Graphene quantum dots in analytical science

Accepted Manuscript Title: Graphene quantum dots in analytical science Author: S. Benítez-Martínez, M. Valcárcel PII: DOI: Reference:

S0165-9936(15)00178-8 http://dx.doi.org/doi:10.1016/j.trac.2015.03.020 TRAC 14476

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Trends in Analytical Chemistry

Please cite this article as: S. Benítez-Martínez, M. Valcárcel, Graphene quantum dots in analytical science, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi:10.1016/j.trac.2015.03.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphene quantum dots in analytical science S. Benítez-Martínez, M. Valcárcel * Department of Analytical Chemistry, University of Córdoba, E-14071 Córdoba, Spain HIGHLIGHTS  Graphene quantum dots are fluorescent nanoparticles with unique properties  Analytical applications of graphene quantum dots  Graphene quantum dots in development of analytical sensors and biosensors  Development of analytical techniques based on graphene quantum dots  Analytical methods needed to determine graphene quantum dots ABSTRACT Graphene quantum dots (GQDs) are small fluorescent nanoparticles with unique properties that make them attractive tools for research in various fields. We review their state of the art in analytical chemistry and summarize their analytical applications. Also, we deal with GQDs as target analytes, a scarcely explored aspect in analytical nanoscience and nanotechnology, and suggest potential future directions for GDQbased analytical research. Keywords: Analytical application Analytical science Biosensor Graphene Graphene quantum dot Nanoscience Nanotechnology Quantum dot Sensor Target analyte *

Corresponding author. Tel./Fax: +34 957 218616. E-mail address: [email protected] (M. Valcárcel)

1. Introduction Graphene has attracted increasing attention among the scientific community ever since it was isolated as a single layer of material from highly oriented pyrolytic graphite (HOPG) in 2004 by Novoselov and Geim using the “Scotch-tape method” [1]. Graphene, with its truly two-dimensional (2D) planar structure, and the thickness of a single atom, consists of carbon atoms arranged in a honeycomb lattice with sp2 hybridization. Its unique properties include extremely high intrinsic mobility of charge carriers, zero band gap, large surface area and high chemical stability. Graphene also exhibits superior mechanical, magnetic, optical and thermal properties [2]. However, it disperses poorly and tends to agglomerate in solvents. Research into this material has grown exponentially in recent years, particularly in material science, physics, chemistry, engineering, and analytical chemistry. Graphene quantum dots (GQDs), which constitute a zero-dimensional photoluminescence (PL) carbon-based nanomaterial consisting of very thin (typically 3–20 nm) graphene sheets that exhibit exciton confinement and quantum-size effect, recently aroused much scientific interest by virtue of their exceptional properties. Although graphene is a zero-band gap 1 Page 1 of 34

nanomaterial – and hence non-luminescent – it has an infinite exciton Bohr radius and affords quantum confinement in finite-sized specimens [3]. The band gap in GQDs is non-zero and can be tuned by altering the size and the surface chemistry of the dots [4]. These nanoparticles (NPs) can be obtained as single-layer, double-layer and multilayer materials [5]. Quantum confinement and edge effects confer them with interesting properties, such as fluorescence (FL) activity, robust chemical inertness, excellent photostability, high biocompatibility and low toxicity. In addition, GQDs exhibit stable PL, resistance to photobleaching, tunable luminescence and high solubility in various solvents. GQDs provide an effective alternative to colloidal inorganic semi-conductive quantum dots (QDs), which have attracted much attention in the past two decades on account of their electronic and optical properties [6] but are highly toxic due to the release of heavy metals, such as cadmium, selenium, tellurium and zinc, from their core and their coating. Although GQDs have been classified as carbon nanodots (C-dots), they differ from them in some respects. Thus, C-dots are quasi-spherical NPs less than 10 nm in diameter, possessing PL properties. However, GQDs are graphene nanosheets in the form of one, two or more layers all less than 10 nm thick and 100 nm in lateral size; also, they usually contain functional groups (carboxyl, hydroxyl, carbonyl, epoxide) at their edges that can act as reaction sites and alter PL emission from the dots by changing their electron density [7]. Quantum yield (QY), which is an important factor for FL materials, ranges from 2% [8] to 46% [9] in GQDs, depending on the particular method of synthesis and whether their surface is passivated [10], reduced [11] or further modified [12].

2. Synthesis Progress in nanoscience and nanotechnology rests heavily on the development of effective methodologies of synthesis allowing new nanomaterials with specific properties shape, size, surface characteristics and inner structure to be obtained. Also, some chemicals allow the properties and the distribution of NPs to be adjusted as needed. Approaches to synthesizing nanomaterials have traditionally been classified as “topdown” or “bottom-up”. 2.1. Top down In top-down approaches, large macroscopic materials (bulk materials) are restructured and externally controlled in order to reduce their size and to obtain a specific shape. The resulting nanosized materials may exhibit very interesting, unique properties differing from those of the starting materials. Top-down syntheses of nanocomponents are usually expensive and slow, require special equipment and critical operating conditions – and toxic organic solvents or strong acids in some cases – and provide low yields, which make them unsuitable for large-scale production [10,13,14]. In addition, they introduce internal stress and faults in the crystallographic network that can lead to surface defects and structural damage – and ultimately to altered surface properties due to the typically large surface area per unit volume of these materials. In any case, top-down approaches to synthesis are the more commonly used in nanoscience and nanotechnology. The main precursors used in top-down syntheses of GQDs include graphene oxide (GO) [15], coal [16], carbon fibers [17], graphite powder [18] or rods [19], single-walled carbon nanotubes (SWCNTs) [20] or multi-walled CNTs (MWCNTs) [21], carbon black [5], graphene [22] and, recently, metal-organic framework (MOF)-derived porous carbon [23]. The precursors are usually subjected to 2 Page 2 of 34

acid, hydrothermal, solvothermal or electrochemical treatment, laser ablation or exfoliation. Acid-based chemical procedures of synthesis use one of several possible acids to cut bulk materials into GQDs. The treatment involves using a concentrated strong acid (e.g., as nitric acid [5,24], mixtures of nitric and sulfuric acids in variable proportions of 3:1–1:3 [15,17] – and sonication in some cases [20,25] – or nitric acid in combination with amidative cutting [18], in addition to temperatures up to 80ºC and solution stirring. Hydrothermal routes for GQD synthesis involve dissolving or dispersing an appropriate carbon-based raw material in water and heating at 180–200ºC at high pressure in a closed container (usually an autoclave) for 2–12 h [19,26–28]. Solvothermal syntheses of GQDs use organic solvents {e.g., dimethylformamide (DMF) [29]}, and heating temperatures and times similar to those of hydrothermal routes. Fig. 1 depicts the hydrothermal treatment of SWCNTs for the production of GQDs. The electrochemical preparation of GQDs requires applying an anodic potential of 1 V for 7 h, 11 h or 15 h to a MWCNT-coated working electrode in order to fracture the micromaterial [30]. Alternatively, GDQs can be obtained by electrolyzing a graphite rod immersed in a 0.1 M NaOH solution with a current intensity of 80–200 mA cm–2 [19] or by cyclic voltammetry (CV) (viz. by electrochemical reaction of a GO film working electrode immersed in a 0.1M PBS solution subjected to a potential of ± 3 V [31]). Recently, GQDs were synthesized by laser ablation with a femtosecond laser (800 nm, 35 s pulses for 20 min) of HOPG in aqueous media [32] and by irradiating graphite powder with an Nd:Yag laser in the presence of benzene (1064 nm, 10 ns pulses for 30 min) [33]. Ultrasound-assisted exfoliation of graphite nanofibers [34], electrochemical exfoliation of graphene [22], organic solvent-assisted exfoliation of graphite NPs [35], and exfoliation and disintegration of graphite flakes and MWCNTs by intercalation of highly reactive potassium between layers and walls, respectively [36], are among the most widely used top-down methods of synthesis for GQDs. A method involving a onestep sonication–redox treatment of GO with KMnO4 and providing GQDs in a high QY in a short time without the need for an acid was recently reported [37]. 2.2. Bottom up Bottom-up routes of synthesis for GQDs assemble basic building blocks with suitable properties, including elemental precursors, such as atoms, molecules or nanoclusters, by controlling their interactions in order to facilitate environment-friendly large-scale production of these nanomaterials [38,39]. Bottom-up approaches to synthesis introduce fewer defects than top-down approaches; also, they afford more uniform chemical composition, and precise control over the shape and size distribution of the product. However, they have been less widely explored than top-down routes. In one bottom-up route, ethylene gas was continuously injected into argon plasma to generate a carbon-atom beam that flowed through a carbon tube for dispersal in a chamber to obtain size-controllable GQDs [40]. Haloaromatic compounds, such as chlorobenzene and dichlorobenzene, have been used as carbon sources for laser-induced photochemical stitching [41]. Thus, the oxidation of polyphenylene dendritic precursors by solution chemistry produces GQDs [3]. These nanomaterials can also be obtained by hydrothermal treatment combined with (a) prior charring of polycyclic aromatic hydrocarbons (PAHs) with a strong acid, such as H2SO4 [42]; or, (b) microwave heating of glucose, sucrose or fructose aqueous solutions [43]. Pyrolysis of L–glutamic acid [44] or citric acid [45] (Fig. 2) above 200ºC provides an easy, fast bottom-up method for the synthesis of highly-fluorescent NPs. Also, a combined top-down/bottom-up

