Two-Dimensional Graphitic Carbon Nitride (g-C3N4) Nanosheets and Their Derivatives for Diagnosis and Detection Applications

The early diagnosis of certain fatal diseases is vital for preventing severe consequences and contributes to a more effective treatment. Despite numerous conventional methods to realize this goal, employing nanobiosensors is a novel approach that provides a fast and precise detection. Recently, nanomaterials have been widely applied as biosensors with distinctive features. Graphite phase carbon nitride (g-C3N4) is a two-dimensional (2D) carbon-based nanostructure that has received attention in biosensing. Biocompatibility, biodegradability, semiconductivity, high photoluminescence yield, low-cost synthesis, easy production process, antimicrobial activity, and high stability are prominent properties that have rendered g-C3N4 a promising candidate to be used in electrochemical, optical, and other kinds of biosensors. This review presents the g-C3N4 unique features, synthesis methods, and g-C3N4-based nanomaterials. In addition, recent relevant studies on using g-C3N4 in biosensors in regard to improving treatment pathways are reviewed.


Introduction
The early detection of the biomarkers of the diseases plays a significant role in their treatment and control. It is essential to detect biomarkers associated with a disease early and with the high precision for diagnosis, treatment, and prognosis of fatal diseases, such as cancer, which causes a high mortality rate yearly, and neurodegenerative disorders [1][2][3][4]. There are some current conventional diagnostic methods, such as blood tests, imaging, and biopsies, which can be expensive and time-consuming with low sensitivity. Moreover, they require trained personnel, limiting their availability to low-income patients [3].
Today, biosensors are used for detection approaches, such as the high-resolution imaging, fast detection, and monitoring of diseases. Biosensors consist of three main components: recognition, signal transducer, and processor, designed to determine specific biomolecules [5]. These biomolecules can be macromolecules, such as nucleic acid and proteins, or small molecules, such as glucose. Various cancer biomarkers, such as BRCA1, BRCA2, CA 15-3, and CA 125 for breast cancer and PSA for prostate cancer, can be detected as well [6]. Various g-C3N4 precursors and the corresponding temperatures for their thermal condensation into g-C3N4-, adapted from reference [50] under the terms and conditions of the Creative Commons Attribution (CC BY) license. (B) g-C3N4 structure, adapted from reference [51] under the terms and conditions of the Creative Commons Attribution (CC BY) license.
In addition, g-C3N4 has been reported to display antimicrobial activity. A number of parameters, including the g-C3N4 band gap, intermediate defect states, dispersed surface area, absorbance in suspension, and charge separation influence its photocatalytic bacterial inactivation [39]. Thus, the modification of this properties influences the production of reactive oxygen species, hence the antibacterial activity. The bactericidal rates of more than 99% have been successfully achieved for eight kinds of foodborne pathogenic bacteria with 8 h incubation in the dark. Cell rupture caused by direct mechanical contact between g-C3N4 and cell membranes has been observed. Molecular dynamics simulations further indicated that the presence of large defects in g-C3N4 enhanced the electrostatic attraction between inherent pores and lipid heads, resulting in enhanced antibacterial activity.
The thermal and chemical stability of biosensors is crucial for long shelf lives. g-C3N4 nanosheets show high thermal stability in the air (up to 600 °C) thanks to the graphitic graphene-like structure with sp 2 bonds between carbon and nitrogen, providing high chemical stability [52]. g-C3N4 has low cytotoxicity and good biocompatibility due to its metal-free structure. Moreover, it has a low production cost, a simple synthesis process, a large specific surface area, easy functionalization, and increased penetration coefficient, allowing the efficient immobilization of molecules in the matrix for biosensing [53]. As g-C3N4 materials are increasingly used in biomedicine, improving their biocompatibility and biodegradability properties is a necessity. Therefore, modifications are applied to enhance the biocompatibility, biodegradability, and further development of g-C3N4 materials. For instance, Kang et al. showed that successfully inserting abundant disulfide bonds into g-C3N4 endowed more biodegradability and biocompatibility, boosting its application in biomedical fields [54]. In another study that was recently conducted for glucose  [50] under the terms and conditions of the Creative Commons Attribution (CC BY) license. (B) g-C 3 N 4 structure, adapted from reference [51] under the terms and conditions of the Creative Commons Attribution (CC BY) license.
