Novel Fluorophores and Optical Chemosensors

## **Chapter 3**

## Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review

*Hu Li and Raffaello Papadakis*

### **Abstract**

Graphene is a two-dimensional allotrope of carbon with a range of highly attractive physicochemical properties suitable for a wide variety of applications. In the context of fluorescence imaging graphene and its derivatives have recently started to gain more attention since they could assist in the enhancement of imaging of cells, tissue, or other biologically relevant samples such as cell organoids for example mitochondria as well as in the imaging of cancer cells, tumors, and various pathogens. This chapter attempts to cover the most relevant, recent advances in this growing research field. Some basic information on the physical and (photo)chemical properties of important members of the graphene family is provided. Additionally, novel approaches involving graphene-based materials (GBMs) in cellular and tissue imaging systems are reviewed. Important examples of contemporary applications of GBMs in cancer detection using fluorescence imaging are also presented. The specific role of graphene (or other GBMs) in each case is explained and analyzed. Finally, future perspectives and novel applications of fluorescent imaging techniques involving GBMs are discussed.

**Keywords:** graphene, fluorescence, imaging, 2D-materials, microscopy, cellular imaging, cancer detection

### **1. Introduction**

### **1.1 Graphene-based fluorescence imaging applications**

Fluorescence imaging is a non-invasive approach that utilizes fluorescent probes to generate photons and provides more sensitivity, specificity, and less harm than other imaging methods [1]. It can be used to monitor cells, tissues, and living organisms *in situ* and analyze specific biomolecules. This is why fluorescence imaging is considered as one of the most essential tools for biomedical research [2]. The fluorescence imaging probes should not be cytotoxic while they should exhibit adequate resistance to photobleaching caused by the natural features of the biological system. In this regard, graphene-based nanomaterials are presently considered as viable alternatives for fluorescence imaging. However, because graphene is a

zero-bandgap material, fluorescence cannot be detected in pristine graphene [3]. Conveniently, numerous studies have successfully fragmented or functionalized 2D graphene into 0D graphene quantum dots (GQDs) or carbon nanodots (CNDs) [4]. GQDs consist of graphene lattices and are commonly employed in fluorescence imaging due to quantum confinement and edge effects. Not only do they exhibit the distinct physical and chemical characteristics of conventional graphene, but they also display excellent biocompatibility, low cytotoxicity, and easy functionalization opportunities [5]. However, the poor fluorescent quantum yields often encountered along with the non-specificity of GQDs restrict their wide application in fluorescence imaging. Various research attempts have demonstrated that surface modification of GQDs can effectively alter their chemical activity, electrical structure, and quantum yield, consequently improving their photoluminescence capabilities [6, 7]. Since the realization of nitrogen doped GQDs [8], heteroatomic doping of GQDs has proven to be an effective technique for increasing quantum yield and tuning photoluminescence wavelength. For instance, in Wang et al. utilized the solvothermal method to synthesize boron-doped GQDs (B-GQDs) and phosphorus-doped GQDs (P-GQDs) [9]. B-GQDs and P-GQDs have emission wavelengths of 460 and 630 nm, respectively. Due to the matched band structure, rapid energy transfer between the blue-emitting B-GQDs and the orange-emitting P-GQDs can result in efficient fluorescence emission in the P-GQDs when the blue-emitting B-GQDs are excited at the ideal excitation wavelength of 460 nm. Furthermore, with effective energy transfer, the quantum yield of P-GQDs improves to 0.48, which is significantly greater than the quantum yield of pure P-GQDs. Similarly In Gong et al. synthesized nitrogen and bromine codoped GQDs (NBr-GQDs) that used a facile deflagration approach for the first time. The quantum yield of NBr-GQDs was up to 52%, with low cytotoxicity, great pH resistance, and stable photoluminescence intensity [10]. Theoretical calculations indicate that N and Br co-doping may reduce the band gap between excited singlet states, significantly improving the photoluminescence performance of GQDs. Recently, Li et al. have also synthesized manganese and boron-nitrogen-doped graphene quantum dots (Mn-BN-GQDs) using the hydrothermal synthesis method and employed them in biosensors [11]. The results reveal that Mn-BN-GQDs have excellent fluorescence characteristics and quantum yield, low cytotoxicity, and high biocompatibility, indicating a promising future for the advancement of bioimaging. On the other hand, graphene oxide (GO), one of the most significant graphene derivatives, has a heterogeneous electrical structure that enables it to emit fluorescence in a particular wavelength range. GO exhibits exceptional features including great mechanical strength, strong photostability, simple surface modification, and photoluminescence that is wavelength dependent. The presence of epoxide functional groups on its surface offers an easily chemically modifiable substrate allowing for the conjugation or interaction with a wide range of biomolecules *via* a variety of covalent/non-covalent interactions, electrostatic forces, absorption, and hydrogen bonding [12]. GO mainly consisting of sp2 and sp3 carbons, generate electron and optical band gaps, allowing it to fluoresce over a broad range of wavelengths and serve as a donor for fluorescence resonance transfer. Moreover, GO possesses effective fluorescence quenching characteristics. Most often, GO acts as the energy acceptor, whereas organic dye acts as the donor. Since the donor's emission spectrum overlaps with the acceptor's absorption spectrum, fluorescence resonance transfer can occur between the two, resulting in fluorescence quenching of the fluorescent dye. In particular, the functional group types, localized domains, lateral dimensions, and solvent dopants can significantly impact the electronic energy transition and fluorescence characteristics of GO [13].

### *Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

Currently, Numerous studies have demonstrated that GO and its derivatives may generate fluorescence signals of various colors. Based on a study by Kalluru, Nano-GO exhibits single-photon excitation wavelength-dependent photoluminescence in the visible and short near-infrared ranges, making it appropriate for multicolor fluorescence imaging *in vivo* [14]. This particular property of GO in a single biological platform makes this nanomaterial a promising candidate for clinical applications in the early diagnosis of a variety of disorders. Wang et al. reported for the first time the metal fluorescence enhancement of GO and discovered that the fluorescence intensity of GO on an Ag substrate was approximately 10 times stronger than that on glass [15]. Furthermore, unlike other fluorescent materials, GO in direct contact with the metal exhibits a high metal fluorescence enhancement without quenching, indicating that GO might be employed as a fluorescent probe for 3D optical imaging and sensing.

The following section provides a comprehensive introduction to the fluorescence imaging mechanism of graphene quantum dots and graphene oxide, as well as some reviews of current achievements in the field of graphene-based fluorescence imaging application. Fundamentally, to increase the performance of graphene and its derivatives in bioimaging applications, it is necessary to properly control their sizes, surface coatings, and components in order to maximize their photoluminescence capabilities. It is anticipated that the unique structure and exceptional capabilities of graphene and its derivatives offer new opportunities for disease detection and clinical treatments, have promising application prospects in fluorescence imaging, and play a crucial role in fostering the growth of the biomedicine industry.

### **2. GO, GQDs and CNDs in fluorescence imaging: why so important?**

### **2.1 Graphene oxide**

Graphene oxide is one of the most commonly used forms of the graphene-based materials (GBMs) family nowadays. This comes as no surprise since it is a very stable multifunctional material with a wide range of superior properties and hence a broad application scope [16]. GO is essentially the oxidized form of graphene which can be produced through a variety of oxidative methods [17–19]. Structurally, GO encompasses a variety of functional groups, the most important being: carboxy, hydroxy, epoxy, and keto- or aldehyde groups. Hydroxy groups can be found both at the edges and the surface of GO, epoxy on the surface areas and the rest mostly at the edges [20]. Their abundance and ratios are sensitive to the preparation method. These functional groups give rise to a quite dipolar character of this GBM and at the same time generate a range of excitation possibilities due to the presence of oxygen as the main heteroatom involving lone pairs of electrons. As a result, transitions such as *n-σ*\* *n-π*\* as well as *π-π*\* are the most important [21]. When it comes to the electronic and emission properties which are of primary interest for applications within fluorescence imaging, GO is known to exhibit a range of very interesting features:

### *2.1.1 Excitation energy dependent fluorescence*

This is a property which is also observed in GQDs. Unlike most of the conventional fluorophores, GO can result in different emission energies (i.e. a variety of colors of emitted light) when photo-excited at different wavelengths (energies) [22]. Specifically, upon increasing excitation wavelength from 325 nm to 650 nm, a red

shift of the GO fluorescent band is observed [23]. This effect in terms of applications in fluorescence imaging is very important since by employing a single material it is possible to obtain a wide range of emitted light colors simply employing different laser energies for excitation.

### *2.1.2 Fluorescence resonance energy transfer (FRET)*

FRET occurs when a donor chromophore, being in its electronically excited state, transfers energy to an acceptor chromophore *via* nonradiative dipole–dipole coupling [24]. The efficiency FRET is proportional to the reciprocal sixth power of the distance between the acceptor and the donor rendering this effect very sensitive to the donor-acceptor distance (see **Figure 1**) [25]. A wide variety of donor and acceptor fluorophore combinations [26] have been reported so far and FRET is considered as an eminent tool for biomolecular research and applications thereof [27, 28]. When it comes to GO, it is well established that GO can function as an efficient energy acceptor (EA) causing the quenching of fluorescence of a variety of energy donor organic fluorophores [29]. The benefits of using GO as an EA are multiple; the variety of functionalization possibilities its structure offers, the large number of binding sites as a result of its size, the high internalization to a wide range of cells, and the very effective energy transfer though a long distance [30] are some of them. In practice, the approach of employing GO, a fluorophore acting as the electron donor, and a functionality, factor, or molecule exhibiting specificity to a target analyte is widely used in many sensor and imaging attempts. Biorelevant analytes and biomarkers such as antibodies, microRNA [31, 32], folate receptor [33] as well as many other examples of analytes [29, 34] have been reported so far.

Additionally, GO can also be employed as an energy donor (ED) in FRET systems involving GO and an acceptor fluorophore. Such systems might involve either organic

#### **Figure 1.**

*Fundamental info about FRET. (A) Illustration of the FRET interaction between a donor and an acceptor fluorophore. (B) Example of successfully donor emission/acceptor absorbance overlapping spectra (C) dependence of FRET efficiency on the distance between the donor and the acceptor.*

*Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

dyes or noble metal nanoparticles acting as EAs corresponding to GO as ED [35, 36]. A variety of bio-relevant sensor applications based on GO/EA FRET systems have been reported so far allowing for the detection of antibodies [35] pharmaceutical screening/imaging, diagnostic tools [36, 37], etc. Some more detailed examples are presented in the next paragraphs.

