A Review on Characterization Techniques for Carbon Quantum Dots and Their Applications in Agrochemical Residue Detection.

2. Noble metal nanomaterials-based fluorescent nanoprobes for sensing and imaging of metal ions Noble metal nanomaterials, such as gold and silver, have frequently been used in the construction of fluorescent nanoprobes for metal ions during the past decade because of their unique optical properties [ 63 - 65 ]. These metal nanomaterials can be categorized into two types based on the size and optical properties: colloid metal nanoparticles (NPs) and metal nanoclusters (NCs). Colloid metal NPs generally refer to materials with diameters more than 10 nm and less than a few hundred nm. Typically, these NPs produce localized surface plasmon resonance (LSPR) and strong light scattering [ 66 - 69 ], however, they usually have poor intrinsic fluorescent quantum yields (QYs) that are not suitable for direct fluorescent sensing of metal ions. Nevertheless, with the ability to quench molecular excited states, colloid metal NPs could function as effective photoluminescence (PL) quenchers in designing fluorescence-based sensors. On the other hand, metal NCs are defined as small metal clusters (e.g., Au, Ag, Cu, and Pt) with size ranges from sub-nanometer to 10 nm in diameter. Specific features of such metal NCs include high fluorescence, excellent photophysical and chemical stability, good biocompatibility, controllable sizes and tunable emissions, and rich surface chemistry for functionalization [ 70 ]. Because of these useful features, fluorescent probes using metal NCs have contributed to the development of many innovative analytical methods for biosensing and bioimaging in vitro and in vivo .

Colloid metal nanoparticles 2.1.1

Detection of metal ions in environmental and biological samples

Colloid metal NPs with unique optical properties have been widely used for the detection of metal ions in environmental and biological samples [ 71 ]. Among these, gold nanoparticles (AuNPs), including nanospheres and nanorods, are especially attractive because of their convenient synthesis, biocompatibility, and novel optical properties. The synthesis of AuNPs can date back to Michael Faraday’s work in 1857, in which the gold hydrosols were prepared by reduction of an aqueous solution of chloroaurate with phosphorus dissolved in carbon disulfide. Since then, numerous methods have been developed to synthesize and functionalize AuNPs, and these methods have been extensively reviewed in the past [ 72 - 74 ]. Although AuNPs display rather weak fluorescence, they can be used in fluorescent sensors as a “super-quencher” for almost all dyes, because the quenching effect of AuNPs is generally several orders of magnitude higher than that of an organic quencher. Therefore, a critical parameter for fluorescent sensing using AuNPs is the change in distance between the fluorophore and AuNP. This sensing can be achieved by linking a fluorophore probe with a target recognition molecule conjugated to the AuNP. Following this principle, AuNPs have been successfully used to construct fluorophore and quencher-based biosensors for a broad range of metal ions [ 75 ]. Among the many classes of molecules utilized as metal recognition elements, organic complexes are one of the most widely used, due to the diversity in their structural types and physico-chemical characteristics for intramolecular charge transfer (ICT). As an example, Wang et al. [ 76 ] reported a turn-on fluorescent sensor for the detection of Pb 2+ in wastewater samples using a fluorophore–AuNP assembly ( Figure 1A ). A fluorescent organic complex, brilliant cresyl blue (BCB), was assembled on negatively charged glutathione (GSH)-modified AuNPs via electrostatic interaction, resulting in fluorescence quenching of the fluorophore. In the presence of Pb 2+ , fluorescent BCB molecules were detached from AuNPs due to the formation of a chelating complex between Pb 2+ and GSH confined on AuNPs, restoring the BCB fluorescence. The limit of detection (LOD) was 0.51 nM, and the potential coexisting ions (e.g. Al 3+ , Ca 2+ , Co 2+ , Cr 2+ , Cu 2+ , Cd 2+ , K + , Na + , Mg 2+ , Mn 2+ , Zn 2+ , Fe 3+ ) induced less than ±5% interference in the detection of Pb 2+ . Similarly, rod-shaped AuNPs functionalized with bisacridinedione complex were applied for Ca 2+ detection by Kim and Park Group [ 77 ]. In other cases, the same group reported a series of fluorometric chemosensors for selective signaling toward Ca 2+ and Mg 2+ , using aza-crown ether acridinedione-functionalized AuNPs as the probes [ 78 , 79 ]. However, these chemosensors cannot differentiate the fluorescent signal from Ca 2+ and Mg 2+ due to similar binding events of crown ethers with these two metal ions. Taking advantage of a similar principle, rhodamine-functionalized AuNPs were also employed to develop “turn-on” fluorescent sensors for sensitive detection of Hg 2+ [ 80 ] and Zn 2+ [ 81 ], with a LOD of 0.32 μM and 0.05 mg/L, respectively. A fluorescence resonance energy transfer (FRET) -based “turn-off” sensor for Cu 2+ has been recently developed using FITC modified AuNPs by Hormozi-Nezhad et al. [ 82 ]. Recently, Wu et al. published an even more sensitive method [ 83 ]. In their method, AuNPs functionalized with catechin were synthesized, and upon addition of Pb 2+ , Pb 2+ –catechin complexes and Pb–Au alloys that formed on the AuNP surfaces allowed the AuNPs to exhibit peroxidase-mimicking catalytic activity, resulting in a strong enhancement in the emission intensity at 588 nm (>100-fold). In spite of substantial progress, AuNP-based probes with high affinity and selectivity for metal ions remains a challenge. Alternatively, functional DNAs are another class of recognition molecules for metal ion sensing.

