Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> 2010 WILEY-VCH Verlag Gm Graphene-based materials are promising building blocks for future nanodevices owing to their superior electronic, thermal, and mechanical properties as well as their chemical stability. However, currently available graphene-based materials produced by typical physical and chemical routes, including micromechanical cleavage, reduction of exfoliated graphene oxide (GO), and solvothermal synthesis, are generally micrometer-sized graphene sheets (GSs), which limits their direct application in nanodevices. In this context, it has become urgent to develop effective routes for cutting large GSs into nanometer-sized pieces with a well-confined shape, such as graphene nanoribbons (GNRs) and graphene quantum dots (GQDs). Theoretical and experimental studies have shown that narrow GNRs (width less than ca. 10 nm) exhibit substantial quantum confinement and edge effects that render GNRs semiconducting. By comparison, GQDs possess strong quantum confinement and edge effects when their sizes are down to 100 nm. If their sizes are reduced to ca. 10 nm, comparable with the widths of semiconducting GNRs, the two effects will become more pronounced and, hence, induce new physical properties. Up to now, nearly all experimental work on GNRs and GQDs has focused on their electron transportation properties. Little work has been done on the optical properties that are directly associated with the quantum confinement and/or edge effects. Most GNRand GQD-based electronic devices have been fabricated by lithography techniques, which can realize widths and diameters down to ca. 20 nm. This physical approach, however, is limited by the need for expensive equipment and especially by difficulties in obtaining smooth edges. Alternative chemical routes can overcome these drawbacks. Moreover, surface functionalization can be realized easily. Li et al. first reported a chemical route to functionalized and ultrasmooth GNRs with widths ranging from 50 nm to sub-10 nm. Very recently, Kosynkin et al. reported a simple solution-based oxidative process for producing GNRs by lengthwise cutting and unraveling of multiwalled carbon nanotube (CNT) side walls. Yet, no chemical routes have been reported so far for preparing functionalized GQDs with sub-10 nm sizes. Here, we report on a novel and simple hydrothermal approach for the cutting of GSs into surface-functionalized GQDs (ca. 9.6-nm average diameter). The functionalized GQDs were found to exhibit bright blue photoluminescence (PL), which has never been observed in GSs and GNRs owing to their large lateral sizes. The blue luminescence and new UV–vis absorption bands are directly induced by the large edge effect shown in the ultrafine GQDs. The starting material was micrometer-sized rippled GSs obtained by thermal reduction of GO sheets. Figure 1a shows a typical transmission electron microscopy (TEM) image of the pristine GSs. Their (002) interlayer spacing is 3.64 A (Fig. 1c), larger than that of bulk graphite (3.34 A). Before the hydrothermal treatment, the GSs were oxidized in concentrated H2SO4 and HNO3. After the oxidization treatment the GSs became slightly smaller (50 nm–2mm) and the (002) spacing slightly increased to 3.85 A (Fig. 1c). During the oxidation, oxygen-containing functional groups, including C1⁄4O/COOH, OH, and C O C, were introduced at the edge and on the basal plane, as shown in the Fourier transform infrared (FTIR) spectrum (Fig. 1d). The presence of these groups makes the GSs soluble in water. A series of more marked changes took place after the hydrothermal treatment of the oxidized GSs at 200 8C. First, the (002) spacing was reduced to 3.43 A (Fig. 1c), very close to that of bulk graphite, indicating that deoxidization occurs during the hydrothermal process. The deoxidization is further confirmed by the changes in the FTIR and C 1s X-ray photoelectron spectroscopy (XPS) spectra. After the hydrothermal treatment, the strongest vibrational absorption band of C1⁄4O/COOH at 1720 cm 1 became very weak and the vibration band of epoxy groups at 1052 cm 1 disappeared (Fig. 1d). In the XPS C 1s spectra of the oxidized and hydrothermally reduced GSs (Fig. 2a), the signal at 289 eV assigned to carboxyl groups became weak after the hydrothermal treatment, whereas the sp carbon peak at 284.4 eV was almost unchanged. Figure 2b shows the Raman