#### *2.1.3 Fluorescence of GO*

The fluorescence band of GO is very broad and thus not suitable for accurate sensor and fluorescence imaging applications. Nonetheless, GO exhibits NIR emission when photoexcited in the NIR region and due to this feature GO is currently finding application in TP microscopy and imaging of biological samples [38, 39]. It is noteworthy that the excitation dependent emission (*vide supra*) allows for use of GO in the two biological wavelength-windows of 1000–1350 nm and 650–950 nm in which biological samples are nearly transparent [40]. This important fact justifies one more important use of GO in biological imaging in addition to FRET applications. Moreover, research attempts to tune the emission colors of GO have been also reported. Mei et al. [41] came out with an oxidative methodology allowing for controlled modifications of GO leading to emissions shifts from brown to cyan with no excitation wavelength alteration. Indeed, by merely varying oxidation reaction times of GO nanosheets was proved to result in controllable and accurate tuning of the emission properties. This approach is thought to introduce new opportunities in cellular imaging as well as in multiplex encoding analysis.

#### **2.2 Graphene quantum dots (GQDs) and carbon nanodots (CNDs)**

A very interesting family of materials belonging to the wide carbon family is the Carbon dots family. Two important classes of carbon dots are Graphene quantum dots (GQDs) and carbon nanodots (CNDs). GQDs and CNDs are currently in the forefront of research in the field of bioimaging and specifically within the research and development of novel fluorescence imaging materials and techniques. The ease of production of CNDs and GQDs, their high structural versatility and the wide range of structure-properties modulation opportunities, as well as their biocompatibility and bright fluorescence are the main reasons associated with their high relevance for bioimaging. Up to date a broad range of production methods have been published.

Several methods have been reported for producing GQDs and CNDs, with solvothermal, [42] microwave-assisted, [43] and electrochemical methods [44] being the most commonly used. The choice of the synthetic method used relies on the target batch-size and desired structure and properties of the final materials. It is currently established that for their production inert conditions e.g., heating at temperatures as low as even 120°C are adequate. As a comparison earlier methods employed drastic conditions involving laser ablation [45] or even temperatures higher than 900°C [46]. Nonetheless, it is well agreed by many researchers that milder production conditions can indeed result in very interesting properties since many of the microwave-assisted and hydrothermal methods leave parts of the molecules being subjected to heat or microwaves respectively, unaffected. Hence, the surface of the GQDs and CNDs produced by this "mild processing" can encompass a variety of functional groups [47, 48].

Up to date a wide variety of GQDs and CNDs have been synthesized *via* combining simple compounds such as citric acid, glucose or mono- or di-saccharides and urea, formamide or amino acids [46]. Another trend is to employ natural occurring sources such as fruit extracts, plant specimens etc. which upon treatment (usually microwave or hydrothermal) yield in GQDs and CNDs with a large variety of properties [49, 50]. This research field is highly active which promotes more sustainable and inexpensive production strategies for these vey important class of GBMs.

Why are GQDs and CNDs so important for fluorescence imaging is strongly associated to their outstanding photophysical properties. GQDs and CNDs exhibit photoluminescence (fluorescence emission by many authors) of often high quantum yields in a variety of media. The photoluminescence can be highly dependent on the size and even the shape of the dots [51] but mainly on the surface defects that they exhibit. In fact, without the surface passivation of carbon dots the observed photoluminescence is very limited. Post functionalization of their surface and/or incomplete "termination" reactions/partial carbonization during their production can efficiently enhance their photoluminescent properties [46]. Probably the most remarkable of the properties of GQDs and CNDs is the excitation-dependent photoluminescence [46]. This property (which also GO exhibits, *vide supra*) has a number of important implications on the potential uses of these materials. Specifically, the fact that different emission colors can be obtained by the same material just by choosing a suitable excitation wavelength, is highly appreciated in bioimaging where the use of multiple fluorophores can lead to complications and misinterpretations of the observed phenomena. Indeed, problems such as overlapping emission spectra, photobleaching, and phototoxicity are often encountered [52–54]. These adverse effects can be minimized or even eliminated when using carbon dots. The low toxicity, high water solubility, and very high stability, of carbon dots render this class of GBMs a very attractive option for bioimaging [46]. In the following paragraphs the use of GQDs and CNDs in contemporary fluorescent imaging technologies and applications is discussed.

### **3. Cellular and tissue imaging systems involving graphene-based materials**

Contemporary cell-imaging methods have facilitated the advancement of a wide range of biologically relevant assays aiming at a variety of therapeutic fields and revolutionized the R&D relating to drug design. Cellular imaging encompasses the application of a system or technology required for the visualization of a single cell, cell population, or subcellular structure. Even though a wide range of technologies, methodologies, and molecules enabling cell imaging do exist, there is a constant need for the development of novel systems of higher accuracy, fidelity, specificity, low cost, low cytotoxicity, and high photo- and chemical stability. In terms of fluorescent molecular materials, many of these requirements are fulfilled by members of the wide family of GBMs. In this section, the most recent developments falling in this area of research and technology are reported.

The fluorescence imaging of biomolecules particularly proteins and DNA is an important field of research and technology that can enable the visualization of proteins in cells and tissues with the use of fluorescent probes. Its importance is rationalized as high in terms of the various opportunities for the studies of localization and dynamics of proteins in living cells and tissues that it can offer (e.g. studies of protein–protein interactions, protein folding, and protein degradation) [55–58].

#### *Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

Indeed, graphene and other GBMs have been shown to act as useful platforms for fluorescent sensing of biomolecules including DNA and a variety of proteins and this opens a variety of opportunities in cell and tissue fluorescent imaging [59–62].

Bovine serum albumin (BSA) is a globular protein of animal origin (cow) that is used in a plethora of biochemical applications due to its stability and lack of interference with biological reactions [63]. Kuchlyan et al. performed a thorough study on the interactions of BSA with GO. The group employed a set of spectroscopic methods such as fluorescence correlation spectroscopy (FCS), Fluorescence Lifetime Imaging Microscopy (FILM), and Circular Dichroism (CD). For the study, BSA was labeled with the bright fluorescent Alexa Fluor 488 (AF488). They concluded that GO exhibits a pronouncedly strong interaction with BSA. GO was proved to have a drastic fluorescent quenching effect on AF488-BSA [64]. On the other hand, Yang et al. recently reported on the advancement of a highly sensitive nanosystem based on GO corresponding to microRNA (miRNA) which can be applied in living cells as well as *in vivo* [65]. GO acted as an efficient quenching agent against a molecular beacon labeled with the bright fluorophore cyanine-5 (Cy5). In the presence of the specifically targeted analyte miRNA, fluorescence was recovered allowing detection in cells or tumor tissue samples at very low levels [65].

Very recently Reagen et al. developed a novel class of GQDs exhibiting nearinfrared (NIR) fluorescence (emission centered at λ = 860 nm) derived from biomass obtained from organic source and prepared through pyrolysis. The prepared GQDs were tested for cell imaging in two distinct cell lines namely RAW 246.7 (macrophage cells) and MCF-7 breast cancer cell line. The results indicated low cytotoxicity as well as substantial internalization through endocytosis. Moreover, the GQDs exhibited a marked aptitude in detecting Hg2+ ions in biological samples enabling NIR fluorescence imaging in cells and toxic heavy metal detection *in vivo* [66]. Such multifunctional GQD-fluorescence imaging systems are currently sought-after. The multifunctional character and multi-use feature of GQDs in cellular imaging has been recently pointed out by a range of research groups [67–69].

Two-photon (TP) microscopy is a fluorescence imaging technique that is particularly well-suited to image-scattering living tissue of up to about 1 millimeter in thickness. It works by shining an intense beam of near-infrared light onto a single point within a sample, inducing simultaneous absorption of two photons at the focal point, where the intensity is the highest [70]. TP microscopy has found nowadays huge applicability in bioscience. Nonetheless, a typical restriction that TP- microscopy/ bioimaging techniques exhibit is that they rely on single-color fluorescence changes. Due to the special emission properties of GQDs among a range of other beneficiary properties (*vide supra* paragraph 2 for details) these nanomaterials have become very attractive for fluorescence imaging. Zhao et al. as early as 2016, reported the development of a dual-emission and TP GQD suitable for imaging applications targeting hydrogen peroxide (H2O2) as an analyte. The fluorescence response of the TP-GQD towards H2O2 was shown to be fast and very specific and renders the mapping of the production of endogenous H2O2 in living cells and deep tissues feasible. This probably constitutes the first published example pertaining to a dual-emission, TP-GQD of high specificity and applicability in cell and tissue fluorescence imaging [71].

In Wang et al. reported on the development of some GQDs through a hydrothermal method utilizing 1,3,6-trinitropyrene and (NH4)2SO3. The resulting GQDs encompassing amino as well as sulfate groups were evaluated in terms of their TP fluorescence efficiency in the context of cellular imaging. A very high TP absorption cross-section was observed and evaluated as significantly higher as compared to

**Figure 2.**

*(a) Image of GQDs at 405 nm excitation. (b) Image of nuclear view red dye at 633 nm excitation. (c) Merged image (obtained with permission from supplementary information of Ref. [72]).*

traditional/conventional fluorophores. The research group further performed tests in a cell line (HeLa cells) and found out that the GQDs were internalized in the cytoplasm providing very bright and clear cell images (see **Figure 2**) [72].

Earlier Sapkota et al. reported the synthesis of GQDs of tunable size and explored their capacity in fluorescence imaging. It was found that GQDs with a size between 15 and 35 nm exhibit vivid fluorescence (quantum yields of 0.64 by average) as well as high TP absorption (TPA) cross sections, which renders these GQDs excellent candidates for fluorescence imaging. Indeed, their use in cellular imaging was evaluated on living epithelial cells and even though internalization was observed, entering the nucleus was not possible [73].

Chen et al. as early as in 2015 advanced an aptameric sensor with nano dimensions based on graphene which is capable of inducing/enhancing the fluorescence activation imaging of cytochrome c (Cyt c; a major mediator in cell apoptosis released from mitochondria) [74]. In order to achieve this, Chen et al. connected a fluorophore-tagged DNA aptamer on graphene nanosheets modified with PEG polymer chains. The fluorescence of the fluorophore is inhibited due to the presence of graphene. Yet, dissociation of the a fluorophore-tagged DNA aptamer from graphene occurring immediately after cytosolic release of Cyt C, triggers the fluorescence and empowers real-time visualization of the Cyt c release kinetics. This nanosensing technology is envisioned to exhibit potential applications in visualization of key molecular factors in apoptotic signaling which are critical for cell biology and clinical theranostics.

