BY-NC-ND 3.0 license Open Access Published by De Gruyter July 22, 2015

Luminescent assays based on carbon dots for inorganic trace analysis

Isabel Costas-Mora, Vanesa Romero, Isela Lavilla and Carlos Bendicho


Carbon dots (CDs) are a recently discovered class of fluorescent nanomaterials with great potential to be applied in the analytical field. CDs have demonstrated to be a promising alternative to conventional organic fluorophores or quantum dots as optical nanoprobes for sensing different chemical species. In this overview, we review the progress in the design of novel nanoprobes based on fluorescent CDs for inorganic trace analysis. Representative examples of CD-based assays are described and the different sensing strategies are discussed.


Carbon dots (CDs) are a new type of fluorescent nanostructured material with a size smaller than 10 nm, which have shown interesting properties for their application as optical nanoprobes. These are also known as carbon nanodots, fluorescent carbon nanoparticles (CNPs), or carbon quantum dots (QDs).

CDs were accidently discovered by Xu et al. (2004) during electrophoretic purification of single-walled carbon nanotubes fabricated from arc-discharge soot. Since then, several synthesis pathways have been proposed for obtaining fluorescent CDs, and application for these was found in several areas, including biological, medical, or chemical fields (Baker and Baker 2010, Silva and Gonçalves 2011, Li et al. 2012, Wang and Hu 2014c, Lim et al. 2015).

Semiconductor nanoparticles, also known as QDs, have been extensively used as optical probes due to their interesting optical properties (Freeman and Willner 2012, Frigerio et al. 2012, Li and Zhu 2013, Costas-Mora et al. 2014b). Nevertheless, QDs possess some limitations related to its potential toxicity since these usually contain toxic elements within their composition such as Cd, Se, Te, or As, among others. Compared to QDs and conventional organic dyes, CDs exhibit superior features in terms of high aqueous solubility and resistance to photobleaching, as well as easy functionalization and low toxicity (Bourlinos et al. 2008, Baker and Baker 2010).

In addition, CDs can be fabricated from simple and nontoxic precursors such as carbohydrates (e.g. glucose, fructose, sucrose). The use of this kind of precursor suggests the low toxicity of this nanostructured material and its potential in different areas related with biology and biochemistry.

In the last years, CDs have gained increasing popularity in a wide variety of areas, including photocatalysis, optoelectronic, and especially optical sensing. In this sense, CDs have been applied for detection of biological (Cai et al. 2015, Han et al. 2015, Ma et al. 2015, Mehta et al. 2015, Nandi et al. 2015, Niu et al. 2015, Wu et al. 2015), organic (Huang et al. 2013 Huang et al. 2014a, Hu et al. 2014, Wei et al. 2014, Zhang et al. 2014a, Zhang et al. 2015), and inorganic (Yin et al. 2013, Liu et al. 2014b, Zhang and Chen 2014c, Zhao et al. 2014a, Cui et al. 2015, Gogoi et al. 2015) species. Regarding inorganic trace analysis, a dramatic increase in the number of publications describing CD-based systems has occurred in the last 2 years (Figure 1).

Figure 1: Evolution of the number of publications involving the use of CD-based assays for inorganic trace analysis.

Figure 1:

Evolution of the number of publications involving the use of CD-based assays for inorganic trace analysis.

To date, several review articles focused on the synthesis procedures, properties, and general applications of CDs have been published (Baker and Baker 2010, Silva and Gonçalves 2011, Li et al. 2012, Wang and Hu 2014c, Lim et al. 2015), but none of them has tackled specific applications of CDs for detection of inorganic species. In this review, we present an insight into CD-based systems for inorganic trace analysis.

Optical properties of CDs

The main feature accounting for the great interest of many researchers concerning CDs lies on their ability to emit fluorescence. In spite of the fact that the exact mechanism involved in the emission of CDs is still unknown, it is generally attributed to the presence of surface defects generated from chemical oxidation or functionalization (Sun et al. 2006). In addition, the presence of carbon-oxygen bonds seems to be necessary for obtaining fluorescent CDs.

There are several synthetic pathways to synthesize fluorescent CDs, which can be classified as top-down and bottom-up methods. The first group includes treatments by arc-discharge, laser ablation, or electrochemical exfoliation, whereas the latter involves thermic carbonization, electrochemical treatment, and different synthesis methods involving microwave and ultrasound energies (Baker and Baker 2010, Silva and Gonçalves 2011, Li et al. 2012, Wang and Hu 2014c).

Generally, application of top-down methods leads to the formation of nonfluorescent CDs, so additional chemical treatments are essential in order to get fluorescent CDs. Therefore, surface activation and subsequent fluorescence emission are achieved through changes in the superficial states of CDs. To this end, there are three different strategies: (i) acid oxidation of nonfluorescent CDs (Xu et al. 2004, Sun et al. 2006, Bourlinos et al. 2008), (ii) doping of oxidized CDs with inorganic salts (Sun et al. 2008, Wang et al. 2009), and (iii) covering the surface of CDs with an organic polymer (Sun et al. 2006, Sun et al. 2008, Wang et al. 2009). On the other hand, most of the bottom-up procedures are based on the simultaneous synthesis and passivation of CDs leading to the formation of fluorescent CDs via a one-step procedure. Among the synthesis pathways proposed for CDs, bottom-up methods are the most widely used due to their simplicity and low cost, as well as ease of controlling the size, shape, and optical properties of synthesized CDs. One of the properties affected by synthesis procedures is the fluorescence quantum yield (QY). This property depends on the synthesis pathway (Sun et al. 2006, Li et al. 2012) and surface ligand nature (Sun et al. 2006, Hu et al. 2009, Peng and Travas-Sejdic 2009).

