Glycol (PEG) (Yang et al. 2009;Wang et al. 2011b).The PEG-modified CDs with all available sizes were found to be safe and biocompatible at any concentration necessary for utilization in every sector including cell imaging.In addition, the PEG1500N-modified CDs were tested in mice in vivo and the result exhibited no substantial harmful effect up to 28 days (Qi et al. 2009).Furthermore, under relatively high concentrations, CDs functionalized with poly-propionylethyleneimine-co-ethyleneimine (PPEI-EI) were significantly nontoxic toward cells.CDs changed with Polyethyleneimine (PEI) were more hazardous than PPEI-EI-modified CDs leading to excessive ethylenimine (EI) units in the PEI.Moreover, when administered at low doses, both pure PAA and PAAfunctionalized CDs proved toxic to cells, at only 50mg/ mL, as per the experiment result.Functional groups with high cytotoxicity as Bi-Propionlylethyleimine (BPEI) can be applied to functionalize CDs even in extreme cases like small concentrations or in a short incubation period, which would be extremely dangerous.
Notwithstanding their photoactivity, it is unknown if cytotoxic effects are valid for photo-induced degradation or if the degradation of CDs happens when illuminated.According to the findings of Yue-Yue et al. normal (HEK-293) and malignant (HeLa and HepG2), human cells are both hazardous to CDs treated with light that degrade into molecules.They tested different concentrations (0, 10, 30, 100, and 300 mg carbon/L) of CDs that had been irradiated with white fluorescent light (60 mol photons/ m 2 /s) for 0.5 (i0.5-CD), 1 (i1-CD), 4 (i4-CD), and 8 (i8-CD) days on three different cell lines (HeLa, HepG2, and HEK-293).Coincidently, CDs that had not been irradiated (N-CDs) came out to be viable the most.The cell viability is seen to increase in HEK-293 cells as the concentration of CDs increased.When HeLa, HepG2, and HEK-293 cells were subjected to the greatest concentration (300 mg carbon/L) of i8-CDs, their survivability dropped to 91, 49, and 90%, respectively.It was assumed that during the irradiation, cytotoxic photolyzed particles may emit or that reactive oxygen species may be produced on CDs surface that leading to light-induced cytotoxicity of CDs (Liu et al. 2021).

Electrochemical luminescence (ECL)
A parameter that is normally used for the measurement purpose of fluorescence emission of semiconductor nanocrystals is Electrochemiluminescence (ECL) (Qi et al. 2009).Coincidentally, the ECL behavior of CDs and QDs (CdSe) is similar.The ECL procedure of CDs is as follows: firstly, CDs can be produced in two states, oxidized (R + ) and reduced (R − ).Secondly, after the elimination of two oppositely charged carriers (R + and R − ) by electron transport, an energized state (R * ) is formed.Finally, through emitting a photon by a radiative pathway, the CDs in the R * condition (excited) have returned to ground level.The R + was found to be more stable than R − owing to a lower cathodic ECL response than the anodic one.Moreover, over time, ECL response has acquired much stability and got employed in several research fields.Strong ECL emission was recorded from CDs produced by electrochemical oxidation of graphite, during cyclic potential between + 1.8 and -1.5 V (Zheng et al. 2009).Research reports unveil the potential use of ECL emission in CDs.Surface states were termed to be the core point of most ECL exhibited in semiconductor nanomaterials and subsequently found to be redshifted in comparison to the peak of photoluminescence.Because ECL is mostly connected with nanoparticle surface state transitions, it is mostly used to study the surface traps that could be implemented in the comparison of PL and ECL before or after the surface modification.

Electrochemical performance and electrolytes used for CDs
When it comes to the electrochemical application and performance of CDs, a number of their good qualities can be underlined, including electrical conductivity, a variety of electrochemically active sites, an extensive surface area, compatibility with different materials, strong plasticity, and environmental durability (Hoang et al. 2019).
For instance, Niu et al. (2018) employed one of the CDs as nano conductive agents called graphene quantum dots (GQDs) instead of regular carbon black (CB) which showed great potential for the creation of an efficient conductive network to increase the specific capacitances of super capacitors.It is possible to build different shapes of electrodes and reduce the transmission paths of ions due to the nanoscale diameters of CDs.Different functional groups (hydroxyl, carboxyl, amine, etc.) on CDs can provide greater capacities, allowing them to be used with various electrode production techniques (inkjet printing) (Zhang et al. 2019).Furthermore, by expediting intermolecular electroconductivity, these large functional groups can provide a significant number of sites for surface modification to improve electrocatalytic activity (Tian et al. 2021).Oxygen reduction reaction (ORR), alcohol oxidation reaction (AOR), oxygen evolution reaction (OER), and hydrogen evolution reaction (HER) are some examples of electrochemical processes in which CDs can significantly improve electrocatalysis (Jana et al. 2020).Table 2 compiles information obtained from a comprehensive body of literature on the electrochemical performance of CDs and their derivatives for supercapacitors.