Wang et al. on the other hand developed an innovative nanosensor employing graphene quantum dots (GQDs) which were conjugated to gold nanoparticles (AuNPs). The nanosensor was found to efficiently serve as a sensor of endogenous biological cyanide ions. This graphene-based nanomaterial further exhibits efficient TP excitation and exploits the drastic quenching efficiency of AuNPs and thus it can accomplish detection cyanide limits as low as 0.52 μM. This, combined with the potential of deep penetration depth of approx. 400 μm render this nanomaterial a perfect candidate for tissue imaging of cyanides [75].

Similarly, Hong et al. also employed a combination of AuNPs and a GBM (GO) for the development of fluorescent imaging/sensing system with specific applicability in monitoring intracellular telomerase activity. Their fluorescence imaging is applicable to a variety of living cells and it was tested toward the aptitude to distinguish normalform cancer cells [76].

*Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

One of the pioneering works reporting GQDs-based nanosystems utilized in TP-induced fluorescence imaging was that by Liu et al. published as early as 2013. The research group produced some N-doped GQDs employing a solvothermal method in which dimethylformamide (DMF) was used both as solvent as well as N-source. The reported GQDs exhibit a marked TP absorption cross-section of nearly 48 kGM (GM: Göppert Mayer units) and were proved to function well even at imaging depth in tissue samples as large as 1800 μm. The development of these GQDs has been an early example of the immense potentials of GQDs in fluorescence bioimaging with applications in the broad biological and biomedical research fields [77].

### **4. GBMs in cancer detection through fluorescence imaging**

Cancer currently constitutes one of the major mortality causes for humans. Ensuring enhanced cancer therapies requires improvement of cancer diagnostic techniques. In recent years, GBMs have found applicability in cancer detection. The wide range of suitable properties and attributes of this broad range of materials are considered for efficient cancer imaging.

Campbell et al. recently reported on the use of GQDs involving amino, hydroxy as well as carboxy functional groups and doped with nitrogen, nitrogen and boron or sulfur, in spectrally distinguishing among healthy and various types of cancer cells. In addition to this, the authors evaluated the pH-responsiveness of the investigated GQDs exploiting their wide range of emitted light wavelengths spanning from the visible to near-infraredred (NIR) part of the electromagnetic spectrum (**Figure 3**) [78].

Wu et al. on the other hand developed a new type of graphene-based nanomaterial functionalized with Anti-EpCAM antibodies and galactose-rhodamine-polyacrylamide

#### **Figure 3.**

*Illustration depicting the structure of GQDs developed by Campbell et al. and the possibility of distinguishing cancer from healthy cells. Lower images depict the variety of colors emitted by the GQDs as a result of their excitation at different wavelengths (excitation-dependent emission). Obtained with permission from supplementary information of Ref. [78].*

nanoparticles with a high aptitude to recognition of hepatocellular carcinoma cells (HCC-CTCs). The presence of graphene is dual here, acting both as a nanoparticle carrier platform as well as a strong quencher of rhodamine's fluorescence. Upon capturing and endocytosis of the aforementioned nanoparticles, fluorescence is recovered and the fluorescence imaging of HCC-CTCs can thus be efficiently achieved (**Figure 4**) [79].

It is important to mention that the aforementioned strategy involving graphene as a quencher of fluorophores connected to agents that can efficiently/specifically bind cancer cells or other bio-targets, has been repeatedly proposed within the context of fluorescence bio-imaging. A variety of recently published attempts towards bioimaging targets of biologically relevant analytes such as DNA [80], ions, [81] antibiotics [82], etc. have recently been reviewed [83].

Nurannabi et al. as early as 2014, developed GQDs bearing -OH and -COOH functionalities with an average size of 5 nm exhibiting significant red photoluminescence when excited at 655 nm (**Figure 5**). Their photoluminescence behavior was shown to enhance visualization of deep tumor tissues in experimental animals. Moreover, the described GQDs were tested *in vivo* as photodynamic therapeutic agents against MDA-MB231 cancer cells and it was proved that they exhibit a marked phototherapeutic activity [84].

In Pramanik et al. reported on the use of aptamer-conjugated GO in TP imaging (TPI) of breast cancer cells (specifically of SK-BR-3 cells). The developed GBM displays a drastic 2-photon absorption and marked photostability after even long irradiation sessions [38].

In Narasimhan et al. reported on the use of GQDs produced through laser ablation for use in both *in vitro* and *in vivo* fluorescence imaging of MCF-7 breast cancer cells. The reported GQDs were bearing hydroxyl and carboxylic acid groups at the edges as well as on their surface and it was observed that uptake of the GQDs by the

#### **Figure 4.**

*Illustration of graphene-based nanomaterial developed for recognition of hepatocellular carcinoma cells (HCC-CTCs). Obtained with permission from supplementary information of Ref. [79].*

*Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

#### **Figure 5.**

*Confocal laser scanning images of MDA-MB231 cells after treatment of cGdots (100 μg/mL). Scale bar: 20 μm (obtained with permission from supplementary information of Ref. [84]).*

MCF-7 cells was feasible through endocytosis rendering imaging of implanted cells in mice possible through an intense red fluorescence observed upon excitation at 610 nm [85].

In Liu et al. proposed the use of graphitic carbon nitride nanosheets as a scaffold allowing for the detection of hyaluronase (HAase). Similarly to the already described strategies for cancer cell/tissue imaging, a cancer marker is targeted as an analyte (in this particular case HAase). Imaging of HAase in cancer tissues can be achieved through the activable two-photon fluorescence of the developed graphitic carbon nitride nanosheets in presence of HAase [86].

Some years earlier Park and coworkers utilized hyaluronic acid (HA) instead of HA-ase to accomplish efficient target-specific delivery of GQDs. In their strategy, the researchers tethered HA to GQDs and thus synthesized brightly fluorescent nanoparticles with an approximate size of 20 nm [87]. These interesting HA-GQD conjugates were tested and it was found that they exhibit strong fluorescence in CD44 overexpressing A549 cells as well as in *in vivo* experiments involving CD44 receptor overexpressing tumor-bearing balb/c female mice. CD44 antigen is a cell-surface glycoprotein involved in cell–cell interactions, cell adhesion and migration. It is the receptor for hyaluronic acid and can also interact with other ligands e.g. matrix metalloproteinases (MMPs) etc. [87]. Its specificity to HA explains the very good emission response of the HA-GQDs by Park and coworkers. As a step further the efficiency of HA-GQDs to specifically curry chemotherapeutic drugs such as doxorubicin to cancer cells was evaluated as high rendering this smart nanomaterial both a good nanosensor and a drug nanocarrier.

In a similar fashion, Goreham et al. investigated the role of GO modification with folic acid in fluorescence lifetime imaging of HaCaT cells. Since folate receptor is an important recognized biomarker [88] currently considered for new diagnostic tools for cancer, modification of graphene oxide with folic acid was considered in this study. The water solubility green fluorescence (upon photoexcitation at 305 nm) and the evaluated low toxicity of GO indicated the high potentials of these graphene family members in cancer fluorescence imaging and corresponding diagnostics [89].

Liu et al. also utilized GO as a perfect platform on which a molecular beacon (a type of oligonucleotide hybridization probe which is capable of specifically detecting the presence of nucleic acids) having a couple of Cyanine-5 (Cy-5) fluorophore units at its both ends. The fluorescence of Cy-5 was quenched both due to self-quenching and fluorescence resonance energy transfer (FRET) caused by GO. With this

nanosystem, the researchers achieved detection of microRNAs even at concentrations a low as 30 pM. This nanoimaging technique was found to be applicable to a variety of cancer tissues and cells [65].

Additionally, Kumawat et al. reported the use of GQDs obtained *via* a green chemistry approach in fluorescence nuclear imaging in a variety of cell lines. It was observed that the reported GQDs even deprived of further support of external targeting agents (such as folic acid, hyaluronic acid etc.) exhibited a drastic propensity to self-localize into cell nuclei [90].

Moreover, Fan et al. investigated the use of some pH-responsive GQDs (pRF-GQDs) which were synthesized *via* an electrochemical method. The pRF-GQDs were found to undergo a fluorescence color transition between green and blue by varying the pH and developed a method which enable distinguishing between healthy and cancer tissues [91]. These are two important recent examples of the immense potentials of GQDs in bioimaging of cancer and cancer diagnosis advancements.

While fluorescence can be readily used in the detection and imaging of a variety of tumor cells, Magnetic resonance imaging (MRI) techniques enhanced through the use of GQDs appear to be very attractive due to the ease of production, size, easy structure modulation as well as multi-purpose character of GQDs [92]. Zhang et al. nearly a decade ago reported the use of GO-gadolinium (GO-Gd) complexes for the enhancement of and quality improvement of the MR-imaging of cancer. They showed that the developed GO-based material not only can operate as an enhancing agent for MRI but furthermore serves as a material for fluorescent imaging with anticancerdrug delivery aptitude (**Figure 6**). This early finding clearly showcases the immense possibilities and versatility of GBMs in bioimaging [93].

More recently and in a similar fashion, Yang et al. reported on the use of surfacemodified GQDs by polyethylene glycol (PEG) which were functionalized with the Gd-DOTA complex (where DOTA stands for tetraazacyclododecanetetraacetic acid) in cancer imaging through MRI and fluorescence imaging. The research group managed to significantly increase the relaxivity by regulating the length of the PEG linkers and hence advanced a novel MR contrast agent with immense potentials within cancer-imaging [94].

#### **Figure 6.**

*Illustration of the Gd-bearing GO-based platform developed by Zhang et al. allowing for enhanced MR-cancer imaging as well as for targeted drug delivery applications. Obtained with permission from supplementary information of Ref. [93]).*

*Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

### **5. Conclusions and future perspectives and uses of fluorescent imaging involving GBMs**

There is a wide range of current and potential future applications of fluorescence imaging, two of the most promising being fluorescence-guided surgery (FGS) [95] and robotic-assisted fluorescence surgery (RAFS) [96]. The applications are numerous while probably the most relevant ones lie in the field of surgical oncology [95, 97]. The idea of using tumor-targeted imaging agents in the context of this developing research and technology area is considered as a promising strategy for intraoperative cancer detection. In this regard, the broad family of GBMs and their attributes can demonstrate an important role as potent fluorescent/emissive materials of low toxicity, biocompatibility and tunable optical properties [46]. A variety of GQDs have been reported so far labelling tumor cells and tissue thus potentially facilitating tumor surgery [91, 98, 99]. Particular interest has been placed on the use on pH-responsive fluorescent GQDs (pRF-GQDs) as transition in the emitted light color depending on the pH can help distinguish healthy tissue from tumors (extracellular microenvironment of tumors exhibits lower pH) [33, 91]. Based on the so-far published research works, it becomes apparent that the multifunctional character of GBMs could facilitate tumor FGS in the future and in general bioimaging and the detection of disease [100, 101].