In addition, g-C 3 N 4 has been reported to display antimicrobial activity. A number of parameters, including the g-C 3 N4 band gap, intermediate defect states, dispersed surface area, absorbance in suspension, and charge separation influence its photocatalytic bacterial inactivation [39]. Thus, the modification of this properties influences the production of reactive oxygen species, hence the antibacterial activity. The bactericidal rates of more than 99% have been successfully achieved for eight kinds of foodborne pathogenic bacteria with 8 h incubation in the dark. Cell rupture caused by direct mechanical contact between g-C 3 N 4 and cell membranes has been observed. Molecular dynamics simulations further indicated that the presence of large defects in g-C 3 N 4 enhanced the electrostatic attraction between inherent pores and lipid heads, resulting in enhanced antibacterial activity.
The thermal and chemical stability of biosensors is crucial for long shelf lives. g-C 3 N 4 nanosheets show high thermal stability in the air (up to 600 • C) thanks to the graphitic graphene-like structure with sp 2 bonds between carbon and nitrogen, providing high chemical stability [52]. g-C 3 N 4 has low cytotoxicity and good biocompatibility due to its metal-free structure. Moreover, it has a low production cost, a simple synthesis process, a large specific surface area, easy functionalization, and increased penetration coefficient, allowing the efficient immobilization of molecules in the matrix for biosensing [53]. As g-C 3 N 4 materials are increasingly used in biomedicine, improving their biocompatibility and biodegradability properties is a necessity. Therefore, modifications are applied to enhance the biocompatibility, biodegradability, and further development of g-C 3 N 4 materials. For instance, Kang et al. showed that successfully inserting abundant disulfide bonds into g-C 3 N 4 endowed more biodegradability and biocompatibility, boosting its application in biomedical fields [54]. In another study that was recently conducted for glucose detection in diabetic patients, the addition of metal co-catalysts (Fe(III), Cu(II)) to the structure via adsorption noticeably enhanced the sensitivity compared to the pristine g-C 3 N 4 [55]. Thanks to its easy functionalization, g-C 3 N 4 can be adapted to various targets with high sensitivity. For instance, a platform based on proton-functionalized ultrathin g-C 3 N 4 nanosheets with a positive charge has been developed for heparin (as a biomolecule with a high negative charge) detection in human serum [56].

Synthesis of g-C 3 N 4 Nanosheets
The classification of the synthesis methods based on the synthesis procedure can be divided into bottom-up and top-down categories. The "bottom-up" approach generally applies small-sized particles to assemble complex structures. However, the "top-down" procedure is based on splitting large-sized and thick bulks into small particles and thin nanosheets [57,58]. The bottom-up procedure includes ionic liquid, supramolecular preassembly, and hydrothermal methods [58]. In the bottom-up approach, g-C 3 N 4 sheets are synthesized on a large scale via thermal polymerization (pyrolysis) or the carbonization of small organic compounds (that contain hydroxyl, carboxyl, carbonyl, and primary amine functional groups) [59], such as melamine, cyanimide, Dicyanamide, or urea [60]. Dante et al. obtained g-C 3 N 4 from the pyrolysis of melamine cyanurate at 650 • C for 50 min (in the crucible with atmosphere condition), which was used for glucose sensing [55]. On the other hand, chemical exfoliation and ultrasonic exfoliation methods have been utilized for the top-down approach. Chemical exfoliation is more common for large-scale production due to its high efficiency and the easier tuning of the g-C 3 N 4 structure [61]. For example, Hatamie et al. used g-C 3 N 4 as a label-free fluoro-sensor to analyze the amount of metronidazole in biological fluids and drug samples. g-C 3 N 4 ultrathin nanosheets were synthesized in bulk via the thermal polymerization method from melamine, possessing a highly π-conjugated structure at 600 • C. The exfoliation procedure was performed through ultrasonication in water media [62].