As is shown in Table 1, the main limitation of CDs resides on the relatively low fluorescence QY, which usually are lower than 15%, so new synthesis and doping procedures for CDs are currently being investigated.

Table 1

Properties of CDs synthesized from different precursors under different synthesis treatments.

Synthesis methodPrecursorSize (nm)Surface ligandQY (%)References
Laser ablationGraphite powder and cement5PEG1500N4–0Sun et al. 2006
Graphite powder and cement5PPEI-EI4–10Sun et al. 2006
Graphite3.2PEG2003–8Hu et al. 2009
Arc-dischargeSWCNTs1.6Xu et al. 2004
Electrochemical treatmentCNTs2.86.4Zhou et al. 2007
Graphite2–102.8–5.2Lu et al. 2009
Graphite50.81Wang et al. 2014b
Glycine2.427.1Wang et al. 2014a
Ethanol2.1–4.34–15.5Deng et al. 2014
Hydrothermal treatmentMWCNTs3Acetone10Cayuela et al. 2013
Lactose1.5Tris12.5Zhang et al. 2013
EG0.7–425Liu et al. 2012c
Glucose7.5PEI3.5Han et al. 2012
Glucose1.8–3.82.4–1.1Yang et al. 2011
Dopamine3.86.4Qu et al. 2013
Sucrose1.8421.6Chen et al. 2013
Sucrose542Bhaisare et al. 2015
Bergamot1050.87Yu et al. 2015
Ultrasonic treatmentGlucose<57Li et al. 2011a
Active carbon5–105Li et al. 2011b
Microwave treatmentCitric acid2.2–3EDA30.2Zhai et al. 2012
Glycerol3.5TTDDA12.02Liu et al. 2011
Glycerol7BPEI7–15.3Liu et al. 2012a
Glycerol2.13.2Chen et al. 2011
Saccharide2.7–3.6PEG3.1–6.3Zhu et al. 2009
Glycerin3–4PEG12Lin et al. 2011
Citric acid12PEI30Salinas-Castillo et al. 2013
[BMIM][Br]2–619.76Zhao et al. 2014a
[BMIM][BF4]2–625.8Zhao et al. 2014a

[BMIM][BF4], 1-butyl-3-methylimidazolium tetrafluoroborate; [BMIM][Br], 1-butyl-3-methylimidazolium bromide; BPEI, branched poly(ethyleneimine); CNTs, carbon nanotubes; EDA, ethylenediamine; EG, ethyleneglycol; MWCNTs, multiwalled carbon nanotubes; PEG, poly(ethyleneglycol); PEI, poly(ethylenimine); PPEI-EI, poly(propionylethyleneimine-co-ethyleneimine); SWCNT, single-walled carbon nanotubes; Tris, tris(hydroxymethyl)aminomethane; TTDDA, 4,7,10-trioxa-1,13-tridecanediamine.

Thus, Bhaisare et al. (2015) synthesized CDs with a QY of 42% by hydrothermal treatment of sucrose, whereas Yu et al. (2015) developed a novel synthesis pathway making use of a plant of Jinhua bergamot as carbon source, which allows obtaining highly fluorescent CDs, with a QY of about 50%.

In addition, Wang et al. (2015c) fabricated organosilane functionalized CDs, which are amphiphilic so that they can display multisolvent dispersibility. These CDs are dispersable in water and other common organic solvents (i.e. dimethyl sulfoxide, methanol, dimethylformamide, acetone, ethanol, tetrahydrofuran, toluene, and hexane). As-prepared CDs have a QY of 51%.

Another synthesis strategy providing highly fluorescent CDs (QY=52%) was proposed by Zhou et al. (2015). These workers used citric acid and Tris as precursors to generate CDs by hydrothermal treatment.

On the other hand, several studies have been carried out for doping CDs so that their optical properties are improved. As occurs with QDs, coating of CD surface with inorganic compounds such as ZnS, ZnO, or TiO2 causes a significant improvement in the fluorescence properties of the CDs (Anilkumar et al. 2011, Shen et al. 2012). As mentioned above, the ability of CDs to emit fluorescence seems to be related to the presence of energy traps or superficial defects. The covering of these traps during passivation allows the radiative recombination of trapped electrons and holes, thus facilitating fluorescence emission. For this reason, the improvement of surface passivation causes the enhancement of CD fluorescence intensity. Among the different doping strategies applied so far, doping with inorganic species and further passivation with organic ligands are the ones providing a relevant increase in fluorescence QY, which can reach values up to 78% (Anilkumar et al. 2011).