In drug delivery
According to many researchers, AuNPs are found to be the most studied nanoparticles in drug delivery systems (DDSs).However, owing to their less ideal biocompatibility and cytotoxicity, their pathological applications are bounded.CDs on the other hand, having high aqueous degradability, praiseworthy biocompatibility, and flexible modification properties in and out of cells, are a superlative competitor in replacing AuNPs for drug delivery purposes.Carbon nanodots have gained much appreciation because of their capacity to serve as a foundation for attaching to several drugs and ligands.In cancer research, CDs are widely used for exposing receptors in imaging and drug delivery for specific cells.CDs are found to inhibit human insulin fibrillation because of their non-toxic nature.It has been found that CDs can resist HepG2 (a liver cancer), MCF-7, and MDA-MB-231 cancer cells (breast cancer) growth in several cases happening as a result of a large number of reactive oxygen species (ROS) generated (Yang et al. 2016a).CDs-Dox (Doxorubicin) complexes functionally persuade apoptosis in adenocarcinoma cells of human lungs as found by Yang et al. (2016b).Dox on the other hand is normally taken for lymphomas, sarcomas, and leukemia.When they investigated the efficiency of both Dox and CDs-Dox, the in vivo efficiency was found to be way higher in the case of CDs-Dox.Further studies proved the ideal drug release profile of CDs and Dox following the first-order release kinetics at slightly acidic pH.Moreover, in normal cells, the toxic effect declined due to receptor-mediated delivery (Wang et al. 2016b).Wang and his colleagues found that the fluorescence of Dox and CDs was put off in a conjugated form, which was put back when they separated within the tumor cell.This can be observed under a confocal microscope (Fig. 16), which directs to the fact that the most dependable measure to release the attached conjugate system is pH-dependent release.Paclitaxel and hyaluronan conjugated CDs expectedly showed the tumor cell dispatching capability; the CDs in the study were imaged and progressed by using near-infrared light, and subsequently, paclitaxel was released in a pH-dependent manner.

