Recent advances in fluorescence probes based on carbon dots for sensing and speciation of heavy metals

3.1 Synthesis of CDs

For the synthesis of CDs, there are a variety of synthetic routes. The desired routes are eco-friendly, size-controllable, convenient, cost-effective, environment friendly, and easy for large-scale production. In general, most of these routes can be categorized into “Top-down” and “Bottom-up” methods. In the “Top-down” approaches, CDs are prepared by breaking down bulk carbon materials such as graphite and carbon nanotubes, graphene, and suspended C-powders by electrochemical oxidation, acidic oxidation, laser ablation, plasma treatment, arc discharge, and so on. In the “Bottom-up” approaches, organic molecules such as glycerol, citric acid, ethylenediamine, amino acids and natural materials such as coffee grounds, soy milk, soybeans [28], grass, eggs, gelatin, pomelo peel, honey, etc., are candidates as carbon sources and precursor to generate CDs by hydrothermal, solvothermal, thermal decomposition, carbonization, ultrasonic and microwave experimental procedures [28]. For both “Top-down” and “Bottom-up” methods, reaction conditions such as selected reaction precursors, solvent, temperature, and reaction time are critical factors that will affect the CD’s chemical structure, size, and QYs [15], [18], [29], [30]. Until now, there is still a great challenge for controllable synthesis of CDs with precise chemical structure, desirable size, and high emission efficiency with high QYs due to the multistep reactions involved and complicate formation mechanisms during nucleation and growth processes. More reviews on the preparation strategies of CDs could be found in publications [12], [31], [32], [33], [34], [35], [36].

Concerning CD-based HM fluorescence sensors, the preparation of CDs with effective and sufficient functional groups is essential to provide binding or reaction sites with HMs. Heteroatom doping and surface modification are feasible methods. For heteroatom doping, carbon materials containing heteroatoms, such as nitrogen (N), sulfur (S), boron (B), silicon (Si), phosphorus (P) are promising candidates as the carbon source or precursor [23], [37], [38], [39], [40], and N- and S-doping of CDs are the most adopted strategies. By adopting different synthetic methods and synthesis conditions, the ratio of heteroatoms with the main element of carbon and the surface chemistry can be manipulated. Sometimes the ratio of the heteroatoms in doped CDs can vary significantly in raw and purified CDs, which shows a significant improvement of the optical properties of CDs. Compared with nondoped CDs, the excitation band of CDs may shift from typical 260–320 nm to longer wavelengths, and the emission band may shift to green, yellow, and red regions. As a result, the ability of antimatrix interference of CDs as HMs sensor was increased, as the natural fluorescence of the sample matrix at low wavelengths could be avoided. The QYs could also be improved with heteroatoms doping, which is beneficial to enhance the sensitivity of CD-based HM sensors. Many functional groups that can bind with HMs could be generated during the doping reaction, although the rational structure design is difficult. If the functional groups on the surface of heteroatoms doped CDs are lack of affinity and selectivity to target HMs, they can be further conjugated with other organic molecular, oligomeric polymers, and biomolecules through elimination, substitution, condensation, dehydration, carbonization, or passivation reactions to obtain specific binding sites for HMs.

3.2 Purification of CDs

For the successful application of CDs as HMs sensors, the purification is an essential and crucial preliminary step because the product of synthesized CDs is usually in a complex mixture containing unreacted starting materials and by-products alongside CDs. The unsuccessful purification will affect the QY, the fluorescent properties, and the ratio of heteroatoms in CDs, leading to significant challenges in understanding the structure and optical properties, generating the reproducible batch-to-batch results of reaction CDs, and further scaling-up application. However, because of their small size (< 10 nm) and the presence of plenty of polar functional groups on the surface, CDs show high colloidal stability in aqueous media and cause difficulty in obtaining high purify CDs. Newly reported purification methods include filtration, column chromatography, centrifugation, precipitation, pH control cloud point extraction [41], solid-phase extraction (SPE) [42], and dialysis. So far, dialysis is the predominant technique used to separate CDs from colloidal solutions using a semipermeable membrane. By choosing suitable molecular weight cutoff of dialysis bags, the small-sized target CDs penetrate through the membrane pores, while the bigger-sized nontarget compounds are kept in the dialysis bag. The main drawbacks of this method include the dilution of the dialyzed CDs solution, and the time-consuming process of dialysis. Centrifugation is another method reported for purification of CDs. Adjustment of centrifuge speeds can provide several fractions of NPs of different masses [43]. An organic solvent such as acetone could be added to separate the fractions in small mass ranges. Size-exclusion chromatography has also been applied to separate synthesized CDs products into several fractions, but the operation is tedious, and the elution conditions need to be carefully optimized. Koutsioukis et al. [42] reported an effective purification method by SPE after organic solvents extraction. CDs were first extracted into an organic solvent such as acetone and ethanol, further adsorbed on the surface of alumina as the solid phase, then eluted from alumina using water. Beiraghi and Najibi-Gehraz [41] applied pH-controlled cloud-point extraction (CPE) to separate two fractions using Triton X-114 as the enrichment phase and water as the back-extraction phase. Additional advances on purification techniques for CDs had been summarized by Kokorina et al. [44].

3.3 Photoluminescence (PL) principles

When CDs are excited under photons, the electrons jump to excited states and then return to the equilibrium states/ground state. The excess energy was released through the radiative process, which generates light emission known as PL. PL is the most attractive and important feature of CDs, determining their optical–optical properties. For fluorescence sensors, the detection is established based on the changes in the optical properties. Thus, a well understanding of PL principles helps design effective sensing strategies of CDs for HMs detection.

CDs with PL emissions could range from deep ultraviolet to near-infrared (NIR). The illumination of PL principles can guide exploring unique and novel applications of CDs, including HMs sensors. Although some controversy exists, four PL principles could be elucidated for CDs: the quantum confinement effect, surface/edge state, molecule state, and cross-link enhanced emission (CEE) effect [37]. The quantum confinement effect is mainly determined by conjugated π domains and cluster size. The emission wavelength was found to decrease with the decrease of the size of CDs, as the energy gap between the valence shell and conduction band widens [38], while the PL emissions increased with the increase of the fused aromatic rings as the bandgap of conjugated π domain narrows. The surface/edge state is related to the synergetic hybridization of the carbon core and connected chemical groups. Some reports found that more functional groups such as C=N/C=O and C–N groups are generated on the surface of the full-colored CDs than the CDs with single emissions. The molecule state relates to organic fluorophore on the surface of the carbon core, which can exhibit PL emission directly. When CDs were prepared at low reaction temperatures, small fluorophore molecules may be formed beside the carbogenic core obtained [39]. The exist of molecular fluorophores is beneficial to the improvement of QYs. CEE effect involved the formation of a new PL center induced by aggregation or cross-link. Yang’s group [40] found that the fluorescence of polyethyleneimine (PEI)-based PDs was amplified by the CEE effect, attributed to the decreased vibration and rotation in crosslinked PEI-based PDs structures. Commonly, the fluorescence of CDs is determined by one or a combination of these principles.