Undoubtedly, N is the doping species most widely used to improve the optical properties of CDs, since experimental procedures to introduce N in the CD structure are relatively fast and simple (Barman and Sadhukhan 2012, Barman et al. 2014, Liu et al. 2014a, Qian et al. 2014a, Tang et al. 2014, Wu et al. 2014b, Yang et al. 2014, Xu et al. 2015b). It must be mentioned that despite these advantages, QY reached are lower than those achieved by the introduction of inorganic compounds. Usually, these vary in the range of 30%–40%, although some works reported QYs of up to 64% (Barman et al. 2014). The QY of doped CDs depends on the introduced species. Yang et al. (2014) investigated the effect of doping CDs with different species over QY. CDs synthesized prior to doping procedure had a fluorescence QY of 6%, whereas the introduction of N, S, or Se caused an enhancement of QY reaching values of 39%, 24% and 19%, respectively. In addition, the introduction of these doping species affects the emission wavelength. Although N is the most extended doping species used up to date, in the last years, some works dealing with doping species such as B (Shan et al. 2014), S (Xu et al. 2015a), P (Zhou et al. 2014), or Si (Qian et al. 2014b) have been published.

Another strategy that allows achieving high fluorescence QY lies on the introduction of two doping species. To date, some combinations have been reported, including N-S (Dong et al. 2013, Ding et al. 2014, Mohapatra et al. 2015), N-P (Barman et al. 2014), N-Al (Wang et al. 2015b), N-B (Barman et al. 2014), and N-Mn (Li et al. 2014). Among the mentioned combinations, QYs obtained by the combinations of N-S, N-P, and N-Mn, which are 54%–73%, 70%, and 83%, respectively, must be highlighted.

Table 2 shows the QYs of doped CDs following the different strategies mentioned above. Therefore, the doping of CDs offers a way to solve the limitation of the low fluorescence QY that is generally reached.

Table 2

Quantum yield of doped CDs with several species and following different strategies.

Doping speciesQY (%)References
ZnO45Sun et al. 2008
ZnS50Sun et al. 2008
ZnS78Anilkumar et al. 2011
TiO270Anilkumar et al. 2011
N32Xu et al. 2015b
N29Barman and Sadhukhan 2012
N35Tang et al. 2014
N15.7Zhang and Chen 2014c
N13–22Teng et al. 2014
N36.3Qian et al. 2014a
N37.4Liu et al. 2014a
N36.5Wu et al. 2014b
N64Barman et al. 2014
N39Yang et al. 2014
S24Yang et al. 2014
S67Xu et al. 2015a
Se19Yang et al. 2014
B14.8Shan et al. 2014
P25Zhou et al. 2014
Si19.2Qian et al. 2014b
N, S54.4Ding et al. 2014
N, S69Mohapatra et al. 2015
N, S73Dong et al. 2013
N, P70Barman et al. 2014
N, Al25.7Wang et al. 2015b
N, B39Barman et al. 2014
N, Mn83Li et al. 2014

A relevant feature of CDs is the strong influence of excitation wavelength over both emission wavelength and fluorescence intensity (Sun et al. 2006, Chen et al. 2011, Li et al. 2011a,b, Han et al. 2012, Lai et al. 2012, Lin et al. 2012, Liu et al. 2012c, Ma et al. 2012, Shen et al. 2012, Zhai et al. 2012, Du et al. 2013, Liu et al. 2013b, Zhang et al. 2013). Therefore, it is possible to change the emission wavelength merely by modification of the excitation wavelength, which allows working in a wide spectral range, i.e. from UV to near-infrared. It must be mentioned that several synthesized CDs have up-conversion properties, so they are able to emit fluorescence at a wavelength shorter than that of excitation (Li et al. 2011a,b, Ma et al. 2012, Liu et al. 2013b, Zhang et al. 2013). The up-conversion properties can be ascribed to multi-photon activation process, in which the simultaneous absorption of two or more photons leads to the emission at a shorter wavelength, which is known as anti-Stokes type emission. This attractive optical property enables promising applications.

As was mentioned, one of the most interesting features of CDs is their high stability. The pH of the medium is one of the main factors influencing both the stability and optical properties of CDs due to the protonation or deprotonation of capping ligands. This limitation is overcome by the selection of the appropriate capping ligand depending on the working pH. To date, several CD-based pH sensors have been developed taking advantage of this limitation (Jia et al. 2012, Mao et al. 2013, Kong et al. 2014, Nie et al. 2014, Pedro et al. 2014, Wu et al. 2014b, Wang et al. 2015a, Jin et al. 2015).

Application of CDs as optical probes for inorganic analysis

The optical properties mentioned in the previous section have been exploited for developing several CD-based optical nanoprobes.

Even though fluorescence is the optical property most widely exploited to develop CD-based probes, their ability to emit chemiluminescence (CL) and electrochemiluminescence (ECL) has also been used to construct novel detection approaches for inorganic species (Figure 2).

Figure 2: Inorganic species studied by different detection techniques involving the use of CD-based assays.

Figure 2:

Inorganic species studied by different detection techniques involving the use of CD-based assays.

As can be observed, most of CD-based nanoprobes developed to date are based on fluorescent sensing, with metal ions such as Cu(II), Hg(III), and Fe(III) being the analytes most widely investigated. Table 3 shows some features of selected works based on the use of CDs as optical probes for the detection of inorganic species.

Table 3

CD-based systems developed for inorganic species detection.