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approach was used to obtain alginate by pyrolysis and ablate the resulting graphitic carbon material with a pulsed laser to produce multilayer graphitic QDs [46]. Using a suitable route of synthesis of the top-down or bottom-up type in combination with appropriate conditions usually allows GQDs with tunable blue, green, yellow or even red luminescence to be prepared. PL emission from these materials commonly depends on size, excitation and solvent. GQDs can be obtained in sizes of 1.5–30 nm and QYs from 1.8–14%. The shape of QGD edges (viz zig-zag or arm-chair) also affects their PL and various other properties. 2.3. Doped and functionalized GQDs The crystalline network of GQDs consists of carbon atoms arranged in a honeycomb lattice, as in graphene. The chemical structure of these NPs is similar to that of GO having oxygen-containing groups (hydroxyl, carboxyl, epoxy, and carbonyl) at their edges [28]. Although GQDs can exhibit strong FL, they are often limited by a poor QY and little tunability. However, inserting heteroatoms, such as nitrogen [23,47] or sulfur [48], into their carbon chains facilitates tuning of their physical and chemical properties. Nitrogen has been widely used for doping carbon nanostructures by virtue of it having a comparable atomic size for bonding to carbon atoms. Sulfur, oxygen [49], boron [50] and fluoride [51] have been less commonly used to obtain doped GQDs. Quantum confinement and edge effects in QGDs allow heteroatoms to penetrate directly into the carbon lattice and to modulate their electronic, optical and chemical properties by creating more reactive sites. Doped GQDs can be prepared via top-down and bottom-up approaches. The most widely used top-down routes for preparing N-doped GQDs (Fig. 3) are hydrothermal [52–54] and solvothermal [55] synthesis from GO using various nitrogen sources, such as ammonia [52,53] ammonium hydroxide [54] or dimethylformamide (DMF) [55]. Cutting pre-oxidized N-doped graphene is another effective hydrothermal treatment for obtaining N-GQDs [56]. N-doped GQDs were also obtained by streaming treatment of MOF-derived porous carbon in the presence of HNO3 [23]. Graphene can be converted into N-GQDs by CV of tetrabutylammonium perchlorate (TBAP) in acetonitrile as electrolyte and nitrogen as source [57]. Cutting graphene directly grown onto a Cu substrate by chemical vapor deposition (CVD) with nitrogen plasma also proved effective for this purpose [47]. Fig. 4 illustrates the process. Worth special note among the bottom-up routes for N-doping of nanodots is the hydrothermal carbonization of citric acid as carbon source in the presence of ammonia [58], dicyandiamide [59,90] or hydrazine [60]. N,S-co-doped GQDs obtained hydrothermally [61,62] or solvothermally [63], and O,N,F-GQDs [49] and graphenefluoroxide QDs [64], have also been reported. There are various chemical-modification methods for modulating GQD properties by introducing different functionalities. Amidation of carboxyl groups at GQD edges has been extensively explored. For example, surface modification with alkylamine was found to increase QY by 205% relative to non-functionalized GQDs [67]. Also, amino groups can tune the FL of GQDs and provide QY values of 16.4–40% [68–70]. Similarly, insertion of aryl groups after a hydrothermal treatment with ammonia improved the QY up to about six times [71]. Surface passivation of GQDs with polyethylene glycol (PEG) by way of the reaction of hydroxyl groups in it with carboxyl groups in the dots boosted GQD PL by 13% [65] or 15% [66]; also, it increased the solubility of graphene. Surface functionalization with small organic molecules, such as alcohols, diamines, thiols, ionic liquids (ILs) and glutathione (GSH) [12,18,20,73], is also useful to modulate the PL of GQDs. Recent studies have shown that surface oxidation [73] or 4 Page 4 of 34

reduction [11,74] effectively alters the PL properties of GDQs and improves their optical performance. Post-synthesis hydrothermal treatments have also proved useful for regulating the PL properties of GQDs obtained by using various routes and bulk materials as a result of their removing epoxy and hydroxyl groups from the GQD surface (Fig. 5) [75]. A combination of doping and functionalization allowed N-doped, amino-functionalized GQDs (NA-GQDs) to be obtained in a single step by thermolysis with glycine as both carbon and nitrogen source [76]. Although these promising methods have proved effective for enhancing the luminescence of GQDs, their underlying mechanism remains unclear.

3. Analytical applications of GQDs Ever since their emergence, GQDs have been widely used for a variety of purposes, including production of photovoltaic devices [77], organic light-emitting diodes [43], fuel cells [78] and drug-delivery systems [79]. In recent years, they also found many applications in analytical chemistry in response to the increasing research interest aroused by their unique properties. Thus, GQDs have emerged as new potential tools for designing and tuning PL sensors and biosensors (probes, electrochemical sensors and hybrid sensors), such as immunosensors and aptasensors. In the past decade, many efforts were made in the development of NP-based sensors and biosensors. In this context, CNTs and graphene are powerful tools in developing biosensors due to their large specific surface area, in which macromolecules can be anchored on their surface and act as binding sites for the recognition of target analytes. They have been also employed in developing electrochemical sensors and less used in optical sensing [80]. However, inorganic semiconductor QDs have attracted considerable interest over the past two decades due to their exceptional optical and electronic properties being widely used in the development of PL and electrochemical sensors. However, these semiconductor crystals show important disadvantages, such as their intrinsic toxicity due to the presence of a heavy-metal core, which make them biologically incompatible. In addition, they are colloids and their coupling to chemicals is difficult and causes problems with the colloidal stability in some applications [81]. Sensor systems based on CNTs, graphene and QDs have been applied in the detection of biomolecules, DNA, proteins, pesticides, heavy metals and gas sensing [80,81]. Compared to these sensing systems, GQDs have been also applied in the determination of a large number of target analytes with comparable sensibility and selectivity and improved some relevant aspects, such as the low dispersion of CNTs and graphene in polymeric matrices and aqueous solutions [80]. Likewise, GQDs are nontoxic, easy to handle and possess optical and electrical properties as QDs. However they have not be fully investigated as sensing materials despite their potential due to their recent emergence. This review reviews the main analytical applications of GQDs since their appearance in the literature, particularly in the development of optical and electrochemical sensing systems. The following sections present their use as surfactants and their applications in Raman spectroscopy and liquid chromatography. Table 1 displays the applications of GQDs in analytical chemistry. We also include details of the types of GQD, precursor used, target analyte, limits of detection (LODs) and real sample used.

3.1. Sensors and biosensors

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The exceptional properties of GQDs have fostered the development of different types of sensor using conventional, doped or functionalized NPs for detecting metal ions, small organic molecules and biomaterials with improved sensitivity, selectivity and specificity. 3.1.1. Photoluminiscence–based sensors FL sensors based on GQDs have so far been used mainly to detect ionic species, small organic molecules or biomaterials. 3.1.1.1. Probe sensors. GQD-based probe sensors have been used to determine Fe3+ [22,23,42,48,58,60,76], Cu2+ [68,82,83], Al3+ [84] and the hazardous species Hg2+ [85, 86], Cr6+ [87] and Ni2+ [88]. Analytical applications of these sensors has focused largely on Fe3+, probably because of its prominent role in biological systems (particularly in regulatory processes). Recently, Xu et al. [23] developed a sensor based on N-GQDs for the selective FLbased detection of Fe3+ in real water samples. Binding of ferric ions to phenolic hydroxyl groups on the GQDs considerably increased the selectivity by promoting photo-induced charge transfer from N-GQDs to Fe3+. Such charge transfer was assumed to perturb the electronic states of the N-GQDs and non-radiative transitions, thereby leading to substantial FL quenching. Li et al. [48] used sulfur-doped GQDs to develop an efficient FL probe for the selective detection of Fe3+ in serum samples. Incorporating S atoms into the carbonaceous skeleton tuned the electronic local density of the dots and promoted coordination of phenolic hydroxyl groups at the edges of S-GQDs to Fe3+, a highly specific interaction responsible for PL quenching in the dots. Fig. 6 illustrates the performance of the Fe3+ sensor and the quenching mechanism of the S-GQDs. Li et al. [76] used NA-GQDs to determine Fe3+in tap water by attenuating the FL emission of the dots. Nitrogen atoms acted mainly as chelating sites for Fe3+ ions and efficiently quenched the FL of the dots as a result. Tam et al. [58] developed a green, inexpensive, very sensitive and selective sensing platform for Fe3+ According to these authors, metal ions may form hydroxides by coordination with hydroxyl groups at the surface and the edges of N-GQDs, and these complexes may restrain recombination excitons and facilitate charge transfer, thereby quenching the FL of the dots. Ananthanarayanan et al. [22] used IL-modified GQDs for the optical detection of 3+ Fe . Incorporating BMIMPF6 into the reaction medium not only improved exfoliation and dispersion of GQDs, but also led to its stacking on NP surfaces, thereby increasing the affinity of ferric ions for the dots via imidazole rings. These authors showed Fe3+ to induce aggregation of BMIM+-GQDs by ferric ions acting as coordinating sites to bridge several functionalized dots. As a result, the FL emission of the dots was quenched. Fig. 6 illustrates the synthesis procedure and detection mechanism. Ju et al. [60] reported a label-free detection platform for Fe3+ in real water samples. They used a FL probe based on N-GQDs obtained by hydrothermal treatment in the presence of hydrazine of GQDs previously prepared by pyrolysing citric acid of ferric ions by N bringing them into close proximity; as a result, the FL emission of the NGQDs decreased with increasing concentration of Fe3+. Detection was enabled through nitrogen doping modulating the chemical and electronic properties of the dots and promoting their complexation with ferric ions. Zhou et al. [42] used the quenching effect of GQDs obtained by carbonizing PAH precursors (pyrene, mainly) for the sensitive detection of Fe3+. The selectivity for ferric ions was ascribed to their specific coordination with phenolic hydroxyl groups at GQD

