Optical bio(sensing) using nitrogen doped graphene quantum dots: Recent advances and future challenges

Accepted Manuscript Optical bio(sensing) using nitrogen doped graphene quantum dots: Recent advances and future challenges Ayub Karimzadeh, Mohammad Hasanzadeh, Nasrin Shadjou, Miguel de la Guardia PII:

S0165-9936(18)30210-3

DOI:

10.1016/j.trac.2018.08.012

Reference:

TRAC 15222

To appear in:

Trends in Analytical Chemistry

Received Date: 15 May 2018 Revised Date:

21 August 2018

Accepted Date: 21 August 2018

Please cite this article as: A. Karimzadeh, M. Hasanzadeh, N. Shadjou, M.d.l. Guardia, Optical bio(sensing) using nitrogen doped graphene quantum dots: Recent advances and future challenges, Trends in Analytical Chemistry (2018), doi: 10.1016/j.trac.2018.08.012. 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.

ACCEPTED MANUSCRIPT Optical bio(sensing) using nitrogen doped graphene quantum dots: Recent advances and future challenges

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Ayub Karimzadeh a, Mohammad Hasanzadeh b*, Nasrin Shadjou c, Miguel de la Guardia d

Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz

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51664, Iran.

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51664, Iran.

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Nanotechnology research center, Uremia University, Uremia, Iran.

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Department of Analytical Chemistry, University of Valencia, Dr. Moliner 50, 46100

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Burjassot, Valencia, Spain.

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Corresponding authors: E-mail address: (*) [email protected]

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ACCEPTED MANUSCRIPT Abstract The exceptional optical properties and the presence of high number of reactive sites make nitrogen doped graphene quantum dots (N-GQDs) powerful tools in analytical nanoscience and nanotechnology. At the same time, their opto-electronics properties make them excellent

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nanomaterials for biomedical analysis aspects. This review aims to explore progress to date various features of N-GQDs for optical bio(sensing) of target analytes. Moreover, as another aim of this review is to provide insight into the intensity based spectroscopic methods which

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are applied in bio(sensing) discussing their advantages and disadvantages. More importantly, we discuss in detail different aspects of the applied optical bio(sensing) (e.g., formation,

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detection techniques, labels and sensitivity). Consequently, we discuss several outstanding properties of the optical methods, research opportunities and the development potential and prospects. We believe that N-GQDs based analytical biosensors need to be developed to detect different target analytes, elaborating microchip systems to develop GQD-based lab-on-

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a-chip devices for separation and detection of bio-targets.

Keywords: Biomedical analysis, cancer, advanced nanomaterial, nitrogen doped graphene

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quantum dots, biosensing

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ACCEPTED MANUSCRIPT 1. Introduction Graphene quantum dots (GQDs), a member of the graphene family, known as a novel type of zero-dimensional luminescent nanomaterials, are small graphene fragments to cause excitation confinement in 3-20 nm particles and quantum-size effect. [1] GQDs is a

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promising new substance from the carbon material family that has been attracting researchers of many fields, such as biomedical sensors, medical imaging, polymer science, solar cells, light emitting diodes, and photoelectrons. Its unique electrical and mechanical properties

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could encourage its usage due to its low cost, high surface area, safety, stable luminescence, excellent biocompatibility, suitable conductivity, and low toxicity. GQDs exhibit

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extraordinary optoelectronic properties as well as excellent biocompatibility and low-cost preparation methods, by which they hold potentials in replacing those well-known metal chalcogenides based quantum dots [2]. Besides, the π–π bonds below and above the atomic plane give graphene exceptional thermal and electrical conductivity, compared with

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conventional semiconductor quantum dots, making GQDs possess their favorable attributes without incurring the burden of intrinsic toxicity [3-7]. The quantum-confinement effect and the variation in density and nature of sp2 sites available in GQDs make their optical

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properties greatly depend on their size so that the energy band gap of GQDs can be tuned by

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modulating their size. [8]

Over the past few decades, quantum dots with steadily increasing research have been occupied a special status in nanomaterial fields and have made considerable progress, but in recent years, graphene based nanohybrid has captured more interest and imagination in scientific research areas. [9] Owing to its extraordinary mechanical properties, biocompatibility, transparency and electrical conductivity, Graphene quantum dots GQDs has obtained a rapid growing as breakthrough tools for multipurpose in various fields of science including photonics, composites, energy, and electronics. Meanwhile, GQDs-based

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ACCEPTED MANUSCRIPT nanomaterials have already shown a promising future in biomedical fields, particularly for diagnostics,[10-14] drug delivery,15 near-infrared (NIR) light-induced photothermal therapy [15-18], in vitro and in vivo bioimaging [19-21]. In addition to those fundamental applications as mentioned above, Ding et al. have reported a GQD-based anticancer drug

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carrier and a signaler for indicating drug delivery, release, and response by providing distinct fluorescence signals at different stages [22]. This research of multifunctional GQDs provides a new direction in future biomedical applications.

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Nitrogen is regarded to be an outstanding element for the chemical doping of GQDs ascribed to its matchable atomic size and five valence electrons for joining with carbon atoms [22].

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Nitrogen doping has been a subsequent way to alter the features of carbon materials varying from activated carbon to graphene. Nitrogen substitution, or substantial N-doping, has been a subsequent procedure to alter graphitic carbon materials for several applications, varying from catalysis to microelectronics [22]. N-doped activated carbon or carbon nanotubel. For

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instance, possess catalytic activities for some significant chemical reactions such as oxygen decrease and dehydrochlorination. In graphene, substitution nitrogen atoms are able to sufficiently alter the electronic construction of the host, supplying an assurance way to tailor

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the transport features of graphene for electronic application.

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Nitrogen-doped graphene quantum dots (N-GQDs) are nitrogen-doped graphene mends (Scheme 1) that exhibit impressive electrocatalytic activities, zero-dimensional N-GQDs are scarcely indicated as one electrocatalyst in fuel cells. The N-GQDs are favorably crystalling with a size of 2.5-8.5 nm of diameter and made up 5-7 layers of thickness. N-GQDs was initially reported by Qu et al., in 2011 by a simple electrochemical approach [22].

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Scheme 1. Schematic illustration of N-GQDs [22]. Redraw by Corel Draw 6.0 software.

To date, great attempts have been made in the preparation of N-GQDs from “top-down” and “bottom-up” approaches [23]. The top-down strategies indicate to producing massive carbon materials into small GQDs accompanied by nitrogen doping, consisting electrochemical [24], hydrothermal or nitrogen plasma manner [25]. Presently, these top-down approaches for

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the manufacture of N-GQDs suffer from low obtainments and troubles in monitoring the morphology and size distribution (Scheme 2). The bottom-up techniques of unit cell

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assembly have advantages over the top-down ways on the tuning of composition, size, and physical features of N-GQDs by ranging the organic precursors and reaction circumstances

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[26].