spectrum of the reduced GSs. A G band at 1590 cm 1 and a D band at 1325 cm 1 were observed with a large intensity ratio ID/IG of 1.26. Second, the size of the GSs decreased dramatically and ultrafine GQDswere isolated by a dialysis process. Figure 3 shows typical TEM and atomic force microscopy (AFM) images of the GQDs. Their diameters are mainly distributed in the range of 5–13 nm (9.6 nm average diameter). Their topographic heights are mostly between 1 and 2 nm, similar to those observed in functionalized GNRs with 1–3 layers. More than 85% of the GQDs consist of 1–3 layers. <s> BIB001 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> Graphene quantum dots (GQDs), which are edge-bound nanometer-size graphene pieces, have fascinating optical and electronic properties. These have been synthesized either by nanolithography or from starting materials such as graphene oxide (GO) by the chemical breakdown of their extended planar structure, both of which are multistep tedious processes. Here, we report that during the acid treatment and chemical exfoliation of traditional pitch-based carbon fibers, that are both cheap and commercially available, the stacked graphitic submicrometer domains of the fibers are easily broken down, leading to the creation of GQDs with different size distribution in scalable amounts. The as-produced GQDs, in the size range of 1–4 nm, show two-dimensional morphology, most of which present zigzag edge structure, and are 1–3 atomic layers thick. The photoluminescence of the GQDs can be tailored through varying the size of the GQDs by changing process parameters. Due to the luminescence stability, nanosecond lifetime, ... <s> BIB002 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> Highly photoluminescent carbon dots with a PL quantum yield of 26% have been prepared in one step by hydrothermal treatment of orange juice. Due to high photostability and low toxicity these carbon dots are demonstrated as excellent probes in cellular imaging. <s> BIB003 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> Oxidization of carbon nanotubes by a mixed acid has been utilized as a standard method to functionalize carbon nanomaterials for years. Here, the products obtained from carbon nanotubes and graphite after a mixed-acid treatment are carefully studied. Nearly identical carbon dot (Cdot) products with diameters of 3–4 nm are produced using this approach from a variety of carbon starting materials, including single-walled carbon nanotubes, multiwalled carbon nanotubes, and graphite. These Cdots exhibit strong yellow fluorescence under UV irradiation and shifted emission peaks as the excitation wavelength is changed. In vivo fluorescence imaging with Cdots is then demonstrated in mouse experiments, by using varied excitation wavelengths including some in the near-infrared (NIR) region. Furthermore, in vivo biodistribution and toxicology of those Cdots in mice over different periods of time are studied; no noticeable signs of toxicity for Cdots to the treated animals are discovered. This work provides a facile method to synthesize Cdots as safe non-heavy-metal-containing fluorescent nanoprobes, promising for applications in biomedical imaging. <s> BIB004 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> An easy approach for large-scale and low-cost synthesis of photoluminescent (PL) graphene quantum dots (GQDs) based on the carbonization of commercially available polycyclic aromatic hydrocarbon (PAH) precursors with strong acid and followed by hydrothermal reduction with hydrazine hydrate is reported. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) characterizations indicate that the size and height of GQDs are in the range of 5–10 nm and 0.5–2.5 nm, respectively. PAH, which has more benzene rings, generally forms GQDs with relatively larger size. The GQDs show high water solubility, tunable photoluminescence, low cytotoxicity, and good optical stability, which makes them promising fluorescent probes for cellular imaging. In addition, the fluorescence of GQDs shows a sensitive and selective quenching effect to Fe3+ with a detection limit of 5 × 10−9m. By combination with the Fe2+/Fe3+ redox couple, the PL GQDs are able to detect oxidant, using H2O2 as an example. This study opens up new opportunities to make full use of GQDs because of their facile availability, cost-effective productivity, and robust functionality. <s> BIB005 