DNA–metal ion interactions based on either DNA mismatches or DNA

G-quadruplexes have been successfully combined with AuNPs for fluorescent detection of Hg 2+ and K + [ 84 ]. Additionally, the use of metal-ion-dependent DNAzymes as the recognition element has helped advance the development of highly selective and sensitive fluorescent nanoprobes for various metal ions. For instance, a Pb 2+ sensor was developed by attaching fluorophore-labeled 8–17 DNAzymes and substrates to AuNPs [ 85 ]. In the presence of Pb 2+ , the sensor underwent fluorescence enhancement as a result of the increase in fluorophore-AuNP distance upon DNAzyme-catalyzed cleavage of the substrate. Similarly, rod-shaped AuNPs coated with positively charged surfactants acted as binders and quenchers for fluorophore-labeled 8–17 DNAzymes and substrates [ 86 ], allowing the detection of Pb 2+ by fluorescence enhancement ( Figure 1B ). Another study used a similar design for Cu 2+ detection using a Cu 2+ -dependent DNA-cleaving DNAzyme via a AuNP-based FRET assay [ 87 ]. Xie et al. [ 88 ] developed a triple-channel optical signal (fluorometric, colorimetric, and resonance light scattering) probe for the detection of Hg 2+ ( Figure 1C ), based on the different interactions of AuNPs with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), the high binding affinity of positively charged acridine orange dye for DNA, and the modulation of the efficiency of energy transfer between fluorescent dyes and AuNPs. Another advantage of AuNPs is that they have a broad quenching ability for almost all fluorophores, which enables a multiplex detection of several metal ions, e.g. in homogeneous solution by the anchoring of multiple recognition elements on AuNPs [ 89 ]. For instance, Kim et al. [ 90 ] developed a multiplex sensing method using aptamer/QD conjugates and AuNPs for Hg 2+ , K + , and adenosine. In this sensor, the fluorescence of QDs was effectively quenched by the AuNPs due to FRET of QDs to AuNPs. In the presence of targets, the QD-conjugated aptamers were detached from AuNPs by target-induced conformational change of aptamers. Consequently, the fluorescence of the QDs was recovered proportional to the target concentration. In addition to fluorescence quenching, AuNPs could serve as fluorescence anisotropy generators for the attached fluorophores due to their large size in comparison to free fluorophores and DNA. Based on this principle, a fluorescence anisotropy sensor was constructed using DNAzyme-functionalized AuNPs for the detection of Cu 2+ and Pb 2+ [ 91 ]. AuNPs can also be used to quench the fluorescence of nanocrystals. As an example, Li et al. [ 92 ] reported a nanometal surface energy transfer (NSET) based sensor for Hg 2+ by using DNA-conjugated QDs and AuNPs ( Figure 1D ). In the presence of Hg 2+ , the QDs and the AuNPs were brought into close proximity, leading to quenching of the fluorescence emission of the QDs. This nanosensor exhibited a LOD of 0.4 and 1.2 ppb toward Hg 2+ in the buffer solution and in river water, respectively. Taking advantage of a similar principle, DNA-conjugated UCNPs and AuNPs were also employed to develop “turn-on” fluorescent sensors for sensitive and simultaneous detection of Pb 2+ and Hg 2+ . In addition to the above AuNP-based fluorescent nanoprobes, using similar design concepts, silver nanoprisms and bimetallic platinum/AuNPs were also successfully transformed into fluorescent sensors for the selective and sensitive detection of Tb 3+ [ 93 ] and Hg 2+ [ 94 ], respectively.