3.4 Sensing mechanism

The sensing applications for HMs detection using CDs were based on the phenomenons that the interactions between HMs and CDs either quench or enhance the fluorescence. The quenching or enhancing effect involve different sensing mechanisms including static and dynamic quenching mechanism, photoinduced electron transfer (PET), energy transfer (ET), inner filter effect (IFE), aggregation-induced emission quenching (ACQ), and aggregation-induced emission enhancement (AIEE) or coordination-induced aggregation (CIA) [45]. The quenching or enhancing of fluorescence intensity may be caused by one or simultaneously two sensing mechanisms, and some characterizations could be tested to verify and confirm the sensing mechanisms.

In terms of kinetic mechanism, static quenching occurs when the molecules form a complex in the ground state, i.e., before excitation occurs, through the interaction between CDs and HMs. In contrast, dynamic quenching happens when excited states return to the ground state by collisions between CDs and HM ions due to ET or charge transfer. To investigate the kinetic mechanisms of static and dynamic quenching, UV-Vis spectra, Stern-Volmer plot, and fluorescence lifetime decay in the absence and presence of HMs by time-correlation single photon counting method are usually investigated. In static quenching mode, the fluorescence lifetimes of CDs will have no change in the absence (τ ₀) and presence (τ) of various concentrations of HMs. The absorption spectra of UV-Vis usually have some changes after the addition of HMs due to the formation of a complex in the ground state. Also, the calculated binding constants between HMs and CDs will reduce with increasing the temperature in the sensing system. On the contrary, in dynamic quenching, τ is shortened after the addition of HMs compared to τ ₀; no changes would be observed from the UV–Vis of CDs with the addition of HMs. Moreover, the increase in binding constant along with the increase of temperature could be calculated, as the higher frequency of effective collisions between CDs and HMs would occur at a higher temperature.

If positively charged HM ions were added into negatively charged CDs solutions, an effective PET process might occur from CDs to HM ions. In that case, the excited state electron transfer from CDs to the lowest unoccupied molecular orbital’s of the HM ions, usually leading to quenching of the fluorescent intensity of CDs.

The ET include förster resonance energy transfer (FRET) and dexter energy transfer (DET). In FRET, the photonic energy of the donor (CDs) is acquired by the acceptor (HMs). The efficiency of ET between CDs and HMs is controlled by the extent of spectral overlapping between the emission of CDs and the absorption by the metal ions, the spatial distance (generally in the range of 10–100 Å) between CDs and HMs [46], and the fluorescence QYs of CDs. The fluorescence lifetime of CDs would decrease during the FRET process. In DET, the electron transfer is caused by redox reaction, not photoinduced. As mentioned, CDs are excellent electron acceptor and electron donator, which could act as oxidants and reductants, respectively. Therefore, DET might occur in the HMs sensing process using CDs as the match between the redox potentials of CDs and HMs is satisfied.

The IFE phenomenon occurs when the absorption spectra of the HMs precisely overlap with the excitation or emission spectra of fluorophore materials (CDs). As a result, the attenuation of the excitation beam or absorption of emitted radiation leads to the apparent fluorescence quenching of CDs. In IFE, no new substance is formed, and the average fluorescence lifetimes of CDs measured do not exhibit significant changes in the presence of HMs. As Cr (VI) has a broad absorption band which can effectively overlap with the emission or excitation spectra of CDs with blue light, the IFE strategy has often been applied to detect Cr(VI) [47], [ 48].

AIEE and CIA are related to the aggregation of CDs caused by temperature, solvent, additives like HMs [49], [50], [51]. When aggregation happens, the intramolecular motion is restricted, which blocks the nonradiative path and activated radiative decay. Characterization methods like transmission electron microscopy (TEM), dynamic light scattering, and small-angle X-ray scattering can be used to prove ACQ or AIEE processes directly. The variations of Zeta-potential and UV-VIS spectra could be measured to know the formation of HM ion–CD complexes indirectly.

4.1 Coordination and chelation

Coordination and chelation are the most common and straightforward strategies to design CD-based HMs sensors. The empirical hard and soft acid and bases (HSABs) principle could be an essential consideration for designing suitable ligands to a specific HM. According to the HSAB principle, soft HMs like Ag(I), Hg(II), Cd(II), and MeHg(I) prefer to form covalent bonds with soft ligands such as –SH, –S–S, and –S–R; transitional metals including Fe(II), Co(II), Ni(II), Cu(II), Zn(II), and Pb(II) trend toward binding borderline ligands like –CO–NH–, >N=H, –C₆H₄NH₂; and hard HMs of Mg(II), Sr(II), Mn(II), Al(III), Cr(III), Fe(III), U(II) have priority to bind with coordinating groups of –OH, –F, –Cl, –PO₄ ³⁻, –OOCR, –OR, –NH₂, –SO₃H, >PO₄, and –NH₂R [52]. Of course, it still needs to notice that the rule is not absolute, especially for transitional HMs. The interaction with hard oxygen/amino groups and soft thiol groups is observed in many complexes. Different coordination structure modes formed like linear or tetrahedral coordination, size-controlled macromolecule ligands involved, and other factors all can affect the association constant. Furthermore, the hardness of different species of the same HM also can vary a lot and needs to adopt different coordination ligands. For instance, As(III) is a typical soft metal, while As(V) is an analog of PO₄ ³⁻ belonging to the hard metal group. Also, considering an abundance of interference ions existed in real sample matrices and most of the synthesized CDs with many oxygen-containing functional groups, the interference caused by nontarget ions needs to be systematically investigated, rendering the sufficient selectivity.

To render the surface of CDs with moieties to bind strongly with HMs, precursors with suitable ligands according to the HSAB principle could be chosen. Mainly heteroatoms like S, N, O, and P could be functionalized as ligand atoms in the form of chemical groups like –SH, –S–S, –NH₂, =NH, –OH, –OPO₃H, or >C=O. These as-prepared CDs could be used as nonlabel probes to detect HMs directly. For instance, thiol groups (–SH) provide an excellent binding affinity to Hg(II), thus was commonly induced to detect Hg(II), even though some drawbacks such as undesired oxidation of sulfides during long-term storage at ambient temperature and possible ineligibility for use in sulfur-rich environments need to be considered and carefully avoided. Ding et al. [49] prepared N-, S-doped CDs using glycerol as the reaction solvent and cystine as the source for C, N, and S. The resulting CDs can specifically interact with Hg(II).

Except for the nonlabeled CDs with suitable ligands as HM sensors, prepared CDs could be further modified with functional groups to increase selectivity via surface chemistry or other interactions like covalent bonding, coordination, π–π interactions, and sol–gel technology. CDs with carboxyl groups were prepared by Ottoor et al. [53] using jeera as a carbon source, then capped with cystamine by carbodiimide chemistry, where 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) is used as an effective crosslinking agent along with N-hydroxysuccinimide (NHS). The cystamine functionalized CDs show much selective quenching response to Cr(VI) compared to unconjugated CDs toward various interference metal ions. The enhancement of selectivity was attributed to the formation of Cr(VI)-cystamine complex, facilitating the charge transfer between cystamine-CDs and Cr(VI). In Kaur et al.’s work [54], surface modification of their as-synthesized CDs with calix[4]arene derivative was done by stirring the reaction mixture of 1,3-disubstituted calix[4]arene and CDs in DMSO at room temperature, FT-IR was used to monitor the course of the substitution at the surface of the CDs. These modified CDs have high selectivity to detect Zn(II) against the other interference metal ions such as Cd(II) and Hg(II) with similar nature. The high selectivity benefits from the structure of calix[4]arene with plentiful rigid cavities of unalterable sizes, which can embrace Zn(II) with exact size.