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> Two types of carbon dots (C dots) exhibiting respective excitation-independent blue emission and excitation-dependent full-color emissions have been synthesized via a mild one-pot process from chloroform and diethylamine. This new bottom-up synthetic strategy leads to highly stable crystalline C dots with tunable surface functionalities in high reproducibility. By detailed characterization and comparison of the two types of C dots, it is proved concretely that the surface functional groups, such as C═O and C═N, can efficiently introduce new energy levels for electron transitions and result in the continuously adjustable full-color emissions. A simplified energy level and electron transition diagram has been proposed to help understand how surface functional groups affect the emission properties. By taking advantage of the unique excitation-dependent full-color emissions, various new applications can be anticipated. Here, as an example, a ratiometric pH sensor using two emission wavelengths of the C dots a... <s> BIB006 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> A simple, low cost, and green method was developed for the synthesis of water-soluble and well-dispersed fluorescent carbon nanodots (CDs) via a one-step hydrothermal treatment of potatoes. The as-prepared CDs exhibit a strong blue fluorescence, with a quantum yield of up to 15%. We further explored the use of these CDs as a novel sensing probe for the label-free, sensitive and selective detection of Fe3+. This is based on strong fluorescence quenching due to the complex formed between the CDs and Fe3+. The detection limit was as low as 0.025 μmol L−1, and different concentrations corresponded to different sensitivities. The linear ranges were 1.0–5.0 μmol L−1 and 5.0–50.0 μmol L−1 at lower and higher concentration ranges respectively, and the recoveries of the spiked water samples were 93.7–101.5%. Therefore, the as-prepared CDs could meet the requirements for the monitoring of Fe3+ in environmental samples. <s> BIB007 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> For the first time, abundant natural carbon-based dots were found and studied in humic substances (HS). Four soluble HS including three humic acids (HA) from different sources and one fulvic acids (FA) were synthetically studied. Investigation results indicate that all the four HS contain large quantities of Carbon-based dots. Carbon-based dots are mainly small-sized graphene oxide nano-sheets or oxygen-containing functional group-modified graphene nano-sheets with heights less than 1 nm and lateral sizes less than 100 nm. Carbon-based nanomaterials not only contain abundant sp2-clusters but also a large quantity of surface states, exhibiting unique optical and electric properties, such as excitation-dependent fluorescence, surface states-originated electrochemiluminescence, and strong electron paramagnetic resonance. Optical and electric properties of these natural carbon-based dots have no obvious relationship to their morphologies, but affected greatly by their surface states. Carbon-based dots in the three HS have relative high densities of surface states whereas the FA has the lowest density of surface states, resulting in their different fluorescence properties. The finding of carbon-based dots in HS provides us new insight into HS, and the unique optical properties of these natural carbon-based dots may give HS potential applications in areas such as bio-imaging, bio-medicine, sensing and optoelectronics. <s> BIB008 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> At present, the actual mechanism of the photoluminescence (PL) of fluorescent carbon dots (CDs) is still an open debate among researchers. Because of the variety of CDs, it is highly important to summarize the PL mechanism for these kinds of carbon materials; doing so can guide the development of effective synthesis routes and novel applications. This review will focus on the PL mechanism of CDs. Three types of fluorescent CDs were involved: graphene quantum dots (GQDs), carbon nanodots (CNDs), and polymer dots (PDs). Four reasonable PL mechanisms have been confirmed: the quantum confinement effect or conjugated π-domains, which are determined by the carbon core; the surface state, which is determined by hybridization of the carbon backbone and the connected chemical