N-GQDs present outstanding physicochemical features and display assurance applications in many areas. still, the synthesis techniques for N-GQDs involve the solution chemistry method, electrochemical method, organic method, acid oxidation [23,24], solvothermal preparation and hydrothermal method [23-25]. Between them, the hydrothermal method is extensively adopted on account of to its simple operation, moderate reaction circumstances and use of partly cheap device. At the same time, in order to prevent organic reagents or costly ionic liquid as a nitrogen source in the synthesis of N-GQDs, the selection of green and 5

ACCEPTED MANUSCRIPT low-price nitrogen sources such as amino acids has become a significant survey direction [24-26]. Scientists discovered that the features of GQDs could be extremely tuned by nitrogen-doping. The several nitrogen doped colloidal graphene quantum dots gained by the solution chemistry

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approach in their survey exhibited a size-reliant electrocatalytic function for the oxygen reduction reaction [10]. Researchers have displayed a technique, in which they first blend GQDs with graphene, a famous carbon support for electrocatalysts, and then co-dope the

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hybrid with boron and nitrogen to gain BN-doped GQD/grapheme hybrid nano-platelets. The out coming hybrid material has showed synergistic impacts to improve the ORR

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electrocatalytic activity [16]. Gong and co-workers. In addition, an efficient two-photon fluorescent N-GQD probe with low cytotoxicity and good photo-consistency for cellular imaging were prepared recently [17]. Researchers of this work illustrated that the nitrogendoping onto GQDs with subsequent electron-donating dimethylamido groups resulted in a red

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shift of its fluorescent emission. The large π-conjugated graphene sheets and the subsequent electron donating impact of dimethylamido facilitated the charge transport efficiency and led

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to strong two-photo-induced fluorescence for N-GQD [18].

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Scheme 2. Schematic illustration of the reaction mechanism of N-GQDs [20]. Redraw by

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Corel Draw 6.0 software.

In this review, the applications of N-GQDs in optical biosensing will be reviewed. Future perspectives and challenges for applying N-GQDs-based materials in biosensing fields will also be covered. This review aims to explore progress to date various features of N-GQDs for optical bio(sensing) of target analytes. Moreover, as another aim of this section is to provide insight into the intensity based spectroscopic methods which are applied in bio(sensing) discussing their advantages and disadvantages. More importantly, we discuss in detail 7

ACCEPTED MANUSCRIPT different aspects of the applied optical bio(sensing) (e.g., formation, detection techniques, labels and sensitivity). Consequently, we discuss several outstanding properties of the optical

2. Why optical bio(sensing) based on N-GQDs

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methods, research opportunities and the development potential and prospects.

The interaction between N-GQDs and certain substances can cause fluorescent intensity of the GQDs to be reduced. According to this principle, a variety of chemical or biological

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sensors can be designed to detect heavy metal ions [27, 28], small organic or inorganic molecules [29, 30], biological molecules [31, 32], etc. For instance, Wang et al. [33] applied

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the GQDs to detect Fe3+ ions. It is found that Fe3+ ions have a significant impact on the fluorescence intensity of GQDs because of the special complexation between Fe3+ ions and the phenolic hydroxyl groups of the GQDs. When the concentration of Fe3+ ions in the solution arrive to 80 ppm, the fluorescence of GQDs is quenched. However, other metal ions

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with the same concentration have little impact on the fluorescence intensity. It shows that the GQD-based sensors to Fe3+ ions are highly selective. Fan et al. [29] investigated using GQDs to detect trinitrotoluene (TNT) in solution, based on the fluorescent quenching of GQDs by

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TNT. It demonstrates that TNT is adsorbed on the surface of the GQDs by a π-π interaction,

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and fluorescence resonance energy transfer is caused by molecular dipole-dipole interaction within a certain distance, reducing the fluorescence intensity of GQDs. Li et al. [34] designed a new type of electrochemical fluorescent sensors to test Cd2+ ions, taking advantage of the electrochemical fluorescence emission properties of the GQDs. Moreover, GQDs have excellent electrical conduction ability, good dispersibility, and a large specific surface area, and is advantageous for the biological molecules to load on its surface. Various spectroscopic methods have been used on the analysis of target analytes. These methods have both advantages and disadvantages. For example, fluorescence based methods

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ACCEPTED MANUSCRIPT are very sensitive to very low concentrations of analyte; also absorption methods are very simple. Recently, near-infrared fluorescence (NIRF) imaging techniques have brought high attentions in non-invasive assessments with high sensitivity and specificity [15-21]. The use of emission on low frequency region (650-900 nm) is of great importance because it provides

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a non-invasive detection. Due to less tissue auto fluorescence, light absorption and penetration of near infrared (NIR) across of tissues, the NIR is extremely used in biomedical recognitions.

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This review aims to explore progress to date various features of N-GQDs for optical bio(sensing) of target analytes. Moreover, as another aim of this section is to provide insight

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into the intensity based spectroscopic methods which are applied in bio(sensing) discussing their advantages and disadvantages. More importantly, we discuss in detail different aspects of the applied optical bio(sensing) (e.g., formation, detection techniques, labels and sensitivity). Consequently, we discuss several outstanding properties of the optical methods,

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research opportunities and the development potential and prospects

2.1. Luminescent and chemiluminescence based bio(sensing)

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Luminescence is an analytical technique and subcategory of intensity based techniques in

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which the analyte may be excited by thermal or optical sources. The main advantage of these methods are less or non-destructiveness and ability to be performed at room temperature. In photoluminescence and phosphorescence techniques, the exciting source is radiation. When a chemical reaction caused excitation of some analytes in the emission range of ultraviolet to infrared wavelengths (10-4-10-8m), the luminescence method called chemiluminescence. Nitrogen doped GQDs displayed better photoluminescence (PL) features, compared to GQDs, as they were combined from citric acid and urea as precursors [35, 36]. Furthermore, the emission color can be tailored from green to yellow by simply altering the solvent in the

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ACCEPTED MANUSCRIPT hydrothermal reaction [37]. The reduction of the emission lifetime (τ) from 14 to 10 ns is proportional to the increase in the emission wavelength from blue and yellow, displaying that the emission initiates from single luminescent sorts specific to each N-GQD [35].The quantum yields of those N-GQDs were stated to be as high as 0.9 (specially blue-emitting

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QDs) ascribed to less hydroxyl and carboxyl groups acting as non-radiative electron-hole recombination centers [35, 36].

Photoluminescent graphene quantum dots (GQDs) have received enormous attention because

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of their unique chemical, electronic and optical properties. In 2014, a series of GQDs were synthesized under hydrothermal processes in order to investigate the formation process and

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optical properties of N-doped GQDs (Scheme 3). Citric acid (CA) was utilized as the carbon source and several amines were utilized as the N source. In this work, N-doped GQDs were prepared using a series of N-containing bases such as urea. Detailed structural and property studies demonstrated the formation mechanism of N-doped GQDs for tunable optical

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emissions. Hydrothermal conditions promote formation of amide between –NH2 and –COOH with the presence of amine in the reaction. The intra-molecular de-hydrolysis between neighbor amide and COOH groups led to formation of pyrrolic N in the graphene framework.

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Combined GQD core materials by using NaOH as dopants. Under basic reaction

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circumstance, the CA molecules self-assembled into sheet structure followed by condensation reactions through intermolecular dehydroxylation, forming nano-crystalline GQDs. The last GQDs have unreacted functional groups such as carboxyl and hydroxyl groups on particle surface. At this point, the gained GQDs display relative low QY of, 10%, excitationindependent PL, and individual exponential lifetime [37].

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Scheme 3: Possible formation process of N-doped GQDs [37].