AnalyteCD synthesis method/precursorCapping ligandEffect on CD emissionLinear rangeLODSensing mechanismReferences
Ag(I)HT/EGFluorescence enhancement0–9×10-5 M3.9×10-7 MReduction of Ag+ to Ag nanoclusters on the surface of CDsGao et al. 2015
ClO-HT/pepperFluorescence quenching1×10-7–1×10-5 M5×10-8 MOxidation of functional groups of CDsYin et al. 2013
1×10-5–3×10-4 M6×10-8 M
ClO-MW/sucroseFluorescence quenching2×10-7–2×10-6 M1.5×10-8 MOxidation of CDsHuang et al. 2014b
CH3Hg+US/fructose-PEG-NaOHPEGFluorescence quenching2.3×10-8–2.8×10-7 M5.9×10-9 MUltrasound-assisted permeation of CH3Hg+ through PEG passivation coating leading to electron transfer processCostas-Mora et al. 2014a
Co(II)HT/cysteineFluorescence quenching10×10-9–1×10-4 M10×10-9 MFormation of CoxSy NPs and aggregation of CDsLi et al. 2015
Cu(II)HT/citric acidBPEIFluorescence quenching1×10-8–1.1×10-6 M6×10-9 MComplex formation between capping ligand and Cu(II)Dong et al. 2012
Cu(II)MW/glucoseBSA-LysFluorescence quenching2×10-12–1.5×10-9 M5.8×10-13 MCoordination of Cu(II) with -NH2 and -COOH groups of lysineLiu et al. 2012b
Cu(II)EC/graphiteTPEAFluorescence quenching1×10-6–1×10-4 M1×10-8 MBinding of Cu(II) and TPEAQu et al. 2012
Cu(II)MW/citric acidPEIFluorescence quenching3×10-7–1.6×10-6 M9×10-8 MSalinas-Castillo et al. 2013
1.2×10-7 M
Cu(II)HT/bamboo leavesBPEIFluorescence quenching3.3×10-7–6.6×10-5 M1.1×10-7 MDisplacement of capping ligand due to its coordination with Cu(II)Liu et al. 2014c
Cu(II)MW/[BMIM][Br]IMD groupsFluorescence quenching5×10-9 MBinding of Cu(II) with N of IMD groupsZhao et al. 2014a
Cu(II)AO/CNPsFluorescence quenching1×10-8–2×10-7 M5×10-9 MCu(II) interaction with C-N groups of CDsYang et al. 2014
Cu(II)HT/OPDOPDFluorescence enhancement2×10-9–8×10-8 M1.8×10-9 MComplex formation of Cu(OPD)2 in the CD surfaceVedamalai et al. 2014
Cu(II)HT/AEAPMSEDA groupsRatiometric0–3×10-6 M3.5×10-8 MCu(II) interaction with superficial EDA groupsLiu et al. 2014b
Cu(II)EQ/graphiteTPEAEnhancement of electric current1×10-6–6×10-5 M1×10-7 MShao et al. 2013
F-MW/glucose-PEGPEGFluorescence quenching1×10-4–1×10-2 M3.1×10-8 MInteraction between F- and CD capping ligandLiu et al. 2013a
Fe(III)Heating of banana peel and further MWFluorescence quenching2×10-6–1.6×10-5 M2.1×10-7 MInteraction of Fe(III) with CD capping ligandsVikneswaran et al. 2014
Fe(III)MW/[BMIM][BF4]OH groupsFluorescence quenching2×10-8 MBinding of Fe(III) with superficial OH groupsZhao et al. 2014a
Fe(III)MW/AA-PEGPEGECL ratio5×10-6–8×10-5 M7×10-7 MZhang et al. 2014b
Fe(III)MW/BergamotFluorescence quenching2.5×10-8–1×10-4 M7.5×10-8 MYu et al. 2015
Hg(II)MW/BergamotFluorescence quenching1×10-8–1×10-4 M5.5×10-9 MYu et al. 2015
Hg(II)HT/citric acidAEAPMSFluorescence quenching0–5×10-6 M1.35×10-9 MComplex formation between amino groups and Hg(II)Wang et al. 2015c
Hg(II)HT/folic acidEGFluorescence quenching0–2.5×10-7 M2.3×10-7 MSuperficial changes of CDsZhang and Chen 2014c
Hg(II)HT/EDTA saltsFluorescence quenching0–3×10-6 M4.2×10-9 MCharge transfer between Hg(II) and CDsZhou et al. 2012
Hg(II)AO/CNPsFluorescence quenching2×10-9–2×10-6 M2×10-9 MHg(II) interaction with superficial SH groupsYang et al. 2014
Hg(II)HT/PEG-NaOHFluorescence quenching0–1×10-8 M1×10-15 MHg coordination with oxygen-rich superficial groups of CDsLiu et al. 2013b
Hg(II)MW/flourFluorescence quenching5×10-10–1×10-8 M5×10-10 MQin et al. 2013
Hg(II)HT/citric acid-EGODNFluorescence recovery5×10-9–2×10-7 M2.6×10-9 MComplex formation of Hg(II)/ODN and subsequent displacement of GOCui et al. 2015
H2O2AO/carbonColorimetry1×10-8– 1×10-5 M1×10-8 MCatalysis of TMB substrate oxidation leading to formation of blue solutionZheng et al. 2013
I-MW/citric acid-EGTurn off-on5×10-7– 2×10-5 M4.3×10-7 MComplex formation and subsequent Hg(II) displacement causing fluorescence recoveryDu et al. 2013
K+HT/candleEDAFRET (fluorescence recovery)5×10-5– 1×10-2 M1×10-5 MInhibition of FRET process due to the binding of the analyte in the place of crown-etherWei et al. 2012