4.2 Electrostatic effect

If the binding sites on the surface of CDs involve ionizable functional groups at certain pH ranges, electrostatic effects may exist between CDs and HMs. For hydroxyl, carboxyl, thiol, sulfonate, and phosphonate groups, they will be deprotonated and negatively charged when the pH value of the medium exceeds their pKa values; For amine, imine, amide, and imidazole groups, they will be positively charged when protonated at pH lower than their pKa. In acidic medium, anionic metal species like Cr(VI) tend to bind better with positively charged groups like amine; cationic metal species reach their maximum binding in a neutral medium when most of the acidic binding groups like carboxyl are deprotonated, and the cationic metals are not precipitated. Therefore, in this strategy, pH conditions will affect sensor performance. Li et al. [40] fabricated Tb(III)-2, 6-pyridinedicarboxylic acid-N-doped CDs (Tb-DPA-CDs) sensor system for detecting Hg(II) in seafood. The quenching efficiency of N-CDs was found to increase in the pH range of 2.0–7.0. That is because the deprotonation of –COOH on the surface of CDs increased with the increase of pH, leading to the negatively charged –COO⁻, which could maximally capture the oppositely charged Hg(II).

4.3 Oxidation-reduction reaction

Photoexcited CDs are reported as excellent electron donors and can reduce metal ions in solution [55], [ 56]. Therefore, an oxidation-reduction reaction could be employed to design CDs based HM sensor. The oxidation–reduction reaction can occur between HMs and CDs, which generate electron transfer and led to nonradiative electron/hole recombination, resulting in the PL changing. Huang et al. [57] prepared fluorescent N-doped GQDs (N-GQDs) to the selective determination of Cr(VI) based on the quenching effect. The quenching mechanism was proved that parts of Cr(VI) were reduced to Cr(III) on the surface of N-GQDs by X-ray photoelectron spectroscopy characterization. The quenching equilibrium reached in 3 min, which means the kinetic behavior of oxidation–reduction reactions between N-GQDs and Cr(VI) was fast. Wang et al. [58] also found that the fluorescence intensity of fabricated CDs was significantly and selectively quenched in the presence of Cr(VI) due to the oxidation–reduction reaction between Cr(VI) and the oxygen-containing groups and S-related species on the surface of CDs.

4.4 DNA aptamer affinity

Aptamer oligonucleotide sequences can selectively bind with HMs to form stable metal-mediated base pairs by groove binding, electrostatic interaction, and intercalation [59], [60], [61], [62]. For instance, DNA with multiple thymine (T) sequences was well known to specifically bind to Hg²⁺ to form a T–Hg²⁺–T structure with two T residues. Their binding undergoes proton substitution reactions to coordinate with Hg(II) via Hg–N bonds. The binding strength of the T–Hg(II)–T complex is even higher than that of the base T-A pair [63]. Thus, T-rich sequences could be applied as recognition molecules to selectively detect Hg(II). DNA-based sensors are rapid, low cost, and suitable for real-time detection due to the stability of DNA. Now, a lot of methods based on DNA detection of HMs have been developed, such as colorimetric detection, electrochemistry, and surface plasmas resonance, fluorescence, and others [64], [ 65]. Fluorescence methods usually involved the chemical conjugating of DNA with a fluorophore, e.g., dyes [64], metal NPs, and quantum dots [66] as a molecular recognition probe. Until now, few works have been reported using CD-based sensors employing aptamer affinity to selectively detect HMs [67].

Carboxyfluorescein (FAM)-DNA macromolecules were strong assembled on the surface of the N-CDs through π–π stacking by Ni et al. [68]. With the aid of 6-mercaptopurine (6-MP), a fluorescence peak at 525 nm of NCDs–ssDNA-6-MP conjugation system was observed. With the further addition of Hg(II), the large system of NCDs–ssDNA-6-MP was broken up due to the formation of stronger T–Hg(II)–T complex, resulting in the quenching of the fluorescence at 525 nm. Thus, the Hg(II) was detected by this quenching effect. Salimi and Hamd-Ghadareh [69] designed a fluorescence aptamer sensor system for quantitation of Hg(II) based on the hybridization of T-rich ssDNA immobilized on Rhodamine B(RB)–CDs (RB–CDs–ssDNA) and gold nanoparticles (AuNPs) modified with complementary aptamer (AuNPs-cDNA). By excitation at 450 nm, RB–CDs–ssDNA nanohybrid showed the dual well-resolved emissions at 580 and 668 nm, respectively. With the addition of AuNPs-cDNA, the affinity binding of DNA strands decreases the distance between RB–CDs–ssDNA and AuNPs-cDNA and produced enhanced FRET pairs, resulting in a decrease of fluorescence. In the presence of Hg(II) as the analyte, the hybridized DNA strand will dehybridized due to the high affinity of ssDNA toward Hg(II), and AuNPs-cDNA was released from the assemblies, resulting in the increase of fluorescent intensity at 580 nm and decrease at 668 nm. The Hg(II) was thus high selectively detected by calculation of the I ₅₈₀/I ₆₆₈ intensity ratio.

The fluorescence of N/Ce-doped CDs prepared by Liang et al. [70] was found to be quenched by As(III)-specified aptamer via the formation of a large CD–aptamer conjugate due to π–π electron stacking, hydrogen bond, hydrophobic force, and intermolecular force. When As(III) was added, As(III) can specifically react with aptamer to form a stable aptamer–As(III) complex, resulting in the restoration of fluorescence intensity in this As(III) sensing system.

Yang et al. [65] labeled the synthesized CDs with DNA as fluorophore and use graphene oxide (GO) as a quencher to quench the fluorescence through π–π stacking interaction. The detection of Hg(II) was realized by measurement of fluorescence recovery in the presence of Hg(II) due to the release of DNA-CDs from GO through the formation of T–Hg(II)–T duplex. Similarly, Srinivasan et al. [71] adopted the MoS₂ nanosheet as a quencher to quench the fluorescence of DNA functionalized CDs and detect the Hg(II) by the recovery of the fluorescence signals. Wei et al. [72] reported an amplified assay for Hg(II) determination based on an ssDNA-labeled FAM-CQDs hybrid. Firstly, the quenched fluorescence of ssDNA-labeled FAM by QCDs was restored in the presence of Hg(II) like other works above. With the further addition of DNase I, a 110% signal increase was obtained over that without DNase I. The reason is that the nuclease of DNase I can cleave the ssDNA–Hg(II) complex and release the Hg(II). The released Hg(II) then binds another ssDNA, and the cycle starts, which leads to amplification of the signal. With this amplification strategy, a 20-fold lower limit of detection (LOD) than that of traditional unamplified homogeneous assays was achieved.