On the other hand, carbon nanomaterials doped with heteroatoms (typically doped by

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nitrogen atoms) can effectively handle the local chemical properties and band gaps of graphene structures, leading to alterations in their electronic features and optical features[38,

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39]. This approach supplies a novel way to gain amine-functionalized nitrogen-doped GQDs with high quantum yield through a facile synthesis technique. At present, the techniques ready for the combination of N-GQDs consist of hydrothermal treatment of carbon sources in the presence of dicyandiamide [40], ammonia [38] or hydrazine [41], subsequent acid treatment of N-doped grapheme [42], the electrochemical technique [43] and the solution chemistry approach. In contrast to other technique, the hydrothermal approach is the most extensively adopted for its plain pertormance and moderate reaction circumstances [44].

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ACCEPTED MANUSCRIPT Recently, Lin and co-workers stated, a facile bottom-up technique for the combination of highly fluorescent N-GQDs has been developed by a one-step pyrolysis of citric acid and tris (hydroxymethyl) aminomethane. The gained N-GQDs emitted subsequent blue fluorescence under 365 nm UV light excitation with a high quantum yield of 59.2%. They showed

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excitation-independent treatment, high persistence photo-bleaching and high ionic power. In addition to the good linear connection between the fluorescence intensity of the N-GQDs and pH in the 2-7, the fluorescence intensity of the N-GQDs could be greatly quenched by the

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addition of a small amount of 2,4,6-trinitrophenol (TNP). A sensitive approach has been developed for the discovery of TNP with a detection limit of 0.30 µM, and a linearity varing

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from 1 to 60 µM TNP could be gained. The approach was highly selective and appropriate for TNP analysis in natural water samples [45].

Tabaraki and Nateghi stated highly luminescent N-GQDs were prepared from ammonia and glucose as nitrogen and carbon sources, respectively. The N-GQDs displayed a subsequent

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analytical potential as sensing examination for silver ions assignment. The best circumstances were assigned as pH 7, N-GQDs focus 1 mg/mL and time 60 min. It proposed that N-GQDs exhibited high sensitivity and selectivity toward Ag+. The linear range of N-GQDs and the

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limit of detection were 0.2-40 µM and 168 nM, respectively. The N-GQDs-based Ag+ ions

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sensor was successfully utilized to the assignment of Ag+ in tap water and real river water samples [46].

2.2. Fluorescence

Fluorescent based sensors have been regarded as practical technique to detect metal ions ascribed to their high sensitivity, rapid discovery and selectivity. However fluorescent semiconductor quantum dots and metal nanoparticles have appealed remarkable attention as fluorescent sensing materials for the discovery of metal ions, they are confined by their toxicity and costly combination technique. Recently, fluorescent N-GQDs have obtained

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ACCEPTED MANUSCRIPT great attention as alternative to semiconductor quantum dots in the discovery of metal ions ascribe to their good photo-stability, outstanding biocompatibility, and low toxicity [47]. GQDs and N-GQDs are attractive fluorophores that are inexpensive, nontoxic, photo-stable, water-soluble, biocompatible, and environmentally friendly. They find extensive applications

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in fluorescent biosensors and chemo-sensors, in which they serve as either fluorophores or quenchers. As fluorophores, they display tunable photoluminescence emission and the “giant red-edge effect”. As quenchers, they exhibit a remarkable quenching efficiency through

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either electron transfer or Förster resonance energy transfer (FRET) process. In this subsection, the origin of fluorescence and the mechanism of excitation wavelength-dependent

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fluorescence of N-GQDs are discussed. Sensor design strategies based on N-GQDs are presented. The applications of these sensors in health care, the environment, agriculture, and food safety are highlighted.

Interestingly, a highly sensitive sensor for discovery of histidine (His) based on the N-GQDs-

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Cu2+ system has been designed. The fluorescence of N-GQDs can be effectively quenched by Cu2+ ascribed to the binding between Cu2+ and functional groups on the surface of N-GQDs. The high affinity of His to Cu2+ enables Cu2+ to be dissociated from the surface of N-GQDs

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and recovering the fluorescence. The sensor showed a sensitive response to His in the focus

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range of 0-35 µM, with a detection limit of 72.2 nM. The suggested technique is successfully utilized to discover His in samples with a recovery range of 96-102% [48]. Also, Chen and co-workers stated dopamine (DA) was sensitively discovered by a label-free fluorescence method using N-GQDs as effective examination. The discovery of DA was also performed on the N-GQDs immersed filter paper. The fluorescence intensity of the N-GQDs was effectively quenched upon addition of DA. There was a good linear connection between the fluorescence intensity of the N-GQDs and DA focus in the range of 1-200 µM, with a detection limit of 0.07 µM. The suggested technique was facile, sensitive, and rapid for DA

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ACCEPTED MANUSCRIPT discovery with simple operation. DA in real serum samples was successfully discovery with acceptable consequences [49]. Kaur et al stated, the carbonization assisted green approach for the invention of N-GQDs. The gained N-GQDs showed good water dispersibility and consistency in the extensive pH range.

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The as combined N-GQDs were utilized as a fluorescent examination for the sensing of explosive 2,4,6-trinitrophenol (TNP) in aqueous medium based on fluorescence resonance energy transfer (FRET),molecular interactions and alter transfer mechanism. The quenching

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efficiency was found to be linear in proportion to the TNP focus within the range of 0-16 µM with discovery limit of 0.92 µM. The presented technique was successfully used to the

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sensing of TNP in tap and lake water samples with acceptable consequence. so, N-GQDs were utilized as a selective, sensitive and turn off fluorescent sensor for the detection of perilous water contaminant i.e. TNP [50].

Interestingly, Zhou and et al prepared an economical hydrothermal method was developed for

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the combination of N-GQDs using citric acid as carbon source and melamine as nitrogen source (Scheme 4). The as-combined N-GQDs possess many outstanding features, such as subsequent blue fluorescence, excitation-independent emission, high mono-dispersity, good

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consistency, and outstanding water solubility with a fluorescence quantum yield of 18%. The

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analytical application of this technique as an effective sensor for direct analysis of metacycline (MTC) revealed its selective response by a fluorescence quenching mechanism. This sensing system exhibits outstanding sensitivity and selectivity toward MTC with the detection limit of 20 nM, which was successfully used in the discovery of MTC in mice plasma [51].

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Scheme 4. Schematic illustration of N-GQDs preparation and MTC detection.

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Liu et al., stated a dopamine-modulated N-GQD system was examined to develop a fluorescent sensor based on a chemical redox mechanism for the facile, sensitive and

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selective discovery of glutathione (GSH) in biological samples. Dopamine could selfpolymerize to poly dopamine-quinone and snap to the surface of N-GQDs to form a thin film in an alkaline environment, leading to quenching of fluorescence ascribed to fluorescence resonance energy transfer. Afterward, GSH can decrease dopamine-quinone, and prevent the

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electron transfer process of N-GQDs and quinone, which results in the recovery of the fluorescence of N-GQDs (Scheme 5). The fluorescence sensor displayed a response to GSH within an extensive focus range of 0.20-85 µM, with a detection limit of 43 nM. The

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fluorescence sensor can discover glutathione quickly with high sensitivity and selectivity in human urine and serum samples. Thus, this sensor was effective for the routine assignment of

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trace glutathione in relatively complex matrices and diagnostic application [52].