groups; the molecule state, which is determined solely by the fluorescent molecules connected on the surface or interior of the CDs; and the crosslink-enhanced emission (CEE) effect. To give a thorough summary, the category and synthesis routes, as well as the chemical/physical properties for the CDs, are briefly introduced in advance. <s> BIB009 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> Abstract Carbon-based photoluminescent nanodot has currently been one of the promising materials for various applications. The remaining challenges are the carbon sources and the simple synthetic processes that enhance the quantum yield, photostability and biocompatibility of the nanodots. In this work, the synthesis of blue photoluminescent carbon nanodots from limeade via a single-step hydrothermal carbonization process is presented. Lime carbon nanodot (L-CnD), whose the quantum yield exceeding 50% for the 490 nm emission in gram-scale amounts, has the structure of graphene core functionalized with the oxygen functional groups. The micron-sized flake of the as-prepared L-CnD powder exhibits multicolor emission depending on an excitation wavelength. The L-CnDs are demonstrated for rapidly ferric-ion (Fe3 +) detection in water compared to Fe2 +, Cu2 +, Co2 +, Zn2 +, Mn2 + and Ni2 + ions. The photoluminescence quenching of L-CnD solution under UV light is used to distinguish the Fe3 + ions from others by naked eyes as low concentration as 100 μM. Additionally, L-CnDs provide exceptional photostability and biocompatibility for imaging yeast cell morphology. Changes in morphology of living yeast cells, i.e. cell shape variation, and budding, can be observed in a minute-period until more than an hour without the photoluminescent intensity loss. <s> BIB010 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> Obtaining tunable photoluminescence (PL) with improved emission properties is crucial for successfully implementing fluorescent carbon nanodots (fCDs) in all practical applications such as multicolour imaging and multiplexed detection by a single excitation wavelength. In this study, we report a facile hydrothermal approach to adjust the PL peaks of fCDs from blue, green to orange by controlling the surface passivation reaction during the synthesis. This is achieved by tuning the passivating reagents in a step-by-step manner. The as-prepared fCDs with narrow size distribution show improved PL properties with different emission wavelengths. Detailed characterization of fCDs using elemental analysis, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy suggested that the surface chemical composition results in this tunable PL emission. Surface passivation significantly alters the surface status, resulting in fCDs with either stronger surface oxidation or N element doping that ultimately determine their PL properties. Further experiments suggested that the as-prepared orange luminescent fCDs (O-fCDs) were sensitive and specific nanosensing platforms towards Fe3+ determination in a complex biological environment, emphasizing their potential practical applications in clinical and biological fields. <s> BIB011 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> A novel graphene quantum dot (GQD)@Fe3O4@SiO2 based nanoprobe was reported for targeted drug delivery, sensing, dual-modal imaging and therapy. Carboxyl-terminated GQD (C-GQD) was firstly conjugated with Fe3O4@SiO2 and then functionalized with cancer targeting molecule folic acid (FA). DOX drug molecules were then loaded on GQD surface of Fe3O4@SiO2@GQD-FA nanoprobe via pi-pi stacking, which resulted in Fe3O4@SiO2@GQD-FA/DOX conjugates based on a FRET mechanism with GQD as donor molecules and DOX as acceptor molecules. Meanwhile, we successfully performed in vitro MRI and fluorescence imaging of living Hela cells and monitored intracellular drug release process using this Fe3O4@SiO2@GQD-FA/DOX nanoprobe. Cell viability study demonstrated the low cytotoxicity of Fe3O4@SiO2@GQD-FA nanocarrier and the enhanced therapeutic efficacy of Fe3O4@SiO2@GQD-FA/DOX nanoprobe for cancer cells. This luminomagnetic nanoprobe will be a potential platform for cancer accurate diagnosis and therapy. <s> BIB012 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> On the basis of the unique physicochemical properties of graphene quantum dots (GQDs), we developed a novel type of theranostic agent by loading anticancer