Alternatively, Song et al. [73] directly used DNA as a carbon source to synthesize CDs by hydrothermal method. The characterizations showed residual thymine (T) and cytosine (C) groups are still existing on the CDs, which could strongly bind with Hg(II) and Ag(I), respectively, resulting in a quenched fluorescence.

More aptamer-based CD sensors for HM detection are summarized in Table 2.

5.1 Turn-on

To date, most of the fluorescent probes in detecting HMs are based on a fluorescence quenching (“turn-off”). For fluorescent probes with turn-off response, interference ions or compounds in the matrix may also quench the fluorescence, lowering the selectivity and limit the practical applications [49]. In comparison, fluorescent probes with turn-on (switch-on or OFF–ON) responses could reduce false-positive signals and the dark background, resulting in increased selectivity. Therefore, it is highly desirable to establish fluorescent ‘turn-on’ probes to detect HMs. AIEE and chelation-enhanced fluorescence (CHEF) effect is most employed to design turn-on probes. The AIEE-based fluorophores show non- or very weak emission in its dissolved state at first. When target HMs were added to form aggregation with the intramolecular motion restriction, the fluorescence turn to show strong emission. The CHEF effect of fluorophores is attributed to the enhancement of fluorophore when some types of complexation with nonquenching HMs are formed. Different strategies such as doping, conjugating, oxidation–reduction reaction, and OFF–On response could be adopted to make turn-on fluorescent sensors. Up to now, turn-on sensors mainly focus on organic dyes, while rarely on fluorescent CDs.

Ding et al. [49] found that the prepared N-, S-doped CDs have a turn-on fluorescence response to Hg(II) based on AIEE, and thus used as selective Hg(II) probe. The aggregation is attributed to the formation of a new complex between CDs and Hg(II) by S–Hg bonds, and the larger percentage of sulfur content of CDs contained, the higher enhancement of the fluorescent intensity caused by AIEE was observed.

In Zhang et al.’s method [50], amine-functionalized CDs were firstly prepared and purified, then conjugated with reduced glutathione (GSH) by the carbodiimide-activated coupling using EDC/NHS. Benefit with free carboxyl and amino groups of GSH to provide binding sites for metal cations, the fluorescence enhancement of resultant GSH-CDs in the presence of Fe(III) ions was observed, and the “turn-on” probe for Fe(III) detection was thus constructed.

Guo et al. [51] designed a fluorescence “turn-on” sensing for Pb(II) detection via CDs immobilized in spherical polyelectrolyte brushes (SPBs) with “OFF–ON” response. The fluorescence of CDs was firstly ”turned off” via electrostatic interaction induced quenching by SPB, then specifically turned on by the addition of Pb(II) via the aggregation of the SPB particles. Zhang et al. [74] also developed a “turn on” fluorescence CDs sensor for selective Hg(II) determination based on bis(dithiocarbamato)copper(II) (CuDTC2)-conjugated CDs (CuDTC₂-CDs). They found that the CuDTC₂ complex at the CDs’ surface could strongly quench the fluorescence of the CDs by a combination of electron transfer and ET mechanism. With the addition of Hg(II), the quenched fluorescence of the CuDTC₂-CDs was switched on because Hg(II) displaced the Cu(II) in the CuDTC₂ complex, and thus shutting down the ET pathway.

Chen et al. [75] constructed a turn-on fluorescence sensor with green luminescent emission to detect Ag(I) based on the oxidation–reduction reaction that occurred on the surface of CDs. Because the reduction potential of Ag(I)/Ag is relatively high (0.7991 V), Ag(I) has precedence over other metal ions to be reduced to Ag⁰, which could enhance the radiative emission of CDs.

In Table 3, a list of turn-on CD-based fluorescence sensors for HM detection is given.

5.2 Ratiometric measurement

Even though most of the fluorescent sensors were designed using a single emission wavelength for quantification by either off or on effect, the drawbacks are obvious. Many analyte-independent factors could affect the fluorescence signals including photobleaching of the fluorophore, light scattering caused by the sample matrix, fluctuations in the excitation source, and the changes of the solution environment. Ratiometric fluorescent (RF) sensors, which detect the analytes by measuring the change of the ratios of fluorescence intensities at two wavelengths, could provide a built-in self-calibration as an internal standard to correct analyte-independent parameters and eliminate the impact of false signals caused by the background fluorescence mentioned above. Therefore, RF measurement is expected to be more reliable and accurate. RF sensors can be designed by reference mode and dynamic mode. In reference mode, a fluorescence channel without a signal change in the presence of target analytes acts as a reference and another channel with signal increase/decrease/shift upon analyte’s presence as a signal reporter. In dynamic mode, two dynamic channels with signal changes in opposite or same directions with the exist of analytes were applied [76]. Fluorescent organic dyes [77], lanthanide elements [78], and quantum dots [79], [ 80] could be employed to design RF-based sensors. Among them, organic fluorophores were most applied but usually needed elaborate molecular design and complex synthesis. RF-based CDs sensors are relatively less reported but are very promising. Different strategies could be utilized to design RF-based CD sensors for HMs detection.

5.2.2 Mixture of different kinds of CDs

The RF-CDs sensor also can be realized by choosing two suitable CDs to mix as a sensor system. Two kinds of CDs with different emission ranges of red and blue were synthesized separately by Yu et al. [84] and mixed to provide reference fluorophore and signal response fluorophore, respectively. The CDs with blue emission (B-CDs) was prepared with gelatin and 3-aminobenzeneboronic acid as carbon sources by hydrothermal method, while the CDs with red emission (R-CDs) was synthesized by phenylenediamine solvothermal method. Upon addition of Hg(II), the fluorescence intensity of B-CDs continuously decreases along with the increasing concentration of Hg(II), while the fluorescence intensity of the R-CDs remains almost unchanged. Zhao et al. [85] reported a similar approach to design RF-CDs as Cu(II) sensor by mixing two color CDs (Figure 1). The mechanism is interesting: Cu(II) ions can efficiently bind to the red CDs (R-CDs) to produce a strong visible absorption, which overlaps the emission of blue CDs (B-CDs). The adsorption of small B-CDs onto the surface of larger R-CDs with existing of Cu(II) in solution was observed by TEM due to the dual-coordinating interactions of Cu(II) with the surface ligands of both R-CDs and B-CDs. As a result, FRET could occur between B-CDs and Cu(II)-R-CDs, leading to quenching of the fluorescence of B-CDs, and unchanging of the fluorescence of R-CDs. Gogoi et al. [86] reported a carbon-based nanohybrid system comprising GSH-functionalized reduced CDs (GSH-f-rCDs) with emission in the violet region as signal reporters and green-emitting GQDs as reference for As(III) detection with a lower detection limit of 0.5 ppb.