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edges. A combination of GQDs and the Fe2+/Fe3+ redox couple was used as an efficient sensing platform for H2O2. Wu et al. [44] developed a label-free GQD-based colorimetric sensor for detecting H2O2. The sensor was used to detect the color change in a GQD solution containing peroxidase substrate ABTS [( 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] from yellow to green as a consequence of the GQD-catalyzed reduction of hydrogen peroxide. Copper ions play a critical role in biological and environmental processes; copper is an essential trace element for plants, animals and humans. However, long-term exposure to high copper levels can cause gastrointestinal, liver and kidney damage. This has fostered the recent development of GQD-based analytical methods for determining copper. Thus, Liu et al. [82] found Cu2+ ions to be easily absorbed on pristine GQDs. Biothiol cysteine was added to the aqueous solution to recover the PL fraction quenched by complexation of Cu2+ ions with GQDs thanks to the ability of cysteine to capture metal ions and suppress charge transfer from GQDs to Cu2+ as a result. This principle facilitated the development of an effective turn-on method for detection and quantitation of Cu2+ in real water samples. Wang et al. [83] developed a FL sensing platform for the highly efficient detection of 2+ Cu . Complexation of Cu2+ by GQDs via a static mechanism resulted in efficient quenching of the FL. Sun et al. [68] used amino-functionalized GQDs (af-GQDs) to sense copper ions by the increased binding affinity and faster chelating kinetics of Cu2+ with N and O widely distributed on the dots. The FL-quenching effect, which was ascribed to static and dynamic phenomena, was possibly caused by non-radiative electron/hole recombination annihilation via an effective electron transfer process. The presence of Al3+ ions in drinking water can have adverse effects on human health. Fan et al. [84] developed a boron-functionalized GQD-based chemosensor for the detection of Al3+. Green luminescent B-GQDs were electrochemically prepared by using a graphite rod (anode) and a Pt foil (cathode) immersed in a borax aqueous solution. The sensor exploited the FL-intensity enhancement of B-GQDs upon interaction with aluminum ions (Fig. 7). The LOD thus obtained, 3.64 µM, was lower than the maximum allowed level set by the World Health Organization. Heavy-metal ions Hg2+, Cr6+, Ni2+ and Cd2+ are hazardous, pervasive pollutants with high toxicity and adverse impacts on human health. A number of GQD-based methodologies for their detection and quantitation in various media have been reported. Thus, Wang et al. [85] developed a GQD-based sensing probe for Hg2+ from blue FL dots. The sensor measured the gradual decrease in FL intensity with increasing Hg2+ concentration. The FL-quenching effect was ascribed to facilitated non-radiative electron/hole recombination annihilation through effective electron transfer quenching and to aggregate-induced quenching caused by the affinity of mercury ions for carboxyl groups at GQD edges. Chakraborti et al. [86] developed a sensor based on the same principle to determine Hg2+ in an aqueous solution. The carbonyl and hydroxyl functional groups in the GQDs were thought to act as binding sites for Hg2+; this, in combination with adsorption of mercury ions at GQD surfaces, may have caused the FL quenching observed via static and dynamic mechanisms. Cai et al. [87] used a FL probe based on strongly blue PL N-GQDs to determine Cr6+ in real water samples. The quenching effect was ascribed to reduction of Cr6+ to Cr3+, which was probably promoted by hydroxyl groups and nitrogen in the dots. Huang et al. [88] reported a metal-ion sensor based on a quenching-recovery strategy for Ni2+ detection. According to these authors, photoinduced electron transfer from the GQDs to metal ions with partly filled d orbitals possibly caused electronic perturbation 7 Page 7 of 34

and non-radiative transitions in the dots, thereby quenching their FL. Introducing a competitive chelator for Ni2+, such as dimethylglyoxime (DMG), as recovery agent afforded the selective detection of nickel ions. This sensing strategy is also useful with Mn2+, Co2+ and Cu2+ ions. The monitoring of anionic species in environmental matrices with GQDs was a subject of some interest in recent years. Hallaj et al. [89] found blue luminescent GQDs to be useful for detecting ClO– ions in the presence of CTAB; the presence of hypochlorite ions caused a proportional increase in chemiluminescence (CL) that was used for analytical purposes. Hypochlorite ions successfully oxidized the dots, thereby enhancing their CL. We assume that the sensing mechanism involves a chemical reaction between GQDs and ClO–. Dong et al. [90] constructed another free-chlorine sensor based on surface-passivated blue GQDs. Strongly-oxidative ClO– ions destroyed the self-passivated surface of the GQDs and quenched their FL as a result. A quantitative pH sensor for environmental and intracellular use was developed by Wu et al. [91] to exploit the FL properties of N-GQDs. The N-doped GQDs were sensitive to pH in the range 1.81–8.96, thus providing a general pH sensor with a wide range of applications from determinations of species in real waters to quantitation of intracellular contents. Bai et al. [92] proposed a rapid, sensitive, specific, pH-dependent off–on GQD-based PL sensor for phosphate ions. Addition of Eu3+ caused GQDs to aggregate through complexation of the ion by carboxyl ions in the dots and promoted an energy transfer that switched off their FL. In the presence of phosphate, Eu3+ ions bonded to oxygen donors of phosphate preferentially over carboxyl groups at GQD edges, thereby facilitating recovery of the quenched FL. Liu et al. [93] developed an off–on sensor based on glutathione-functionalized GQDs (GQDs@GSH) as a FL probe for adenosine triphosphate (ATP) and phosphatecontaining molecules in general. The sensing mechanism was based on quenching of the FL of GQDs@GSH in the presence of Fe3+ by GSH acting as a ligand for ferric ions and enabling effective electron transfer between Fe3+ and the dots as a result. Phosphatecontaining molecules acted as complexing units for Fe3+ – phosphate ions have a high affinity for iron ions, with which they easily form Fe–O–P bonds – and facilitated recovery of the FL of the functionalized dots. The quenching effect was attributed to a dynamic mechanism. Regarding small organic molecules and biomaterials, Zhu et al. [37] used GQDs to detect alkaline phosphatase (ALP). They took advantage of the known ability of these dots to coordinate with Cu2+ and the affinity of pyrophosphate (PPi) for Cu2+ to design a GQD-based sensor for detecting ALP where chelation of Cu2+ and PPi induced changes in the FL of the dots. In the general sensing procedure, ALP at variable concentrations was incubated with PPi in Tris-HCl at 37°C for 60 min. Then, Cu2+ was added to the solution and incubation allowed to continue for 10 min. Finally, a GQD solution was added to the mixture containing ALP, PPi and Cu2+, and incubated for a further 30 min at room temperature. Upon addition of ALP, PPI was hydrolyzed into Pi by destroying the PPi–Cu2+ complex. Copper ions thus released reacted with hydroxyl and carboxyl groups in the GQDs and promoted energy transfer. The resulting quenching in the FL of the dots was used to determine ALP with an LOD of 17 pM. The quenching effect of Cu2+ allowed these dots to be directly used after 30 min of incubation to detect Cu2+. Zhang et al. [94] designed a simple turn-on method for the PL sensing of amino acids based on the quenching effect of the formation of aggregates by interaction of Eu3+ with GQDs, as previously described [91]. GQDs exhibited both up-conversion and down-conversion PL properties. The PL fraction lost was recovered through the competition of amino acids with the nanodots for Eu3+. This enabled the sensitive 8 Page 8 of 34

detection of glutamic acid (Glu) and aspartic acid (Asp) with an LOD of 0.19 µM (upconversion) or 0.32 µM (down-conversion) for the former, and an LOD of 0.18 µM or 0.32 µM, respectively, for Asp. Ju et al. [59] reported a FL turn-on sensing system for the detection of glutathione. The system was based on the PL quenching of N-GQDs by Hg2+ via electrostatic interactions and electron energy transfer between them. The addition of glutathione allowed the PL of the nanodots to be recovered through the increased affinity of mercury ions for S atoms in glutathione. Introducing Hg2+ in the sensing system allowed Li et al. [95] to develop a GQD-based sensor for detecting melamine. Coordination of mercury ions with the analyte quenched the blue FL of the dots – neither mercury nor melamine by itself caused any significant changes in GQD emission. The LOD achieved with this turn-off sensing mechanism was 0.12 µM, which is comparable with those of other reported methods. Wu et al. [96] developed a FL turn-on detection system using GQDs to detect biothiols GSH, Cys and homocysteine (Hcy). Complexation of Hg2+ ions quenched the FL of GQDs. Addition of a specific biothiol molecule to a GQD–Hg2+ mixture caused the dots to bind mercury ions via Hg–S bonding interactions between the thiol functional group and Hg2+. Subsequently, the GQD–Hg2+ complex was dissociated, Hg2+ ions were removed from GQD surfaces and the emission intensity of the dots restored as a result. GQDs allowed Li et al. [97] to develop a sensor for 2,4,6-trinitrophenol (TNP), in which this explosive substance formed a complex with the GQDs through strong π–π stacking interactions. The dots acted as the donor and TNP as the acceptor in a FL resonance-energy transfer (FRET). The presence of the nitro group in TNP proved essential to attenuate the FL of the dots, and so that of hydroxyl and nitro groups on the phenol ring. A few years before, Fang et al. [98] had accomplished the ultrasensitive detection of 2,4,6-trinitrotoluene (TNT) in solution with the aid of multicolor GQDs prepared by oxidation of GO with strong acids. The underlying mechanism involved an energy transfer similar to that in TNP; thus, FRET upon binding of TNT to the surface of GQDs as acceptors suppressed the FL of the nanodots. Li et al. [99] found o-, m- and p-dihydroxybenzene (DHB) to be oxidized to a benzoquinone in the presence of H2O2 and horseradish peroxidase (HRP) with effective quenching of the blue FL of GQDs obtained from pyrolyzed citric acid. Benzoquinones are good electron acceptors able to transport electrons from the conduction band to the valence band of the excited state of GQDs and hence to attenuate their FL. According to these authors, quenching was the consequence of a static mechanism rather than a dynamic mechanism. Sun et al. [100] used the same synthesis and oxidizing method for the sensitive detection of phenol in various types of water by resonance-light scattering (RLS) spectroscopy. Hydrogen peroxide in combination with HRP induced the oxidation of phenol to quinone intermediates interacting electrostatically with GQDs to form molecular aggregates that enhanced their RLS spectral band (310 nm). Zhang et al. [50] used B-GQDs prepared by hydrothermal cutting of boron-doped graphene in a PL probe for the selective label-free sensing of glucose. Their approach was based on the abnormal aggregation-induced PL enhancement resulting from the formation of rigid structures of B-GQDs and glucose (Fig. 8), probably by reaction of the two cis–diol units in glucose with the two boronic acid groups on B-GQDs – which restricted intramolecular rotation and increased PL as a result. In another boronic GQD sensor for the determination of glucose [101], APBA-GQDs were obtained by hydrothermal treatment of GO and post-functionalization with 3aminobenzeneboronic acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl) 9 Page 9 of 34