drug doxorubicin (DOX) to GQD’s surface and conjugating Cy5.5 (Cy) dye to GQD though a cathepsin D-responsive (P) peptide. Such type of agents demonstrated superior therapeutic performance both in vitro and in vivo because of the improved tissue penetration and cellular uptake. More importantly, they are capable of functioning as probes for programmed tracking the delivery and release of anticancer drug as well as drug-induced cancer cell apoptosis through GQD’s, DOX’s, and Cy’s charateristic fluorescence, respectively. <s> BIB013 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> A biocompatible and cell traceable drug delivery system Graphene Quantum Dots (GQD) based, for the targeted delivery of the DNA intercalating drug doxorubicin (DOX) to cancer cells, is here reported. Highly dispersible and water soluble GQD, synthesized by acidic oxidation and exfoliation of multi-walled carbon nanotubes (MWCNT), were covalently linked to the tumor targeting module biotin (BTN), able to efficiently recognize biotin receptors over-expressed on cancer cells and loaded with DOX. Biological test performed on A549 cells reported a very low toxicity of the synthesized carrier (GQD and GQD-BTN). In GQD-BTN-DOX treated cancer cells, the cytotoxicity was strongly dependent from cell uptake which was greater and delayed after treatment with GQD-BTN-DOX system with respect to what observed for cells treated with the same system lacking of the targeting module BTN (GQD-DOX) or with the free drug alone. A delayed nuclear internalization of the drug is reported, due to the drug detachment from the nanosystem, triggered by the acidic environment of cancer cells. <s> BIB014 </s> Graphene-Based Materials for Biosensors: A Review <s> Figure 5. Schematic illustration and photoluminescent mechanism of carbon-based dots, including graphene quantum dot (GQD), carbon quantum dot (CQD), carbon nanodot (CND), compared to the quantum dots made of semiconductor (SQD). <s> This work reports a convenient and real-time assay of alkaline phosphatase (ALP) in living cells based on a fluorescence quench-recovery process at a physiological pH using the boron-doped graphene quantum dots (BGQDs) as fluorophore. The fluorescence of BGQDs is found to be effectively quenched by Ce3+ ions because of the coordination of Ce3+ ions with the carboxyl group of BGQDs. Upon addition of adenosine triphosphate (ATP) into the system, the quenched fluorescence can be recovered by the ALP-positive expressed cells (such as MCF-7 cells) due to the removal of Ce3+ ions from BGQDs surface by phosphate ions, which are generated from ATP under catalytic hydrolysis of ALP that expressed in cells. The extent of fluorescence signal recovery depends on the level of ALP in cells, which establishes the basis of ALP assay in living cells. This approach can also be used for specific discrimination of the ALP expression levels in different type of cells and thus sensitive detection of those ALP-positive expressed cells (for example MCF-7 cells) at a very low abundance (10±5 cells mL-1). The advantages of this approach are that it has high sensitivity because of the significant suppression of the background due to the Ce3+ ion quenching the fluorescence of BGQDs, and has the ability of avoiding false signals arising from the nonspecific adsorption of non-target proteins because it operates via a fluorescence quench-recovery process. In addition, it can be extended to other enzyme systems, such as ATP-related kinases. <s> BIB015
The GQDs can be made up of a single atom of graphite layer or graphene chopped into fragments in a range of 2-20 nm. The disk feature of GQDs is very tiny, analogous to the dot BIB010 BIB002 . Upon either stimulus by UV light or electrical energy, the excitons would be confined to the quantized energy levels and release photoluminescent light in a narrow band. Interestingly, the major material precursors are not only the infinite graphene sheet and the functionalized graphene, but some organic molecules are also applicable. For the top-down methods the hydrothermal cutting produces several nanosized sheets while the bottom-up methods use direct pyrolysis and carbonization of the organic precusors. The nano-to micro-sized fragments of GO from hydrothermal cutting were passivated by placing -C=O, -OH and -COOH groups on the surface BIB001 . Suvarnaphaet BIB010 achieved high quantum yield of