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Scheme 5. Schematic illustration of the developed turn-on fluorescence sensor based on N-

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GQD [52]. Redraw by Corel Draw 6.0 software.

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Also, Cai et al. developed a new method for selective assignment of Cr(VI) in environmental

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water samples was developed based on its quenching impact on the fluorescent N-GQDs. The gained N-GQDs with oxygen-rich functional groups exhibited a subsequent blue emission with a quantum yield of 18.6%, which was 7 times greater than that of GQDs. Ascribed to the selective coordination to Cr(VI), the N-GQDs can be utilized as a green and facile sensing platform for label-free sensitive and selective discovery of Cr(VI) ions in aqueous solution and real water samples. Compared to GQDs, the N-GQDs as a fluorescent probe promises much improved selectivity for sensing of Cr(VI). The N-GQDs fluorescence examination display a sensitive response to Cr(VI) in an extensive focus range of 0-140 mM with a

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ACCEPTED MANUSCRIPT discovery limit of 40 nM. The N-GQDs-based fluorescence technique was successfully utilized to selectively discover Cr(VI), and discriminate it and Cr(III) as well in aqueous samples [53]. Fang et al stated, the N-GQD examination displayed a selective and sensitive fluorescence

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improvement response to Al3+, the mechanism might be the formation of a complicated between Al3+ and N-GQD completed the photo-induced electron transfer (PET) process of NGQD itself. The pKa value of N-GQD was also assigned to be 4.4 by capillary

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electrophoresis. In pH 4.0 PBS solution, there was a good linear connection between the fluorescence intensity and the logarithm of focus of Al3+ in the range of 2.5 -75 µmol L-1, the

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limit of detection was 1.3 µmol L-1. This “Off - On” fluorescence technique had been used to precise quantification of aluminium in hydrotalcite tablets. What more, the fluorescence switch feature of N-GQD was confirmed by regulating with Al3+ and EDTA, and the examination was also used for discovery Al3+ in living cells ascribed to its outstanding

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biocompatibility [54].

Nitrogen doping has been a subsequent technique to modulate the features of carbon materials for several applications, and N-GQDs has obtained considerable enthusiasm

solid-phase

combination

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3,4-dihydroxy-L-

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one-pot

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because of their special chemical, optical features, and electronic. Shi et al introduce a facile

phenylalanine (L-DOPA) as the N source and citric acid as the carbon source (Scheme 6). Moreover, a highly efficient fluorosensor based on the as-prepared N-GQDs was developed for the discovery of Hg2+ because of the effective quenching impact of metal ions by nonradiative electron transport. This fluorosensor exhibits high sensitivity toward Hg2+ with a detection limit of 8.6 nM. The selectivity tests reveal that the fluorescent sensor is particular for Hg2+. Most significantly, the practical use of the sensor based on N-GQDs for Hg2+ discovery was successfully illustrated in river-water samples [55].

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Scheme 6. Possible formation process of the N-OGQDs and the sensing principle of the N-

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OGQDs based fluorescent probe for Hg2+ [55].

Yan and co-workers proposed a relatively facile and environment-friendly one pot technique to prepare water-soluble N-GQDs by utilizing glycine and citric acid as nitrogen and carbon

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sources respectively. The as-prepared N-GQDs have an exhibit blue fluorescence with a high

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quantum yield of 28.1%, which is far higher than the quantum yield of GQDs simply prepared by citric acid. In addition, owing to the coordination happening between Hg2+ and functional groups on surface of N-GQDs, the fluorescence of N-GQDs was quenched efficiently. This fluorescence “turn-off” process makes it possible to label-free sensitive and selective discover Hg2+ with a detection limit of 0.032 µM. With the addition of Cys and GSH, the fluorescence of the N-GQDs/Hg2+ system is recovered slowly because of their selective affinities with Hg2+ through Hg-S bonding interactions. Correspondingly, the fluorescence “turn-on” process happens. The application of N-GQDs in the discovery of Hg2+ 18

ACCEPTED MANUSCRIPT and biothiols in real samples is also successfully illustrated. Detection limit of 0.034 µM for glutathione and 0.036 µM for cysteine. Most significantly, the N-GQDs based fluorescence technique was successfully used to control Hg2+ in real water samples and biothols in serum samples [56].

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For complete our discussion in this section, Table 1 summarized recent reports on the optical sensing of some ions based using N-GQDs and its derivatives [34, 45-51, and 56-63]. This table shows that N-GQDs -based sensor constitute one of the most fascinating current issues

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in the nanotechnology field. Although the percentage of sensing methods involving the use of N-GQDs is still low, as discussed above, this table highlights the attractive possibilities that

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N-GQDs can offer in this research area (environmental and metal analysis). The advances in the synthesis of N-GQDs and in the availability of methods to functionalize their surface have given rise to a wide range of options that can minimize the limitations of optical sensing methods.

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The different examples described throughout this sub-section demonstrate that the use of NGQDs is a suitable strategy to increase the sensitivity and response time of sensing methods. Also, the wide variety of optical properties of N-GQDs has been extensively used to improve

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the sensitivity of optical sensing methods. The use of N-GQDs as matrices has allowed or

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facilitated the development of very sensitive sensing assays. In addition, N-GQDs that simultaneously present two or more properties are a suitable option, as it has been discussed above.

Table 1. Summarized recent reports on the optical sensing of some ions based using NGQDs. Probe N-GQDs/Hg2+ N-GQDs-NCNFs N-GQDs N-GQDs/GC

Detection limit (µM) 0.036 3 53 0.0002

Linear range (µM) 5-30 20-117

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Analyte Cysteine Nitrite H 2O 2 TNT

Ref [56] [61] [62] [60]

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0.2-40 25-375 0.3-5 1-60 0.01-0.25

0-35 1-200 0-16 0.1-157.6

H 2O 2 Ag+ H 2O 2 Glucose PPI Glutathione TNP CAP MTC Hg2+ His DA TNP GSH

[59] [46] [57] [62] [34] [56] [45] [58] [51] [55] [48] [49] [50] [63]

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N-GQDs-NC-pd-HNS N-GQDs N-GQDs-Au-NPs N-GQDs N-GQDs -AEu3+ N-GQDs/Hg2+ N-GQDs N-GQDs N-GQDs N-GQDs N-GQDs-Cu2+ N-GQDs N-GQDs AgNPs-N-GQDs

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N-GQDs results in enhanced fluorescence performance and a wider range of applications in photocatalysis, sensors, bioimaging, etc. Recently, Xu et al stated, a water-soluble and well-

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crystallized N-GQD has been gained by using a MOF-obtained carbon (ZIF-8C) as a novel source of graphitic sheets. The preparation is based on a quick, eco-friendly and efficient acid vapor cutting method, which is unlike from previously reported solution chemistry ways. The as-prepared N-GQD is photoluminescent and exhibits an excitation-independent behavior.

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Because of the presence of O-functional groups on the surface, the gained N-GQD material could be utilized as a fluorescent sensing examination for highly selective discovery of Fe3+ ions with a detection limit of 0.08 mM [64].