Electrolyte
CDs-Dox-transferrins conjugation is used to indicate tumor cell death for pediatric patients compared to the free drug alone along with bypassing the blood-brain barrier.In addition, for bioimaging purposes transferrin is quite remunerative owing to its natural capacity to shift iron, which results in a cleaner tumor outline in MRI reports, aiding in surgical resection, particularly when magnetic CDs are conjugated together (Łubgan et al. 2009).Folic Acid (FA) exposes higher potential in detecting cancerous cells while CDs are functionalized with FA devolving endocytosis at a greater frequency compared to ordinary cells.FA readily conjugates with carboxylrich CDs due to having amines attached to aromatic rings along with several carboxyl groups.The mechanism uses 1-ethyl-3-(-3-dimethyl aminopropyl) carbodiimide hydrochloride/(N-hydroxysuccinimide) (EDC/NHS) chemistry.Hyaluron-conjugated CDs are often equally functionalized for drug and gene carriers.The toxicity in CDs is surprisingly decreased and characteristics are maintained greatly by the conjugation.
Another research on gemcitabine (Gem) conjugated CDs showed positive outcomes.Patients with mesotheliomas, pancreatic malignancies, cervical cancers, ovarian cancer, lung cancer, or other complicated situations are usually administered (Gem).CDs impede the growth of tumors as well as exhibit prevention of metastasis after successfully being conjugated with Gem making a complete full-proof drug delivery vehicle (Yang et al. 2011).
Chung et al. ( 2020) created a carbon dots/hydroxyapatite (CD-HAP) nanoparticle as an acetaminophen drug carrier (mild pain killer).CDs were made here using the hydrothermal approach from a bio-waste precursor, Fig. 16 Confocal microscopy demonstrates that Doxorubicin is released from CDs-Dox conjugate at 6 h post-treatment inside the cell, wherein, the conjugate regains the fluorescent capacity; the scale bar is 20 µm (Wang et al. 2016b) sugarcane bagasse char.In recent years, scholars have focused their attention on the combined application of CDs and HAP.The HAP-decorated CDs (CD-HAP) exhibit good biocompatibility and solubility, as well as an enhanced luminous characteristic.Because of their qualities such as controlled drug release, beneficial trackable drug release, and excellent targeting ability, they are commonly used in the pharmaceutical industry as quality drug delivery vehicles (Chung et al. 2020).The surface area of the sample mainly had a large impact on drug loading capacity.The acetaminophen loading is easier and has higher adsorption capacity when the surface area is bigger.Hence, CD-HAP-40 implies the best loading performance as it has the highest surface area.The loading of acetaminophen onto the CD-HAP happens through physical surface adsorption (Fig S7 ).Zheng et al. (2014) reported the production of CD-Oxaylation (CD-Oxa) by fabricating fluorescent CDs with oxaliplatin, an anticancer drug where the carboxyl groups of oxaliplatin derivatives combine together in a condensation process along with amino molecules on the CDs surface enhancing its effectiveness to cancer resistance.In the reducing environment (hepatic cancer cells), the drug is released as demonstrated in the case of assynthesized CD-Oxa.Moreover, it is proved that CDs are biocompatible with ordinary fibroblast cells; on the other hand, CD-Oxa has issues with hepatic cancer cells for the cytotoxic effect.For both in vivo and in vitro operations, the research team utilized CD-Oxaylation as an imageguided delivery of drugs.They observed that CD-Oxa injected intratumorally successfully killed tumor cells in the hepatic tumor cell of xenograft tumor mice models that assisted in diminishing tumor size without altering the surrounding environment.Using data on the distribution of the CDs-Oxa by monitoring the FL signal of the CDs, the proper dosage and timing of medicine can be perfectly traced.
On consequent practice, Feng et al. ( 2016) fabricated image-guided drug delivery centered on the precursor's molecules (citric acid and diethylenetriamine) using cisplatin (IV) pro-drug-loaded charge convertible CDs [CDs-Pt(IV)@PEG-(PAH/DMMA)].Further functionalizing of CDs with PEG-poly-(allylamine) and polydimethyl maleic acid (PEG-(PAH/DMMA)) resulted in the generation of charge convertible CDs.The demonstration in vitro signified the high therapeutic capability of charge-adaptable nano-carriers in comparison with normal physiological conditions.Their reports also suggested that for the xenograft mice model, intravenous administration of the nanoformulations showed higher tumor cell suppression capabilities with less systemic toxicity.Thakur et al. (2014) reported CDs with antibiotics and Gum Arabic (GA) as a prelude via a microwave-assisted method, which was used as a theranostic agent for controlled drug release, enhanced antimicrobial activity, and clear bioimaging.In their study, a wide-spectrum medication, ciprofloxacin hydrochloride was delivered to patients via CDs (Cipro@CDs), a synthetic CD that was affixed to the surface.In comparison to (1.2 mM) unconfined ciprofloxacin, Cipro@CDs showed good biocompatibility in that physiological conditions controlled the ciprofloxacin released from CDs.This conjugation of Cipro@CDs was also used to observe units of yeast live with the fluorescent microscope as it showed much precious antimicrobial effect opposing both gram-positive and gram-negative microorganisms.
Later, Liang et al. (2020) eventually came up with a pH serial dual sensitive drug delivery system that was contrasted by three different nanoparticles, such as chitosan (CS), CDs, and hyaluronic acid (HA).Finally, mesoporous silica nanoparticles (MSNs) were prepared.The outcome of their experiment indicated that the doxorubicin (DOX) (the model drug selected for their study) could be discharged from MSNs when glutathione (GSH) and pH are serially boosted.Low toxicity and high drug loading efficiency were found when the conjugation of MSN-CS@HA-CDs NPs was utilized (up to 32.5%).This nano platform proved fetal for the liver cancer cells along with high target ability.This research successfully initiated the era of a desirable anticancer drug delivery System.