Imaging of metal ions within cells and in vivo

The detection and imaging of metal ions within cells and animals are of great significance for medical and biological studies. However, compared with the large amount of work on metal ion detection in vitro using colloid metal NP-based fluorescent probes, there are only very limited examples of colloid metal NP-based fluorescent sensors for metal-ion detection in living cells. Colloid metal NPs (e.g. AuNPs) can be easily incorporated into living cells by direct uptake or by any transfection mechanism. Likewise, the quantum mechanical effects, such as photoluminescence emission or plasmon resonance in AuNPs can make AuNPs excellent candidates for a different kind of low cytotoxicity intracellular imaging. A significant advantage of using AuNP-based probes in intracellular imaging is that they can be used as delivery agents to facilitate entry of other material, such as DNA [ 95 ] and organic dyes [ 96 , 97 ], into cells. Using this feature to their advantage, the Lu group [ 95 ] has recently developed a fluorescent nanoprobe for intracellular UO 2 2+ imaging. A 13-nm AuNP was used as an agent for efficient cellular delivery of dye-labeled uranyl-specific 39E DNAzyme. The fluorescence of a Cy3 fluorophore modified at the 5’ end of the substrate strand was quenched by both the AuNP and by the molecular quencher modified at the 3’ end of the substrate strand ( Figure 2A ). In the presence of UO 2 2+ , the DNAzyme cleaved the substrate strand, thus releasing the shorter Cy3-labeled product strand and thereby increasing the fluorescence. This sensor was the first DNAzyme-based probe of metal ions in live cells ( Figure 2B ). In addition, Liu et al. [ 96 ] developed a AuNP-based FRET assay for detection of Hg 2+ . Rhodamine B isothiocyanate (RBITC) was modified on the surface of AuNPs and the fluorescence was quenched by AuNPs ( Figure 2C ). Due to higher affinity of Hg 2+ toward isothiocyanate (ITC), binding with ITC induced detachment of RBITC from the AuNP surface, thus triggering a remarkable enhanced fluorescence of RBITC. This sensor has shown high sensitivity in monitoring Hg 2+ in complex samples, such as in river water and live cells ( Figure 2D ), with a LOD of 3.8 nM and 10 μM, respectively. Similarly, Lee et al. [ 97 ] proposed a Cu 2+ nanosensor based on its effect on the fluorescence of a boradiazaindacene (BODIPY) fluorophore, while using AuNPs as agents to promote cellular uptake. Bradley and co-workers [ 98 ] recently reported that Pd NPs trapped within polystyrene microspheres can enter cells and mediate a variety of Pd 0 -catalysed reactions, such as allylcarbamate cleavage and Suzuki–Miyaura cross-couplings. Cells were loaded with fluorescently labeled Pd 0 microspheres and the intracellular formation of a carbon–carbon cross-coupled product based on Pd 0 -mediated synthesis of an anthofluorescein rhodamine-based dye offered the fluorescent Pd 2+ imaging in HeLa cells ( Figure 3 ).

Metal nanoclusters

Fluorescent metal NCs have emerged as a new class of fluorophores because of several advantageous features [ 99 - 102 ] such as good fluorescence properties and biocompatibility, excellent photostability, sub-nanometer size, and ease of synthesis. The synthesis, biofunctionalization, and applications of metal NCs have been extensively reviewed elsewhere [ 17 , 84 , 103 - 107 ]. In the following section, we focus on the recent advances in the application of fluorescent metal NCs in metal ion sensing and imaging areas.