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Interestingly, a highly greenish-yellow fluorescent N-GQDs have been combined by the onepot hydrothermal technique utilizing graphene oxide (GO) and lysine (Lys) as a carbon and

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nitrogen source, respectively. The as-prepared N-GQDs with a high quantum yield of 13.2% possessed outstanding optical features also the profits of environmental friendliness, lowprice and simple operation. AEu3+-modulated N-GQDs off-on fluorescent examination for pyrophosphate (PPi) discovery was made with a detection limit of 0.074 µM. The examination was selective and sensitive towards PPi. The discovery process was plain in design and easy to perform, which offered a ‘‘mix and detect’’ protocol for PPi analysis. This

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ACCEPTED MANUSCRIPT work widens the applications of N-GQDs with versatile functionality and reactivity in clinical diagnostics and as biosensors [65]. Significantly, Ju et al. [66] synthesized highly blue luminescent nitrogen-doped graphene quantum dots (N-GQDs) by a facile one-step hydrothermal treatment of citric acid and

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dicyandiamide. A quantum yield (QY) as high as 32.4% is achieved at an excitation wavelength of 350 nm. It is found that such N-GQDs with a high QY can be used as efficient fluorescent probes for the detection of glutathione (GSH). In the detection, the

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photoluminescence (PL) intensity of the N-GQDs could be quenched by mercuric ions due to the strong electrostatic interaction and electron transfer between N-GQDs and Hg(II). Upon

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the addition of GSH, the PL intensity of N-GQDs can be recovered owing to the preferred combination of Hg(II) and GSH by forming a Hg(II)-S bond. Under optimal conditions, this fluorescence turn-on sensing system exhibits excellent sensitivity and selectivity for GSH determination with a detection limit of 87 nM. Importantly, the N-GQDs-Hg(II) system can

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be successfully applied for visualizing the intracellular GSH in live HeLa cells due to bright luminescence, low cytotoxicity, and good biocompatibility. Furthermore, they designed a novel colorimetric sensor for more sensitive detection of GSH based on the intrinsic

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peroxidase-like activity of Ag nanoparticles supported on the nitrogen-doped graphene

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quantum dots (Ag NPs-N-GQDs) [67]. The Ag NPs-N-GQDs nanocomposites can catalyze the oxidation of 3,3′5,5′-tetramethylbenzidine (TMB) to oxTMB with the presence of H2O2 along with a visual color variation. As GSH can reduce oxTMB, the UV-Vis absorption intensity of oxTMB shows a strong dependence on the concentration of GSH. The novel Ag NPs-NGQDs-TMB-H2O2 system exhibits more high sensitivity and selectivity for the detection of GSH with a lower detection limit of 31 nM and a wide detection linear range of 0.1-157.6 µM. This novel sensor system shows great potential application for GSH detection in blood serum sample and easy to-make analytical approaches in the future.

21

ACCEPTED MANUSCRIPT In summary, it is important to point out that the different type of luminol compound such as luciferase, pyropheophorbide, 1,2-Dioxetane, N-(4-aminobutyl)-Nethylisoluminol were used as reporter in the luminescence and chemiluminescence based biosensors for detection of analytes. In fluorescence based biosensors for detection of target analytes, fluorescent

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polymer coated microsphere, aggregation induced emission, fluorescein amidite, enhanced green fluorescent protein, monomeric red fluorescent protein, cerulean, quantum dots, 7amino-4-trifluoromethylcoumarin, fluorescein isothiocyanate, oligodeoxyfluoroside and

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quinone, were used as fluorescent reporters and the compounds polyethylene glycol, quencher-tether-ligand, AuNPs, microsphere, and dabcyl, were used as fluorescent quenching

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agents.

Simplicity in the synthesis of bio-probe or biosensor for detecting of target analytes is very critical. In comparison with EC/ECL, intensity based optical methods are mostly simple and approximately they have easy construction steps and fast response time. Rapid

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bioanalysis or low response time is another essential parameter in the design and development of optical biosensors for detection of target molecules. Also, the selectivity of proposed biosensors for detection of target analytes based on optical methods is another

EP

important issue which need to be addressed. Sensitivity is the most critical factor on the

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construction of intensity based biosensors and bioprobes for the detection of target analytes in real samples and cancer cells. Multiplex analysis or simultaneous determination of different viruses is vital for distinguishing analytes. This ability may to help to physicians for prescribing different and precise drugs for patients. Among the intensity based methods and imaging bioprobes developed for quantification of target analytes, only minor of prepared optical based biosensors were used in simultaneous model. We hope new type of optical based biosensors will be applied at in vivo and clinical laboratories as commercial kits and especially helps to secure and increase of survival time of patients.

22

ACCEPTED MANUSCRIPT All of above discussed articles show that optical based methods were valuable and sensitive for the in vivo or in vitro detection of analytes. Although, ability for working in in vitro condition is more acceptable. Also the facility of usage in animal models or human body is very critical. Unfortunately, none of the constructed optical biosensors has ability to

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application in human body. Also, minor type of developed biosensors are able to detect the target analyte. To the best of our knowledge, there is no report about the clinical application of the above discussed articles which need to address by researchers. After solve

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of above mentioned problems, it seems that optical based biosensors and bio-probes could be used as commercial bio-devices in clinical laboratories and disease diagnosis.

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Emerging of novel nanomaterials used for optical based biosensing of analytes, yet many challenges require to be solved by effective contribution scientists including chemistry, physics, biology and medicine. We are still far from real optical based biosensors that can operate in an automatic mode in living organisms. However, the progress in nano and

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microsystem technology, including nanomotors, may open the way to real novel optical sensors and even to a new class of immunosensors that could combine diagnostic and therapy in the so called theranostic devices. This can probably significantly shorten the time from

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protein sensing (diagnostic) to therapy (i.e. cancer cell fighting) that would improve an

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individual life and ensure an improved safety and security. Many different optical based biosensors for detection of various analytes provide sensitive and specific techniques. However generally require multiple steps and time-consuming procedures such as labeling and sample treatment prior to detection. Labeling methods are not suitable in some cases, because labeling materials may occupied the important binding sites or cause steric hindrance, resulting in false information. Above discussed articles show that, optical methods are powerful analytical techniques for direct monitoring of analytes.

23

ACCEPTED MANUSCRIPT Therefore, optical based biosensors could be suitable method for sensing target molecules with appropriate features. Most of the applications for the detection of target analytes rely on laboratory-based instrumentation for color, fluorescence, and even chemiluminescence. Cellphone (CP)-based

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technologies with smart applications are a new trend in bioanalytical applications. A compact colorimetric and fluorometric reader, as well as a CP-based compact EC sensing platform, have been reported. Indeed, it is feasible for colorimetric readout from a 96-well MTP using a

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CP device and the measurement of a whole emission spectrum of any light emitter using a CP-based fluorimeter. Together with graphene, as a sensing interface, the CP-platform

widespread applications for POC.