Biosensors
CDs are a very promising candidate for biosensor agents due to their high photostability, excitation-dependent multicolor emission, good biocompatibility, good water solubility, superior cell permeability, and finally flexible surface modification capability.This property of CDs could be utilized widely in the monitoring metals and ions in biomedical sectors like the presence of iron, and copper along with the detection of several nucleic acids and their pH levels.Through π-π interactions of CDs as fluorescent quenchers based on adsorption of the fluorescent single-stranded DNA (ssDNA) probe, nucleic acid biosensing can contribute to biosensing science.A target DNA can be detected by this mechanism.For that, a double-strand DNA (dsDNA) is synthesized from the hybridized ssDNA with its target.From the surface of CDs, fluorescence is emitted with the desorption of replicated dsDNA which is the key factor for the sensing of target DNA (Li et al. 2011d).
For sensing and imaging mitochondrial H 2 O 2 , a powerful and versatile Fluorescence resonance energy transfer (FRET) probe based on CDs was used.CDs served as both the carrier and source of energy for the detecting mechanism.The further experiment ended up in the fabrication of a nanoprobe by a mitochondrion binding site ligand triphenylphosphonium (TPP) and a pharmaceutical formulation intermediate structure and PFI, a versatile H 2 O 2 identifying segment, which was covalently linked with the probe into CDs (Fig. 17).Then PFI moieties on CDs transformed space in presence of H 2 O 2 , a FRET-based ratiometric probe for H 2 O 2 was generated as a nano-platform (Du et al. 2014).Qian et al. (2014) discovered the use of hydroquinone and SiCl 4 -derived CDs as Fe 3+ , H 2 O 2 , and melamine sensors.Between Si-doped CDs and H 2 O 2 , the electron transport mechanism immensely helped in the determination of H 2 O 2 .On the contrary, Fe 3+ was determined to undertake the operation of both electron and energy transfer principles.Following melamine addiction, the stabilization of adducts between H 2 O 2 and melamine regained the fluorescence and so served as a sensitive detector for melamine.Dong et al. (2012) successfully demonstrated a novel system for the determination of Cu 2+ ion centered CDs modified with BPEI or simply (BPEI@CDs).Surface amino groups of BPEI-CDs would capture the Cu 2+ ion from the complex, which results in CD fluorescence quenching by the inner filter effect.The intricate structure was found reliable, fast, and of high selectivity for Cu 2+ ion recognition as low as 6 nM of concentration, and eventually at an effective range of 10 to 1100 nM.These CD-based new strategies made pathologists not only rely on the use of QDs and organic dyes.They are now capable of applying meteoric, to-thepoint, cheap, and eco-friendly strategies.

Bioimaging
The qualities of its aqueous solubility, superior photobleaching resistance, lower toxicity, and distinctive luminescent nature make CDs a promising candidate for bioimaging applications.Yang et al. (2018) managed to synthesize F-doped CDs from the combination of sodium fluoride, urea, and citric acid.They experimented with Fig. 17 FRET-based ratio metric sensing of mitochondrial H 2 O 2 in living cell by the nano-probe (Du et al. 2014) imaging in both in vivo and in vitro conditions.CDs fluoresced red when the cytoplasmic glioma C6 cell line was stimulated at 530 nm.The group of researchers took a nude mouse-bearing xenograft tumor for in vivo assessment and observed the 530 nm excitation and 600 nm emission wavelengths for whole-body fluorescence.Due to the EPR effect, the CDs were most likely to accumulate within the tumor region.Yang et al. (2009) manufactured PEGylated CDs for in vivo imaging systems of various organs, such as the liver, kidney, and bladder.Significant contrast was observed in the image of PEGylated CDs, which were subsequently injected into the mice model.A strong fluorescent image of the injected CDs was formed in vivo that later concerning their biocompatibility, would offer a wide application range in bioimaging and pathological studies (Fig. S8).
The thermal degradation of ultrafine water-dispersible Gd(III)-doped CDs with a dual MRI/FL feature was recently revealed by a group of scientists who made a revolutionary change in bioimaging with MRI (Bourlinos et al. 2012).When compared to commercial Gadovist, the produced nanoconjugate showed brilliant fluorescence in the visible spectrum and T 1 -weighted MRI contrasted dramatically with negligible cytotoxicity.As a result, the Gd-doped CDs could be used in biomedicine for multifunctional image analysis.In addition, MRI and Fluorescence modalities can be used in tandem to facilitate picture analysis.
Another investigation used the pyrolysis of complex organic molecules as a starting substrate to make (Srivastava et al. 2012) iron oxide doped CDs (IO-CDs) in the presence of microscopic Fe 3 O 4 nanoparticles for MR/ FL multi-imaging.FL signals in spleen slide samples and contrast-enhanced MRI images were observed using both T1 and T2 mice models after intramuscular infusion of IO@CDs.The nano-composite, on the other hand, was biocompatible and showed no signs of cytotoxicity.
Qian et al. ( 2014) created 20% QY silicon-doped CDs (SiCDs) and utilized them in imaging HeLa cells.Excitation-dependent fluorescent behavior, superior photobleaching resistance, and negligible cytotoxicity towards HeLa cells were all exhibited in the SiCDs.The cell proliferation was shown to be greater than 80% at concentrations of up to 100 g/mL of SiCDs.The cytoplasmic portion of HeLa cells was tagged with SiCDs.The segregated sp 2 carbon clumps and defective phases well within the crystallographic core were the sources of CDs' fluorescence.Due to the creation of a greater interface region and more discrete sp 2 carbon clumps inside the graphitic core, Si doping increased the fluorescence nature of CDs.Jiang et al. (2016b) investigated the use of Si-doped CDs coupled with dopamine (Si-CDs@DA) for imaging HeLa cells and assessing intracellular Ag + .Most importantly, up to a concentration of 100 g/mL, Si-CDs@ DA was reported to be nontoxic to HeLa cells (Jiang et al. 2016b).