carbodiimide (EDC). In the presence of glucose, the FL of the GQDs was quenched through the formation of a negatively-charged boronated complex between glucose molecules and APBA on the dots. Despite the Coulombic repulsion between APBA and GQDs, APBA-GQDs were efficiently cross-linked by covalent anchoring of glucose. The efficient FL quenching observed may therefore have been caused by the surface states formed by stretching of the APBA–GQD interface resulting from elastic stress introduced by both electrostatic repulsion and covalent cross-linking. Various monosaccharides, including glucose in aqueous solutions, were detected by using FL GQDs in combination with a boronic acid-substituted bipyridinium salt (BBV) [102]. Dots negatively charged through the presence of polar (carboxyl and hydroxyl) surface groups and cationic BBV efficiently quenched the FL of the dots and simultaneously acted as glucose receptors through a combination of affinity sensing and electrostatic interactions. Electrostatic attraction between the dots and BBV facilitated excited-state electron transfer from the carbon NPs to bipyridinium by formation of a ground-state complex and the resulting quenching of their PL. Addition of glucose to the system converted boronic acid into tetrahedral anionic glucoboronate esters that offset the net positive charge in cationic BBV, thereby reducing the quenching efficiency and restoring the PL initially lost. This label-free sensing system was also sensitive to other saccharides, its sensitivity decreasing in the following sequence: fructose » galactose » glucose. Benítez–Martínez et al. [103] developed a sensor for phenols in olive oil. Phenols were extracted from olive oil, preconcentrated, redissolved and mixed with GQDs for FL-quenching-based quantitation. The quenching effect was ascribed to energy transfer from the dots to the phenols, which were assumed to hold π–π staking and non-covalent interactions with oxygen-containing groups at GQD edges. The sensing system was used to determine the total phenol contents of olive oils. Yang et al. [28] proposed using GQDs extracted from reduced GO with the ozonation pre-oxide method in a sensing platform for pyrocatechol. The FL of the dots decreased with increasing concentration of pyrocatechol. Non-covalent interactions of the electrostatic, hydrogen-bonding and π–π stacking types between oxygen-containing groups and pyrocatechol led to an energy transfer that quenched the FL. The growing industrial use of NPs and their incorporation into commercial products (e.g., cosmetics, foods, drugs and electronic devices) have increased human and environmental exposure to nanomaterials. Developing effective analytical methods to monitor the presence of nanomaterials has therefore become indispensable for detecting relevant concentrations – which typically fall in the ng/L range – separating NPs and removing potential interferents from samples. There are several effective methods for this purpose, some of which use GQDs to analyze nanostructures. Thus, BenitezMartinez et al. [104] proposed a GDQ-based sensor for the determination of GO in environmental samples by its quenching the FL of the dots. GO was retained in a cellulose membrane from spiked river-water samples and recovered by sonicating the membrane. Attenuation of the PL was ascribed to energy transfer between the two types of carbon NP (non-covalent interactions and π–π stacking, mainly). Liu et al. [105] studied FL quenching between GQDs and gold NPs (AuNPs), which are two unbound structures, upon simple mixing. Based on FL and UV–Vis absorption spectra, the authors assumed static and dynamic quenching to co-exist in their system. Thus, GQD FL was assumed to be quenched by citrate-coated AuNPs in two possible ways, namely: (a) by forming a GQD–AuNP complex; or, (b) by interacting from a distance – without contacting the GQDs – in such a way that some dots were attached onto AuNP surfaces whereas others remained monodispersed in water. Both GQDs and AuNPs were negatively charged, the former through hydroxyl and carboxyl groups at their edges, and the latter with citrate. An increased concentration of GQDs caused 10 Page 10 of 34

AuNPs to grow into larger structures through the replacement of citrate ions by dots; however, an increased concentration of AuNPs caused them to self-aggregate. The results of this study were thought to be potentially useful with a view to developing GQD/AuNP-based sensors. Most of the above sensors use doped or functionalized GQDs to improve selectivity and sensitivity, but comparatively few have relied on pristine GQDs for this purpose. 3.1.1.2. Hybrid fluorescence sensors based on GQDs. This section summarizes the uses of hybrid systems based on GQDs as PL-sensing platforms. GQDs can easily form composites with other types of NP (mainly carbon and metallic nanostructures) or even biomaterials, by virtue of the large number of reactive sites provided by oxygencontaining functional groups at their edges – or, in some cases, on their surface. He et al. [106] used hemin-functionalized GQDs to monitor glucose in blood and detect H2O2. Dots were coupled to hemin simply by absorption onto GQD surfaces through electrostatic and π–π stacking interactions, facilitated by the anionic charge of the dots in water and the cationic charge of hemin. Formation of nanocomposites with hemin reduced the FL of the dots by about 9% as a result of changes in their surface state through self-assembly. The ensuing sensing system proved very sensitive to hydrogen peroxide; thus, addition of H2O2 to the platform led to considerable FL attenuation of the hemin–GQD signal through the suppression of surface passivation by free radicals formed in the reaction of hemin with the medium. Li et al. [107] developed a method for determining hydroquinone at trace levels with a GQD–enzyme hybrid system. GQDs were diluted and mixed under vigorous stirring with a solution of the mixture for the enzyme-catalyzed reaction (viz., hydroquinone– HRP–H2O2) and incubated for 8 min. Hydroquinone was converted into 1,4– benzoquinone in the presence of H2O2 and the peroxidase. Adding the hydroquinone– HRP–H2O2 mixture to a GQDs solution caused the FL of the NPs to be considerably quenched. There was reasonable evidence suggesting that benzoquinone quenched the FL of the dots through electron transfer of their surface states. Wang et al. [108] used GQDs as PL probes for protein kinase sensing based on the selective aggregation of phosphorylated peptide–GQD conjugates triggered by coordination with Zr4+ ions. covalently bonded peptide–GQD conjugates were obtained by mixing EDC (N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride), GQDs, NHS (N-hydroxysuccinimide) and the peptide under shaking. For detection, peptide–GQD solution, Tris buffer, CK2 (casein kinase II) and ATP were mixed, diluted with ultrapure water and incubated to effect the phosphorylation reaction. Subsequent addition of Zr4+ induced aggregation of the phosphorylated peptide–GQD conjugate through coordination with carboxyl groups on the GQD surface. The fact that titration of the peptide–GQD conjugate with Zr4+ ions did not alter its PL was ascribed to covalent bonding in it. In the presence of ATP, CK2 efficiently catalyzed the phosphorylation of the peptide–GQD conjugate. Adding Zr4+ to the phosphorylated peptide–GQD solution quenched PL. The effect was ascribed to an energy or electron transfer resulting from aggregation of the dots through coordinate– covalent interactions between Zr4+ ions and phosphate groups on peptide–GQD surfaces. The fact that PL intensity decreased with increasing concentration of CK2 was used to determine protein kinase. Ran et al. [109] showed GQDs decorated with silver NPs (AgNPs) to be useful for the label-free detection of Ag+ and biothiols (GSH, Cys and Hcy). The dots were prepared by microwave-assisted acid cleavage of GO. Electrostatic attachment of Ag ions onto their surface caused their FL to be quenched through formation of AgNP/GQD hybrids. Addition of Ag+ to the hybrid solution reduced the FL intensity through charge transfer, and addition of Cys resulted in a further decrease due to its 11 Page 11 of 34

bonding to the hybrid nanostructure. The AgNP-decorated sensing platform was used for the selective detection of GSH, Cys and Hcy – three important biothiols in biological processes and medical diagnosis – based on the formation of Ag–S bonds by the thiols on the surface of the AgNPs. All biothiols attenuated the FL of the AuNP/GQD hybrid system. Zhou et al. [110] developed a GQD-based FL sensor for the determination of pnitrophenol (4-NP) in water samples, using a molecularly-imprinted polymer (MIP) hybridized with the dots. Silica-coated dots were obtained by using a simple hydrothermal method and the composite by anchoring a MIP layer onto silica-coated GQDs by using 3-aminopropyltriethoxysilane as functional monomer and tetraethoxysilane as cross-linker. MIP-coated GQDs exhibited FL emission after conjugation. MIP increased the selectivity for 4-NP. Bringing 4-NP into contact with MIP-coated GQDs caused NH2 groups at their surface to act as binding sites for the phenol via hydrogen-bonding interactions, and the FL of the hybrid system to be considerably quenched through a resonance-energy transfer from GQDs to 4-NP as a result. The FL intensity decreased markedly with increasing concentration of 4-NP. Biosensing of trypsin used a method of Li et al. [111], in which GQDs were selfassembled by induction of cytochrome c (Cyt c), an electron-transfer cationic protein forming complexes with anionic GQDs. Coordination of Cyt c to GQDs strongly quenched their FL emission through adsorption of GQDs on Cyt c, which also promoted aggregation of GQDs by electrostatic attraction. However, the addition of trypsin substantially increased the FL of the NPs through the hydrolysis of trypsin fragmenting Cyt c and suppressing their FL quenching as a result. Also, Fe3+ present in Cyt c was reduced to Fe2+, which led to further FL recovery. In addition, trypsin cleaved peptide bonds in Cyt c, and released lysine and arginine residues that effected the chemical reduction of GQDs to r-GQDs, thereby increasing the FL intensity even further. 3.1.2. Electrochemical sensors 3.1.2.1. Modified electrodes. GQDs have proved useful for developing FL sensors and biosensors by virtue of their stable luminescence and unique optical properties. By contrast, GQDs have scarcely been used to construct electrochemical sensors despite their well-known exceptional properties as electron carriers and acceptors. The few such sensors reported to date were developed by modifying others of a different nature. Thus, Raushani et al. [112] reported an electrochemical sensor based on GQDs for the detection of persulfate (S2O82–). For this purpose, GQDs were coated onto the surface of a glassy carbon electrode (GCE) using the drop-casting method. The resulting GCE/GQDs electrode was activated and additionally coated with riboflavin (RF). The modified electrode, GCE/GQDs/RF, exhibited direct electron transfer and an excellent electrocatalytic response to persulfate. The electrochemical performance of the sensor was examined by CV and chronoamperometry (CA). The GQDs provided support for riboflavin, which acted as mediator in shuttling electrons between S2O82– ions and the working electrode, and facilitated electrochemical regeneration following electron exchange with the ions. The GCE/GQDs/RF-modified electrode was found to operate with no detectable changes in peak height after 100 cycles. A GQD-modified gold electrode was used by Zhang et al. [113] to detect H2O2. Nanodots were assembled on a gold electrode by using cysteamine as cross-linker. Cysteamine molecules were chemically bonded to the electrode surface and carbodiimide esters at GQD edges reacted with amino groups in cysteamine to form amide bonds. CV and CA measurements revealed that CA was more suitable for assessing the catalytic response of the GQD/Au electrode to H2O2. The cathodic current