### **Acknowledgements**

The authors are grateful to Dr. K. Tzavlaki for fruitful discussions on bioimaging.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Acronyms and abbreviations**


### *Fluorescence Imaging – Recent Advances and Applications*


### **Author details**

Hu Li1,2 and Raffaello Papadakis3 \*

1 Shandong Technology Centre of Nanodevices and Integration, School of Microelectronics, Shandong University, Jinan, China

2 Department of Materials Science and Engineering, Uppsala University, Uppsala, Sweden

3 TdB Labs, Uppsala, Sweden

\*Address all correspondence to: rafpapadakis@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

### **References**

[1] Baker SN, Baker GA. Luminescent carbon nanodots: Emergent nanolights. Angewandte Chemie International Edition. 2010;**49**:6726-6744. DOI: 10.1002/anie.200906623

[2] Zhu S, Meng Q, Wang L, Zhang J, Song Y, Jin H, et al. Highly photoluminescent carbon dots for multicolor patterning, sensors, and bioimaging. Angewandte Chemie International Edition. 2013;**52**:3953-3957. DOI: 10.1002/anie.201300519

[3] Kavitha MK, Jaiswal M. Graphene: A review of optical properties and photonic applications. Asian Journal of Physics. 2016;**25**:809-831

[4] Xin G, Meng Y, Ma Y, Ho D, Kim N, Cho SM, et al. Tunable photoluminescence of graphene oxide from near-ultraviolet to blue. Materials Letters. 2012;**74**:71-73. DOI: 10.1016/j. matlet.2012.01.047

[5] Li L, Wu G, Yang G, Peng J, Zhao J, Zhu J-J. Focusing on luminescent graphene quantum dots: Current status and future perspectives. Nanoscale. 2013;**5**:4015-4039. DOI: 10.1039/ C3NR33849E

[6] Li H, Han S, Lyu B, Hong T, Zhi S, Xu L, et al. Tunable light emission from carbon dots by controlling surface defects. Chinese Chemical Letters. 2021;**32**:2887-2892. DOI: 10.1016/j. cclet.2021.03.051

[7] Ai L, Shi R, Yang J, Zhang K, Zhang T, Lu S. Efficient combination of G-C3N4 and CDs for enhanced photocatalytic performance: A review of synthesis, strategies, and applications. Small. 2021;**17**:2007523. DOI: 10.1002/ smll.202007523

[8] Li Y, Zhao Y, Cheng H, Hu Y, Shi G, Dai L, et al. Nitrogen-doped graphene quantum dots with oxygenrich functional groups. Journal of the American Chemical Society. 2012;**134**:15- 18. DOI: 10.1021/ja206030c

[9] Wang G, He P, Xu A, Guo Q, Li J, Wang Z, et al. Promising fast energy transfer system between graphene quantum dots and the application in fluorescent bioimaging. Langmuir. 2019;**35**:760-766. DOI: 10.1021/acs. langmuir.8b03739

[10] Gong L, Sun J, Zheng P, Liu Y, Yang G. Yellow fluorescent nitrogen and bromine Co-doped graphene quantum dots for bioimaging. ACS Applied Nano Materials. 2021;**4**:8564-8571. DOI: 10.1021/acsanm.1c02004

[11] Li B, Xiao X, Hu M, Wang Y, Wang Y, Yan X, et al. Mn, B, N co-doped graphene quantum dots for fluorescence sensing and biological imaging. Arabian Journal of Chemistry. 2022;**15**:103856. DOI: 10.1016/j.arabjc.2022.103856

[12] Aliyev E, Filiz V, Khan MM, Lee YJ, Abetz C, Abetz V. Structural characterization of graphene oxide: Surface functional groups and fractionated oxidative debris. Nanomaterials. 2019;**9**:1180

[13] Lai S, Jin Y, Shi L, Zhou R, Zhou Y, An D. Mechanisms behind excitationand concentration-dependent multicolor photoluminescence in graphene quantum dots. Nanoscale. 2020;**12**:591-601. DOI: 10.1039/C9NR08461D

[14] Kalluru P, Vankayala R, Chiang C-S, Hwang KC. Nano-graphene oxide-mediated In vivo fluorescence imaging and bimodal photodynamic and photothermal destruction of tumors. Biomaterials. 2016;**95**:1-10. DOI: 10.1016/j.biomaterials.2016.04.006

[15] Wang Z, Zheng L, Cheng Q, Li X, Huang L, Lu Y. Metal-enhanced fluorescence of graphene oxide sheets. Analytical and Bioanalytical Chemistry. 2022;**414**:3625-3630. DOI: 10.1007/ s00216-022-04001-x

[16] Razaq A, Bibi F, Zheng X, Papadakis R, Jafri SHM, Li H. Review on graphene-, graphene oxide-, reduced graphene oxide-based flexible composites: From fabrication to applications. Materials. 2022;**15**(3):1012. DOI: 10.3390/ma15031012

[17] Marcano DC, Kosynkin DV, Berlin JM, Sinitskii A, Sun Z, Slesarev A, et al. Improved synthesis of graphene oxide. ACS Nano. 2010;**4**(8):4806-4814. DOI: 10.1021/nn1006368

[18] Chen J, Yao B, Li C, Shi G. An improved hummers method for ecofriendly synthesis of graphene oxide. Carbon. 2013;**64**:225-229. DOI: 10.1016/j. carbon.2013.07.055

[19] Sun L. Structure and synthesis of graphene oxide. Chinese Journal of Chemical Engineering. 2019;**27**(10):2251-2260. DOI: 10.1016/j. cjche.2019.05.003

[20] Dimiev AM, Eigler S, editors. Graphene Oxide: Fundamentals and Applications. John Wiley & Sons, Ltd; 2016

[21] Gao W, editor. Graphene Oxide. Reduction Recipes, Spectroscopy, and Applications. Springer Cham; 2015. DOI: 10.1007/978-3-319-15500-5

[22] Li M, Cushing SK, Zhou X, Guoc S, Wu N. Fingerprinting photoluminescence of functional groups in graphene oxide. Journal of Materials Chemistry. 2012;**22**:23374-23379

[23] Zheng P, Prof W, N. Fluorescence and sensing applications of graphene oxide and graphene quantum dots: A review. Chemistry, an Asian Journal. 2017;**12**(18):2343-2353. DOI: 10.1002/ asia.201700814

[24] Helms V. Fluorescence resonance energy transfer. In: Principles of Computational Cell Biology. Weinheim: Wiley-VCH; 2008. p. 202

[25] Schneckenburger H. Förster resonance energy transfer–what can we learn and how can we use it? Methods and Applications in Fluorescence. 2019;**8**(1):013001. DOI: 10.1088/2050-6120/ab56e1

[26] Bajar BT, Wang ES, Zhang S, Lin MJC, B. T. A guide to fluorescent protein FRET pairs. Sensors. 2016;**16**(9):1488. DOI: 10.3390/s16091488

[27] Algar WR, Hildebrandt N, Vogel SS, Medintz IL. FRET as a biomolecular research tool — Understanding its potential while avoiding pitfalls. Nature Methods. 2019;**16**:815-829. DOI: 10.1038/ s41592-019-0530-8

[28] Kaur A, Kaur P, Ahuja S. Förster resonance energy transfer (FRET) and applications thereof. Analytical Methods. 2020;**12**:5532-5550. DOI: 10.1039/ D0AY01961E

[29] Tian F, Lyu J, Shi J, Yang M. Graphene and graphene-like twodenominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications. Biosensors & Bioelectronics. 2017;**89**:123-135. DOI: 10.1016/j. bios.2016.06.046

[30] Prabakaran G, Velmurugan K, David CI, Nandhakumar R. Role of *Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

Förster resonance energy transfer in graphene-based nanomaterials for sensing. Applied Sciences. 2022;**12**(14):6844. DOI: 10.3390/ app12146844

[31] Nitu FR, Savu L, Muraru S, Stoian I, Ionită M. Label-free homogeneous microRNA detection in cell culture medium based on graphene oxide and specific fluorescence quenching. Nanomaterials. 2021;**11**(2):368. DOI: 10.3390/nano11020368

[32] Guo Y, Xu H, Li Y, Wu F, Li Y, Bao Y, et al. Hyaluronic acid and Arg-Gly-asp peptide modified graphene oxide with dual receptor-targeting function for cancer therapy. Journal of Biomaterials Applications. 2017;**32**(1):54-65. DOI: 10.1177/0885328217712110

[33] Battogtokh G, Ko YT. Graphene oxide-incorporated pH-responsive folatealbumin-photosensitizer nanocomplex as image-guided dual therapeutics. Journal of Controlled Release: Official Journal of the Controlled Release Society. 2016;**234**:10-20. DOI: 10.1016/j. jconrel.2016.05.007

[34] Cruz SMA, Girão AF, Gonçalves G, Marques PAAP. Graphene: The missing piece for cancer diagnosis? Sensors. 2016;**16**(1):137. DOI: 10.3390/s16010137

[35] Kim J, Park S-J, Min D-H. Emerging approaches for graphene oxide biosensor. Analytical Chemistry, 2017;**89**(1):232-248. DOI: 10.1021/acs. analchem.6b04248

[36] Kwak S-Y, Yang J-K, Jeon S-J, Kim H-I, Yim J, Kang H, et al. Luminescent graphene oxide with a peptide-quencher complex for optical detection of cellsecreted proteases by a turn-on response. Advanced Functional Materials. 2014;**24**(32):5119-5128. DOI: 10.1002/ adfm.201400001

[37] Shi Y, Dai H, Sun Y, Hu J, Ni P, Li Z. Fluorescent sensing of cocaine based on a structure switching aptamer, gold nanoparticles and graphene oxide. The Analyst. 2013;**138**(23):7152-7156. DOI: 10.1039/c3an00897e

[38] Pramanik A, Chavva SR, Fan Z, Sinha SS, Nellore BPV, Ray PC. Extremely high two-photon absorbing graphene oxide for imaging of tumor cells in the second biological window. The Journal of Physical Chemistry Letters. 2014;**5**(12):2150-2154. DOI: 10.1021/jz5009856

[39] Li D, Xue L, Zhu Z, Zhao X, Qian J. Graphene oxide nanoparticles for two-photon fluorescence imaging of zebrafish. Optical and Quantum Electronics. 2016;**48**(11):519. DOI: 10.1007/s11082-016-0783-8

[40] Tsai M-F, Chang S-HG, Cheng F-Y, Shanmugam V, Cheng Y-S, Su C-H, et al. Au nanorod design as light-absorber in the first and second biological near-infrared windows for in vivo photothermal therapy. ACS Nano. 2013;**7**(6):5330-5342. DOI: 10.1021/ nn401187c