AO, acid oxidation; EC, electrochemical treatment; HT, hydrothermal treatment; MW, microwave treatment; US, ultrasound treatment; AA, ascorbic acid; AEAPMS, N-(β-aminoethil)-γ-aminopropyl methyldimethoxy silane; [BMIM][BF4], 1-butyl-3-methylimidazolium tetrafluoroborate; [BMIM][Br], 1-butyl-3-methylimidazolium bromide; BPEI, branched poly(ethylenimine); BSA, bovine serum albumin; CNPs, carbon nanoparticles; EDA, ethylenediamine; EG, ethyleneglycol; GO, graphene oxide; IMD, imidazole; Lys, lysine; NP, nanoparticle; ODN, oligodeoxyribonucleotide; OPD, o-phenylenediamine; PEG, poly(ethyleneglycol); PEI, poly(ethylenimine); TMB, 3,3,5,5-tetramethylbenzidine; TPEA, N-(2-aminoethyl)-N,N’,N’-tris(pyridine-2-yl-methyl)ethane-1,2-diamine).

CDs as fluorescent probes

Besides sensing approaches making use of quenching or fluorescence enhancement, assays based on fluorescence resonance energy transfer (FRET), ratiometric, reversible and up-conversion nanoprobes have also been developed. Figure 3 shows relevant sensing mechanisms involved in the use of CDs as fluorescent nanoprobes to detect inorganic species.

Figure 3: Processes involved in the sensing mechanisms using CD-based assays.

Figure 3:

Processes involved in the sensing mechanisms using CD-based assays.

Probes based on changes of CDs fluorescence

As is shown in Figure 2, most CD-based fluorescent probes use direct measurements of fluorescence changes caused by the interaction with the target analyte.

The most widely observed effect is the fluorescence quenching of CDs when these interact with inorganic species. This effect is observed in 90% of the CD-based systems developed to date. Analytes mostly investigated are Cu(II), Hg(II), and Fe(III).

Recently, Zhao et al. (2014a) reported a novel fluorescent assay using CDs synthesized from two different ionic liquids (i.e. 1-butyl-3-methylimidazolium tetrafluoroborate, [BMIM][BF4], and 1-butyl-3-methylimidazolium bromide, [BMIM][Br]). The use of different precursors leads to the formation of different functional groups. CDs fabricated from [BMIM][Br] have imidazol groups in the CD surface, whereas those synthesized from [BMIM][BF4] are rich in hydroxyl surface groups. These CDs obtained from [BMIM][Br] have a great selectivity for Cu(II), whereas those fabricated from [BMIM][BF4] allow the selective detection of Fe(III).

As was mentioned, several novel methods have been developed for the sensitive detection of Cu(II). Among them, the work performed by Dong et al. (2012) must be highlighted since it allows the detection of Cu(II) at picomolar levels (LOD=0.58 pM). This assay is based on the use of CDs functionalized with bovine serum albumin (BSA) and lysine. In the presence of Cu(II), the fluorescence intensity of the system was quenched probably due to the coordination of Cu2+ with the carboxyl group and amino group of lysine and glycine of partial uncoated CD-BSA. The applicability of this approach for Cu(II) detection in hair and water samples was demonstrated.

As indicated above, Hg(II) has also been detected using CD-based systems. Among the developed methods, those relying on changes in fluorescence intensity are mostly employed for detection.

The fabrication of N-doped CDs from folic acid, which acts as C and N source at the same time, has been reported (Zhang and Chen 2014c). As-prepared CDs display great sensitivity toward Hg(II), thus allowing its detection.

Another interesting method was designed by Zhou et al. (2012) for detection of Hg(II) and biothiols in complex matrices. In this system, the presence of Hg(II) causes the fluorescence quenching of CDs due to a charge transfer process. Initial fluorescence intensity is recovered when CD-Hg interact with biothiols due to the great affinity of thiol groups toward Hg(II), which causes the displacement of Hg(II) from the CD surface.

Liu et al. (2013b) have reported an extremely sensitive assay for the detection of Hg(II). In this assay, a detection limit of 1 fM is reached using CDs synthesized from poly(ethylene glycol) (PEG) and NaOH. In addition, the applicability of the proposed method was demonstrated in different water samples. Other assays showing high sensitivity for Hg(II) detection were developed by Qin et al. (2013) and Yang et al. (2014). The former group synthesized CDs from flour to generate a fluorescent probe that allows reaching a detection limit (LOD) of 0.5 nM Hg(II). In the second paper, CDs doped with different heteroatoms including N, S, or Se were tried for detecting metal ions. The obtained results suggest that N-doped CDs display a great sensitivity toward Cu(II), whereas S-doped CDs are suitable for Hg(II) detection. LODs were 5 and 2 nM for Cu(II) and Hg(II), respectively.