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was found to increase with increasing concentration of H2O2. The electrode proved to be reusable more than 20 times with at least 90% of its current response. Razami et al. [114] reported a carbon ceramic electrode (CCE) modified with GQDs as a GOx substrate for the electrochemical sensing of glucose. A GQD solution obtained by hydrothermal treatment of GO was cast onto the CCE surface and dried at room temperature to obtain a homogeneous GQD-CCE that was activated potentiostatically. Also, a GOx solution was casted onto the GQDs|CCE surface to obtain the GOx–GQDs|CCE. CV measurements revealed that GOx catalyzed the reduction of oxygen. Addition of glucose decreased the peak current through oxygen being reduced on the electrode surface via the enzyme-catalyzed reaction between the oxidized form of GOx and glucose. The decrease in current was proportional to the glucose concentration added. The modified electrode was reusable with a peak current almost 95% of the initial response over 200 cycles. 3.1.2.2. Electrochemiluminescence (ECL) sensors. Electrochemical sensors (particularly probe-type sensors) based on GQDs have been used mainly to detect metal ions. Thus, Chen et al. [115] designed an ECL sensor to detect hexavalent chromium in environmental water samples. Their sensing system exploited the excellent ECL of GQDs in the presence of S2O8–. The co-reactant system, GQD/S2O8–, gave a strong cathodic signal that was considerably quenched by Cr(VI). The nanodots and S2O8– were reduced to negatively charged GQDs and SO4•– radicals, respectively, electron transfer between them leading to excited-state GQDs that gave an ECL signal upon return to the ground state. Like PL signals, ECL signals can be quenched through static and dynamic mechanisms. Because the dots were deactivated by contact with Cr(VI) – which decreased ECL in the co-reactant system – these authors assumed a dynamic mechanism for the ECL-quenching effect. Li et al. [116] developed a Cd2+ ECL sensor based on greenish-yellow GQDs. They preceded Chen et al. in investigating the ECL performance of GQDs with S2O8– as coreactant and proposed a mechanism described elsewhere [112], by which strongly oxidizing SO4•− radicals and GQD•− radicals were produced by electrochemical reduction of S2O82 − and GQDs, respectively. The Cd2+-sensing system was based on the coordination of metal ions with N, and carboxyl and hydroxyl groups, and on the resulting aggregation of GQDs decreasing their ECL. Addition of an effective masking agent, such as Cys, allowed the fraction of ECL quenched by other metal ions to be recovered – by exception, the ECL signal remained attenuated in the presence of Cd2+. 3.1.2.3. Hybrid electrochemical sensors. Mazloum-Ardakani et al. [117] used a modified GCE to simultaneously detect GSH, uric acid (UA) and tryptophan (Trp). The GCE was modified by immersion in a GQD solution at room temperature for 12 h. The resulting GQD/GCE was cycled 15 times in PBS at pH 7 to reduce oxygen-containing groups in the dots and obtain an ER-GQDs/GCE, which was immersed in a HClAu4 solution and subjected to electrochemical deposition of AuNPs in order to obtain a new, Au/ERGQDs/GCE. Finally, this electrode was immersed in a 4-(((4mercaptophenyl)imino)methyl)benzene-1,2-diol (MIB) aqueous solution to improve its catalytic activity and obtain the final, MIB/Au/ERGQDs/GCE. The presence of ERGQDs and AuNPs on the electrode increased its active surface area. MIB, which can produce the redox reaction of GSH, was oxidized at the MIB/Au/ERGQDs/GCE surface. GSH electrocatalytic activity was assessed from CV and CA measurements. The high charge-detaching efficiency of ERGQDs and AuNPs promoted electron transfer from the electrode surface to GSH; also, the self-assembled monolayer of MIB resulted in improved LODs. The fact that GSH, UA and Trp were oxidized via independent mechanisms at the MIB/Au/ERGQDs/GCE allowed their simultaneous 13 Page 13 of 34

determination. Peak currents in differential pulse voltammetry (DPV) increased linearly with increasing concentration of GSH, UA and Trp. Muthurasu et al. [118] reported an electrochemical biosensor based on HRPfunctionalized GQDs for detecting H2O2. The dots were green PL spherical particles possessing oxygen-rich (hydroxyl and carboxyl) functional groups. The presence of these active sites facilitated anchoring of HRP to GQDs via a peptide-coupling reaction to form an amide linkage. The resulting HRP-GQDs were immobilized onto a GCE by drop-casting, followed by dipping in a solution of chitosan, which acted as a binder to prevent leaching of HRP. Performance in H2O2 detection was assessed by monitoring the enzyme activity towards H2O2 reduction in an aqueous phosphate-buffer solution via CV and CA measurements. The enzyme-modified electrode exhibited a redox peak corresponding to the Fe(III)/Fe(II) redox reaction in heme groups present in the enzyme. Addition of H2O2 resulted in a proportional increase in reduction current. The analytical performance of the sensing system was assessed via CA measurements, using current– concentration plots that exhibited two linear segments for H2O2. Hou et al. [119] reported the electrochemical detection of malachite green (MG) with a GQD/AuNP-modified GCE. After a GQD solution was dropped onto the GCE surface, the resulting GQD/GCE was immersed in an HAuCl4 solution and the AuNPs obtained were electrodeposited onto the GDQ/GCE following electrochemical reduction of chloroauric acid by application of a negative potential. Multilayer Au-GQDs were obtained by alternate doping of GQDs and electrodeposition of AuNPs. CV measurements revealed that the modified electrode possessed acceptable catalytic activity. The current signal was stronger with four layers than with a single layer. Measurements also exposed a redox reaction in MG, probably due to mutual transformation of the dye and poly(malachite green). Also, DPV measurements revealed that the oxidation peak current was greater with four layers than one layer. The oxidation peak current, which was proportional to the concentration of MG, was used as the analytical signal. 3.2. Immunosensing and aptasensing 3.2.1. Immunosensors Immunological sensors are based on recognition of the coupling of an antigen with an antibody to form an antigen–antibody complex. An antigen or antibody immobilized onto a sensing surface will cause a signal change upon formation of a complex with the corresponding antibody or antigen. Highly-sensitive immunosensors can thus be constructed using enzymatic reactions involving fixed enzyme-labelled antigens. Immunosensors have great potential in clinical analysis thanks to the specificity of immunological reactions. Immunosensors can be competitive or non-competitive, and homogeneous or heterogeneous. Non-competitive immunoassays are usually more sensitive and more specific than competitive. Immunosensors can be classified as electrochemical, optical, piezoelectric, thermometric or magnetic, according to the type of transduction [120]. Li et al. [121] reported a paper-based ECL immunodevice consisting of nanoporous gold–chitosan hybrids and GQD-functionalized Au@Pt composites for the detection of carcinoembryonic antigen (CEA). Green GQDs were immobilized on aminofunctionalized Au@Pt with a core–shell structure, and monoclonal signal antibodies (McAb2) were attached and GOx was added to form labelled McAb2/GQD/Au@Pt bioconjugates. Monoclonal capture antibodies (McAb1) were immobilized onto a 3D origami ECL inmunodevice to prepare the working zone. For the ECL-assay procedure, the ECL inmunodevice was incubated with the sample solution and then with GOx/McAb2/GQD/Au@Pt bioconjugate. An amplified ECL signal was obtained 14 Page 14 of 34