[41] Mei Q, Chen J, Zhao J, Yang L, Liu B, Liu R, et al. Atomic oxygen tailored graphene oxide nanosheets emissions for multicolor cellular imaging. ACS Applied Materials & Interfaces. 2016;**8**(11):7390-7395. DOI: 10.1021/ acsami.6b00791

[42] Huo Y, Xiu S, Meng L-Y, Quan B. Solvothermal synthesis and applications of micro/nano carbons: A review. Chemical Engineering Journal. 2023;**451**(138572):138572. DOI: 10.1016/j. cej.2022.138572

[43] De Medeiros TV, Manioudakis J, Noun F, Macairan J-R, Victoria F, Naccache R. Microwave-assisted

synthesis of carbon dots and their applications. Journal of Materials Chemistry C. 2019;**7**(24):7175-7195. DOI: 10.1039/c9tc01640f

[44] Rocco D, Moldoveanu VG, Feroci M, Bortolami M, Vetica F. Electrochemical synthesis of carbon quantum dots. ChemElectroChem. 2023;**10**(3):e2022011. DOI: 10.1002/celc.202201104

[45] Sun Y-P, Zhou B, Lin Y, Wang W, Fernando KAS, Pathak P, et al. Quantumsized carbon dots for bright and colorful photoluminescence. Journal of the American Chemical Society. 2006;**128**(24):7756-7757. DOI: 10.1021/ ja062677d

[46] Jelinek R. Carbon Quantum Dots: Synthesis, Properties and Applications. 1st ed. Switzerland: Springer International Publishing; 2016

[47] Bhunia SK, Maity AR, Nandi S, Stepensky D, Jelinek R. Imaging cancer cells expressing the folate receptor with carbon dots produced from folic acid. Chembiochem: A European Journal of Chemical Biology. 2016;**17**(7):614-619. DOI: 10.1002/cbic.201500694

[48] Tang L, Ji R, Cao X, Lin J, Jiang H, Li X, et al. Deep ultraviolet photoluminescence of water-soluble self-passivated graphene quantum dots. ACS Nano. 2012;**6**(6):5102-5110. DOI: 10.1021/nn300760g

[49] Chahal S, Macairan J-R, Yousefi N, Tufenkji N, Naccache R. Green synthesis of carbon dots and their applications. RSC Advances. 2021;**11**(41):25354-25363. DOI: 10.1039/d1ra04718c

[50] Hill S, Galan MC. Fluorescent carbon dots from mono- and polysaccharides: Synthesis, properties and applications. Beilstein Journal of Organic Chemistry. 2017;**13**:675-693. DOI: 10.3762/bjoc.13.67

[51] Kim S, Hwang SW, Kim M-K, Shin DY, Shin DH, Kim CO, et al. Anomalous behaviors of visible luminescence from graphene quantum dots: Interplay between size and shape. ACS Nano. 2012;**6**(9):8203-8208. DOI: 10.1021/nn302878r

[52] Zhang Y, Zheng Y, Tomassini A, Singh AK, Raymo FM. Photoactivatable fluorophores for bioimaging applications. ACS Applied Optical Materials. 2023;**1**(3):640-651. DOI: 10.1021/ acsaom.3c00025

[53] Qiu X, Xu J, Cardoso Dos Santos M, Hildebrandt N. Multiplexed biosensing and bioimaging using lanthanide-based time-gated Förster resonance energy transfer. Accounts of Chemical Research. 2022;**55**(4):551-564. DOI: 10.1021/acs. accounts.1c00691

[54] Pandey S, Bodas D. High-quality quantum dots for multiplexed bioimaging: A critical review. Advances in Colloid and Interface Science. 2020;**278**:102137. DOI: 10.1016/j.cis.2020.102137

[55] Zhu H, Hamachi I. Fluorescence imaging of drug target proteins using chemical probes. Journal of Pharmaceutical Analysis. 2020;**10**(5):426-433. DOI: 10.1016/j. jpha.2020.05.013

[56] Berezin MY, Achilefu S. Fluorescence lifetime measurements and biological imaging. Chemical Reviews. 2010;**110**(5):2641-2684. DOI: 10.1021/ cr900343z

[57] Yu L, Lei Y, Ma Y, Liu M, Zheng J, Dan D, et al. A comprehensive review of fluorescence correlation spectroscopy. Frontiers in Physics. 2021:**9**. DOI: 10.3389/fphy.2021.644450

[58] Terai T, Nagano T. Small-molecule fluorophores and fluorescent probes for *Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

bioimaging. Pflugers Archiv: European Journal of Physiology. 2013;**465**(3):347- 359. DOI: 10.1007/s00424-013-1234-z

[59] Lu C-H, Yang H-H, Zhu C-L, Chen X, Chen G-N. A graphene platform for sensing biomolecules. Angewandte Chemie (Int. Ed.). 2009;**48**(26):4785- 4787. DOI: 10.1002/anie.200901479

[60] Coreas R, Castillo C, Li Z, Yan D, Gao Z, Chen J, et al. Biological impacts of reduced graphene oxide affected by protein corona formation. Chemical Research in Toxicology. 2022;**35**(7):1244-1256. DOI: 10.1021/acs. chemrestox.2c00042

[61] Yildiz G, Bolton-Warberg M, Awaja F. Graphene and graphene oxide for bio-sensing: General properties and the effects of graphene ripples. Acta Biomaterialia. 2021;**131**:62-79. DOI: 10.1016/j.actbio.2021.06.047

[62] de Lázaro I, Vranic S, Marson D, Rodrigues AF, Buggio M, Esteban-Arranz A, et al. Graphene oxide as a 2D platform for complexation and intracellular delivery of siRNA. Nanoscale. 2019;**11**(29):13863-13877. DOI: 10.1039/c9nr02301a

[63] Bovine serum albumin. Meyler's Side Effects of Drugs. Amsterdam, Netherlands: Elsevier; 2016. p. 1045

[64] Kuchlyan J, Kundu N, Banik D, Roy A, Sarkar N. Spectroscopy and fluorescence lifetime imaging microscopy to probe the interaction of bovine serum albumin with graphene oxide. Langmuir: The ACS Journal of Surfaces and Colloids. 2015;**31**(51):13793-13801. DOI: 10.1021/acs.langmuir.5b03648

[65] Yang L, Liu B, Wang M, Li J, Pan W, Gao X, et al. A highly sensitive strategy for fluorescence imaging of MicroRNA in living cells and in vivo based on graphene oxide-enhanced signal molecules quenching of molecular beacon. ACS Applied Materials & Interfaces. 2018;**10**(8):6982-6990. DOI: 10.1021/ acsami.7b19284

[66] Reagen S, Wu Y, Liu X, Shahni R, Bogenschuetz J, Wu X, et al. Synthesis of highly near-infrared fluorescent graphene quantum dots using biomassderived materials for in vitro cell imaging and metal ion detection. ACS Applied Materials & Interfaces. 2021;**13**(37):43952-43962. DOI: 10.1021/ acsami.1c10533

[67] Dutta Chowdhury A, Ganganboina AB, Tsai Y-C, Chiu H-C, Doong R-A. Multifunctional GQDsconcanavalin a@Fe3O4 nanocomposites for cancer cells detection and targeted drug delivery. Analytica Chimica Acta. 2018;**1027**:109-120. DOI: 10.1016/j. aca.2018.04.029

[68] Thakur M, Kumawat MK, Srivastava R. Multifunctional graphene quantum dots for combined photothermal and photodynamic therapy coupled with cancer cell tracking applications. RSC Advances. 2017;**7**(9):5251-5261. DOI: 10.1039/ c6ra25976f

[69] Yang Y, Tang S, Chen D, Wang C, Gu B, Li X, et al. Multifunctional redemission graphene quantum dots with tunable light emissions for trace water sensing, WLEDs and information encryption. Colloids and Surfaces. A, Physicochemical and Engineering Aspects. 2021;**622**(126593):126593. DOI: 10.1016/j.colsurfa.2021.126593

[70] Suan D, Hampton HR, Tomura M, Kanagawa O, Chtanova T, Phan TG. Optimizing fluorescence excitation and detection for intravital two-photon microscopy. Methods in Cell Biology.

2013;**113**:311-323. DOI: 10.1016/ B978-0-12-407239-8.00014-8

[71] Zhao W, Li Y, Yang S, Chen Y, Zheng J, Liu C, et al. Target-activated modulation of dual-color and twophoton fluorescence of graphene quantum dots for in vivo imaging of hydrogen peroxide. Analytical Chemistry. 2016;**88**(9):4833-4840. DOI: 10.1021/acs.analchem.6b00521

[72] Wang L, Li W, Li M, Su Q, Li Z, Pan D, et al. Ultrastable amine, sulfo cofunctionalized graphene quantum dots with high two-photon fluorescence for cellular imaging. ACS Sustainable Chemistry & Engineering. 2018;**6**(4):4711-4716. DOI: 10.1021/ acssuschemeng.7b03797

[73] Sapkota B, Benabbas A, Lin H-YG, Liang W, Champion P, Wanunu M. Peptide-decorated tunablefluorescence graphene quantum dots. ACS Applied Materials & Interfaces. 2017;**9**(11):9378-9387. DOI: 10.1021/ acsami.6b16364

[74] Chen T-T, Tian X, Liu C-L, Ge J, Chu X, Li Y. Fluorescence activation imaging of cytochrome c released from mitochondria using aptameric nanosensor. Journal of the American Chemical Society. 2015;**137**(2):982-989. DOI: 10.1021/ja511988w

[75] Wang L, Zheng J, Yang S, Wu C, Liu C, Xiao Y, et al. Two-photon sensing and imaging of endogenous biological cyanide in plant tissues using graphene quantum dot/gold nanoparticle conjugate. ACS Applied Materials & Interfaces. 2015;**7**(34):19509-19515. DOI: 10.1021/acsami.5b06352

[76] Hong M, Xu L, Xue Q, Li L, Tang B. Fluorescence imaging of intracellular telomerase activity using enzymefree signal amplification. Analytical

Chemistry. 2016;**88**(24):12177-12182. DOI: 10.1021/acs.analchem.6b03108

[77] Liu Q, Guo B, Rao Z, Zhang B, Gong JR. Strong two-photon-induced fluorescence from photostable, biocompatible nitrogen-doped graphene quantum dots for cellular and deep-tissue imaging. Nano Letters. 2013;**13**(6):2436-2441. DOI: 10.1021/ nl400368v

[78] Campbell E, Hasan MT, Gonzalez Rodriguez R, Akkaraju GR, Naumov AV. Doped graphene quantum dots for intracellular multicolor imaging and cancer detection. ACS Biomaterials Science & Engineering. 2019;**5**(9):4671-4682. DOI: 10.1021/ acsbiomaterials.9b00603