3.2. Synthesis of g-C 3 N 4 -Based Composites g-C 3 N 4 properties can be enhanced through its fabrication with other materials into composites. In the modification techniques, metal loading is critical for increasing the potential application of g-C 3 N 4 biosensors due to outstanding electrochemical qualities. Metal/g-C 3 N 4 composites are produced with solvothermal treatment, photo-deposition, precipitation, and thermal polymerization methods [63]. Generally, there are numerous ways to prepare g-C 3 N 4 -based nanocomposites. The simple pyrolysis method, solution (sonication) mixing, the hydrothermal method, the simple calcination method, the hydrolysis method, sol-gel, and microwave irradiation are some synthesis methods that have been applied in the formation of nanocomposites based on g-C 3 N 4 -and have been utilized for different applications [43]. The pyrolysis method is a common way to produce g-C 3 N 4based composites in diagnosis applications where the mixture of the precursor of g-C 3 N 4 and the other components is calcinated in a crucible for a while with a specific heating rate and initial temperature to prepare the nanocomposite. Then, the product is cooled at 25 • C. For example, a sensitive electrochemical sensor for dopamine detection was fabricated by firstly preparing calcium stannate (CaSnO 3 ) nanoparticles from CaCl 2 and SnCl 2 .2H 2 O via the hydrothermal method, then CaSnO 3 -gC 3 N 4 nanohybrid was produced through the pyrolysis of melamine, (NH 4 ) 2 SO 4 , and CaSnO 3 mixture at 550 • C in a crucible [64]. In another study for glucose detection, Cu(II)-Fe(III)-g-C 3 N 4 was prepared through the sonication method (2 h sonication of a suspension of 416 mg of g-C 3 N 4 in a 20 mL aqueous solution containing Cu(II) and Fe(III) ions), which led to the adsorption of ions on the g-C 3 N 4 structure [55]. A highly selective glucose-sensing (in human blood) biosensor based on ultrathin g-C 3 N 4 nanosheets doped with niobium (Nb) metal was synthesized by the pyrolysis method from urea [65]. A biosensor for 4-nitrophenol detection was developed by Vinoth et al. 4-nitrophenol is a very poisonous chemical compound released into the water during the production of some drugs, dyes, and leather, posing human health at high risk.

g-C 3 N 4 -Based Surface Plasmon Resonance (SPR) Biosensors
Surface plasmon resonance (SPR) sensing is a powerful probe of the interplays between protein-ligand, protein-DNA, protein-protein, and protein-membrane binding [67]. SPR biosensors are a very effective tool for measuring many biomarkers [68]. The main advantages of these biosensors are their fast response and ability to detect various analytes concurrently [69]. Moreover, among various new techniques available, SPR biosensors are the best optical biosensors for label-free, fast, and in situ diagnosis of molecules [40]. SPR is a physical optics phenomenon that can detect biomarkers because of the high sensitivity of surface plasmons to the dielectric medium [70]. In these biosensors, receptors are immobilized on the metal surface, interacting with the analytes and leading to dielectric alteration. This phenomenon affects the resonance condition of surface plasmons with specific surface plasmon waves (SPWs), allowing the transmission of photon's energy to the surface plasmons at the resonance angle resulting in the decrease of the light reflectance and thus the SPR curve [71]. Based on the characteristic of light, the SPR biosensors can be categorized into angular, wavelength, or intensity-modulated systems [72][73][74]. The Kretschmann configuration is the most recent version of SPR based on attenuated total reflection [54]. At an angle, part of light energy is transmitted to the surface plasmon, and the reflectance can be shown in the angular scanning.
The presence of adsorbed molecules on the biosensor surface varies the refractive index, and the SPR angle is changed accordingly [75].
Two-dimensional (2D) materials with large surface areas, such as g-C 3 N 4 , can act as the sensitive layers for SPR [40]. Duan et al. designed a surface plasmon resonance (SPR) biosensor based on a 2D nanocomposite of g-C 3 N 4 nanosheets and molybdenum disulfide quantum dots (MoS 2 QDs), adorned with chitosan-stabilized Au nanoparticles (CS-AuNPs) to detect prostate specific antigen (PSA) selectively. In this work, the MoS 2 QDs easily aggregated and reduced the sensitivity, but as a support for MoS 2 QDs, the g-C 3 N 4 nanosheets improved the biosensing performance for PSA detection. Additionally, the MoS 2 QDs@ g-C 3 N 4 @ CS-AuNPs-based SPR aptasensor showed a very low limit of detection (LOD), 0.77 ng·mL −1 , with good linearity range at PSA concentrations in the range of 1.0-250 ng·mL −1 [40].