Figure 1:

(A) Transmission electron microscopy (TEM) image of small carbon dots with blue emission (b-CDs) adsorbed onto lager Red-CDs after the addition of Cu(II) ions; (B) schematic illustration of the conjugation of blue-CDs and red-CDs through a Cu(II) ions bridge. (C) Schematic of the ratiometric fluorescent (RF) sensor designed to determine Cu(II) by mixing red-CDs and blue-CDs; Reprinted from Zhao et al.’s work with kind permission of American Chemical Society [85]. Copyright (2017) American Chemical Society.

5.2.3 Combination of CDs with other fluorophores

The design of the RF-CD–based sensing system for HMs detection can also combine CDs with other fluorophores such as organic dyes, metal clusters, quantum dots, and MOFs, as long as they can be excited at the same time. The combination can be the direct mixture of CDs and other fluorophores, postmodification of CDs with other fluorophores, or covalent bonding between CDs and fluorophores. CDs can be applied as either reference fluorophore or signal response fluorophore.

A dual-signal RF sensor of CDs/AuNCs nanosystem was developed by He et al. [87] by simply mixing CDs with Au nanoclusters (AuNCs) to detect Ag(I). CDs and AuNCs have no interaction with each other. CDs served as reference fluorophore, and Au NCs served as a recognition unit with turn-on response. Wu et al. [88] developed a specific RF probe by adding Rhodamine B (RhB) as a reference to CDs as a sensing signal for sensitive detection of Hg(II). He et al. [89] synthesized nitrogen and cobalt(II) codoped CDs encapsulated in europium MOFs (CDs@Eu-MOFs) as a RF sensor for the detection of Cr(VI). The fluorescence intensity of the CDs decreased and the intensity of the Eu-MOFs remained in the increase of Cr(VI) concentration. Lu et al. [90] embedded CDs and AuNCs into classical MOFs of ZIF-8 to form CDs/AuNCs@ZIF-8 nanocomposites for RF detection of Cu(II) and CDs served as reference. Mahmoudi et al. [91] combined blue emission CDs (B-CDs) and thioglycolic acid (TGA)-capped yellow emissive cadmium telluride quantum dots (TGA–CdTe–QDs) to consist of ratiometric fluorescence probe for Hg(II) detection. With the exposure of Hg(II) ions to the probe, the fluorescence of TGA–CdTe–QDs was selectively quenched and red-shifted, while the blue emission of CDs remained constant as an internal standard.

Wang et al. [92] conjugated FTIC dye with PEI-modified CDs to fabricate CDs-FITC composites as Cu(II) RF sensor and observed that the spectral peaks both at 412 nm (for CDs) and 518 nm (for FITC) gradually decreased with the increase of Cu(II). Tan et al. [93] conjugated a europium complex onto the surface of CDs to form dual-fluorophore NPs of CDs–BHHCT–Eu(III) (Figure 2) and applied them in the RF detection of Cu(II) with emission at 615 nm as signal and 410 nm as reference.

Typical RF CD-based fluorescence sensors for HMs detection are listed in Table 4.

Figure 2:

Preparation of carbon dots (CDs)-BHHCT-Eu(III) dual-fluorophore system. Reprinted from Tan et al. works with the kind permission of The Royal Society of Chemistry [93]. Copyright (2014) The Royal Society of Chemistry.

Table 4:

Summary of RF CD-based sensors for HMs detection.

HMs	RF mode	CD sensor system	LODs	Sample	Reference
Cr(VI)	Mode 1	CDs at 430 nm(D), 510 nm(D)	0.4 μM	Textile, steel, industrial wastewater	[150]
Hg(II)	Mode 1	CDs at 406 nm, 678 nm	9 nM	Serum, water	[81]
Cu(II)	Mode 1	CDs at 412 nm, 553 nm	23 nM	Water	[151]
Ag(I)	Mode 1	CDs at 532 nm, 619 nm	0.27 μM	Cell imaging	[82]
Pb(II)	Mode 1	CDs at 477 nm, 651 nm	0.055 μM	NA	[152]
Fe(III)	Mode 1	CDs with multicolor from violet to red	10 nM	Water	[83]
Cu(II)	Mode 2	B-CDs, R-CDs	37 nM	Water	[85]
As(III)	Mode 2	GSH-f-rCDs(S)+GQDs(R)	0.5 ppb	NA	[86]
Hg(II) Cr(VI)	Mode 3	CDs (R)+ BSA-Au NCs(S) BSA-Au(R) + CDs (S)	1.85 nM 5.34 nM	Water, human blood	[107]
Hg(II)	Mode 3	BSA-CDs(R)+ AuNCs(S)	0.73 nM	Water	[153]
Ag(I)	Mode 3	CDs(R)+AuNCs(S)	2 nM	NA	[87]
Cd(II)	Mode 3	CDs(R)+AuNCs(S)	32.5 nM	NA	[154]
Cu(II)	Mode 3	CDs(R)+ Au NCs(S)	0.33 nM	Serum	[90]
Hg(II)	Mode 3	CDs(S)+SiNCs	7.63 nM	NA	[155]
Zn(II)	Mode 3	Fe doped CDs(S)+ Au NCs(R)	0.1 μM	NA	[156]
Cr(VI)	Mode 3	RhB -doped silica nanoparticles(R)+CDs(S)	1.3 ng/mL	Water	[89]
Hg(II)	Mode 3	CDs(S)+ RhB dye(R)	25 nM	serum	[88]
Fe(III)	Mode 3	CDs(S)+RhB dye(S)	0.73 μM	NA	[157]
Cu(II)	Mode 3	CDs(R)+ RhB dye(S)	2 ppb	Water	[158]
Hg(II)	Mode 3	CDs(S)+ RhB dye(R)	30 nM	Water	[159]
Hg(II)	Mode 3	NCDs(S)-RhB(S)@COF	15.9 nM	NA	[130]
Cr(VI)	Mode 3	Eu-MOFs(R)+ Co doped CDs(S)	0.21 μM	Water	[160]
Hg(II)	Mode3	CDs(R)+ CdTe-QDs(S)	4.6 nM	Water	[91]
Cu(II)	Mode 3	CDs(R)+CdTe-QDs (R)	0.36 nM	Water	[161]
Hg(II)	Mode 3	CDs(S)+ CdTe@SiO2(R)	0.47 nM	Water	[162]
Fe(III)	Mode 3	CDs(R)+CdSe QDs(R)	5.4 μM	Cell imaging	[163]
Hg(III)	Mode 3	CDs(R)+ CdSe @ ZnS QDs (S)	0.1 μM	Water	[164]
Cu(II)	Mode 3	CDs(R)+Mn‐doped ZnS QDs(S)	33.1 nM	NA	[165]
Cu(II)	Mode 3	CDs(R)@Eu-DPA MOFs(S)	26.3 nM	NA	[166]
Cu(II)	Mode 3	CDs(R)/QDs(S)@ZIF-8	1.53 nM	Water	[167]
Cu(II)	Mode 3	PEI-CDs+FTIC dye	7 nM	Serum, yogurt	[92]
Cu(II)	Mode 3	CDs(R)–BHHCT–Eu(III)(S)	4 nM	Water	[93]

1. Mode 1: CDs with multichannel emissions; Mode 2: Mixture of different kinds of CDs; Mode 3: Combination of CDs with other fluorophores; ND, not discussed; NA, not analyzed; MOFs, metal-organic frameworks; GQDs, graphene quantum dots; HM, heavy metal; RF, ratiometric fluorescent; PEI, polyethyleneimine.