Yu et al. (2015) used a plant of Jinhua bergamot as carbon source to obtain highly fluorescent CDs with a QY of about 50%. As-prepared CDs were applied as optical probes for Hg(II) and Fe(III) detection through fluorescence quenching effects. LODs were 0.075 μM and 5.5 nM for Fe(III) and Hg(II), respectively.

Another CD-based system that allows the detection of Hg(II) at nanomolar levels was recently developed by Wang et al. (2015c). This system is based on CDs functionalized with organosilane and allows achieving a LOD as low as 1.35 nM.

Moreover, our group developed a novel assay for the sensitive and selective detection of CH3Hg+ (Costas-Mora et al. 2014a). This method is based on the integration of synthesis and detection processes in a single step, which is accomplished by the use of high-intensity ultrasound energy. The applicability of the proposed method for CH3Hg+ detection was demonstrated by analyzing different reference certified materials of water and marine animal tissues. Some remarkable features of the method are its simplicity, sensitivity, and rapidity. A LOD of 5.9 nM CH3Hg+ was reached with this assay.

Besides metal ions, other inorganic species have been detected by CD-based systems. Among them, H2O2 is one of the most widely studied. B-doped CDs were used for H2O2 and glucose detection (Shan et al. 2014). One of the most relevant features lies in the increased sensitivity achieved due to the CD doping process. The LOD reached with B-doped CDs is 10 mM. Yeh et al. (2013) have reported another CD-based system for H2O2 and glucose detection. In this work, reduced graphene oxide (RGO) with incorporated CDs (CDs@RGO) is employed to accomplish the recognition event. Fluorescence quenching of CDs is produced due to its interaction with reactive oxygen species (ROS). Nevertheless, the presence of H2O2 in the reaction medium inhibits the ROS effect, and thus, a fluorescence enhancement is observed. A LOD of 140 nM H2O2 is reached.

In spite of the fact that fluorescence enhancement caused by inorganic analytes is scarcely observed, there are some relevant approaches reported to date.

Gao et al. (2015) observed that Ag(I) caused the fluorescence enhancement of CDs, which can be attributed to the reduction of Ag(I) to generate silver nanoclusters on the CD surface. The LOD obtained following the above strategy was 320 nM of Ag(I).

Probes based on FRET processes

Until now, FRET processes have been scarcely used to detect inorganic species. A novel sensor has been described by Cui et al. (2015) using CDs functionalized with oligodeoxyribonucleotide (ODN) for the detection of Hg(II). When CDs are in the presence of GO, fluorescence quenching is produced due to a FRET process. Nevertheless, when Hg(II) is introduced in the mixture, the displacement of GO occurs due of the formation of a complex between Hg(II) and thymine from ODN, so the recovery of fluorescence is produced. With this detection strategy, a LOD of 2.6 nM is achieved.

Another work based on FRET was conducted by Wei et al. (2012). This system uses CDs in conjunction with an ion-selective crown ether and graphene to develop a FRET system that is highly selective for K+ detection. CDs act as donor species, whereas graphene does it as acceptor ones. Therefore, FRET is inhibited by the presence of K+ due to a competitive process between the ammonium group of crown-ether and the target analyte, hence causing the fluorescence recovery of CDs.

Ratiometric probes

Ratiometric probes are based on the calculation of a fluorescence ratio of the quenched and constant reference fluorescence signals. It allows correction of some instrumental variations and provides improved sensitivity.

A ratiometric probe to detect Hg(II) was reported by Cao et al. (2013) using CDs and QDs as emitting species. The solution containing blue-emission CDs and red-emission CdSe/ZnS QDs exhibits dual emissions at 436 nm and 629 nm under a single excitation wavelength. The interaction of Hg(II) with QD functional capping groups causes the fluorescence quenching of QDs, whereas CD fluorescence remains constant, so Hg(II) can be monitored by fluorescence color evolution from red to blue. This strategy constitutes a simple way for visual detection of Hg(II).

In addition, Yan et al. (2015) reported a novel nanohybrid ratiometric fluorescence probe for NO2 determination. This system uses blue-colored CDs in conjunction with red-colored QDs. CD fluorescence is not affected by NO2, whereas it causes the fluorescence quenching of QDs. This leads to a distinguishable color change from orange-red to blue in the presence of NO2, which allows achieving a LOD of 19 nM. Moreover, this system allows the visual detection of NO2 with a LOD of 1 ppm.

Lastly, a FRET-based ratiometric probe for H2O2 detection was recently fabricated by Wu et al. (2014a). Herein, CDs act as energy donor and recognition element for H2O2. It was demonstrated for the first time that this FRET-based nanoprobe serves to detect H2O2in vivo using zebrafish as a vertebrate model. It was found that CDs are accumulated in the abdominal region of the zebrafish, and the majority of them are excreted within 4 h without causing apparent toxicity. The LOD of the method is 0.5 μM H2O2.

Reversible probes

In recent years, reversible systems have emerged as a new detection strategy, which is characterized by its high selectivity. These systems involve two steps: (i) fluorescence quenching and (ii) fluorescence recovery.

Regarding CD-based reversible probes, it should be mentioned that a few systems based on the use of turn off-on strategies have been developed to date.