Analysis
Several suggestions have been offered, but scientists in pathology investigations are still struggling to identify the actual mechanism for CDs' chemo-sensing of diverse substances.It is promising that after the practice of modification of CDs for -CvO -OH, -COOH, -NH 2 , etc. enriched surface CDs can build coordination complexes with electron-deficient entities by donating their lone pair of electrons.
Complexation is mainly responsible for the change in color and aggregation.With the addition of Fe 3+ , colorless banana peel-derived N-doped CDs became yellow which was described by Atchudan et al. (2020).They proposed that due to the development of a complex between surface constituents, such as -COOH, -OH, -NH 2 , this color change is evident (Fig. 18).The complexation is proved by the Fourier transform infrared (FT-IR) analysis that displays the shift of -COOH, -NH 2 , and -OH bands towards higher wave numbers when reacted with Fe 3+ ion.
The movement of an electron from the LUMO (lowest unoccupied molecular orbital) of CDs to the reagent is frequently alluded to as electron transfer-oriented sensing/analysis by CDs (Omer et al. 2019).In addition to the ferric ion solution, N-doped aminophenol-based CDs in a colorless solution appeared purple.The attribution of this color shift to the transfer of an electron from the LUMO of CDs to the d-orbital of the Fe 3+ ion (Fig. S9) has been developed by electrochemical investigations based on cyclic voltammetric measurements.
Oxidation-reduction processes are used in the colorimetric detection of numerous chemical and biological entities.Zhao et al. (2015b), for example, described the fading of the yellowish asparagine-based silver nanoparticles (AgNPs) topped CDs due to a redox reaction amongst AgNPs and S 2− in the aqueous phase premised on UV/Vis.spectral and zeta potential measurements.CDs serve as a stabilizer for reductants in this situation.The reaction is given below: CD scaped with metallic nanoparticles has also put great emphasis on metal sensing.Studies indicated colorimetric detection of thiocyanate ions using a detection device comprised of hydrothermally generated CDs from histidine in PBS-buffer maintained Au-nanoparticles since this probe demonstrated a blue to pink optical change for SCN − .The amino group's nitrogen atom created a link for the attachment of AuNPs to the CD membrane (AuNPs-CDs), and the color shift was due to the interaction of S of SCN − with this Au particle (Zhao et al. 2015b).
Ascorbic acid-based CDs have been implemented for colorimetric pH determining of HCl-NaOH oriented system.As the pH range shifted from 3.5 to 10.2, a gradual color change was noticed from grey to brown (Pedro et al. 2014).Further investigation led to the development of a pH-measuring calorimetric probe founded on 1,2,4-triaminobenzene-derived CDs that showed a noticeable visual effect from purple-red to orange, then yellow, for pH 3 to 13.The progressive color change is caused by the gradual ionization of the -OH and -COOH groups of CDs to O-and COO-when the primary intensity of sensing media rises.
Paraxon (an Organophosphate-OP) has been noted to range in hue from colorless to yellow, employing folic acid-based calorimetric probe-derived CDs with DL of 0.4 µg mL −1 (1.45 nM) (Wang et al. 2018).On the other hand, in Fructus Lycii-derived CDs tipped with silver NPs, a yellow to red visual shift for phoxim another organophosphate (OP) is detected, with a limit of detection (LOD) value tenfold lower (0.04 M) than that of the preceding one.The evident reason for this is that in acidic media, phoxim containing cyano and ethoxy functionalities attain a positive charge; contrastingly, in the same medium, moieties in CDs containing -OH, -COOH, and -NH 2 become negatively charged.
Another research showed the identification technique of cymoxanil (a fungicide) colorimetrically by employing a CDs-AgNPs system possessing a very small determination limit of 3nM (Jiang et al. 2019).A determination process similar to that of phoxim was also observed in this case.The aspiring positively charged group of cymoxanil was -NH 2 at optimum pH as AgNPs stabilized with citrate ceaseing to be oppositely charged group.This phenomenon finally resulted in two opposite-charged moieties' electrostatic interactions that were observed by the redshift in absorption spectra.