g-C 3 N 4 -Based Electrochemical Biosensors
Electrochemical biosensors have been recognized as powerful diagnostic tests over the past years thanks to their unique advantages, such as simplicity, high sensitivity, and accuracy [76]. Three vital components are necessary to develop electrochemical biosensors: (I) a bioreceptor to link with analyte, (II) an electrode, and (III) a read-out system [77]. An electrochemical sensor requires a working reference and an auxiliary electrode; the working electrode in the electrochemical biosensor acts as a transducer in the reaction between the bioreceptor and the analyte. It generates a biological signal which changes into an electronic signal and is processed with high sensitivity [78]. On the other hand, Ag/AgCl-based reference electrode is kept at the site of the reaction to maintain a particular potential. The auxiliary electrode links the electrolytic solution and must be conductive; thus, gold or platinum are suitable candidates [79]. Some electrochemical methods for marker detection include voltammetric techniques (cyclic, square wave, or stripping), impedimetric, and amperometry. Of these techniques, cyclic voltammetry (CV) is preferred [77].
In an electrochemical biosensor, an electrode is the main component for immobilizing electron motion and biomolecules [80]. Nanomaterials have piqued attention due to their unique electronic characteristics [81]. The carbon allotropes can be applied as electrodes due to their effective electron transfer rate and high active surface area. Additionally, carbon nanostructured materials are significant in research due to their unparalleled properties, such as chemical stability and good conductivity [82]. g-C 3 N 4 is a polymeric semiconductor with a specific structure and high stability, making it a good nanocomposite for electrochemical biosensors [83]. g-C 3 N 4 is known as the most thermal stable allotrope of carbon nitrides [84], which can be used in the diagnosis system based on its catalytic ability [85]. Due to the low electron conductivity of g-C 3 N 4 , it has been used with other materials to enhance its surface conductivity. The g-C 3 N 4 derivatives can electrically connect to the redox center of biomolecules on the surface of the electrode. The electronic integration of the g-C 3 N 4 with various carbon types notably increases the surface area and conductivity [85]. The chemical exfoliation of bulk g-C 3 N 4 has been used to develop g-C 3 N 4 nanosheets for the detection of neurotransmitters, such as dopamine (DA). Kathiresan et al. developed a glassy carbon electrode (GCE) doped with bulk g-C 3 N 4 . The electrochemical activation of bulk g-C 3 N 4 was performed with a potential of 1.75 V in neutral pH conditions (pH 7.0). In the electrode oxidation reaction, the two-electron process is followed by the transfer of two protons, resulting in 5-HTquinoneimine. Figure 2 illustrates the redox reaction. Oxidation leads to the transfer of protons to form 5-HTquinoneimine and the reduction occurs in the quinone group on 5-HT quinoneimine [86].
In an electrochemical biosensor, an electrode is the main component for immobili electron motion and biomolecules [80]. Nanomaterials have piqued attention due to unique electronic characteristics [81]. The carbon allotropes can be applied as electr due to their effective electron transfer rate and high active surface area. Additionally bon nanostructured materials are significant in research due to their unparalleled pro ties, such as chemical stability and good conductivity [82]. g-C3N4 is a polymeric semi ductor with a specific structure and high stability, making it a good nanocomposit electrochemical biosensors [83]. g-C3N4 is known as the most thermal stable allotrop carbon nitrides [84], which can be used in the diagnosis system based on its catalytic ity [85]. Due to the low electron conductivity of g-C3N4, it has been used with other m rials to enhance its surface conductivity. The g-C3N4 derivatives can electrically con to the redox center of biomolecules on the surface of the electrode. The electronic inte tion of the g-C3N4 with various carbon types notably increases the surface area and ductivity [85]. The chemical exfoliation of bulk g-C3N4 has been used to develop gnanosheets for the detection of neurotransmitters, such as dopamine (DA). Kathires al. developed a glassy carbon electrode (GCE) doped with bulk g-C3N4. The electroch ical activation of bulk g-C3N4 was performed with a potential of 1.75 V in neutral pH ditions (pH 7.0). In the electrode oxidation reaction, the two-electron process is follo by the transfer of two protons, resulting in 5-HTquinoneimine. Figure 2 illustrates th dox reaction. Oxidation leads to the transfer of protons to form 5-HTquinoneimine the reduction occurs in the quinone group on 5-HT quinoneimine [86].