Detection of metal ions in environmental and biological samples

Metal ions, particularly heavy metal ions, have always been a significant threat to human health and the environment. Many sophisticated methods for rapid, sensitive, and selective detection of heavy metal ions, such as Hg 2+ [ 108 , 109 ], Cu 2+ [ 110 , 111 ], and Pb 2+ [ 112 , 113 ], have been developed using metal NC-based fluorescent probes. One example is a “turn off” sensing system developed for the detection of Hg 2+ , which takes advantage of Hg 2+ ’s ability to form strong metallophilic bonds with the high percentage of Au + or Ag + present on the surface of NCs, leading to efficient fluorescence quenching of NCs. Xie et al. [ 114 ] developed a one-pot route of synthesis to prepare fluorescent AuNCs using bovine serum albumin (BSA) as the template. The BSA-AuNCs consisted of 25 gold atoms and gave an intense red emission (640 nm) when excited at 480 nm. A highly selective and ultrasensitive detection of Hg 2+ , based on fluorescence quenching of Au nanoclusters by the Hg 2+ -Au + interaction, was then developed with a LOD of 0.1 ppb, which is lower than the maximum contamination level (MCL) (2.0 ppb) of mercury in drinking water defined by the United States Environmental Protection Agency (EPA). In a complementary effort, the Lu group developed a new method for the selective detection of Hg 2+ that uses lysozyme-stabilized AuNCs as fluorescent probes [ 115 ]. In our method, the selective fluorescent quenching of AuNCs by Hg 2+ was found to originate from gold clusters and not by the lysozyme-Au + complex. Similarly, Wang et al. [ 116 ] also developed a method that could be used in the selective determination of Hg 2+ by developing a facile approach to synthesize red-emitting fluorescent AgNCs using lysozyme as scaffold. Additionally, luminescent Au/Ag NCs coating with various ligands, including peptide [ 117 ], glutathione [ 116 ], pepsin [ 109 ] and other molecules [ 118 - 121 ], were also reported for the highly sensitive and selective detection of Hg 2+ or CH 3 Hg + . In additional to fluorescence quenching detection methods, turn-on fluorescence methods for rapid, easy, and reliable screening of Hg 2+ were also developed using DNA-protected AgNCs [ 122 , 123 ]. Copper was also detected using fluorescent metal NCs [ 111 ]. For instance, Gui et al. [ 110 ] reported the synthesis of human serum albumin (HSA)-stabilized fluorescent Au/Ag core/shell NCs for highly sensitive and selective sensing of Cu 2+ ( Figure 4A ). The Au/Ag NCs’ photoluminescence (PL) quenching in the presence of Cu 2+ was ascribed to the reduction of Cu 2+ to Cu + , and the subsequent interaction between Cu + and Ag (around Au/Ag NCs). Although Hg 2+ at a higher concentration (e.g. 5 μM) had a potential influence on the Cu 2+ sensor, this influence could be distinctly differentiated because Hg 2+ -induced aggregation of HSA-stabilized Au/Ag NCs resulted in PL quenching. Later, Liu et al. [ 124 ] proposed a hydrothermal synthesis of polyelectrolyte -stabilized Ag NCs as selective and ultrasensitive indicators for the simultaneous detection of Hg 2+ and Cu 2+ , with the addition of ethylenediaminetetraacetate (EDTA) as the masking agent ( Figure 4B ). Lead is a highly toxic metal ion that is often encountered in the environment due to its use in batteries, gasoline, and pigments [ 125 ].

Fluorescence quenching or enhancing of metal

NCs by lead ions have been developed for screening Pb 2+ in contaminated lake water samples [ 126 , 127 ] and in DMSO [ 128 ]. In addition, an aggregation-induced fluorescence quenching strategy based on coordination of ferric ions and dihydroxyphenylalanine-capped AuNCs has also been applied to the detection of Fe 3+ , with a LOD of 3.5 μM that is close to the MCL (5.4 μM) of Fe 3+ permitted in drinking water by the U.S. EPA. Similarly, other metal ions, such as Ag + [ 129 , 130 ], Pd 2+ [ 129 ], Co 2+ [ 131 ], Fe 3+ [ 132 ], Al 3+ [ 133 ], Cr 3+ [ 134 ], Cr 6+ [ 134 ], have also been successfully detected using metal NCs in the past five years.