Therapy
Recently CDs have been used in many therapeutic processes induced by photodynamic effect and because of their biocompatibility, greater hydrophilicity, and controllable photoluminescence, hydrophobic photosensitizers can be delivered into cells.
Modified nanosurfaces conjugated with CDs such as glycol-coated CD/MnO 2 nanocomposites were fabricated by Chen et al. (2018) in 2018 in the tumor microenvironment (TME) for pH/H 2 O 2 -responsive PDT (photodynamic therapy) effect where the Hypoxia, Acidosis, and H 2 O 2 levels were increased.In an acidic H 2 O 2 (pH 6.5) solution, MnO 2 catalyzes the breakdown of H 2 O 2 into O 2 resulting in the formation of O 2 under hypoxia upon light radiation that finally ends up demonstrating a fine PDT efficiency in comparison to the neutral condition.Hela cell lines and 4-T1 tumor-carrying mice likewise showed a low pH-dependent PDT effect and excellent output was guaranteed only due to the reason of this nanocomposite polyethylene glycol (PEG)-coated CD/MnO 2 .
Previous study created NIR-triggered photodynamic and photothermal therapy in association with chemotherapy (DOX loaded) using magneto-fluorescent carbon dots coupled with folic acid and riboflavin (GP-Rf-FA-FeN@CDs) (Zhang et al. 2017).In this study, the PDT performance of the nanoformulations was noticeably increased solely due to the photosensitivity of CDs triggered by the conjugation of riboflavin.An excellent exhibition of NIR light absorption by GP-Rf-FA-FeN@ CDs-DOX is a notable part of the study run by Zhang and his coworkers.Moreover, due to the photothermal heating ability, GP-Rf-FA-FeN@CQDs-DOX was utilized for NIR-responsive drug (DOX) release.The latest synergistic chemo phototherapeutic effects of GP-Rf-FA-FeN@CDs-DOX resulted in a safe trouble-free drug delivery system for cancer treatment.
Synthesis of CDs-Ce6-HA, hyaluronic acid-modified CDs conjugated with chlorine-Ce6 (Ce6, a photosensitizer) increased the chance of suppressing melanoma cells in tumor mice.In this study, Beack and his colleagues targeted this therapy for skin cancer treatment (Beack et al. 2015).It is noticed that Ce6 in conjugation with CDs increased the photodynamic reaction of Ce6 resulting in more singlet oxygen-free Ce6.Under laser irradiation, transdermal application of CDs-Ce6-HA managed to suppress B16F10 melanoma cells effectively in vivo.Yang et al. (2016a) then managed to produce Mg/N doped CDs to use as a carrier of Ce6, which increased the PDT effect, subsequently followed by a high FRET efficiency.However, the study showed the production of singlet oxygen twice for the case of Mg/N doped CDs allowing for a significant enhancement in the PDT effect in HepG2 cells compared to pure Ce6.Zhang et al. (2020) prepared a new nanomaterial combining PDT/starving therapy/photothermal therapy (PTT) and checkpoint-blockade immunotherapy to enhance tumor treatment.They formed unique immune-adjuvant nano-agents (γ-PGA@GOx@Mn, Cu-CDs) by integrating the gamma-glutamyl transferase (GGT) enzyme-induced cellular uptake polymer-poly (γ-glutamic acid) (γ-PGA), self-supplied oxygenator nanodots and finally a glucose-metabolic reaction agentglucose oxidase (GOx), Mn along with CDs doped with Cu as a photosensitizer.γ-PGA@GOx@Mn, Cu-CDs nanoparticles (NPs) are likely to target the cancer cell after completing the long-term retention period at the tumor acidic microenvironment.Under laser irradiation, this NP exhibited both photothermal and photodynamic effects.Interestingly enough, the harmful by-product hydrogen peroxide (H 2 O 2 ) from the nanoreactors would deliberately perish tumor hypoxia following the increase of in vivo PDT.The treatment efficiency was significantly enhanced by combining the NPs-based starving-like therapy/PDT/PTT and check-point-blockade therapy.
Gadolinium (Gd) has long been utilized as a contrast agent and radio-sensitization enhancer in MRI.The Gd ions released by complexes build up in the body and are unable to be digested.By blocking calcium channels, Gd ions can induce significant biological damage, including nephrogenic system fibrosis in patients with renal failure.A group of researchers generated Gd-doped CDs employing simple hydrothermal carbonization of gadopentetic acid (Gd-DTPA) as the gadolinium source and glycine as the interfacial agent.Gd-doped CDs could reduce Gd leakage even under extreme biological circumstances due to the nontoxicity of carbon cages.These multipurpose theranostic nanostructures with effective metabolic pathways in vivo and better longitudinal relaxation efficiency have a huge potential as MRI contrast agents.More crucially, Gd-doped CDs operate as radiosensitivity boosters, revealing a novel way to overcome solid tumor radiation (Du et al. 2017).