[79] Wu C, Li P, Fan N, Han J, Zhang W, Zhang W, et al. A dualtargeting functionalized graphene film for rapid and highly sensitive fluorescence imaging detection of hepatocellular carcinoma circulating tumor cells. ACS Applied Materials & Interfaces. 2019;**11**(48):44999-45006. DOI: 10.1021/acsami.9b18410

[80] Wang H-B, Ou L-J, Huang K-J, Wen X-G, Wang L-L, Liu Y-M. A sensitive biosensing strategy for DNA detection based on graphene oxide and T7 exonuclease assisted target recycling amplification. Canadian Journal of Chemistry. 2013;**91**(12):1266-1271. DOI: 10.1139/cjc-2013-0285

[81] Sharma MD, Rayalu SS, Kolev SD, Krupadam RJ. Graphene/fluorescein dye-based sensor for detecting As(III) in drinking water. Scientific Reports. 2021;**11**(1):17321. DOI: 10.1038/ s41598-021-96968-3

[82] Youn H, Lee K, Her J, Jeon J, Mok J, So J-I, et al. Aptasensor for multiplex detection of antibiotics based on FRET *Fluorescence Imaging Enhanced by Members of the Graphene Family: A Review DOI: http://dx.doi.org/10.5772/intechopen.113228*

strategy combined with aptamer/ graphene oxide complex. Scientific Reports. 2019;**9**(1):7659. DOI: 10.1038/ s41598-019-44051-3

[83] Zheng X, Zhai R, Zhang Z, Zhang B, Liu J, Razaq A, et al. Graphene-oxidebased fluoro- and chromo-genic materials and their applications. Molecules. 2022;**27**(6):2018. DOI: 10.3390/molecules27062018

[84] Nurunnabi M, Khatun Z, Reeck GR, Lee DY, Lee Y-K. Photoluminescent graphene nanoparticles for cancer phototherapy and imaging. ACS Applied Materials & Interfaces. 2014;**6**(15):12413-12421. DOI: 10.1021/am504071z

[85] Narasimhan AK, Lakshmi BS, Santra TS, Rao MSR, Krishnamurthi G. Oxygenated graphene quantum dots (GQDs) synthesized using laser ablation for long-term real-time tracking and imaging. RSC. Advances. 2017;**7**(85):53822-53829. DOI: 10.1039/ c7ra10702a

[86] Liu J-W, Wang Y-M, Zhang C-H, Duan L-Y, Li Z, Yu R-Q, et al. Tumortargeted graphitic carbon nitride nanoassembly for activatable twophoton fluorescence imaging. Analytical Chemistry. 2018;**90**(7):4649-4656. DOI: 10.1021/acs.analchem.7b05192

[87] Nahain A-A, Lee J-E, In I, Lee H, Lee KD, Jeong JH, et al. Target delivery and cell imaging using hyaluronic acid-functionalized graphene quantum dots. Molecular Pharmaceutics. 2013;**10**(10):3736-3744. DOI: 10.1021/ mp400219u

[88] Coney LR, Tomassetti A, Carayannopoulos L, Frasca V, Kamen BA, Colnaghi MI, et al. Cloning of a tumorassociated antigen: MOv18 and MOv19 antibodies recognize a folate-binding

protein. Cancer Research. 1991;**51**(22):6125-6132

[89] Goreham RV, Schroeder KL, Holmes A, Bradley SJ, Nann T. Demonstration of the lack of cytotoxicity of unmodified and folic acid modified graphene oxide quantum dots, and their application to fluorescence lifetime imaging of HaCaT cells. Mikrochimica Acta. 2018;**185**(2):128. DOI: 10.1007/ s00604-018-2679-8

[90] Kumawat MK, Thakur M, Gurung RB, Srivastava R. Graphene quantum dots for cell proliferation, nucleus imaging, and photoluminescent sensing applications. Scientific Reports. 2017;**7**(1):15858. DOI: 10.1038/ s41598-017-16025-w

[91] Fan Z, Zhou S, Garcia C, Fan L, Zhou J. pH-responsive fluorescent graphene quantum dots for fluorescenceguided cancer surgery and diagnosis. Nanoscale. 2017;**9**(15):4928-4933. DOI: 10.1039/c7nr00888k

[92] Lu H, Li W, Dong H, Wei M. Graphene quantum dots for optical bioimaging. Small. 2019;**15**(36):e1902136. DOI: 10.1002/smll.201902136

[93] Zhang M, Cao Y, Chong Y, Ma Y, Zhang H, Deng Z, et al. Graphene oxide based theranostic platform for T1-weighted magnetic resonance imaging and drug delivery. ACS Applied Materials & Interfaces. 2013;**5**(24):13325- 13332. DOI: 10.1021/am404292e

[94] Yang Y, Chen S, Li H, Yuan Y, Zhang Z, Xie J, et al. Engineered paramagnetic graphene quantum dots with enhanced relaxivity for tumor imaging. Nano Letters. 2019;**19**(1):441- 448. DOI: 10.1021/acs.nanolett.8b04252

[95] Mieog JSD, Achterberg FB, Zlitni A, Hutteman M, Burggraaf J, Swijnenburg R-J, et al. Fundamentals and developments in fluorescence-guided cancer surgery. Nature Reviews. Clinical Oncology. 2022;**19**(1):9-22. DOI: 10.1038/ s41571-021-00548-3

[96] Lee Y-J, van den Berg NS, Orosco RK, Rosenthal EL, Sorger JM. A narrative review of fluorescence imaging in robotic-assisted surgery. Laparoscopic Surgery. 2021;**5**:31-31. DOI: 10.21037/ ls-20-98

[97] Egloff-Juras C, Bezdetnaya L, Dolivet G, Lassalle H-P. NIR fluorescence-guided tumor surgery: New strategies for the use of indocyanine green. International Journal of Nanomedicine. 2019;**14**:7823-7838. DOI: 10.2147/IJN.S207486

[98] Chen J, Liu C, Zeng G, You Y, Wang H, Gong X, et al. Indocyanine green loaded reduced graphene oxide for in vivo photoacoustic/fluorescence dual-modality tumor imaging. Nanoscale Research Letters. 2016;**11**(1):85. DOI: 10.1186/s11671-016-1288-x

[99] Cui F, Ji J, Sun J, Wang J, Wang H, Zhang Y, et al. A novel magnetic fluorescent biosensor based on graphene quantum dots for rapid, efficient, and sensitive separation and detection of circulating tumor cells. Analytical and Bioanalytical Chemistry. 2019;**411**(5):985-995. DOI: 10.1007/ s00216-018-1501-0

[100] Han Q, Pang J, Li Y, Sun B, Ibarlucea B, Liu X, et al. Graphene biodevices for early disease diagnosis based on biomarker detection. ACS Sensors. 2021;**6**(11):3841-3881. DOI: 10.1021/acssensors.1c01172

[101] Fahmy HM, Abu Serea ES, Salah-Eldin RE, Al-Hafiry SA, Ali MK, Shalan AE, et al. Recent progress in graphene- and related carbonnanomaterial-based electrochemical biosensors for early disease detection. ACS Biomaterials Science & Engineering. 2022;**8**(3):964-1000. DOI: 10.1021/ acsbiomaterials.1c00710

### **Chapter 4**

## Optical Chemosensors: Principles, Chemistry, Strategies, and Applications

*Mohamed Yahya, Müjgan Yaman and Zeynel Seferoğlu*

### **Abstract**

Chemosensors for anions and cations detections have been extensively used in several disciplines, including pharmacology, environmental science, biology, and chemistry. This field which is a division of supramolecular chemistry has been known for more than 150 years. It deals with chemosensors that recognize and detect anions and cations via optical or electrochemical signals. Today, a sustainable variety of chemosensors are established to detect both anions and cations. Additionally, chemosensors can be used to construct a sensory device and extract, and separate anions and cations. Chemosensors can detect toxic anions such as fluoride and cyanide as well as cations like mercury. Thus, chemosensors have become an attractive area of supramolecular chemistry. This chapter focuses on both colorimetric and fluorometric optical chemosensors and their application for anions and cations detections.

**Keywords:** chemosensors, colorimetric, fluorescence, anions and cations detection, optical chemosensors

### **1. Introduction**

Czarnick defines the chemosensor as a "molecule of abiotic origin that signals the presence of matter or energy [1]." Furthermore, chemosensors are molecule receptors that can sense and precisely interact with an analyte and generate a response as a detectable signal. The signal can be optical or electrical. It consists of a detection group and a signaling moiety. The detection group is responsible for selectivity and binding efficiency. While converting information into a detectable signal is due to the signaling moiety.

Recently, considerable studies focused on chemosensors because of their susceptible photophysical properties to the environment. The change in the optical signals can provide data on the chemical parameters such as pH and analytes concentration [2]. Chemosensors are extensively investigated to detect heavy metal ions [3, 4]. Optical Chemosensors are classified into colorimetric and Fluorometric chemosensors. The colorimetric receptors display the advantage of naked-eye detection of color changes, making the process simple.

On the other hand, the Fluorometric chemosensors are more chosen for the ratiometric response because of the ratio between the intensity of two emissions which allows for correcting the analytic concentration of the sensor and the effects of environment like temperature and polarity [5]. Today the demand is high for chemosensors that are selective towards specific harmful ions. The toxicity of certain anions and cations for humans as well as animals has motivated researchers to design chromophores that are selective to a specific anion or cation [6–8]. Several international agencies prohibit numerous heavy metal ions because of their toxicity and non-biodegradability, resulting in their accumulation in the environment [9, 10]. Nowadays, chemists and scientists are attempting to design chemosensors that can be used for environmental and industrial sample analysis.

Fluorometric chemosensors have attracted increasing intention for detecting selective anion or cation due to:


In this contribution, both colorimetric and fluorometric chemosensors will be discussed, focusing on fluorometric receptors. The most crucial applications for Fluorimetric chemosensors for detecting Cyanide and Fluoride are also briefly given.

### **2. Optical chemosensors**

Optical sensors are investigation techniques that detect light intensity. They modify the receptor's photophysical properties upon analyte (guest) binding to the receptor. Depending on the sensor type, these modifications are characterized in UV–visible and fluorescence spectroscopy instruments. The chemosensors can be classified into colorimetric chemosensors, fluorometric chemosensors, or fluorescent chemosensors.

### **2.1 Colorimetric chemosensors**

A colorimetric chemosensor is defined as the color change that occurs after the receptor's binding with a specific analyte [11]. After binding the receptor and the analyte, the chemosensor's signaling unit shows a color change [12]. Colorimetric chemosensors have attracted significant interest due to the possibility of obtaining qualitative and quantitative data via the naked eye [13] without referring to any complex techniques [14].