through the efficient catalysis by GOx of the oxidation of glucose to in situ generated H2O2. Incubating the inmunodevice with CEA increased the ECL signal. Loading GQDs onto Au@Pt further amplified the ECL signal and the resulting sensitivity. Yang et al. [122] reported a GQD-coated porous PtPd nanochain (pPtPd)-labelled ECL immunosensor using gold–silver nanocomposite-functionalized graphene for the detection of tumor markers in serum. To this end, AgNPs were attached to a PVPcoated graphene sheet and post-surface decorated with AuNPs to obtain a GN–Ag–Au hybrid nanomaterial that was deposited onto a GCE surface. Then, the primary antibodies were dropped onto the modified GCE. Separately, GQDs and pPtPd were conjugated (pPtPd@GQDs) through NH3+ groups present at the porous nanochains and COO– groups in the GQDs. This was followed by incubation with the secondary antibodies (Ab2) to obtain pPtPd@GQDs/Ab2. Next, the ECL immunosensor was incubated with the sample (CA199) at different concentrations and, finally, with pPtPd@GQDs/Ab2 to obtain a sandwich-type structure. The anodic ECL of the assay was measured in the presence of tripropylamine (TAP) as co-reactant. The ECL intensity of the immunosensor increased with increasing concentration of CA199. Wang et al. [123] explored the detection of avian leucosis virus subgroup J (ALV-J) by using a doubly-assisted signal-amplification electrochemical immunosensor based on GQD–apoferritin-encapsulated CuNPs. A bare GQD-modified GCE was incubated with Ab1 and then with variable concentrations of ALV-J. Next, previously prepared Fe3O4@GQDs/Ab2–Cu-apoferritin/BSA bioconjugates were dropped onto the electrode surface and incubated. After the sandwich-type assembly was formed, Cu was released from the apoferritin cavity for detection by DPV. The dots were used for both conjugation of ALV-J Ab1 and immobilization of ALV-J Ab2. Al-Ogaidi et al. [124] developed an optical immunoassay for the detection of ovarian biomarker CA-125 based on CL resonance-energy transfer (CRET) to GQDs immobilized by electrostatic attraction onto an amino-modified glass chip. Fig. 9 depicts the immunoassay and the detection principle behind it. Capture antibodies (cAb) were covalently linked to the dots via amide conjugation. In the absence of the antigen (CA-125), HRP catalyzed the production of reactive oxygen species (ROS) from H2O2 that oxidized luminol to its singlet dianion and produced excited electrons generating blue CL upon returning from the excited state to the ground state. In the presence of the antigen, exposure of the antibody–antigen complex formed (GQD-cAb + CA-125) to Ab-HRP led to the sandwich-type structure (GQD-cAb + CA-125 + Ab-HRP), where HRP was in close proximity to the GQDs. The fact that the dianion involved in the HRP-catalyzed reaction was close to the dots facilitated resonance-energy transfer between the two; this quenched the CL of the dots to an extent inversely proportional to the CA-125 concentration. Zhao et al. [125] reported a fluoroimmunoassay based on the interaction of graphene and GQDs for the sensitive detection of human immunoglobulin G (IgG). In this immunosensing strategy, graphene acts as an acceptor and mouse anti-human IgG (mIgG, antibody)-conjugated GQDs as donors. The addition of graphene to a mIgG– GQD solution, π–π stacking interactions between graphene and GQDs, and non-specific binding interactions of mIgG with the graphene surface combined to cause a luminescence resonance-energy transfer (LRET) that quenched the luminescence of the dots. However, the addition of human IgG, which was probably bound to mIgG via specific antibody–antigen interactions, restored the luminescence previously lost by increasing the distance between mIgG–GQD and the G surface. 3.2.2. Aptasensors Aptasensors are sensing platforms modified with oligonucleotides previously selected in vitro by using a combinatorial method that exhibits high affinity and high 15 Page 15 of 34

specificity for a particular ligand. The presence of the ligand induces a conformational change that triggers an oligonucleotide molecular recognition event. The variety of potential ligands available from ions to whole cells makes them very attractive for not only therapeutic agents and controlling expression of genes, but also molecular design of receptor-sensing phases. Their use as molecular receptors has the advantage that they can be subjected to extreme conditions and undergo denaturation cycles without losing their affinity for their ligands. Aptasensor-based analysis has evolved continuously with detection schemes ranging from label-free methods such as surface-plasmon resonance (SPR) and quartz-crystal microbalance (QCM) measurements to label-dependent methods using electrochemical, FL or CL measurements, or field effect transistors, for example. At present, electrochemical and optical aptasensors prevail. Aptasensors have a greater potential than antibodies and can be useful for biomarker detection, cancer diagnosis, and detection of pathogens and small molecules to assure food safety and monitor environmental pollution [126]. In this context, Sheng Qian et al. [127] reported the simultaneous detection of DNA and thrombin based on GQDs and GO, and regulated by an off–on process. Dots were synthesized via a microwave-assisted chemical-oxidation route. QY and sensitivity were improved by preparing 1,2-ethylendiamine-functionalizated GQDs (eGQDs) and reduced GQDs (rGQDs), which differed in color and in maximum emission. eGQDs were condensed with a DNA probe to obtain ssDNA–eGQDs, whereas rGQDs and thrombin aptamer were used to prepare a thrombin probe (TA-rGQDs). This afforded separate detection of DNA and thrombin, and their simultaneous detection. For detection of the individual analytes, ssDNA–eGQDs or TA–rGQDs were mixed with GO for adsorption onto its surface through electrostatic attraction and π–π stacking interactions in order to construct the assemblies ssDNA–eGQD/GO or TA–rGQD/GO, respectively. Formation of the assemblies quenched the FL of the probe through electron transfer between GQDs and GO. Addition of DNA or thrombin caused the probe to be desorbed from the GO surface through rupture of the non-covalent interaction between GQDs and GO. Then, the target DNA (tDNA) was hybridized with ssDNA–eGQD/GO to obtain double-stranded (ds)DNA-GQDs by specific base pairing, whereas thrombin formed a complex with its probe through its aptamer specifically binding to thrombin exosite I via TT loops formed through a combination of hydrophobic and polar interactions. Release of the two probes from the GO surface led to apparent recovery of the FL. For the simultaneous detection of DNA and thrombin, both probes were assembled to GO at the same time so that the dual nanosensor would synchronously respond to the presence of the two analytes thanks to the FL recovery induced by the release of their complexes from GO. Sheng Qian et al. [128] previously reported another selective, sensitive FL-sensing platform for the individual detection of DNA based on FRET between GQDs and GO. To this end, GQDs were reduced to rGQDs with sodium borohydride and used to construct a single-stranded (ss) labelled DNA probe. The detection mechanism was the same as that described above for DNA and involved absorption of the ssDNA–rGQD probe onto the GO surface through π–π stacking interactions and electrostatic attraction, which quenched the FL of the probe. Addition of tDNA led to the formation of dsDNA– rGQDs, which were thus released from the GO surface; this restored the previously lost FL by rupturing π–π stacking interactions and electrostatic attractions in ssDNA– rGQDs. This approach [129] was also used for DNA detection with a complex sensing system based on GQDs and oxidized MWCNTs (o-MWCNTs). The sensing mechanism was similar to that described in the previous two references [127,128] for individual DNA detection except that the substrate used to quench the FL of the GQD probe was o-MWCNTs rather than GO.

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A GQD-based platform for the detection of DNA was developed by Zhao et al. [130] in the form of a probe consisting of a GQD-modified pyrolytic graphite (PG) electrode coupled to specific sequences of ssDNA molecules. The PG electrode was coated by dripping on a GQD solution and drying at room temperature to obtain a uniform film on its surface. Then, the GQD-modified electrode was immersed in Tris-HCl buffer containing ssDNA. For electrochemical (DPV) measurements, the target was added to the solution containing ssDNA and incubated for 1 h before immobilization, and TrisHCl buffer at pH 6 containing [Fe(CN)6]3–/4– was used as electrolyte. Adding the complementary ssDNA to the probe caused the self-assembly of a double helix structure and increased the peak current of [Fe(CN)6]3–/4, which suggests that the formation of dsDNA disrupted attachment of the probe onto the modified electrode surface and enhanced its electrochemical response. The LOD thus obtained for the target complementary ssDNA was 100 nM. The ssDNA probe used was also a thrombin aptamer, so, consistent with the above principle for DNA, the electrochemical signal increased with increasing thrombin concentration. The results confirmed the prominent role of GQDs in this sensing system; the PG electrode was unable to distinguish conformational changes in the ssDNA probe. Liu et al. [131] developed an electrochemical DNA sensor by using a modified GCE and peroxidase-like magnetic ZnFe2O4/GQD nanohybrid mimicking an enzyme label (Fig. 10). The electrode was modified by depositing graphene sheets onto the GCE surface, followed by casting of Pd nanowires onto the electrode. The modified electrode was immersed in an ssDNA solution and treated with 6-mercapto-1-hexanol (MCH) to obtain a well-aligned DNA monolayer. Next, the electrode was hybridized with tDNA and then with ZnFe2O4/GQD–ssDNA. The ZnFe2O4/GQD nanohybrid in the sensing system exhibited better peroxidase-like catalytic performance than HRP, possibly as a result of a synergistic effect of the individual activities of ZnFe2O4 and GQDs. The nanohybrid formed facilitated electron transfer from the GQDs to ZnFe2O4. The small size, more intact aromatic structure, and richness in peripheral carboxyl groups and unpaired electrons at GQD edges were also assumed to enhance the catalytic activity of the nanohybrid. DPV measurements revealed that the reduction peak current increased with increasing tDNA concentration. The average reduction peak current of the DNA biosensor was linearly proportional to the logarithm of the tDNA concentration. Lu et al. [132] proposed an aptasensor for the detection of ATP based on the ECL of blue GQDs. A solution containing ssDNA1 was dropped onto an Au-electrode surface and treated with MCH to obtain a well-aligned DNA strand. The resulting electrode was immersed in a solution containing hybrid nanostructures consisting of SiO2 nanospheres, GQDs and ssDNA2 (SiO2/GQDs/ssDNA2) in addition to variable concentrations of ATP. In the presence of ATP, ssDNA1 and SiO2/GQD-modified ssDNA2 formed a stable complex that facilitated immobilization of SiO2/GQDs onto the electrode surface and obtaining an ECL signal. However in the absence of ATP, interactions between the two fragments were weaker, and so was the ECL signal. Anodic ECL was observed by using H2O2 as co-reactant. 3.3. Other analytical applications The unique properties of GQDs conferred by their nm size have been exploited to develop novel analytical methods and improve existing ones. This section describes selected chromatographic and spectrometric methods of this sort. Wang et al. [133] reported a screening method for detection and identification of radical-scavenging natural antioxidants based on a free radical reaction and liquid chromatography with tandem mass spectrometry (LC-MS/MS). Amine-functionalized GQDs were used to load free radicals onto the complex system. Detection was 17 Page 17 of 34