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doubtlessly provides cost-effective mobile healthcare and personalized medicine tools with

The clinical significance of optical based biosensors deserves a brief comment here as the clinicians begin monitoring target molecule levels in the patients before the treatment to

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establish a baseline amount. The test will be repeated during and after treatment to assess changes over time. There is also a need to devise and validate prospective multiplexing strategies, enabling the precise determination of target molecules in a sample together with

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the presence of interfering agents. With the improved diagnosis, specific treatment can be

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offered to the patients to attain better treatment outcome. Different aspects of the biosensors, such as type of nanomaterial, injection and detection method, labels, signal amplificators and the corresponding sample matrix, the sensitivity, etc. have been discussed in detail. Consequently, several outstanding properties of the immunosensors and their research opportunities as well as the development potential and prospects can be discussed. Additionally, the summarized examples of nanomaterial-based biosensors reported so far in the literature, could be evaluated with their advantages and limitations in order to stress their potential for future developments in this field.

24

ACCEPTED MANUSCRIPT The use of nanomaterial would most likely result in enhanced analytical characteristics of the optical based biosensors, particularly with respect to operational stability, life-time, and operation in harsh conditions. While reported results are exciting and exhibit great potential for future applications, new

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breakthroughs are still required for optical based biosensing. For example, many of the new optical based biosensors built have not yet been investigated in vivo. So, important information regarding circulation properties in blood and accumulation in liver or other

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tissues needs to be acquired, and eventual problems emerging from those studies will need to be addressed. Although the fast advancement of optical based biosensors highlights their

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future applications in diverse scientific fields, the major impacts of such technologies appear to be in early detection of disease. Of these applications, detection of analytes and importantly its early diagnosis seems to provide an interesting approach for the development of

novel

target-based

therapies

(e.g.,

using

electrochemical/photonic

and

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photoelectrochemical probes for detection of target molecules).

Since many of optical based biosensors have been only developed in laboratory so far, it will be a major challenge to introduce a technology allowing rapid production of large numbers of

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sensors with relatively low cost and high quality specifications. Such technology is

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prerequisite for any successful commercial application. It is worth mentioning that nanomaterial based biosensors can be used as quantitative analytical tools for detection of virus in different analytes, thus evidencing that optical based biosensors could be competitive with ELISA.

3. Conclusions and future perspectives (Unresolved problems, challenges and controversies)

25

ACCEPTED MANUSCRIPT In this review, recent research progress of N-GQDs based nanomaterials, focusing on their optical biosensing applications was summarized. One of the key difficulties facing researchers aiming to develop N-GQDs for nano-medical applications is to obtain high-quality products, the existing synthesis method generally allows

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small-scale production of N-GQDs which have a wide size distribution. It is necessary to find easy purification methods and seek out novel methods to achieve a high yield that does not require the removal of starting materials. Since the size and shape have a massive impact on

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the physicochemical properties of N-GQDs, mass theoretical and practical research must focus on improving synthetic method if the N-GQDs based fluorescence detection methods

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gradually replace the traditional detection technique used in laboratory inspection. More studies concerning the application of N-GQDs based immunosensing are needed compared with the non-immunosensing. Because of the small number of research on bio(sensing) based on N-GQDs, more techniques should be involved in this area and novel

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protocols for detection of cell lines, cancer biomarkers, and disease should be developed, since that the synthetic of the new technologies will bring significant input to ultrasensitive bio(sensor)s relevant to diagnostics, and therapy of cancer.

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Understanding the photoluminescence (PL) properties of N-GQDs is still poor, although

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some possible mechanisms have been proposed, such as size effect, surface modification, and doping with other elements. In spite of the achievement of N-GQDs with different colored PL properties, including PL in the near-infrared region, the quantum yields of most N-GQDs are still at a low level, so the improvement of N-GQDs is imperative because of their restricted application in bio(sensing) from their lower quantum yields. Metal-enhanced fluorescence may be considered for quantum-yield escalation and a study has proved its possibility. Although advances are exciting and encouraging, the use of N-GQDs for bio(sensing) applications is still in infancy, with a lot of challenges remaining. A new strategy for surface

26

ACCEPTED MANUSCRIPT modification is needed to be developed for application in bio(sensing). With their uniform size, excellent PL and high quantum yields, N-GQDs will no doubt be used in more creative applications. In summary, N-GQDs emerge as a novel nanomaterial platform for biosensing, effective collaboration between multiple disciplines including chemistry, physics, biology,

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and medicine must be implemented.

Distinct spectral characteristics of N-GQDs make them very beneficial for not only fluorescence based biosensing of hepatitis but also the electrochemical-based approaches.

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The secondary surface modifications of the nanomaterials open ways to engineer the desired surface mechanical and chemical properties. As a result of surface modification, the

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multiplex analyte sensing could be carried out while in compare with the previously developed methods, different sensing agents could be attached on the surface of only one substrate. For example, various dye acceptors could be assembled to the surface of the same of N-GQDs donor vice versa. Despite of the various advantages of QDs, the auto-

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fluorescence of biological environments is the main restrict of the using of N-GQDs in biological environments which is raised from applied excitation source. However, in upconverting based sensors the auto-fluorescence is comparatively resolved where the

EP

excitation wavelength is located in the IR or NIR region. Also, due to the non-toxic nature of

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Si based materials, the reported methods are increasing. However, there are still some faults to make the reported works applicable in clinical issues. The first is the better figures of merit of the reported methods i.e. their linearity, stability of methods, limit of detection and interfering agents. The second is the designing of easy to use procedures for point-of-care device fabrication to diagnosis more fast detection even in beside of patient. The third is the specificity of the developed methods which in our points of view is the major topic in sensing field.

27

ACCEPTED MANUSCRIPT The potential incorporation of N-GQDs in commercial biosensor increases human exposure daily. N-GQDs have been defined as low toxic, biocompatible NPs; however, their easy dispersion due to their high water solubility, N-GQDs could accumulate in the human body (organs and tissues). It is therefore necessary to develop new analytical methods that allow

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the determination of N-GQDs in complex matrices, such as the biological samples. There is no doubt that new strategy for surface modification need to be developed for application in biosensing. With their uniform size, excellent PL and high quantum yields, N-GQDs will no

SC

doubt be used in more creative applications. Although the advances are exciting and encouraging, the use of N-GQDs for biosensing applications is still in infancy, with a lot of

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challenges remaining. In summary, N-GQDs emerge as a novel nanomaterial platform for biosensing, yet many challenges need to be solved by effective collaborations crossing multiple disciplines including chemistry, physics, biology and medicine. Considering the unique properties of N-GQDs and the current challenges for developing

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smart (bio)sensor, our perspectives on N-GQDs based (bio)sensor in the near future are as follows:

a) Sensors based on the up-conversion photoluminescence of N-GQDs

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b) Surface-enhanced Raman spectroscopy (SERS) sensors based on N-GQDs

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c) Quantum yield improvement of N-GQDs d) Functionalization of N-GQDs with metal and bimetal nanoparticles and enhancement of ECL signals

In summary, some issues remain for further studies: (a) The mechanisms deduced from the optical properties of the N-GQDs generated vary, so a study of the detailed mechanism for photoluminescence in N-GQDs would be highly significant; Two-photon fluorescence imaging or up-conversion fluorescence imaging, with advantages including a larger penetration depth, a minimized tissue auto fluorescence 28

ACCEPTED MANUSCRIPT background, the avoidance of harmful UV or blue excitations and the reduction of photo damage in bio-tissues, has received a great deal of attention for its promising applications in both

basic

biological

research

and

clinical

diagnostics.

Thus,

up-conversion

photoluminescence will prompt the use of GQDs as a tool in biosensing.