As electrodes/supercapacitors
Because of distinct electrical characteristics and their vital role in hosting various functional groups on the surface, CDs, one class of CQDs, have drawn more interest in the field of energy storage.Recently, CDs have gained popularity either functionalization by additive or derivative material in electrode materials, enhancing the energy density of supercapacitors.Researchers from all over the world are trying to show a great potential of CDs as an application in electrochemical supercapacitors (Xiao et al. 2021).For example, Chen et al. (2016) successfully applied C60 powder as a precursor to producing CDs which is a amorphous microstructure (size < 10 nm), and UV-Vis-NIR indicated that the two picks (~ 260 nm, ~ 206 nm) demonstrated the n-π* transition (C = O) and π-π* evolution (aromatic C = C) respectively to validate the perfect preparation of CDs whereas the photoluminescence (PL) emission effect justifies this formation.His research groups found a result of a voltage limit from 0-0.9 V by using cyclic voltammetry (CV) curves at different scan rates.At current densities of 5 A/g and 8 A/g, they were successful in producing specific capacitances of 106 F/g and 84.4 F/g, respectively (Chen et al. 2016).To create composite materials, Yu et al. (2021) uniformly deposited CDs over activated carbons derived from lignin, revealing an internal surface embellished with CD binding sites.After the introduction and increment of CDs, it was interestingly observed that the times of the charging and discharging gradually increased, and the high capacitance of the CDs@AC-11 electrode, which was double that of the AC electrode (125.8F/g), could be achieved at 0.15 A/g (Yu et al. 2021).

Plants and agriculture
Among different applications, one of the most significant at present time is CDs application in the field of plants and agriculture because of its outstanding properties such as higher biocompatibility, and lower concentration of environmental toxicity.CDs treatments or CDs performance, in case of the physiological process of plants and increment of agricultural production, plays a significant role in food production.Enhancement of plant growth, photosynthesis, bio functional contribution on nitrogen fixation, nano-fertilizer, delivering SiRNA in the model plant, in situ imaging (bioimaging), biotic or abiotic stress resistance, disease resistance etc. are recent discoveries by researchers for sustainable developments of agro system for minimizing nutrition (Li et al. 2018b(Li et al. , 2019;;Chandra et al. 2014;Das et al. 2014b).For instance, Kou et al. (2021) for the first time explained clearly how yield, nutrition capability, and quality are correlated to CDs.In his study, a hydrothermal process was used to produce CDs in which L-cysteine and glucose were combined with CDs to apply lettuces and tomatoes in a hydroponic solution consisting of all necessary nutrients liquids to examine the mechanism and its development (biofunctional effects) of plants.They got an excellent result in terms of seed germination rate, development of seedlings, and enhancement of plant maturity.In this assessment, actually, CDs improved the activity of photosynthesis as well as boosted the absorbance of macro or micro minerals which ultimately could increase the rate of germination, elongate (hypocotyl) the seedling and root enhancement, and as a result, yield and mature plant development in case of nutritional qualities appeared.Table 3 summarizes the information obtained from comprehensive reports on plant systems in which CDs play a key role as a plant growth enhancer.CDs also proved their potential as a nano fertilizer in the case of sustainable agricultural production.For example, to produce CDs (nitrogen 20%) based nanofertilizer, Wang et al. (2019b) used thiourea and citric acid as precursors followed by a solid-state method.They used mung beans as plant species to investigate the effects of CDs.They found that relying on the higher content of Nitrogen with CDs was more efficient than those of urea which showed strong potential as a nano fertilizer (luminescent).One of the most significant results came out when mung bean seeds were cultivated in 0.2 mg/mL aqueous solution of the CDs as opposed to the control that used pure water; they produced 17.45% more bean sprouts.

Overview of noteworthy key information of materials used for CD synthesis and surface modification
Nowadays researchers are trying surface modifications of CDs for different application purposes.In this review, we mainly focused on the covalently assisted mechanism, interaction reaction techniques, chemical complexation, and sol-gel mechanism.Amide coupling mechanism, silylation, and esterification sulfonylation copolymerization which are classified as covalent modification systems, are well-established modification techniques for researchers of CD surface modification to apply different applications.The hydrothermal route, pyrolysis, chemical oxidation technique, microwave method or microwaveassisted hydrothermal process, and electrochemical are the most popular protocols to synthesize CDs.Table 4 summarizes the information of materials types treated as well as their synthesis of CDs which are sorted from different literature reports based on interaction reaction routes.These reaction mechanisms play a significant role in surface modification, which greatly influences the application.
Actually, in the overall discussion in the case of CD surface modifications and synthesis, we mostly focused on the organic nature of these materials.In the overall discussion, the synthesis procedure of CDs with different functional groups significantly affects its morphology like a uniform, monodisperse, amorphous, spheroidal, sometimes quasi-spherical, semi-crystalline in which the size ranges from 1.5 nm to 450 nm were reported in different articles.sp 2 or sp 3 hybridized mechanism is normally established in CDs nucleation when different functional groups like carboxylic acid (-COOH), an amino group (-NH 2 ), epoxy/ether (-O-), hydroxyl (-OH), carbonyl group (-CO-), etc. are attached in the surface of CDs (D' Angelis do E. S. Barbosa et al. 2015;Sun et al. 2017;Liu et al. 2014).