Imaging of metal ions within cells and in vivo

There are many advantages to using fluorescent metal NCs as an imaging agent in cells and in vivo , including their unique functionality, ease in conjugation, biocompatibility, large Stoke shift, long lifetime, as well as their photo- and chemical stability [ 99 - 102 ]. A common strategy for intracellular metal ion imaging is based on the analyte-specific quenching of fluorescence NCs probes. The analyte, if present, can quench the fluorescence of the NCs, resulting in a decreased fluorescence signal. Shang et al. [ 135 ] developed an intracellular Hg 2+ sensor in HeLa cells by using NIR-emitting dihydrolipoic acid (DHLA)-capped AuNCs. Furthermore, fluorescent AuNCs synthesized using BSA was also applied for the sensing of Cu 2+ in live cells [ 136 ]. In the same way, Wang et al. developed a method for Pb 2+ detection in aqueous solutions and in living cells via NP aggregation by the synthesis of a pH-responsive copper NC functionalized with GSH [ 137 ]. Another smart strategy for intracellular Hg 2+ imaging was developed by Pu et al. [ 138 ] in which a FRET-based hybrid complex nanoprobe using blue-fluorescent conjugated oligomer-substituted polyhedral oligomeric silsesquioxane (POSSFF) and red-fluorescent metal NCs as the energy donor and acceptor, respectively. Because of the specific metallophilic Hg 2+ interaction on the NC surface, the fluorescence of the complex was significantly quenched by Hg 2+ rather than other metal ions ( Figure 5 ). Moreover, the whole-cell permeability of the complexes and the preserved ion-selective FRET in cells make these complexes effective for multicolor intracellular sensing of Hg 2+ . Very recently, Han et al. [ 139 ] developed a single fluorescent probe based on the combination of AuNCs, FITC, and 7-diethylaminocoumarin-3-carboxylic acid with ratiometric fluorescence signals. This NC-based nanoprobe showed high selectivity, sensitivity, and accuracy for Cu 2+ , and was successfully applied in intracellular imaging and sensing of pH and Cu 2+ , as well as used in ROS regulation of pH and Cu 2+ changes in macrophage cells.

5. Carbon Materials-based fluorescent nanoprobes for sensing and imaging of metal ions Carbon nanomaterials can take several shapes and the best known are: fullerene (C60), carbon nanotubes (CNTs): both single-walled (SWCNT) and multiple-walled (MWCNT), nanodiamonds, carbon nanofibers, graphene (which comprises single molecule layered sheets of graphite), and carbon dots (CDs) [ 239 ].