6.1 Single HM detection

Until now, most efforts have been made to improve the sensitivity and selectivity of the CD-based fluorescence sensors to detect single HMs either based on turn-on or turn-off effect [94], [ 95]. Devi et al. [96] reviewed many applications on the determination of single HM in water.

6.2 Multiple HMs detection

To date, CD-based fluorescence sensors designed for the detection of two or more kinds of metal ions simultaneously have seldom been reported due to the lack of separation ability of fluorescence detection. From recent publications focusing on multi-HMs detection, the following strategies have been adopted for multi-HMs sensing.

6.2.3 Differentiation by CDs contained multifluorophores

Multichannel emissions of CD-based sensing systems also could be constructed through assigning two or more types of fluorophores in the sensing system to detect multi-HMs. Zhang et al. [105] mixed two types of multiemission CDs to form nanohybrids for the determination of Pb(II) and Hg(II). Interestingly, these two CDs were synthesized from the same natural biomass with and without the addition of Na₂CO₃, respectively, during solvothermal treatment. In the presence of Pb(II), the decrease in fluorescence at 493 nm and the increase in fluorescence at 653 nm were observed, and a peak intensity ratio of I ₆₅₃/I ₄₉₃ was recorded to determine Pb(II) concentration. With the existence of Hg(II), fluorescence intensity at 611 nm decreased with a redshift occurring and no changes at 491 nm. Therefore, the I ₆₁₁/I ₄₉₁ ratios were calculated to determine Hg(II) concentrations. Shen et al. [106] mixed as-prepared three kinds of CDs from different initial materials, which had different specific responses to three metal ions (Hg(II), Fe(III), and Cu(II)), and developed a smartphone-based three-channel ratiometric fluorescence device for simultaneous determination of these three target metal ions. Su et al. [107] designed a dual emissive multicolorful fluorescent nanoprobe of Au NCs-CDs (gold nanocluster-CDs) to simultaneously detect two ions of Hg(II) and Cr(VI) (Figure 3). When Hg(II) was present without Cr(VI), the signal peak of Au NCs at 625 nm decreased, and the reference peak of CDs at 444 nm remain consistent, leading to the fluorescence color changing from pink to blue under UV light. In the presence of Cr(VI) without Hg(II), the fluorescence emission of CDs was quenched, while the emission of Au NCs was stable and was used as an internal reference. When both ions were exposed to the Au NCs-CDs, the dual-emissions of the Au NCs-CDs were all decreased.

Figure 3:

The schematic illustration of the preparation of gold nanoclusters (Au NCs)-carbon dots (CDs) and detection for metal ions of Hg(II) and Cr(VI). Reprinted from Su et al.’s work with the kind permission of Elsevier [107]. Copyright (2018) Elsevier.

6.2.4 Discrimination by restoration reagent

If the fluorescence of CDs is quenched by multiple metal ions simultaneously, the restoration reagent could be added to discriminate each ion further. Gogoi et al. [108] successfully developed a CD-based sensor to detect Hg(II) and Cu(II) in parallel by an on–off–on process with the addition of vitamin C and trisodium citrate, respectively (Figure 4). With the addition of trisodium citrate, the quenched fluorescence from the Cu(II)-CDs mixture was recovered, while the fluorescence from Hg(II)-CDs did not recover, which was attributable to the fact that the Cu(II)–SC adduct was more stable than the Cu(II)–CDs adduct. Similarly, the fluorescence of CDs quenched by Hg(II) ions could be recovered with vitamin C, while the quenched fluorescence of Cu(II)–CDs remained unchanged. The reason was that ascorbic acid (AA) was capable of reducing Hg(II) to Hg(I) but not Cu(II), leading to the recovery of fluorescence of Hg(II)–CDs mixture by freeing the CDs, while Cu(II)–CDs mixture remained in the quenched state.

Figure 4:

Simultaneously sensing of Cu(II) and Hg(II) by fluorescence ON–OFF–ON process with the addition of restoration reagents. Reprinted from Zhao et al.’s work with the kind permission of The Royal Society of Chemistry [108]. Copyright (2019) The Royal Society of Chemistry.

6.3 Speciation of HMs

Speciation of HMs is often more important than the determination of total HMs considering their toxicity and bioavailability. However, although there has been tremendous progress in developing fluorescent probes to detect the total concentration of HMs, few publications have reported on the discrimination of different species of a particular HM. Most of these previous publications reported on the speciation of Cr(III) and Cr(VI) by CD-based fluorescence methods, probably because the chemical behavior of Cr(VI) and Cr(III) exhibits relatively large differences in solution [57]. Li et al. [114] developed a simple approach based on calcination treatment of diethylenetriaminepentaacetic acid to obtain CDs with a high QY of approximately 53.7%. The fluorescence of CDs could be quickly and efficiently quenched by Cr(VI) rather than by Cr(III) based on an IFE. Su et al. [115] prepared CDs by using graphene oxide powder through hydrothermal methods and found that Cr(III) could quench the CDs because of electrostatic adsorption of Cr(III) on graphene oxide derivatives (GQDs) and the strong chelation between Cr(III) and the –COOH and –OH groups on the surface of GQDs. As AA and acid phosphatase could reduce Cr(VI) to Cr(III), the indirect detection of AA and acid phosphatase was proposed with the addition of Cr(VI). In Yang et al.’s approach [116], Cr(VI) was detected based on the selective quenching effect, and concentrations of Cr(III) were calculated by subtraction of Cr(VI) concentration from the total Cr concentration, which was detected after the addition of KMnO₄ to oxidize all possible Cr(III) in the sample solution to Cr(VI). More publications on the speciation of Cr are summarized in Table 6.

Beside Cr, there have been very few reports on the speciation of other HMs. Li et al. [117] used lanthanide coordination polymers doped CDs (CDs@AMP-Tb) to detect Fe(II) and Fe(III) species based on the experimental phenomenon that CDs could be quenched explicitly by Fe(II), and Fe(III) could be detected after being reduced to Fe(II) with the help of a reducing agent of NH₂OH·HCl. Recently, Costas-Mora et al. [118] reported in situ ultrasound-assisted syntheses of CDs as optical nanoprobes to selective recognize methylmercury species (Figure 5). The mixture of d-fructose, PEG, NaOH, and EtOH was subjected to ultrasound irradiation for 60 s to obtain the target CDs. The selectivity of the assay was attributed to the different abilities of Hg(II) and MeHg(I) species to interact with CDs encapsulated in PEG. CH₃Hg(I) with hydrophobic property could easily cross the PEG coating, come into contact with CDs and lead to fluorescence quenching, while Hg(II), a hydrophilic Hg species, could not interact in this way with CDs, and no fluorescence quenching was observed.