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(b) Since the GQDs derived from large graphene-based materials are heterogeneous in size and morphology, we expected that GQDs will be obtained from small molecules via appropriate methods of synthesis; the size, shape, morphology, degree of oxidation and

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thickness of GQDs undeniably affect the overall performance of bimolecular immobilization. Exploration of the preparation of high-quality GQDs with uniform size and morphology is

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still ongoing and should be developed. Synthesis of uniform GQDs can help increase the repeatability required for accurate (bio)sensing. Hence, new methods for preparing inerratic GQDs need to be developed. 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

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as graphene, GQDs will be incorporated in commercial (bio)sensor, especially those relevant to electronics, such as nano-electrochemical systems (NEMS). (c) In spite of the achievement of N-GQDs with different colored PL properties, including PL

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in the near-infrared region, the quantum yields of most N-GQDs are still lower than 20%, so

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the improvement of GQDs is imperative because of their restricted application in (bio)sensing from their lower quantum yields. Metal-enhanced fluorescence may be considered for quantum-yield enhancement and a study has proved its feasibility; and, last but not least, GQDs have shown great potential in (bio)sensing for its low toxicity in vivo. But the productivity is quite low in spite of the hydrothermal route, electrochemical method or chemical oxidation and reduction. Therefore, investigation of how to increase the productivity of N-GQDs with high quantum yield is of urgency. The intensity of photoluminescence is very important for a photoluminescence sensor. Unfortunately, like 29

ACCEPTED MANUSCRIPT CDs, the Quantum yield of N-GQDs is usually very low. Therefore, the preparation of NGQDs with a high QY is demanding and important. To this end, many methods for modulating the fluorescence emission spectra of GQDs and improving its QY have been reported. Zhuet al. used NaBH4 as reducing agent to convert greenish-yellow luminescent N-

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GQDs synthesized through a microwave assisted method, to bright blue luminescent NGQDs with a higher QY. Some of research groups reported that the luminescence of GQDs was greatly enhanced by increasing sp2 domains and electron-donating groups in GQDs

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through photochemical reduction and the hydrothermal method. Up to now, even the highest QY of N-GQDs is 73%, the QY of most GQDs ranges from 2 to 22.9%, which is much lower

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than that semi-conductive QDs. The QY improvement of N-GQDs is challenging for their practical applications and requires a great deal of devoted effort.

(d) Understanding the PL properties of GQDs is still poor, although, to date, some possible mechanisms have been proposed, such as size effect, surface modification, and doping with

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other elements; Surface-enhanced Raman spectroscopy (SERS) is expected to have a major impact as a sensitive analytical technique and tool for fundamental studies of surface species. The well-organized assembly of zero dimensional (0D) N-GQDs into 1D nanotube (NT)

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arrays and their significant potential as a new metal-free platform for efficient SERS

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applications have been reported. The emergence of N-GQDs NTs can be used to reduce the substantial cost of SERS sensors by using low-cost carbon materials instead of expensive noble metal substrates.

(e) Toxicity of nanomaterials is one of the major challenges of their applications in science and biotechnology. N-GQDs have the potential to be remarkably successful in the field of nanobiotechnology due to their excellent opto-electrical properties, and extremely low cytotoxicity. Currently used quantum dots (CdS, PbS, ZnS, ZnSe, HgTe, Ag2S, Ag2Se, CuInS2, CuInSe2, InAs and InP) are composed of toxic metals which may cause problems for

30

ACCEPTED MANUSCRIPT their use in biological systems. However, GQDs are increasingly being employed to provide more efficient and less toxic alternatives compared with currently used quantum dots. Studies on human breast cancer have shown that N-GQDs can easily find their way into the cytoplasm and do not interfere with cell proliferation, which indicate that they are non-toxic

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materials. More attention should be paid to the safety of N-GQDs by studying their long term toxicity, cellular-uptake mechanism, intracellular and in vivo metabolic pathway, which is critical and inevitable for applications of in vivo biosensing. Similar to many other

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nanomaterials used in biosensing, the toxicity of graphene is closely associated to its surface functionalization. As-prepared GO, although water soluble, is not stable in physiological

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solutions and causes dose-dependent toxicity both in vitro and in vivo. In marked contrast, GQDs with biocompatible coatings exhibits excellent stability in the presence of highconcentration salts and proteins, and appears to be less toxic in vitro and in vivo. The in vivo biodistribution of N-GQDs is also highly dependent on its biosensing. However, how the size

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of GQDs, which is another important parameter, affects the in vivo behaviors of N-GQDs requires further investigation. A lot more systematic explorations are demanded in order to fully understand the in vivo long-term fate and toxicology of N-GQDs at different doses in

EP

various animal models before any clinical applications of this novel nanomaterial. In general,

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with the wide range of morphologies, coatings, and hybrid structures available for biosensing, more detailed and longer-term studies are required before serious in vivo biomedical NGQDs applications are implemented. (f) The optical properties of N-GQDs greatly depend on their size due to the quantumconfinement effect, and the variation in density and nature of sp2 sites available in N-GQDs. Thus, the energy band gap of N-GQDs can be tuned by changing their size. (g) Compared with the non-bio(sensors) (bio)sensing applications of N-GQDs, more studies concerning the application of N-GQDs in (bio)sensing are needed. Because of the small 31

ACCEPTED MANUSCRIPT amount of research on the development of bio(sensors) based on N-GQDs, more methods/techniques should be involved in this area and researchers should be developing novel protocols for detection of cell lines, cancer biomarkers and disease. Also, the integration of the new technologies will, without doubt, bring significant input to

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ultrasensitive bio(sensors) relevant to diagnostics, control and therapy of cancer.

(h) In the literature concerning the development of clinical bio(sensors) , a lack in the study

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of selectivity of GQDs-based bio(sensors) was verified. When the selectivity is tested, it is possible to determine if the method is completely able to quantify accurately an analyte in the

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presence of interferences, and thus, such figure of merit is crucial to be studied. There is no doubt that new strategy for surface modification need to be developed for application in (bio)sensing. With their uniform size, excellent PL and high quantum yields, N-GQDs will no doubt be used in more creative applications. Although the advances are exciting and encouraging, the use of N-GQDs for (bio)sensing applications is still in infancy,

TE D

with a lot of challenges remaining. In summary, N-GQDs emerge as a novel nanomaterial platform for (bio)sensing, yet many challenges need to be solved by effective collaborations

AC C

EP

crossing multiple disciplines including chemistry, physics, biology and medicine.

Finally, we conclude that there is a bright opportunity for further advances and developments of biosensors and related devices based on N-GQDs, especially through further miniaturization and integration into lab-on-chip systems. The design of implantable biosensors with the ability to monitor cell lines in vivo and in real time is promising for the application of biosensors, even though it is yet to be explored. Scientists therefore have a great deal to do to address the performance of biosensors for cell detection in the future. Acknowledgements 32

ACCEPTED MANUSCRIPT We gratefully acknowledge the partial financial support by Tabriz University of Medical Sciences.

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ACCEPTED MANUSCRIPT [37] D. Qu, M. Zheng, L. Zhang, H. Zhao, Z. Xie, X. Jing, R.E. Haddad, H. Fan, Z. Sun, Formation mechanism and optimization of highly luminescent N-doped graphene quantum dots, Sci. rep. 4 (2014) 5294. [38] Y. Dai, H. Long, X. Wang, Y. Wang, Q. Gu, W. Jiang, Y. Wang, C. Li, T.H. Zeng, Y.