There are some works that exploit the fluorescence quenching caused by Cu(II) and the further recovery of CD fluorescence to develop novel CD-based reversible probes (Hou et al. 2013, Zong et al. 2014). On the one hand, Zong et al. (2014) reported a new method for Cu(II) and L-cysteine detection by a turn off-on strategy. Quenching of CD fluorescence is due to Cu(II) adsorption onto the CDs surface (off). The addition of L-cysteine causes the fluorescence recovery of CDs (on) as a result of its great affinity toward Cu(II), which is displaced from CD surface. Herein, LODs for Cu(II) and L-cysteine are 23 and 0.34 nM, respectively.

On the other hand, a novel reversible sensor for S2- detection has been reported by Hou et al. (2013). This system is based on the use of CDs whose capping ligand has great affinity toward Cu(II). When it is present in the sample, Cu(II) binds to CD capping ligand, causing fluorescence quenching. Nevertheless, the subsequent addition of S2- causes the displacement of Cu(II) to form CuS and, in turn, the recovery of CDs fluorescence.

In addition, Teng et al. (2014) synthesized N-doped CDs from pyrolyzed flour to develop a turn off-on system. This is based on the quenching of CD fluorescence caused by the presence of Fe(III) as a result of an electron transfer process and the fluorescence recovery due to the interaction with L-cysteine, which also causes a shift in the emission wavelength.

Another turn off-on CD-based system was described by Du et al. (2013). This assay is focused on iodide detection using CDs whose fluorescence was previously quenched by Hg(II). Therefore, the formation of a complex between iodide and Hg(II) causes the elimination of Hg(II) from CD surface and, thus, the recovery of CD fluorescence. This system allows detection of iodide in urine samples at very low concentrations, with a LOD of 430 nM of iodide being achieved.

Moreover, Wang et al. (2015d) synthesized polyaniline/CD nanocomposites, which are nonfluorescent due to the quenching caused by the presence of polyaniline. Nevertheless, the addition of Hg2+ caused the release of polyaniline from CDs due to the strong binding affinity between Hg2+ and amino groups and, in turn, the fluorescence restoration of CDs. This strategy allows reaching a LOD of 0.8 nM.

Recently, Gu et al. (2015) designed an off-on fluorescent probe to detect Au(III) and glutathione. The introduction of Au(III) on fluorescent CDs leads to fluorescence quenching due to the formation of the complex Au(III)/CD. Nevertheless, when biothiols are added to the mixture, fluorescence restoration occurs, being especially significant for glutathione. Following this strategy, LODs of 0.48 and 2.02 μM for Au(III) and glutathione are reached.

Up-conversion probes

As was mentioned above, many synthesis approaches give rise to CDs that are able to emit fluorescence at low wavelength when they are excited at larger wavelength. This phenomenon is known as up-conversion fluorescence.

An assay using the down- and up-conversion properties of CDs was proposed by Yin et al. (2013) to determine hypochlorite. The authors found two linear ranges, which allow the determination of hypochlorite in a wide concentration range (i.e. 0.1–10 and 10–300 μM of hypochlorite). As in the work mentioned above, the sensitivity of the CD-based assay depends on the excitation wavelength. LODs of 0.05 and 0.06 μM were achieved using down- and up-conversion properties, respectively.

CDs as chemiluminescent probes

To date, only a few works have been reported based on the use of CDs in conjunction with CL to detect inorganic species.

Recently, Zhao et al. (2014b) demonstrated the ability of CDs functionalized with branched poly(ethyleneimine) to detect Fe(III) in alkaline medium by direct measurements of CL. It was found that Fe(III) is retained onto the CD surface and it acts as oxidant species, thus causing the enhancement of the CL signal. This new method allows the selective and sensitive detection of Fe(III), with a LOD of 66.7 nM being reached.

In addition, Lin et al. (2015) proposed the use of peroxynitrous acid-carbonate-CDs system for nitrite sensing. In this system, CDs act as energy acceptor. This method allows achieving a LOD of 5.0 nM of nitrite.

Lastly, some works based on the use of ECL have been reported. Zhang et al. (2014b) developed a novel method where a dual peak of ECL is used to detect Fe(III). The use of ECL dual peak allows achievement of higher sensitivity in comparison with those based on the use of single-peak. Therefore, a linear range of 5–80 μM and LOD of 0.7 μM were achieved. The main feature of the proposed system is the minimization of the typical instability of ECL methods to detect metal ions, since Fe(III) determination is performed after establishing the ratio of ECL peaks.

An ECL probe based on the use of Ag-CD nanocomposite was described by Wang et al. (2014d) to detect S2-. The affinity of the target analyte and Ag attached to CDs was the responsible for ECL changes and the subsequent ability of the proposed system to detect S2-.

Other strategies for detection of inorganic species

A minor group of detection strategies making use of CDs are commented in this section. Regarding colorimetric detection, Zheng et al. (2013) designed a CD-based system for sensitive determination of H2O2, reaching a detection limit of 10 nM. This method is based on the catalytic effect produced by H2O2, which leads to the formation of a colored solution. In addition, the applicability of the proposed system in biological samples was demonstrated.

On the other hand, Shao et al. (2013) developed a novel method based on the use of CDs functionalized with N-(2-aminoethyl)-N,N′,N′-tris(pyridine-2-yl-methyl)ethane-1,2-diamine) (TPEA) for detection of cerebral Cu(II) using an electrochemical assay. CDs-TPEA were immobilized in the electrode showing a great selectivity toward Cu(II).