Figure 5:

Schematic mechanism of selective speciation of MeHg(I) in presence of Hg(II). Reprinted from Bendicho et al.’s work with the kind permission of the American Chemical Society [118]. Copyright (2014) American Chemical Society.

7.1 Paper-based devices

Inspired by the classical success of pH test paper, the fluorescent test papers have been explored by assembling or printing the fluorescent probes onto a piece of paper-based substrates in the visual assays to detect HMs. The filter paper, anion exchange filter paper, cation exchange filter paper, silica gel filter paper, and cellulose nanofibrils paper are potential candidates to be tested with optimal paper surface. The observation of the variations of fluorescence brightness and color with test samples could be achieved by the naked eyes with or without the aid of a simple ultraviolet (UV) lamp. The colorimetric signals could be further collected using a smartphone, which is a portable technology found everywhere today. Like pH test paper, fluorescent test papers also have advantages such as low cost, convenience, easy operation, fast response, and portability, which is especially suitable for on-site detection applications in environmental, medical, and food testing. Meanwhile, some limitations also exist: the construction of paper-based devices is often complicated; the stability and sensitivity of such solid-phase fluorescence detection devices are usually lower than that of liquid-phase detection; most of the reports can only successfully observation of the variations of fluorescence brightness [119] or color changes in the narrow range [84]; usually only semiquantitative of target analytes could be achieved by naked eyes and hence it is not easy to tell the slight changes of brightness and color variations caused by a small dosage of analytes. The quantitative analysis needs the aid of a camera to collect images and software such as Image J to extract signal intensities from the images for further calculation. In this review, some typical applications of CD-based paper devices for HMs detection and efforts to overcome some limitations are discussed.

Lu et al. [120] printed N-doped CDs to fabricate portable paper strips as sensitive fluorescent probes for Cr(VI). Yan et al. [121] hybridized CDs and Au NCs for printing on cellulose acetate circular filter paper to detect Hg(II), which produced a distinct fluorescence color evolution from pink to blue under UV lamp excitation. The cyan CDs and red GSH/DTT-QDs were mixed and printed on filter paper by Zhou et al. [122] as As(III) detection sensors, and the ratio of 1:5 of emission intensity of cyan to red was confirmed to achieve the highest dosage-sensitive ability for visual quantification of As(III) (Figure 6). The detection was based on the aggregation-induced quenching mechanism. The red fluorescence of GSH/DTT-QDs was effectively quenched with As(III), whereas the cyan fluorescence was not, resulting in the change of ratiometric fluorescence and a wide range of color variations in response to the dosage of As(III), thus exhibiting a similar performance like pH test paper.

Figure 6:

Schematic illustration of fluorescence paper strips for the determination of As(III) with a similar performance like pH test paper. (A) Visualization mechanism of As(III) using glutathione (GSH)/DTT-QDs as fluorescent sensory probe and carbon dots (CDs) as an internal standard probe; (B, C) transmission electron microscopy (TEM) images of GSH/DTT-QDs before and after the addition of As(III); (D) Fluorescent spectra of mixing glutathione (GSH)/DTT-QDs/CDs (20/10 μL in 1.5 mL of Tris HCl buffer, pH 7.4) with the addition of As(III). Reprinted from Zhang et al.’s work with the kind permission of the American Chemical Society [122]. Copyright (2016) American Chemical Society.

Gogoi and Patir [108] developed a filter paper–based microfluidic device loaded with nitrogen-doped CDs (NCDs), vitamin C(Vit C), and trisodium citrate (SC) to simultaneously detect multiple metal ions of Hg(II) and Cu(II) ions. As shown in Figure 7, three channels (a, b, c) were dispersed with NCDs, NCDs plus Vit C, and NCDs plus SC, respectively. After drying at room temperature, the filter paper–based microfluidic device was used as a sensing platform under 365 nm UV light. When Hg(II) only was added in the reaction zone, the regions a and c showed no fluorescence, while region b showed fluorescence restoration. When Cu(II) only was added, the regions a and b showed no fluorescence, while region c showed fluorescence restoration. If Hg(II) and Cu(II) both existed, just the fluorescence of region a was quenched, while the fluorescence signals of both regions b and c were recovered. The concentrations of Hg(II) and Cu(II) ions could be detected by calculating the fluorescence intensity in the images captured by a digital camera using the ImageJ software.

Figure 7:

Schematic illustration of filter paper–based microfluidic device to parallel detect multiple metal ions of Hg(II) and Cu(II) ions. Reprinted from Zhao et al.’s work with the kind permission of The Royal Society of Chemistry [108]. Copyright (2019) The Royal Society of Chemistry.

Xu et al. [123] integrated N-CD paper chip, smartphone detection setup, and software application of the Android system together and developed a portable platform for selective detection of Hg(II). In this platform, nitrogen-doped CDs (N-CDs) were immobilized on the filter paper chip for sensing Hg(II). The smartphone detection setup was 3D printed and composed of three components: a smartphone case to fix the smartphone, a black box with two ultraviolet LED light sources (350–370 nm), a rechargeable battery, a power switch, and a smartphone camera. A self-developed software application was applied for the analysis of the fluorescence of N-CDs decorated paper chip. As a sensing platform, the detection limit of 10.7 nM for Hg(II) detection was achieved based on the quenching effect. A paper-based fluorescence sensor with instrument-free signal quantitation was developed for selective detection of Hg(II) by Henry et al. [124]. The quantitation results were obtained by calculating the quenching distance on the filter paper after the addition of Hg(II) by simple measurement with a ruler. With heating preconcentration, the detection limit for this distance-based paper strip with naked-eye detection of Hg(II) was lowered to 5 ug/L, which was sufficient for monitoring Hg(II) in drinking water.

Xu et al. [125] developed a novel microfluidic paper analytical devices (μPADs) by in situ CDs and AuNPs sequential patterning techniques (Figure 8). Combining the two reading regions of CDs and AuNPs, this μPADs provide reliable measurement for serum Fe(III) and serum ferritin simultaneously in whole blood. Fe(III) was detected in the CDs reading region by quenching effect, and ferritin was measured in AuNPs by ELISA reaction. This technique provides a possibility of an iron health test in real clinic applications for blood analysis.

Figure 8:

Schematic illustration of microfluidic paper analytical devices (μPADs) for the measurement of serum iron and serum ferritin in whole blood. Reprinted from Xu et al.’s work with the kind permission of the American Chemical Society [125]. Copyright (2016) American Chemical Society.

7.2 Sensor arrays

A fluorescent sensor array is a differential sensing platform for analytes (usually qualitative detection) in a complex environment. Fluorescence array–based sensing systems are fast, portable, and inexpensive, promising in chemical and biological sensing research. Unlike conventional ”lock and key” type-specific sensor systems, sensor arrays usually contain a series of nonselective sensors with different chemical properties, and target analytes can be differentiated and recognized by analysis of their nonspecific responses to each sensor element. These different responses generate fingerprint patterns, which are later analyzed by statistical methods to recognize the patterns, classify the data, and detect unknown samples. Input data can be fluorescence intensities or the color indices of R, G, and B. To construct a sensor array, a set of sensors should be firstly designed and prepared. Compared to other fluorescence sensors such as metal oxides, semiconductor nanocrystals, and fluorescent dyes, CDs are more attractive materials to be used in sensor arrays because of their simple synthesis procedures. However, as a set of sensors needs to be synthesized, the whole preparation of the sensor array is relatively time-consuming. To date, few publications have reported CD-based sensor arrays for differentiating metal ions.