### **2.2 Fluorometric chemosensors**

### *2.2.1 General principle: design of chemosensor*

One of the most valuable response methods for optical readout is fluorescence. The fluorescence chemosensors have attracted considerable interest due to their sensitivity, selectivity, quick response time, on-site and real-time detection, straightforward

*Optical Chemosensors: Principles, Chemistry, Strategies, and Applications DOI: http://dx.doi.org/10.5772/intechopen.105968*

**Figure 1.** *Fluorescence sensing (a) A spaced model (b) An integrated model.*

performance, flexibility, and present low molar estimate of the analyte. In the case of fluorometric chemosensors, binding the analyte to the receptor leads to a change in fluorescence behavior [15]. In 1867, Goppelsroder reported the first fluorescent chemosensor to detect aluminum ions via the formation of a morin chelate which is strongly fluorescent.

The fluorescent chemosensor allows the detection at the picomolar scale; however, the colorimetric sensors can only detect concentration at micromolar levels. The fluorometric chemosensor's advantages are mainly due to the proportionality of the emitted fluorescence to the analyte concentration. In contrast, in absorbance measurements, the concentration of the analyte and the absorbance are proportional, which is associated with the ratio between intensities measured before and after the beam passes through the sample.

The essential parts of the fluorescent chemosensors are the recognition and signaling moieties, as given in **Figure 1**. Accordingly, a fluorescent chemosensor is developed by connecting a receptor (ionophore) to a fluorophore responsible for converting the recognition into the photophysical signal (like spectra, fluorescence quantum yield, and lifetime).

The photophysical properties can alter by binding a particular analyte to the receptor, leading to a fluorescence signal, with either an enhancement or quenching of fluorescence. The binding of the analyte to the receptor leads to an enhanced fluorescence intensity called turn-on chemosensor [16, 17], whereas the analyte bounding to the fluorophore results in a decrease of fluorescent intensity quenching called turn-off chemosensor [18, 19].

Depending on the connection of fluorophore and receptor, two models of fluorescent chemosensors can be defined: spaced (**Figure 1(a)**) and integrated model (**Figure 1(b)**). The spaced model offers a design where the fluorophore is connected to the receptor via spacer and signaling moieties that prevent conjugations. In the integrated model, the fluorophore and receptor are conjugatively connected to each other; in this model, the receptor is a part of the π-electron system of the fluorophore.

### *2.2.2 Photo-induced electron transfer*

The fluorescence quenching is due to a photo-induced electron transfer (PET); the normal state returns when a non-luminescent process follows the PET process [10].

#### **Figure 2.**

*Molecular orbital diagram of the photo-induced electron transfer process (PET).*

The PET process could explain the "on–off" switching of fluorescent chemosensors based on the molecular orbital theory. The basic design of the florescent chemosensors based on PET is a fluorophore- spacer- receptor. This model has a spacer separating the fluorophore from the receptor, consequently electronically disconnecting the π-electron systems from the fluorophore and receptor. The PET process is divided into two types: reductive PET and oxidative PET, depending on the electron acceptor or donor links to the fluorophore and receptor [11]. However, in this study, only the reductive PET will be investigated. The design of the fluorescent chemosensors is given in **Figure 2**. The reductive PET is defined as the system where the fluorophore is reduced, whereas the receptor is oxidized. In this type of PET process, the fluorophore provides an electron acceptor, and the receptor serves as an electron donor. The electron photo-excitation from HOMO to LUMO occurs through the reductive PET process. Consequently, the fluorophore and the analyte react and cause the occurrence or elimination HOMO and LUMO "adjacent" orbital, resulting in the fluorescence quenching or enhancement, respectively (**Figure 3**).

### *2.2.3 Intra and intermolecular charge transfer*

Valeur et al. were the first to introduce the intramolecular charge transfer (ICT) process for cation sensing [12]. ICT process is defined as an excited state where the conjugation of an electron-donating unit (such as –NH2, –NMe2, –OCH3) to an electron-accepting (like >C=O, –CN) unit in one molecule is shown to rise a "pushpull" π- electron system [13], which have been widely applied for cation sensing. A blue shift in the absorption spectrum is observed when the electron donor interacts with the analyte, decreasing the electron-donating character. However, the analyte reacts with the electron acceptor part leading to a red shift due to a developed ICT (**Figure 4**). Furthermore, alterations in the fluorescence lifetimes and quantum yields are also detected. Thus far, many fluorescent imaging molecules are obtained from the ICT process by modifying either the electron donor, electron acceptor capacity, or π-conjugation degree of the fluorophores to react with the target analyte.

#### **Figure 4.**

*Schematic representation of ICT chemosensor in the sensing process.*

### *2.2.4 Excited-state intramolecular proton transfer*

Excited-state intramolecular proton transfer (ESIPT) has been implemented to design fluorescent chemosensors due to their distinctive and exceptional spectral sensitivity to the environmental medium. ESIPT process is based on a proton transfer from a proton donor (hydroxyl or amino unit) to an acceptor unit (carbonyl oxygen or imine nitrogen) atom in the excited state of a fluorophore which is facilitated by an intramolecular hydrogen bond [20]. A graphical interpretation of the ESIPT process is illustrated in **Figure 5**.

Due to the change between the enol and keto form in the system based on ESIPT, the photochemical reactivity of the excited molecules is drastically reduced, leading to improved photostability. Additionally, a significant Stokes shift is observed. Hence, the ESIPT process fits the design of fluorescent chemosensors that necessitates spectral shift for selective detection.

### *2.2.5 Fluorescence resonance energy transfer*

Fluorescence Resonance Energy Transfer (FRET) is another type of fluorescence modulation. The FRET process is a transfer of energy between a pair of fluorophores

**Figure 5.**

*Schematic representation of excited-state intramolecular proton transfer system.*

that operate as energy donors and acceptors, respectively [21, 22]. The FRET depends on the interaction distance between the electronically excited states of two chromophores that the excitation energy is non-radiatively relocated from a donor to an acceptor by non-radiative dipole–dipole coupling (**Figure 6**). The vibrational transitions in the donor and the acceptor are approximately equivalent. If the FRET is operative in the molecules, they are typically applied to improve the stokes shift artificially. For sensing applications, the emission of the donor at relatively short wavelengths leads to triggering the acceptor emission at longer wavelengths with a ratio of the fluorescence intensities of the donor and acceptor emissions regulated by the target analytes. Elevated FRET efficacy is obtained when the extensive spectral overlaps with the donor emission and the acceptor absorption spectrum [23]. Several FRET-based fluorescent chemosensors were developed following the groundbreaking endeavors for the ratiometric detection of metal ions like Hg2+ [24], Zn2+ [25].

**Figure 6.** *Schematic representation of FRET chemosensor in the sensing process.*

### **3. Applications of fluorescent chemosensors**

Cyanide (CN− ) and Fluoride (F− ) play an essential role in life and are deemed one of very poisonous anions and harmful to human health. They are used in industrial activities like acrylic fiber manufacturing, metallurgy, electroplating, fibers synthesis, and gold extraction. Absorption by the lungs, exposure to the skin, food, and contaminated drinking water are all exposition methods to CN− and F− intoxication. **Table 1** summarizes the World Health Organization (WHO) and European (EU) guidelines for anions in drinking water. The WHO set the provisional maximum tolerable daily intake limits to 0.07 mg/L and 1.5 mg/L for CN− and F− , respectively, for drinking water. It is consequently of paramount importance to be able to establish their concentration in water at such low levels precisely. The detection of anions is generally effectuated via analytic techniques such as Ionic chromatography. However, these techniques are complex and expensive, where the need to develop new techniques for anions detection that are both accurate and low cost. Recently, fluorescent chemosensors have attracted considerable attention as a promising alternative for anions sensing.

The fluorescent chemosensors have been used in numerous fields, such as biology, physiology, pharmacology, and environmental sciences. Fluorescent chemosensors to detect environmentally critical contaminants such as anions (CN− , F− ) or cations (Hg+ , Pb2+ …) have been widely investigated.

Keleş et al. reported the synthesis of 3,5-dinitro-(*N*-phenyl)benzamide (DNBA) and it is used as a simple colorimetric and fluorimetric chemosensor for selective determination of CN*−* in organic (DMSO and ACN) and aqueous solutions (DMSO/ H2O: 8:2, v/v) [26]. The colorimetric and fluorimetric color intensity changed after adding CN due to the interaction between CN*−* and DNBA. The UV/Vis and fluorescence spectrometry defined the interaction mechanisms between DNBA and CN*−*. Furthermore, spectroscopic results showed that CN*−* interacts with three different mechanisms; deprotonation, nucleophilic aromatic substitution, and formation of benzisoxazole ring. **Figure 7** displays the detection mechanism after adding CN*−* in a DMSO/H2O medium.

Zheng et al. studied the use of naphthalimide-based (NIMS) as a fluorescent sensor for F− [27]. The authors reported that F- detection occurs via a desilylation reaction (**Figure 8**), resulting in a colorimetric/fluorometric spectral response with a broad absorption around 229 nm and a change in color from yellow to blue. Furthermore, it was found that NIMS have high selectivity, rapid response, and sound sensitivity for F− . NIMS is a promising sensor for F− detection and quantification in toothpaste.


#### **Table 1.**

*WHO and EU drinking water standards for anions.*

#### **Figure 7.**

*CN− detection mechanism reported by Keleş et al. [26].*

**Figure 8.** *Mechanism of F<sup>−</sup> detection using NIMS sensor.*

Sourav Bej et al. reported the synthesis of an oxene-based chemosensor (HyMa) via Knoevenagel condensation to detect multi-analytes such as HSO4 − , CN−, and F− [28]. The authors reported a detection limit of around 38 ppb, 18 ppb, and 94 ppb for HSO4 − , CN− , and F− respectively. Furthermore, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) displayed a low cytotoxicity and membrane permeability, making it an attractive material for bio-imaging. The mechanism of CN− detection is given in **Figure 9**.

Deng et al. reported another chemosensor for the detection of CN<sup>−</sup> . TCNT fluorescent displaying an aggregation-induced emission was synthesized. A red fluorescent emission (596 nm) and significant stokes shift (148 nm) were observed, leading to an increased sensitivity to CN− (detection limit = 0.38 μmol L*−*<sup>1</sup> ) [29]. The detection was due to a nucleophilic attack of CN<sup>−</sup> on the vinyl group. Furthermore, the authors proved that TCNT could be used for bioimaging in live organisms.