Carbon dots and graphene QDs

Carbon dots (CDs) are synthesized as either carbogenic dots or carbon nanoparticles (CNPs) [ 15 , 239 , 240 ]. Compared to semiconductor QDs, these luminescent carbon-based nanomaterials demonstrate non-blinking fluorescence emission, excellent water solubility, and non-toxic response [ 240 ]. Because of these properties, CDs are widely used as fluorescent probes for the detection of metal ions [ 241 - 243 ]. For example, Guo et al. [ 244 ] developed a simple, one-step hydrothermal method for the synthesis of highly fluorescent CNPs with a high quantum yield (68%) and good photo-stability, and can be used to detect Hg 2+ in an aqueous solution. CDs have also been used to detect other ions such as Sn 2+ , Cu 2+ , Ag + , and Al 3+ [ 245 ]. Recently, Yuan et al. [ 246 ] reported a new fluorescence turn-on nanosensor for the selective detection of Hg 2+ with bis(dithiocarbamato)copper(II) (CuDTC 2 ) functionalized CNPs ( Figure 15A ). They synthesized amine-coated CNPs and conjugated CuDTC 2 complexes on their surfaces through condensation between carbon disulfides and nitrogen atoms in the surface amine groups. The conjugated CuDTC 2 complex on the surface of CNPs can effectively quench the fluorescence of CNPs due to the combination of electron and energy transfer. The addition of Hg 2+ can lead to recovery of the fluorescence of CNPs because the conjugated Cu 2+ is replaced by Hg 2+ , cutting off the energy transfer pathway ( Fig. 15A ). The nanosensor can detect Hg 2+ with a LOD as low as 20 nM. Interestingly, the nanosensor can be fabricated onto paper and acts as a portable Hg 2+ nanosensor. This fluorescence turn-on nanosensor can eliminate disturbance from the detection medium and could possibly be developed for the detection of other metal ions with CNPs. Ratiometric fluorescent probes have recently attracted interest because of their high accuracy. Cao et al. [ 247 ] have constructed a ratiometric fluorescent nanosensor for Hg 2+ ( Figure 15B ) through simple mixing of the blue-emission CNPs with red-emission carboxylmethyldithiocarbamate modified CdSe@ZnS QDs (GDTC-QDs). The hybrid nanosensor showed dual emissions at 436 nm and 629 nm at a single excitation wavelength (365 nm). Because of the strong chelating ability of GDTC to Hg 2+ , the fluorescence of GDTC-QDs in the hybrid nanosensor can be effectively quenched by Hg 2+ , but the fluorescence of CNPs remained constant, resulting in a continuous fluorescence color change from red to blue with an increase of the concentration of Hg 2+ . This nanosensor exhibited a LOD of 0.1 μM. This method can effectively eliminate disturbance from the detection medium because the fluorescence of CNPs can be used as the standard signal. More importantly, this strategy can easily be adapted for the detection of other metal ions just by changing the ligands on the surface of QDs. The above mentioned F-CNPs can detect Hg 2+ in aqueous solution with proper surface modification. CDs show size dependent photoluminescence and upconversion luminescence properties due to a multi-photon process, which leads to anti Stokes type emission [ 242 ]. Due to the biocompatibility of the carbon materials, they can be readily used in biological applications [ 242 ]. The Tian group integrated an organic molecule specific for Cu 2+ ions into a hybrid system composed of carbon and CdSe/ZnS QDs ( Figure 16A ), allowing for a selective and sensitive ratiometric strategy for intracellular sensing and imaging of Cu 2+ [ 248 ]. The fluorescent probe can monitor Cu 2+ in a concentration range from 5 - 200 μM in a physiological pH environment. Following uptake, the particles were found to reside in various intracellular compartments ( Figure 16B ). After exogenous Cu 2+ source treatment, the fluorescence emission color of the probe turned from green-yellow to red. These initial experiments on live cells demonstrated the great potential for CD-based dual-emission hybrid sensors in the investigation of fundamental biological processes. Later, Castillo et al. [ 