Liu et al. [126] fabricated seven kinds of CDs (CD1, CD2, CD3, CD4, CD5, CD6, and CD7) for the differentiation of six kinds of HMs (Ag(I), Cd(II), Cr(II), Fe(III), Hg(II), and Pb(II)). Using the principal component analysis method, the hexasensor array could be simplified into a binary sensor array with CD3 and CD5, which already could differentiate six HM ions clearly with established fingerprints. This binary sensor array was successfully applied to identify unknown metal ions in fetal calf serum solutions and local tap water solutions. Also, Chen et al. [127] synthesized three kinds of N doped CDs with various surface states of different amino acids and employed as-prepared CDs in sensor arrays to distinguish six metal ions based on their diverse quenching responses to six metal ions. The discrimination accuracy of unknown samples was found to be 100% at concentrations as low as 10 μM. Furthermore, this sensor array could well recognize the relative complex binary and ternary mixtures of three metal ions.

7.3 Hydrogels or polymers films

Hydrogels or polymer films possess 3-dimensional matrices with porous nature, which allow small molecules and ions to penetrate the hydrogels via diffusion. CDs with sizes comparable or larger than the pore sizes of the hydrogels can be entrapped in the polymer networks. The use of hydrogels incorporated with functionalized fluorescent CDs as sensing systems could overcome the disadvantages of solid printing platforms such as particle aggregation, uneven analyte adsorption, and the possible problem of liquid platforms of shorter shelf life. Therefore, it is considered as potentially a good sensor platform.

Cheng et al. [128] embedded synthesized PEI-modified CDs into the microcrystalline cellulose (MCC) matrix to prepare a fluorescent smart hydrogel. PEI-CDs was found to have strong hydrogen bonding with MCC with rich oxygen- or nitrogen functional groups. In the prepared hydrogels, PEI-CDs acted as the fluorescent sensor to selectively detect Fe(III), and MCC as the gel matrix could eliminate the self-quenching of the fluorescent sensor. Truskewycz et al. [129] prepared a novel hydrogel composite of CDs/PVP/ZnO as an effective Cr(VI) sensing platform with a LOD of 1.2 μM. This hydrogel composite was cheap to manufacture and could be injected into a 96 well plate for high-throughput environmental diagnostic applications. Wang et al. [130] dispersed prepared N-doped CDs and Rhodamine B covalent organic frameworks (NCDs-RhB@COF) nanocomposites into agarose gels to make hydrogel sensor films that could be used for semiquantitative visual detection of Hg(II) under a UV lamp.

Wang et al. [131] attempted to impregnate the prepared yellow-emitting CDs into polymer films as y-CDs/polymer composite Cu(II) sensors to overcome the aggregation-induced instabilities of CDs in sensing performance. They compared the performance of two kinds of polymer films: (a) carboxymethylcellulose/polyvinylalcohol and (b) chitosan. Film (b) was found to be more sensitive than film (a) and thus was thus more suitable as a Cu(II) sensor. The detection limit of the sensor was 10 nM (i.e. 1.3 ppm). Zhang et al. [132] grafted CDs onto a poly(vinylidene fluoride) (PVDF)-g-PAA composite membrane through amidation reaction to prepare a PVDF-g-PAA-CDs composite sensor membrane with stable fluorescence and found that Cu(II), Hg(II), and Fe(III) could quench the fluorescence.

7.4 Ion-imprinted fluorescence polymers

Ion-imprinted polymers (IIPs) are an important branch of molecularly imprinted polymers, which can specifically recognize template ions. Ion imprinted strategy is a desirable and effective method to increase the selectivity to detect analytes in complex matrices. Combining the high selectivity of IIPs with the high sensitivity of CD-based fluorescence sensing is a promising approach but have seldom been used for the determination of targets HMs. The whole procedure to fabricate CD-based IIPs includes the synthesis of CDs, imprinted polymer preparation, and template elution, which is relatively time-consuming and complicated. Usually, one kind of ion-imprinted fluorescence polymers can only detect one target ions. Multitemplate imprinting for multi-HMs is a good strategy for high-throughput detection of HMs.

Liu et al. [101] fabricated Cr(III) and Pb(II) imprinted fluorescent sensors by a simple one-pot sol-gel method using blue and red dual emission CDs for signal output. CDs were first synthesized using a one-step hydrothermal method using citric acid and ethylenediamine as precursors in formamide–water (V:V = 1:3) binary systems. The prepared CDs were then mixed with Cr(III) and Pb(II) ions together with tetraethylorthosilicate and cetyltrimethylammonium bromide (CTAB) in alkaline condition and reacted at 70 °C for 3 h to afford white solid polymer particles with fluorescence. To remove template ions and CTAB micelles, EDTA and ethanol were used as the eluting solvent. Compared with nonimprinted polymers, the quenching efficiency of imprinted polymers was much higher, and the imprinting factors, defined as the ratio of the quenching constants, were calculated to be 6.7 and 4.5 for Cr(III) and Pb(II) detection, respectively. In Lu et al.’s work [133], CD-based IIPs with mesoporous structured dual-templates and dual-reference ratio peaks by one-pot synthesis method for simultaneous fluorescence detection of Fe(III) and Cu(II) were developed (Figure 9). The prepared IIPs had fluorophores of CDs at 420 nm and fluorophores of CdTe QDs at 620 nm. In the presence of Fe(II), CDs were quenched while CdTe QDs were used as a reference. For the detection of Cu(II), CdTe QDs were used for the response signal, while CDs were used as a reference. As a result, the dual-reference IIPs probe could simultaneously detect Cu(II) and Fe(III), while maintaining sensitivity and selectivity with enhanced detection efficiency. Similarly, the same group developed dual-template IIPs for simultaneous detection of Ag(I) and Pb(II) using CDs and AuNCs as fluorescence signal output [134]. Table 7 lists a summary of solid-state CDs-based fluorescence sensing platforms.

Figure 9:

The fluorescence spectra of the ion-imprinted (A) and nonimprinted (C) fluorescence polymers under exposure to different concentrations of Cu(II). (B) and (D) are the plots of (I ₄₂₀)₀/I ₆₂₀ as a function of the Cu(II) concentration. (E) shows the photos of IIP detection solutions under 365 nm UV light, while (F) shows the photos of NIP detection solutions under 365 nm UV light. (G) Schematic illustration of the preparation of dual template ion-imprinted dual reference ratiometric fluorescent (RF) probes to detect Fe(III) and Cu(II). Reprinted from Xu et al.’s work with the kind permission of The Royal Society of Chemistry [133]. Copyright (2019) The Royal Society of Chemistry.