**Figure 9.** *The detection mechanism of CN− by HyMa.*

**Figure 10.** *The detection mechanism of CN− by 3TT.*

#### **Figure 11.**

*The detection mechanism of CN− .*

A donor-π-acceptor structure was synthesized by Sun et al. for the CN<sup>−</sup> detection (3TT). The detection of CN− occurs via nucleophilic addition to the β-conjugated position of the barbituric acid of 3TT (**Figure 10**). The authors reported a naked-eye detection with high anti-interference ability and quick response. The detection limit was found to be 39.9 nM [24]. Furthermore, 3TT exhibited exceptional performance in the solid-state, and the 3TT-based test filter paper strips were applied to quickly and effectively detect CN− in water with naked eyes. Likewise, the 3TT was effectively applied to rapidly detect CN− in actual water samples, sour seeds, and food samples, proving its good ability for practical applications in our daily life.

Coumarin-spiropyran dyad having a hydrogen pyran moiety [2] was synthesized by [25]. The structure has an off–on type fluorescent receptor for detecting CN− . Because of the delocalization of π-electrons over the molecule, the receptor has nearly no fluorescence with a quantum yield <0.01. The nucleophilic addition of CN- to the spirocarbon of the molecule leads to a rapid opening of the spirocycle (**Figure 11**). Hence the localization of π-electrons on the coumarin moiety and a strong light-blue fluorescence occurs at 459 nm, and a high quantum yield (0.52) is obtained. The detection limit was low and equal to 1.0 μM.

### **4. Conclusions**

Since Goppelsröder stated the first fluorescent chemosensors selective for Al3+, the chemosensors field has progressed extensively. Notably, the last 50 years witnessed massive development in the field of fluorometric chemosensors. Hence, numerous chemosensors have been designed by researchers over the last years. Chemosensors

present a promising technology for detecting toxic anions and cations in an aqueous medium. This contribution gave a summary of the chemosensors' chemistry and their applications. Finally, some examples of cyanide and fluoride, among other examples, detection were given.

### **Acknowledgements**

This research was supported by Gazi University. This work was a part of Dr. Mohamed Yahya thesis of Reference No: 10451335.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Mohamed Yahya1,2\*, Müjgan Yaman3 and Zeynel Seferoğlu1,4

1 Faculty of Science, Department of Chemistry, Gazi University, Ankara, Turkey

2 Department of Chemistry, University of Nevada, Reno, NV, USA

3 Faculty of Science, Department of Chemistry, Eskisehir Osmangazi University, Eskisehir, Turkey

4 Technological Dyes and Materials Application and Research Center (TEBAM), Gazi University, Ankara, Turkey

\*Address all correspondence to: myahya@umr.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Czarnik AW. Supramolecular chemistry, fluorescence, and sensing. In: Fluorescent Chemosensors for Ion and Molecule Recognition UTC; 1993. p. 1-9. [cited June 9, 2022]. DOI: 10.1021/ bk-1993-0538.ch001

[2] Liu S, Wang YM, Han J. Fluorescent chemosensors for copper(II) ion: Structure, mechanism and application. Journal of Photochemistry and Photobiology C Photochemistry Reviews. 2017;**32**:78-103

[3] Patil NS, Dhake RB, Ahamed MI, Fegade U. A mini review on organic chemosensors for cation recognition (2013-2019). Journal of Fluorescence. 2020. cited 10 May 2022;**30**(6): 1295-1330. Available from: https:// link.springer.com/article/10.1007/ s10895-020-02554-7

[4] Kowser Z, Rayhan U, Akther T, Redshaw C, Yamato T. A brief review on novel pyrene based fluorometric and colorimetric chemosensors for the detection of Cu2+ Vol. 5, Materials Chemistry Frontiers. Royal Society of Chemistry; 2021 [cited June 10, 2021]. p. 2173-2200. Available from: https://pubs. rsc.org/en/content/articlehtml/2021/qm/ d0qm01008a

[5] Kollur SP, Shivamallu C, Prasad SK, Veerapur R, Patil SS, Cull CA, et al. Recent Advances on the development of chemosensors for the detection of mercury toxicity: A review. 2021;**8**(10):192. [cited May 10, 2022] Available from: https://www.mdpi. com/2297-8739/8/10/192/htm

[6] Li MM, Huang SY, Ye H, Ge F, Miao JY, Zhao BX. A new pyrazolinebased fluorescent probe for Cu2+ in live cells. Journal of Fluorescence. 2013 [cited 10 May 2022;**23**(4):799-806. Available from: https://link.springer.com/ article/10.1007/s10895-013-1203-0

[7] Ma S, Wang Y, She M, Wang S, Yang Z, Liu P, et al. Design strategies and progress on xanthene-based fluorescent probe for metal ions. Reviews in Analytical Chemistry 2017. cited 10, May 2022;**36**(2):20160024. Available from: https://www.degruyter.com/ document/doi/10.1515/revac-2016-0024/ html?lang=en

[8] Kaur N, Kaur G, Fegade UA, Singh A, Sahoo SK, Kuwar AS, et al. Anion sensing with chemosensors having multiple NH recognition units. TrAC Trends in Analytical Chemistry. 2017;**95**:86-109

[9] DIRECTIVE 2002/95/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 27 January 2003 on the restriction of the use of certain hazardous substances in electrical and electronic equipment. Official Journal of the European Union.

[10] WHO (2004) Guidelines for Drinking Water Quality. 3rd ed. Vol. 1. Geneva: World Health Organization; 2004. p.188. [cited May 10,2022]. Available from: https://books.google.com.tr/ books?hl=en&lr=&id=SJ76COTm-nQC& oi=fnd&pg=PR15&dq=World+Health+Or ganization+(2004)+Guidelines+for+ drinking-water+quality+(3rd+ed)+vol+1, +Geneva,+188&ots=V9tWpfPa0d&sig= fItvRqdL4WJrZH\_jkE0SnoZxrn0&redir\_ esc=y#v=onepage&q&f=false

[11] Wang M, Leung KH, Lin S, Chan DSH, Kwong DWJ, Leung CH, et al. A colorimetric chemosensor for CU2+ ion detection based on an iridium(III) complex. Scientific Reports. 2014;**4**(1):1-7

[12] Kaur P, Kaur S, Singh K. Colorimetric detection of cyanide in water using a highly selective Cu2+ chemosensor. Inorganic Chemistry Communications. 2009;**12**(10):978-981

[13] Dong X, Wang R, Liu G, Liu P, Pu S. A novel sensitive sensor for Cu2+ and multi-switch based on a diarylethene with a 2-(2′-hydroxyphenyl) benzothiazole unit. Tetrahedron. 2016;**72**(22):2935-2942

[14] Park GJ, Lee JJ, You GR, Nguyen L, Noh I, Kim C. A dual chemosensor for Zn2+ and Co2+ in aqueous media and living cells: Experimental and theoretical studies. Sensors and Actuators B: Chemical. 2016;**223**:509-519

[15] Wang JH, Liu YM, Dong ZM, Bin CJ, Wang H, Wang Y, et al. New colorimetric and fluorometric chemosensor for selective Hg2+ sensing in a near-perfect aqueous solution and bio-imaging. Journal of Hazardous Materials. 2020;**382**:121056

[16] Zeng S, Li SJ, Liu TT, Sun XJ, Xing ZY. A significant fluorescent "turn-on" chemosensor for Al3+ detection and application in real sample, logic gate and bioimaging. Inorganica Chimica Acta. 2019;**495**:118962

[17] Gupta A, Kumar N. A review of mechanisms for fluorescent "turn-on" probes to detect Al3+ ions. RSC Advances. Royal Society of Chemistry. 2016;**6**:106413-106434

[18] Riis-Johannessen T, Schenk K, Severin K. Turn-off-and-on: Chemosensing ensembles for sensing chloride in water by fluorescence spectroscopy. Inorganic Chemistry. 2010;**49**(20):9546-9553

[19] Wu D, Sedgwick AC, Gunnlaugsson T, Akkaya EU, Yoon J, James TD. Fluorescent chemosensors: The past, present and future. Chemical Society Reviews. Royal Society of Chemistry. 2017;**46**:7105-7123

[20] Yang Y, Zhao Q, Feng W, Li F. Luminescent chemodosimeters for bioimaging. Chemical Reviews. American Chemical Society. 2013;**113**:192-270

[21] Fan J, Hu M, Zhan P, Peng X. Energy transfer cassettes based on organic fluorophores: Construction and applications in ratiometric sensing. Chemical Society Reviews. 2013;**42**(1):29-43

[22] Kim JS, Quang DT. Calixarenederived fluorescent probes. Chemical Reviews. American Chemical Society. 2007;**107**:3780-3799

[23] Feng Y, Cheng J, Zhou L, Zhou X, Xiang H. Ratiometric optical oxygen sensing: A review in respect of material design. Analyst. Royal Society of Chemistry. 2012;**137**:4885-4901

[24] Sun T, Niu Q, Li Y, Li T, Hu T, Wang E, et al. A novel oligothiophenebased colorimetric and fluorescent "turn on" sensor for highly selective and sensitive detection of cyanide in aqueous media and its practical applications in water and food samples. Sensors and Actuators B: Chemical. 2018;**258**:64-71

[25] Shiraishi Y, Nakamura M, Hayashi N, Hirai T. Coumarin-spiropyran dyad with a hydrogenated pyran moiety for rapid, selective, and sensitive fluorometric detection of cyanide anion. Analytical Chemistry. 2016 [cited June 9, 2022]. 88(13):6805-6811. DOI 10.1021/acs. analchem.6b01279

[26] Keleş E, Aydıner B, Nural Y, Seferoğlu N, Şahin E, Seferoğlu Z. A new mechanism for selective recognition of

*Optical Chemosensors: Principles, Chemistry, Strategies, and Applications DOI: http://dx.doi.org/10.5772/intechopen.105968*

cyanide in organic and aqueous solution. European Journal of Organic Chemistry. 2020;(30):4681-4692. [cited June 6, 2022]. DOI: 10.1002/ejoc.202000342

[27] Zheng X, Zhu W, Ai H, Huang Y, Lu Z. A rapid response colorimetric and ratiometric fluorescent sensor for detecting fluoride ion, and its application in real sample analysis. Tetrahedron Letters. 2016;**57**(52):5846-5849

[28] Bej S, Das R, Mondal A, Saha R, Sarkar K, Banerjee P. Knoevenagel condensation triggered synthesis of dual-channel oxene based chemosensor: Discriminative spectrophotometric recognition of F−, CN– and HSO4− with breast cancer cell imaging, real sample analysis and molecular keypad lock applications. Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy. 2022;**273**:120989

[29] Deng K, Wang L, Xia Q, Liu R, Qu J. A turn-on fluorescent chemosensor based on aggregation-induced emission for cyanide detection and its bioimaging applications. Sensors and Actuators B: Chemical. 2019;**296**:126645

## Section 3