Recently, Gogoi et al. (2015) developed a solid sensing platform for on-site detection of heavy metals. To this end, CDs were immobilized in an agarose hydrogel film. The presence of different metal ions causes color changes, i.e. Cr6+, green; Cu2+, blue; Fe3+, brown; Pb2+, white; and Mn2+, tan brown. The achievement of low LODs should be highlighted, i.e. 1 pM for Cr6+, 0.5 μM for Cu2+, and 0.5 nM for Fe3+, Pb2+, and Mn2+.

Detection of inorganic species in biological systems

Several fluorescent nanoparticles, including QDs, metallic nanoclusters, and other CNPs, have been used for imaging and detection in biosystems. In this sense, the use of CDs as optical probes has greatly increased within the last few years due to their nontoxic nature.

Recently, Mohapatra et al. (2015) developed a novel method for Hg(II) determination in water and living cells using nitrogen and sulfur co-doped CDs. Hg(II) interacts with sulfur to cause fluorescence quenching. This sensing strategy allows reaching a LOD of 0.05 nM of Hg(II). The suitability of the reported method for Hg(II) detection in living cells was demonstrated by evaluation of N,S-CD cytotoxicity in HaCaT cells.

In addition, a new method based on the use of CDs functionalized with TPEA was developed for imaging and detection of Cu(II) in cells. In this system, TPEA acts as selective recognition unit for Cu(II), and the binding of the target analyte causes the fluorescence quenching of CDs (Qu et al. 2012). Another work focused on the detection of intracellular Cu(II) was recently developed by Vedamalai et al. (2014). In this work, the formation of the Cu(OPD)2 complex in the CD surface causes the fluorescence enhancement and color change from yellow to orange.

Gong et al. (2015) used N-doped CDs for Fe(III) sensing and cellular imaging. The presence of Fe(III) causes the fluorescence quenching of CDs, a LOD of 1.8×10-7 M being reported. Another approach centered on the detection of Fe(III) was recently described by Zhou et al. (2015), in which a LOD of 0.32 μM for Fe(III) is reached.

Moreover, Salinas-Castillo et al. (2013) developed a novel CD-based system to detect Cu(II) using fluorescence measurement generated by down- and up-conversion. This system offers low cytotoxicity and it is suitable for intracellular detection and imaging of Cu(II) in biological species. LODs of the proposed system vary with the excitation wavelength used. LODs of 0.09 and 0.12 μM Cu(II) are achieved by excitation with UV light (down-conversion) and NIR irradiation (up-conversion), respectively. On the other hand, the use of UV light as excitation source does not prevent the strong interference caused by Fe(II), whereas the use of NIR excitation allows the selective detection of Cu(II).

Among ratiometric probes, the system developed by Liu et al. (2014b) should be highlighted. These workers proposed a ratiometric strategy for Cu(II) detection at low concentrations. This probe is based on the use of CDs and silica nanoparticles functionalized with Rhodamine B. The proposed system showed two different bands upon excitation at the same excitation wavelength, one in the blue region corresponding to CD emission and the other in the red region corresponding to Rhodamine B. CDs have surface ethylenediamine groups, which have a great affinity toward Cu(II), hence acting as recognition element. The binding of Cu(II) with the capping ligand causes the fluorescence quenching of CDs, whereas the Rhodamine band remains unchanged, so a ratiometric signal is obtained. A LOD 35.2 nM Cu(II) was achieved using this strategy. This system provided good results when it was applied for imaging and detection of Cu(II) in cells and water samples.


Since discovery of CDs around 10 years ago, these fluorescent nanomaterials have emerged as a suitable alternative to conventional organic dyes and QDs for optical sensing. A variety of simple synthetic pathways for obtaining fluorescent CDs from natural starting materials have been proposed so far. Given their low cost, ease of synthesis and functionalization, high stability, and nontoxicity, CDs show a great potential to be exploited in several fields. Among them, CDs have encountered application in the analytical field as optical probes for the detection of several species. Remarkable progress in the application of CDs for inorganic trace analysis has been achieved in the last years.

To date, several detection strategies based on fluorescence and CL changes have been proposed with this aim. Thus, fluorescent, chemiluminescent, FRET-based, reversible, and up-converted fluorescence probes have been designed. As a result of their interesting properties, it is expected that CDs will play an increasing role in the analytical field in the near future. The extension of CD applicability as optical probes for the sensitive and selective detection of inorganic species still needs further investigation, especially adequate functionalization strategies and further studies on sensing mechanisms.

Corresponding author: Carlos Bendicho, Facultad de Química, Departamento de Química Analítica y Alimentaria, Área de Química Analítica, Universidad de Vigo, Campus As Lagoas-Marcosende s/n, 36310 Vigo, Spain, e-mail:


Financial support from the Spanish Ministry of Economy and Competitiveness (project CTQ2012-32788) and the European Commission (FEDER) is gratefully acknowledged. V. Romero thanks the Spanish Ministry of Education, Culture and Sports, for predoctoral research grant.


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Received: 2015-2-26
Accepted: 2015-6-24
Published Online: 2015-7-22
Published in Print: 2015-12-1

©2015 by De Gruyter

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