Research gaps and future work scope
Though CDs are independently used in several large-scale facilities nowadays, the competency of CDs modified surfaces is imperialized most often, and the potentiality is huge to be addressed now.The pleasant photosensitive CDs made their place as one of the very exciting nanoparticles for emergent chemical, biomedical, and biochemical fields with the maturity of controlling bioimage, chemical sensors, drug delivery, and potentiality and displaying far beyond the regular outputs.This paper is mainly organized with the main modification processes of CDs with other nanoparticles and their surfaces for various functionalities that dramatically influence the activities of biomedical, chemical, and technology fields.Basically, the PL and FL characteristics of CDs are well addressed as prudent and sky-touching research gigs for researchers all around the globe.In the future, the potential use of CDs is assumed to make a mesmerizing change in humanity due to their vast use as a life savior.The use of CDs is not very rare even today, considering their properties of resistance in adverse conditions, precise biomonitors, promising semiconductors, etc., however,   Medical diagnostics, catalysis, photovoltaics, optical-gain (Wang et al. 2011a) this is not the end of this era.Modified surfaces with CDs are those significant nano-surfaces that will allow another innovative era of medical science, chemical/ mechanical industry, focusing on extremely economic, facile and effective in nature.Although a lot of achievements have been made with CDs modified surfaces, the modified surfaces with CDs have yet been explored for drug delivery purposes.Silver nanoparticles are replaced with CDs and modified surfaces with CDs, and they are found heavily effective in insulin fibrillation; resistance to breast cancer and a specific type of liver cancer, however, they are not yet employed for noble purposes.Deep research in vivo and in vitro is essential for extending its maturity in medical science (Hoang et al. 2019).Though phosphorodiamidate morpholino oligomers (PMO)-CDs can potentially be used as an active tool to diagnose early breast cancer with the help of FET (Field Effect Transistor Biosensor) looking at the presence of miRNA in plasma samples, the data of sensitivity is only counted for 1 μM to 100 μM.The detection sensitivity could be improved by nanoparticle-based DNA probes.Thus, that cannot be taken into consideration for a foolproof procedure to enhance the best output.Further research methods are needed to be applied to image the presence of these highly proliferating carcinogens (Li et al. 2022).

Conclusions
A plethora of modification methods of CDs is described in the whole article along with the characteristic properties of these processes and applications followed by the properties of the modified conjugates in the whole article.Modifications are carried out in several methods.However, the amide coupling reaction is the most significant, as it contributes to the amide structure's stability, and CDs' surfaces are rich in carboxyl and amino molecules.The silylation procedure surrounds CDs and another fluorophore in a silicon coating, which can develop CDs' water solubility and selectivity, and considerably lower their cytotoxicity, making it useful in organisms.Eventually from the studies, it is clear that the esterification, sulfonylation, and copolymerization reaction, and sol-gel methods are such reaction processes that are difficult to carry out.All of these strategies boosted the optical performance of CDs by utilizing the physicochemical properties of target molecules on the surface of the CDs that, in addition, revealed a viable link between nanoparticles and biological systems.Regarding properties like cytotoxicity, photostability, and biocompatibility, the CDs-based nanomaterial-modified surfaces exhibited excellent in vivo and in vitro performance.CDs are the best additive for cancer and tumor drugs and therapeutic methods for their versatile nature.
Modification of CDs may potentially lead to their usage in a range of biosensing and nanomaterial science sectors.Now it is high time to shift carbon dots modification and preparation to the industrial level.This will, however, broaden the way of agents based on CD for biomedicine that show high-quality sensing, analysis, therapeutic methods, and drug delivery techniques than the currently used agents.Undoubtedly, CDs retain some promising therapeutic applications that will play a significant role in the biomedical sector very soon.

Fig. 5 Fig. 6
Fig.5Procedure of CDs modification of nanomaterial surface and formation of immunoassay(Mohammadi et al. 2018)

Fig. 8
Fig. 8 Modification of CDs by esterification reaction and detection of Ag + (Algarra et al. 2014)

Fig. 12
Fig. 12Modification principles of CDs nanomaterials by π interaction and detection of Ag +(Jiang et al. 2016a)

Fig. 18
Fig. 18 Complexation between CDs and the metal ion Photoluminescent CDs

Table 1
(Shi et al. 2015)erent covalent modification reactions associated with CDs surface modification(Shi et al. 2015) i) Common but unstable.

Table 3
Overall information about the effects of CDs on plant growth system

Table 4
Overall information about the precursor of CDs, types, synthesis route, QY, and applications