249 ] developed a fluorescent nanosensor for Cu 2+ detection based on upconversion fluorescence of CDs using UV and NIR as excitation sources. The CDs were prepared by using a one-step microwave method via pyrolysis of citric acid at low temperature and in the presence of polyethylenimine (PEI). The CDs were highly selective for Cu 2+ detection. However, at an excitation wavelength of 350 nm, Fe 3+ ions resulted in a dramatic decrease in fluorescence of CDs, attributed to an inner filter caused by its strong absorption at the mentioned excitation wavelength. However, this interference could be eliminated when the excitation was at 850 nm, because Fe 3+ did not suffer inner filter at this excitation wavelength, a useful advantage of this upconverting NPs. In addition, these fluorescent nanosensors showed low cytotoxicity and good cell permeability, thus they could be successfully used for sensing and imaging of Cu 2+ in living cells. Recently, Graphene QDs (GQDs) and their derivatives, including graphene oxide QDs (GOQDs) and reduced graphene QDs (rGOQDs) have been presented [ 250 ], and used as fluorescent probes for biomolecular sensing and cellular bioimaging applications [ 251 , 252 ] due to their unique photoluminescence features. GQDs were used to detect Ag + [ 253 ], Hg 2+ [ 254 ], Cu 2+ [ 255 , 256 ], Cr 6+ [ 257 ], Ni 2+ [ 258 ], and Fe 3+ [ 259 ]. The Lee group [ 260 ] reported a green synthesis method for graphitic carbon QDs (GCQDs) as a fluorescent sensing platform for the highly sensitive and selective detection of Fe 3+ ions. The high sensitivity of GCQDs could be attributed to the formation of complexes between Fe 3+ ions and the phenolic hydroxyls of GCQDs. The Li group [ 258 ] carried out a systematic evaluation of the quenching effect of commonly encountered transition metal ions (Sc 3+ , Cr 3+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Ru 3+ , Ag + , Cd 2+ , and Hg 2+ ) on the PL of GQDs. With GQDs as both the chelator and fluorophore and EDTA as the competitive chelator, quenching-recovery performance of the above metal ions on the photoluminescence of GQDs can be categorized into non-quenching (Sc 3+ , Zn 2+ , Ag + , Cd 2+ , Hg 2+ ), quenching-recovering (Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ ), and quenching-non-recovering groups (Cr 3+ , Fe 3+ , Ru 3+ ). To further improve the recognition specificity, ssDNA aptamer modified probes were introduced to these systems. Li et al. [ 261 ] developed the water-soluble GO sheets, which were functionalized with a ssDNA aptamer and, in the absence of Hg 2+ ions, exhibited a strong fluorescence emission at 600 nm under an excitation of 488 nm. When Hg 2+ ions were present, a rigid hairpin-shaped dsDNA structure was formed due to the coordination of T-Hg 2+ -T, Because the Hg 2+ ions were very close to the surface of the GO sheet, electrons can transfer from GO to the Hg 2+ ions along the duplex DNA channel, resulting in quenching of the fluorescence emission of GO. Thus, the detection mechanism was based on “turn-off” of the fluorescence of GO. Liu et al. [ 262 ] reported a photoluminescent GO array on which heavy metal ion-specific DNA aptamers were immobilized so that sensitive and multiplex heavy metal ion detection was performed utilizing electron transfer between the photoluminescent monolayer GO and the captured metal ion. The Qu group [ 263 ] reported amino-functionalized GQDs (afGQDs) with a high QY (16.4%), which was generated by hydrothermal treatment of greenish-yellow fluorescent GQDs (gGQDs). Due to the fact that Cu 2+ ions have a higher binding affinity and faster chelating kinetics with N and O on the surface of afGQDs than with other transition metal ions, the selectivity of afGQDs for Cu 2+ is much higher than that of gGQDs ( Figure 17A ). Furthermore, amination converted the surface charge of the GQDs from negative to positive, which made it easy for the GQDs to be taken up by cells. Using afGQDs as a fluorescence probe, the profiling of Cu 2+ in living cells was successfully realized ( Figure 17b ).