the GO-like structure from limeade and applied it for rapid sensing of heavy-metal ions, for bioimaging, and it was tested for biocompatibility and found to be non-toxic to human kidney cells. Dong BIB008 also found nano-sized GO−like structures synthesized from humic substances in the presence of nitrogen-containing groups such as -NH2 and C=N on the surface of the carbon-based dots causing an enhancement in quantum yield. Natural and artificial aromatic carbon substances have also been investigated enabling a fine tuning of crucial optical properties, such as tunable photoluminescence. These aromatic carbon substances can be employed as effective labeling molecules as recently reported, for example, polycyclic aromatic hydrocarbon (PAH) BIB005 , chloroform (CHCl3) and diethylamine (DEA) BIB006 , orange juice BIB003 , potato BIB007 , citric acid and linearstructured polyethyleneimine (LPEI) polymer BIB011 . The multicolor emission spectra were due to the different sizes and nature of the surface functional groups, i.e., C=O and C=N. To obtain controllable quantum effects the size can be reduced down to a few nanometers, and the carbon core structure can be chemically modified. Thus the as-prepared carbon-based dots always contain sp 2 -and sp 3 -hybridized structures leading them to be modified by the oxygen/nitrogen functionalized groups and protein modification. The fluorescence properties and their mechanism arises from the localization of electron-hole (e-h) pairs which form excitons analogous to conventional QDs. The conjugated π- The GQDs can be made up of a single atom of graphite layer or graphene chopped into fragments in a range of 2-20 nm. The disk feature of GQDs is very tiny, analogous to the dot BIB010 BIB002 . Upon either stimulus by UV light or electrical energy, the excitons would be confined to the quantized energy levels and release photoluminescent light in a narrow band. Interestingly, the major material precursors are not only the infinite graphene sheet and the functionalized graphene, but some organic molecules are also applicable. For the top-down methods the hydrothermal cutting produces several nanosized sheets while the bottom-up methods use direct pyrolysis and carbonization of the organic precusors. The nano-to micro-sized fragments of GO from hydrothermal cutting were passivated by placing -C=O, -OH and -COOH groups on the surface BIB001 . Suvarnaphaet BIB010 achieved high quantum yield of the GO-like structure from limeade and applied it for rapid sensing of heavy-metal ions, for bioimaging, and it was tested for biocompatibility and found to be non-toxic to human kidney cells. Dong BIB008 also found nano-sized GO−like structures synthesized from humic substances in the presence of nitrogen-containing groups such as -NH 2 and C=N on the surface of the carbon-based dots causing an enhancement in quantum yield. Natural and artificial aromatic carbon substances have also been investigated enabling a fine tuning of crucial optical properties, such as tunable photoluminescence. These aromatic carbon substances can be employed as effective labeling molecules as recently reported, for example, polycyclic aromatic hydrocarbon (PAH) BIB005 , chloroform (CHCl 3 ) and diethylamine (DEA) BIB006 , orange juice BIB003 , potato BIB007 , citric acid and linear-structured polyethyleneimine (LPEI) polymer BIB011 . The multicolor emission spectra were due to the different sizes and nature of the surface functional groups, i.e., C=O and C=N. To obtain controllable quantum effects the size can be reduced down to a few nanometers, and the carbon core structure can be chemically modified. Thus the as-prepared carbon-based dots always contain sp 2 -and sp 3 -hybridized structures leading them to be modified by the oxygen/nitrogen functionalized groups and protein modification. The fluorescence properties and their mechanism arises from the localization of electron-hole (e-h) pairs which form excitons analogous to conventional QDs. The conjugated π-domains in the carbon-core structure arising from modifying surface chemistries and defects are also additionally promoted BIB009 . The strategies for chemical synthesis are used to stabilized the products be a probe for specific applications in in vivo bioimaging and biomarkers BIB010 BIB004 BIB002 , to improved drug delivery BIB012 BIB013 BIB014 , and to be integrated for biosensors BIB015 .