performed with and without preliminary exposure of the samples to specific free radicals on the functionalized GQDs; the former enabled charge transfer between free radicals and antioxidants, and formation of a conductive interface that improved the interfacial electron-transfer kinetics of the screening system with little resistance. The difference in chromatographic peak area in presence and absence of pre-exposure was used to identify potential antioxidants. The ensuing method afforded the simultaneous assessment of antioxidant power against free radicals, and the identification of antioxidants in a complex plant matrix. Antioxidants were identified by MS/MS and comparison with standards. Some 14 compounds were found to possess potential antioxidant activity and 4,5,6,7-tetrahydroxyflavone was identified as that with the highest free radical scavenging ability. Cheng et al. [134] used GQD-assembled NTs (GQD-NTs) for surface-enhanced Raman spectroscopy (SERS). The dots were obtained by electrochemical oxidation of graphene. Application of an appropriate potential for several hours caused them to deposit spontaneously onto the nanochannels of a gold-foil-supported anodic aluminum oxide membrane and form NT arrays. Upon assembly, GQDs lost their O-related groups and GQD-NTs became insoluble in the aqueous medium. The abundant hydrogen atoms terminated on GQD surfaces within the NTs were thought to play a prominent role in promoting efficient charge transfer and enabling the SERS effect. The potential of GQD-NTs as substrates for SERS applications was assessed with rhodamine 6G (R6G), a highly Raman-active molecule. R6G molecules were absorbed onto GQD-NTs simply by soaking. Their vibrational frequencies were similar to those on graphene and metalbased SERS substrates. An overall 40–74-fold enhancement was obtained. The capabilities of GQD-NTs for determining 2,4-dinitrotoluene (2,4-DNT) were investigated and a 59-fold enhancement in the Raman intensity for the NO2 stretching mode was obtained relative to that for the Si reference. The SERS effect of the GQDs was ascribed to a chemical mechanism. GQDs have also been used as surfactants to obtain Pickering emulsions and novel polymer particles. Yang et al. [135] obtained highly luminescent GQDs by removing oxygen-containing groups from GQD surfaces. To this end, they applied a thermal treatment to dots prepared by chemical oxidation of CX-72 carbon black. For surfactant procurement, the dots were reduced in different degrees and dissolved in oil/water emulsions. Only GQDs reduced for long times formed a surfactant and were useful as polystyrene (PS) colloidal particles and PS-b-polybutadiene (PS–b–PB) particles with great potential for bioimaging and environmental applications.

4.

GQDs as analytes

Current progress in analytical nanoscience and nanotechnology is the result of (a) nanomaterials being useful tools to develop new analytical methodologies, and improve well-established analytical processes, methods and techniques; and, (b) NPs and nanostructures being potential target analytes. The analytical determination of nanomaterials requires methods capable of detecting and quantifying very small amounts of material in various types of samples (complex biological and environmental matrices included). However, few such methods have been reported to date, and only two with GDQs as analytes. One was developed by Fuhuno et al. [136], who investigated the size-dependent luminescent properties of GQDs using LC. They obtained PL GQDs by chemical oxidation of pitch-based carbon fibers and separation by size-exclusion HPLC. The dominant features of the dots exhibited moderate changes in relative intensity depending on the overall size of the dots and, especially, in the emission at ~600 nm, 520 nm, 440 nm and 330 nm. Differences in PL were ascribed to differences in density, shape and size of sp2 fragments available in the GQD solution. 18 Page 18 of 34

The optical properties of the dots were controlled via size-based separation and their emission was ascribed to quasi-molecular PL from fragments consisting of small aromatic rings bearing oxygen-containing functional groups. Recently, Benítez-Martínez et al. [137] developed a new strategy for preconcentration and determination of GQDs obtained by pyrolysis of citric acid in environmental and tap-water samples. The ensuing methodology was based on the retention of GQDs in a quaternary amine-functionalized solid-phase extraction (SPE) cartridge from which they were eluted with 0.25 M NaOH prior to fluorimetric analysis. The authors assumed ionic interactions between GQDs bearing negative charge in oxygen-containing groups at their edges and positively-charged quaternary amine groups. Releasing the GQDs from the sorbent for elution required altering their charge. The LOD thus achieved was 7.5 µg·L–1 and recoveries were 84.4–99.3%.

5.

Conclusions and future prospects

GQDs are graphene family FL NPs that have proved easy to obtain. Their exceptional optical and electronic properties and the presence of high number of reactive sites make GQDs powerful tools in analytical nanoscience and nanotechnology. At the same time, their opto-electronics properties make them excellent nanomaterials for a very wide variety of applications. We have proved that these NPs have great potential in the development of analytical sensors and biosensors. Nonetheless, the greatest difficulty is still to obtain high-quality GQDs. The existing methods of synthesis do not allow large-scale production of GQDs and, moreover, those obtained generally have a wide size distribution and low QYs. By contrast, it is very advantageous to have different routes of synthesis that achieve GQDs with different excitation and emission wavelengths, and different diameters and lateral sizes, in line with the needs of each individual research or application. At present, the mechanisms involved in luminescent, optical and electronic properties, those relevant to the performance of sensors and biosensors and those derived from doping and functionalization are not fully understood. In this context, we expect new theoretical and experimental studies to clarify this issue, as well as establishing the significant differences between GQDs and other carbon-based FL NPs, such as carbon dots. Likewise, we expect that the synthesis methods will be improved in order to obtain high yields and more efficient NPs. We also believe that, as well as graphene, GQDs will be incorporated in commercial products, especially those relevant to electronics, such as batteries, solar cells and nanoelectrochemical systems (NEMS). In the analytical chemistry field, we expect that GQDs will be used in several areas, not only sensing. They could be employed as sorbents in extraction procedures [mainly SPE and solid-phase microextraction (SPME)] favoring interaction with analytes due to their aromatic character and the presence of functional groups at their surfaces and edges. GQDs could be also used as adsorbents for organic compounds and metals, and be employed as stationary phases in LC. Moreover, GQD-based analytical techniques need to be developed to detect different target analytes, elaborating microchip systems to develop GQD-based lab-on-a-chip devices for separation and detection of macromolecules, DNA, proteins and others bio-targets. The hybridization of GQDs with other organic or inorganic materials [e.g., polymers, CNTs, and NPs (carbon or metallic)] through covalent binding could considerably extend the possibilities for GQD-sensor development. There is still much work to do to understand and to utilize fully GQDs in sensor development and applications. The potential incorporation of these GQDs in commercial products increases environmental and human exposure daily. GQDs have been defined as low toxic, biocompatible NPs; however, as a consequence of their potential environmental release 19 Page 19 of 34

and their easy dispersion due to their high water solubility, GQDs could accumulate in aquatic ecosystems and the human body (organs and tissues). It is therefore necessary to develop new analytical methods that allow the determination of GQDs in complex matrices, such as the biological and environmental ones. Acknowledgment The authors would like to express their gratitude to the Junta de Andalucía for the financial support (Project FQM 4801). S. Benítez-Martínez also wishes to thank the Junta de Andalucía for the award of a Research Training Fellowship. References [1] K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I. V. Grigorgieva, A.A. Firsov, Electric field effect in atomically thin carbon films, Science 306 (2004) 666-669. [2] A.K. Geim, Graphene: status and prospects, Science 324 (2009) 1530-1534. [3] X. Yan, X. Cui, L.S. Li, Synthesis of large, stable colloidal graphene quantum qots with tunable size, J. Am. Chem. Soc. 132 (2010) 5944-5945. [4] S. Kim, S.W. Hwang, M.K. Kim, D.Y. Shin, D.H. Shin, C.O. Kim, S.B. Yang, J. H. Park, E. Hwang, S.H. Choi, G. Ko, S. Sim, C. Sone, H.J. Choi, S. Bae, B.H. Hong, Anomalous behaviors of visible luminescence from graphene quantum dots: interplay between size and shape, ACS Nano 6 (2012) 8203-8208. [5] Y. Dong, C. Chen, X. Zheng, L. Gao, Z. Cui, H. Yang, C. Guo, Y. Chi, C.M. Li, One-step and high yield simultaneous reparation of single- and multi-layer graphene quantum dots from CX-72 carbon black, J. Mater. Chem. 22 (2012) 8764-8766. [6] C. Frigerio, D.S.M. Ribeiro, S.S.M. Rodrigues, V.L.R.G. Abreu, J.A.C. Barbosa, J.A.V. Prior, K.L. Marques, J.L.M. Santos, Application of quantum dots as analytical tools in automated chemical analysis: A review, Anal. Chim. Acta 735 (2012) 9-22. [7] S.H. Jin, D.H. Kim, G.H. Jun, S.H. Hong, S. Jeon, Tuning the photoluminescence of graphene quantum dots through the charge transfer effect of functional groups, ACS Nano 7 (2012) 1239-1245. [8] M.L. Mueller, X. Yan, J.A. McGuire, L. Li, Triplet states and electronic relaxation in photoexcited graphene quantum dots, Nano Lett. 10 (2010) 26792682. [9] H. Tetsuka, R. Asahi, A. Nagoya, K. Okamoto, I. Tajima, R. Ohta, A. Okamoto, Optically tunable amino-functionalized graphene quantum dots, Adv. Mater. 24 (2012) 5333-5338. [10] J. Shen, Y. Zhu, X. Yang, J. Zong, J. Zhang and C. Li, One-pot hydrothermal synthesis of graphene quantum dots surface-passivated by polyethylene glycol and their photoelectric conversion under near-infrared light, New J. Chem. 36 (2012) 97-101. [11] Y. Feng, J. Zhao, X. Yan, F. Tang, Q. Xue, Enhancement in the fluorescence of graphene quantum dots by hydrazine hydrate reduction, Carbon 66 (2014) 334339. [12] P. Luo, Z. Ji, C. Li, G. Shi, Aryl-modified graphene quantum dots with enhanced photoluminescence and improved pH tolerance, Nanoscale 5 (2013) 7361-7367. [13] L.A. Ponomarenko, F. Schedin, M.I. Katsnelson, R. Yang, E.W. Hill, K.S. Novoselov, A.K. Geim, Chaotic dirac billiard in graphene quantum dots, Science- 320 (2008) 356-358. 20 Page 20 of 34