Carbon nanotubes and graphene oxide

While some carbon-based materials are inherently fluorescent, other carbon materials, such as fullerene, carbon nanotubes and graphene, are powerful fluorescence quenchers [ 264 , 265 ]. Zhang et al. [ 266 ] presented a sensitive and selective fluorescent sensor for Hg 2+ detection that works based on the noncovalent assembly of single-walled carbon nanotubes (SWNTs) and dye-labeled T-rich ssDNA containing thymine–thymine (T–T) mismatches that shows high selectivity for Hg 2+ against other metal ions, owing to the formation of T–Hg 2+ –T base pairs ( Figure 18A ). Similarly, the Sun group [ 267 ] reported water-soluble nano-C60 to be an effective fluorescent sensing platform for the detection of Ag + because of the substantial dye fluorescence quenching that occurs when the fluorescently labeled ssDNA probe adsorbs on nano-C60 ( Figure 18B ). In contrast, in the presence of Ag + , cytosine-Ag + -cytosine (C–Ag + –C) coordination induced the probe to fold into a hairpin structure, which did not adsorb on nano-C60 and thus retained the dye fluorescence. This sensing system exhibited a LOD as low as 1 nM and had a high selectivity against other metal ions. The Qu group [ 268 ] have developed a reusable DNA SWNT-based fluorescent sensor for highly sensitive and selective detection of Ag + and cysteine (Cys) in aqueous solution. SWNTs can effectively quench the fluorescence of dye-labeled single-stranded DNA due to their strong π–π stacking interactions. However, upon incubation with Ag + , stable duplex formation can be induced, mediated by C–Ag + –C coordination chemistry, which has been further confirmed by DNA melting point studies. This weakens the interactions between DNA and SWNTs, activating the sensor fluorescence. On the other hand, because Cys is a strong Ag + binder, it can remove Ag + from C–Ag + –C base pairs and deactivates the sensor fluorescence by rewrapping the dye-labeled oligonucleotides around the SWNT. In addition to the low concentrations of heavy metal ions, detection of these metal ions in real samples is often complicated by the presence of other metal ions, making their determination a difficult task. To overcome this limitation, Wang et al. [ 264 ] developed a novel aptamer biosensor based on MWCNT long-range energy transfer for sensitive, selective and multicolor fluorescent detection of Hg 2+ , Ag + and Pb 2+ ions in homogeneous solution. Three-color nanosensors can rapidly and simultaneously detect these three metal ions in a single solution. This MWCNT-based sensing platform exhibited high sensitivity and selectivity toward Hg 2+ , Ag + and Pb 2+ versus other metal ions, with a LOD of 15 nM for Hg 2+ , 18 nM for Ag + and 20 nM for Pb 2+ . Since its discovery, graphene has been widely used in bioassays [ 269 - 271 ]. GO is an extraordinary optical sensing material that can serve either as an energy acceptor or as an energy donor for a fluorophore. Functional DNAs [ 272 - 275 ], such as aptamers [ 276 ], have been used as metal-ion specific receptors for sensitive and selective metal ion detection [ 277 , 278 ]. For instance, the Ye group [ 279 ] has developed a ssDNA–GO architecture probe for multiplex detection of sequence-specific DNA, thrombin, Ag + , Hg 2+ and cysteine, with a LOD of 1 nM, 5 nM, 20 nM, 5.7 nM and 60 nM, respectively ( Figure 18C ). Another example came from the Fan group [ 280 ] who developed a mix-and-detect fluorescent sensor for Ag + by using a silver-specific oligonucleotide (SSO) probe that can be coupled with the ability of GO to specifically adsorb and quench single stranded fluorogenic DNA probes. For this sensor, a FAM labeled SSO containing cytosine (C)-rich nucleic acids separated by a spacer was used as a fluorescence probe for Ag + . In the absence of Ag+, the SSO was in a flexible single strand state. Upon addition of Ag + , the complexation of Ag + with the cytosine bases of SSO yielded a rigid hairpin structure. Then, GO was added to selectively adsorb the unbound SSO and quench its fluorescence, while the Ag + complexed with SSO remained free and its fluorescence was retained. Through this method, the fluorescence intensity of SSO provided a quantitative readout for Ag + . In yet another example, Zhang et al. [ 281 ] reported a graphene oxide (GO)-based fluorescence Hg 2+ analysis using DNA duplexes of poly(dT) that allows rapid, sensitive, and selective detection via stable T–Hg 2+ –T complexes. Finally, Li et al. [ 282 ] developed a rapid, sensitive and selective fluorescent sensor for detection of Pb 2+ based on a Pb 2+ -induced G-quadruplex on GO. In addition to aptamers described above, DNAzymes have been investigated due to their high metal ion specificity [ 283 - 289 ]. Various optical sensors for metal ions were developed using graphene and DNAzymes [ 290 - 294 ]. For instance, the Yu group [ 292 ] constructed a graphene DNAzyme-based sensing system for amplified fluorescence “turn-on” detection of Pb 2+ . The 5′ end of the substrate strand is labeled with the fluorophore carboxyfluorescein (FAM), which was hybridized with the DNAzyme strand to form a DNAzyme-substrate hybrid containing a large ssDNA loop (containing 15 bases) which can bind to the surface of the GO and thus induce quenching of the labeled FAM fluorophore. Upon addition of Pb 2+ , the DNAzyme was activated which induces cleavage of the substrate strand at the single RNA site into two separate strands. This releases a short FAM-linked oligonucleotide fragment, a related longer oligonucleotide fragment, and the DNAzyme strand. The DNAzyme strand can hybridize with another substrate strand and thus induce a second cycle of cleavage upon binding of Pb 2+ , providing an amplified detection signal for Pb 2+ (LOD of 300 pM). A novel label free fluorescent Cu 2+ sensor based on internal DNA cleavage and an extrinsic fluorophore in a graphene/DNAzymes complex was also designed [ 295 ], with a LOD of 2 nM. Combining the fluorescent properties with fluorescence quenching ability of carbon materials, Wei et al. [ 296 ] designed a FRET sensor which could be used for measuring the concentration of K + with high selectivity (e.g. 37-fold against Na + ) and tunable dynamic range (0-200 mM) via energy transfer from CDs (the donor) to graphene (the acceptor) ( Figure 18D ). For this sensor to function, energy transfer was induced when CDs and graphene were brought into appropriate proximity by covalently aminated CDs and noncovalently functionalized graphene with 18-crown-6 ether (18C6E). The FRET process was inhibited because of competition between K + and ammonium for 18C6E, which had high K + selectivity.