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[41] M. Hasanzadeh, N. Hashemzadeh, N. Shadjou, J. Eivazi-Ziaei, M. Khoubnasabjafari, A.

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(2012) 103107.

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upconversion luminescence of N-doped graphene quantum dots, Appl. Phys. Lett. 101(10)

[43] M. Hasanzadeh, F. Mokhtari, N. Shadjou, A. Eftekhari, A. Mokhtarzadeh, V. JouybanGharamaleki, S. Mahboob, Poly arginine-graphene quantum dots as a biocompatible and nontoxic nanocomposite: Layer-by-layer electrochemical preparation, characterization and noninvasive malondialdehyde sensory application in exhaled breath condensateMater. Sci. Eng. C. 75 (2017) 247-258.

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ACCEPTED MANUSCRIPT [44] Q. Li, S. Zhang, L. Dai, L.-s. Li, Nitrogen-doped colloidal graphene quantum dots and their size-dependent electrocatalytic activity for the oxygen reduction reaction, J. Am. Chem. Soc. 134(46) (2012) 18932-18935. [45] L. Lin, M. Rong, S. Lu, X. Song, Y. Zhong, J. Yan, Y. Wang, X. Chen, A facile

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synthesis of highly luminescent nitrogen-doped graphene quantum dots for the detection of 2, 4, 6-trinitrophenol in aqueous solution, Nanoscale. 7(5) (2015) 1872-1878.

[46] R. Tabaraki, A. Nateghi, Nitrogen-Doped Graphene Quantum Dots: “Turn-off”

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Fluorescent Probe for Detection of Ag+ Ions, J. fluorescence 26(1) (2016) 297-305.

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Spectrochim. Acta. Part. A. 189 (2018) 195-201.

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2251.

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graphene quantum dots and visible paper-based test strips, Anal. Methods 9(15) (2017) 2246-

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[50] M. Kaur, S.K. Mehta, S.K. Kansal, A fluorescent probe based on nitrogen doped graphene quantum dots for turn off sensing of explosive and detrimental water pollutant, TNP in aqueous medium, Spectrochim. Acta. Part. A. 180 (2017) 37-43. [51] C. Zhou, X. He, D. Ya, J. Zhong, B. Deng, One step hydrothermal synthesis of nitrogendoped graphitic quantum dots as a fluorescent sensing strategy for highly sensitive detection of metacycline in mice plasma, Sensors. Actuators. B. Chemical 249 (2017) 256-264.

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ACCEPTED MANUSCRIPT [52] Z. Liu, Y. Gong, Z. Fan, A dopamine-modulated nitrogen-doped graphene quantum dot fluorescence sensor for the detection of glutathione in biological samples, New. J. Chem. 40(10) (2016) 8911-8917. [53] F. Cai, X. Liu, S. Liu, H. Liu, Y. Huang, A simple one-pot synthesis of highly

media, RSC. Adv. 4(94) (2014) 52016-52022.

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fluorescent nitrogen-doped graphene quantum dots for the detection of Cr (VI) in aqueous

[54] B. Y. Fang, C. Li, Y. Y. Song, F. Tan, Y. C. Cao, Y. D. Zhao, Nitrogen-doped graphene

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quantum dot for direct fluorescence detection of Al3+ in aqueous media and living cells, Biosens. Bioelectron. 100 (2018) 41-48.

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[55] B. Shi, L. Zhang, C. Lan, J. Zhao, Y. Su, S. Zhao, One-pot green synthesis of oxygenrich nitrogen-doped graphene quantum dots and their potential application in pH-sensitive photoluminescence and detection of mercury (II) ions, Talanta. 142 (2015) 131-139. [56] Z. Yan, X. Qu, Q. Niu, C. Tian, C. Fan, B. Ye, A green synthesis of highly fluorescent

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nitrogen-doped graphene quantum dots for the highly sensitive and selective detection of mercury (II) ions and biothiols, Anal. Methods. 8(7) (2016) 1565-1571. [57] N. Shadjou, M. Hasanzadeh, A. Omari, Electrochemical quantification of some water

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soluble vitamins in commercial multi-vitamin using poly-amino acid caped by graphene

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quantum dots nanocomposite as dual signal amplification elements, Anal. Biochem. 539, (2017) 70-80.

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ACCEPTED MANUSCRIPT nanospheres with high slectrochemical sensing performance in cancer detection, ACS. Appl. Mater. Interfaces. 8(34) (2016) 22563-22573. [60] Z. Cai, F. Li, P. Wu, L. Ji, H. Zhang, C. Cai, D.F. Gervasio, Synthesis of nitrogen-doped

chem. 87(23) (2015) 11803-11811.

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graphene quantum dots at low temperature for electrochemical sensing trinitrotoluene, Anal.

[61] L. Li, D. Liu, K. Wang, H. Mao, T. You, Quantitative detection of nitrite with N-doped graphene

quantum

dots

decorated

N-doped

carbon

composite-based

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electrochemical sensor, Sens. Actuators. B. 252 (2017) 17-23.

nanofibers

[62] L. Lin, X. Song, Y. Chen, M. Rong, T. Zhao, Y. Wang, Y. Jiang, X. Chen, Intrinsic

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peroxidase-like catalytic activity of nitrogen-doped graphene quantum dots and their application in the colorimetric detection of H2O2 and glucose, Anal. Chim. Acta. 869 (2015) 89-95.

[63] J. Ju, R. Zhang, W. Chen, Photochemical deposition of surface-clean silver nanoparticles

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on nitrogen-doped graphene quantum dots for sensitive colorimetric detection of glutathione, Sens. Actuators. B. 228 (2016) 66-73.

[64] H. Xu, S. Zhou, L. Xiao, H. Wang, S. Li, Q. Yuan, Fabrication of a nitrogen-doped

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graphene quantum dot from MOF-derived porous carbon and its application for highly

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selective fluorescence detection of Fe3+, J. Mater. Chem. C. 3(2) (2015) 291-297. [65] L. Lin, X. Song, Y. Chen, M. Rong, T. Zhao, Y. Jiang, Y. Wang, X. Chen, One-pot synthesis of highly greenish-yellow fluorescent nitrogen-doped graphene quantum dots for pyrophosphate sensing via competitive coordination with Eu3+ ions, Nanoscale. 7(37) (2015) 15427-15433. [66] J. Ju, R.Z. Zhang, S.J. He, W. Chen, Nitrogen-doped graphene quantum dots-based fluorescent probe for the sensitive turn on detection of glutathione and its cellular imaging. RSC Adv. 4 (2014) 52583-52589.

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ACCEPTED MANUSCRIPT [67] J. Ju, R.Z. Zhang, W. Chen, Photochemical deposition of surface clean silver nanoparticles on nitrogen-doped graphene quantum dots for sensitive colorimetric detection

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of glutathione, Sens. Actuators B Chem. 228 (2016) 66-73.

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N-GQDs provide a platform for development of optical (bio)sensors. Recent advances on the optical (bio)sensors N-GQDs were addressed.

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Optical application of GQDs on biosensor technology was discussed.