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Captions Fig. 1. Preparation of GQDs by hydrothermal treatment of SWCNTs. Reproduced, with permission, from reference 20. Fig. 2. Synthesis of GQDs and GO. The black dots in GO represent oxygen atoms. Reproduced, with permission, from reference 45. Fig. 3. Structure of N-GQDs. The red, dashed circles highlight three substitutional N atoms. The missing C atom in the middle represents a potential structural defect. Reproduced, with permission, from reference 54. Fig. 4. Production of N-GQDs by using nitrogen plasma. Reproduced, with permission, from reference 47. Fig. 5. Hydrothermal treatment of GQDs. Reproduced, with permission, from reference 76. Fig. 6. (a) Fluorescence quenching mechanism of the S-GQDs in the presence of Fe3+ and (b) the electron transfer process from S-GQDs to Fe3+. (c) Fluorescence spectra of S-GQDs (5 μg/mL) with different concentration of Fe3+. The inset in (c) shows photographs of S-GQD aqueous solutions with different concentration of Fe3+ under UV irradiation (365 nm). (d) The curve of the fluorescence quenching values ΔF vs Fe3+ concentration ranging from 0 to 1.60 μM. The inset in (d) is the linear calibration plot for Fe3+ detection. Reproduced, with permission, from reference 48. Fig. 7. Ground state structures of one luminescent unit of B-GQDs in the presence (a) and absence (b) of borax obtained by theoretical calculation with density function theory. (c) The size distribution of B-GQDs in the presence (left) and absence (right) of borax determined by DLS. The structure illustration of B-GQDs in the presence (d) and absence (e) of borax, respectively. Reproduced, with permission, from reference 83. Fig. 8. (a) Schematic representation of the boron-doped graphene quantum dots (BGQDs). (b) Proposed “aggregation-induced PL increasing” mechanism for the glucose-specific sensing by BGQDs. Reproduced, with permission, from reference 50. Fig. 9. Scheme for the detection of ovarian biomarker CA-125 and principle behind the immunoassay. Reproduced, with permission, from reference 123. Fig. 10. Use of ZnFe2O4/GQDs to mimic a trace label for the electrochemical detection of DNA. Reproduced, with permission, from reference 130.

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Table. 1. Analytical applications of graphene quantum dots GQDs Type of sensor Type

Target analyte

LOD

Real sample

Ref.

Fe3+

0.08 µM

Tap water

[23]

Fe3+

4.2 nM

Human serum

[48]

Fe

3+

0.1 µM

Tap water

[76]

Fe

3+

NM

NM

[58]

Fe

3+

7.22 µM

NM

[22]

3+

90 nM

Lake water

[60]

5 nM – 0.33 µM

NM

[42]

River water

[82]

2+

0.226 µM

NM

[83]

2+

6.9 nM

Living cells

[68]

Precursor

PHOTOLUMINESCENCE SENSORS Probe sensor

N–GQDs S–GQDs NA–GQDs N–GQDs +

BMIN –GQDs

MOF-derived porous carbon Graphite Glycine Citric acid 3D graphene

N–GQDs

Citric acid

Fe

GQDs

PAHs

GQDs

Graphite fibers

Fe3+ H2O2 Cu2+

GQDs

Re-oxidized GO

Cu

Af–GQDs B–GQDs GQDs

Cu

Al3+

Graphite road Graphene

3.64 µM

Drinking Water

[84]

2+

10 µM

NM

[85]

2+

Hg

GQDs

Citric acid

Hg

3.36 µM

NM

[86]

N–GQDs

Citric acid and ammonia

Cr6+

40 nM

[87]

GQDs

Carbon fibers

Ni2+, Mn2+, Co2+, Cu2+

GQDs

Glucose

ClO–

4.1 µM (Ni2+) 30 µM

Wastewater Lake water River water Domestic sewage NM Tap and pool water

[89]

[88]

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GQDs

Glucose

ClO–

50 µM

Tap water

[90]

N–GQDs

Dicyandiamide (DCD)

pH sensor

1.81–8.96

waters

[91]

0.1 µM

Complex environmental samples Cell lysates and human serum plasma NM

[92]

Bovine serum

[94]

NM

[59]

3–

GQDs

Graphene sheets

PO4

GQDs@GSH

Citric acid and GSH

ATP

22 µM

GQDs

GO

GQDs

GO

N–GQDs

Citric acid and DCD

ALP Cu2+ Glu Asp GSH

17 pM 13 nM 0.19 µM 0.18 µM 87nM

GQDs

GO

Melamine

0.12 µM

NM

[95]

GQDs

Citric acid

[96]

Citric acid

5 nM 2.5 nM 5 nM 91 nM

Bovine serum

GQDs

GSH Cys Hcy TNP

Lake water

[97]

GQDs

GO

TNT

2.2 µM

NM

[98]

GQDs

Citric acid

[99]

Citric acid

20 nM 80 nM 30 nM 2.2 µM

Rain and tap water

GQDs

o-DHB m-DHB p-DHB Phenol

Waste and lake water

[100]

B-GQDs

Boron doped graphene

Glucose

0.03 mM

NM

[50]

APBA–GQDs

GO

Glucose

5.0 µM

Rat brain microdialysate

[101]

GQDs

Graphene sheets

NM

NM

[102]

GQDs

Citric acid

Fructose Galactose Glucose Gallic acid Oleuropein

0.09 mg·L-1 0.12 mg·L-1

Olive oils

[103]

GQDs

GO

Pyrocatechol

NM

NM

[28]

[93] [37]

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GQDs

Citric acid

GO

35 µg·L-1

River water

[104]

GQDs

XC–72 Carbon black

AuNPs

NM

NM

[105]

Colorimetric sensor

GQDs

L-glutamic acid

H2O2

20 µM

NM

[44]

Hybrid fluorescence sensor

Hemin-GQDs

Citric acid

H2O2

0.1 µM

Human serum

[106]

HRP–GQDs

Citric acid

Hydroquinone

0.084 µM

Lake water

[107]

Biological system

[108]

Phosphorylated peptide– GQDs AgNPs–GQDs

Graphene

MIP–GQDs Cyt c–GQDs

Protein kinase

-

0.03 U·mL 1

3.5nM 4.1 nM 6.2 nM 4.5 nM 9.0 mg·L-1

Human plasma

[109]

GO

Ag+ GSH Cys Hcy p–nitrophenol

River water

[110]

GO

trypsin

33 ng·mL-1

NM

[111]

GQDs coated on GC electrode (GCE/GQDs) GQDs/Au

Citric acid

S2O82–

NM

[112]

GO

H2O2

0.1 µM 0.02 µM 0.7 µM

[113]

GQDs/CCE

GO

Glucose

GO

ELECTROCHEMICAL SENSORS Modified Electrodes

Electrochemical sensors

Hybrid electrochemical sensors

GQDs/

S2O82–

GQDs/

S2O82–

CX-72 carbon black

Cr

1.73 µM

Human breast adenocarcinoma cell line MCF-7 Human plasma

6+

20nM

River water

[115]

2+

13nM

NM

[116]

[114]

GO

Cd

MIB/Au/ERGQDs/GCE

GO

[117]

GO

9nM 5.5 µM 6.5 µM 530.85 nM

Blood serum

HRP–GQDs/GCE

GSH Uric acid Tryptophan H2O2

NM

[118]

GQDs/AuNPs/GCE

Graphene

Malachite green

0.1 µM

Salmon muscle

[119]

IMMUNOSENSING AND APTASENSING

33 Page 33 of 34

Immnunosensors

0.6 pg·mL-1

Serum samples

[121]

0.96 mU·mL-1 115 TCID50·mL-

Serum samples

[122]

NM

[123]

CA-125 ovarian biomarker Human IgG

0.08 U·mL-

Human blood plasma

[124] [125]

6.7 nM 7.9 nM 75 pM

Human serum Cell culture fluid NM NM

[128]

100 nM

McAb2/GQD/Au@Pt

GO

pPtPd@GQDs/Ab2

Citric acid

Carcinoembryonic antigen CA199 tumor marker

Fe3O4@GQDs/Ab2–Cuapoferritin/BSA

GO

Avian leucosis virus

1

GQDs

Aptasensors

GO

GQDs/Graphene

GO

eGQDs rGQDs GQDs

Graphite powder Graphite powder

DNA Thrombin DNA

GQDs

Graphene sheets

DNA

ZnFe2O4/GQDs SiO2/GQDs

GO

DNA

1

10 ng·mL-1

[127]

NM

[130]

-17

Human serum

[131]

-12

6.2x10 M

GO

ATP

1.5x10 M

NM

[132]

Radix Scutellariae and Licorice extracts NM

[133]

NM

[135]

OTHER ANALYTICAL APPLICATIONS Liquid Chromatography tándem mass spectrometry Raman Spectroscopy

af–GQDs

Graphite powder

Antioxidants

NM

GQDs nanotubes

Graphene

Surfactant

GQDs

CX–72 carbon black

Rhodamine 6G 2,4-dinitrotoluene NM

10-9M NM NM

[134]

NM, Not Mentioned; ALP, Alkaline phosphatase; Asp, Aspartic acid; ATP, Adenosine triphosphate; Cys, Cysteine; DCH, Dicyandiamide; DHB, Dihydroxybenzene; Glu, Glutamic acid; GO, Graphene oxide; GSH, Glutathione; Hcy, Homocysteine; PAH, Polycyclic aromatic hydrocarbon; TNP, 2,4,6trinitrophenol; TNT, 2,4,6-trinitrotoluene

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