g-C 3 N 4 -Based Photoelectrochemical (PEC) Biosensors
The photoelectrochemical (PEC) detection method is a hopeful technique for biological assays [135], which is also a low-cost approach to transforming chemical energy into electricity under a flash of light [136], and PEC biosensors have become prominent due to their capability of biomolecules diagnosis. This method has had much consideration because of its high sensitivity, simplicity, and fast response [137]. In the PEC diagnosis system, light is used as an excitation source [138], allowing for a high sensitivity with low background signals [136]. The PEC cell includes three main components: (a) a lightharvesting semiconductor, (b) a metal electrocatalyst, and (c) adequate electrolytes among the working electrode and auxiliary electrode to generate PEC signals using redox reaction. Upon illumination, the redox reactions lead to a signal between the working and the auxiliary electrodes [139].
PEC biosensors use wide bandgap semiconductors as photoactive materials [63], changing optical energy to electrical and chemical energy [140]. g-C 3 N 4 is a responsive photocatalyst with a bandgap (2.7 eV) [141]. Additionally, one of the promising approaches is a photocatalytic reaction which can absorb visible light [82]. g-C 3 N 4 , as an inorganic polymeric semiconductor, possesses a graphite-like layer structure [142]. So, PEC biosensors show advantages over electrochemical and optical biosensors with high sensitivity and low cost. Hence research in the PEC biosensor for analyte detection has increased. Biomarkers detected using photoelectrochemical biosensors are summarized in Table 2.  For instance, Li et al. developed a PEC biosensor based on coral-like g-C 3 N 4 nanostructures to detect the metronidazole biomarker. Although metronidazole is a common antibacterial drug, it causes carcinogenic and genotoxic issues. Hence, the sensitive and facile detection of metronidazole's residues in typical oral medicine samples is an effective approach in health care. According to the results, coral-like g-C 3 N 4 nanostructures in the biosensor platform boosted the facility of signal amplification in the PEC sensing [168]. In the other study, Mao et al. applied the photosensitive CuO-g-C 3 N 4 nanostructures as an efficient photocathode in the PEC sensing of aflatoxin B1 (as a food contaminator and class 1 carcinogen). The conjugation of CuO to g-C 3 N 4 efficiently extended the optical absorption toward the visible region. The CuO-g-C 3 N 4 nanocomposite enhanced the PEC signaling for the sensitive detection of aflatoxin B1 [145].

g-C 3 N 4 -Based Fluorescent Biosensors
Fluorescent biosensors have been used in biological assays, owing to their high sensitivity, simple readout systems, lower response time, and visualization [175]. Fluorescent biosensors possess a specific ability to monitor biological cell targets [176,177]. Fluorescence spectroscopy has been widely applied to determine cancer and heavy metal ions [178,179]. Accordingly, the important advantages of this type of biosensor are that it is non-invasive, its capability to use fluorescence intensity, and its fluorescence lifetime. Additionally, using fluorescent nanomaterials, biomarker diagnosis can be highly selective and sensitive [180]. Fluorescent biosensors function by absorbing electromagnetic radiation, which is absorbed by fluorophores or fluorescently labeled molecules. Fluorescent biosensors can be divided into four types according to the signal-producing technique, including FRET (Forster Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging), FI (Fluorescence Intensity and its change), and FCS (Fluorescence Correlation Spectroscopy) [181]. The fluorescence biosensors have a single signal for detection and can easily be disturbed by environmental and instrumental conditions [182]. In luminescence, light is produced by excitation without increasing the temperature. Fluorescence is a type of luminescence that occurs over a short period and is created by electromagnetic excitation [183]. Moreover, in fluorescence, the time interval between absorption and emission is short [184]. Figure 3 shows the various schemes of fluorescent reagent-less protein-based biosensors [185].  Nanomaterials have introduced an attractive method of developing low-cost and portable fluorescent devices [186]. In recent decades, a new group of 2D nanomaterials has attracted research attention. g-C3N4 nanosheets supply an iterating choice for bioimaging and bioprobes applications [187,188] . Additionally, the N-contain structure for the g-C3N4 nanosheet provides the potency for coordination with proton or metal ions [189]. The mentioned unique characteristics of g-C3N4 nanosheets make this useful for developing fluorescent biosensors or bioprobes. Table 3 shows some of the developed fluorescent biosensors for detecting different biomarkers. Nanomaterials have introduced an attractive method of developing low-cost and portable fluorescent devices [186]. In recent decades, a new group of 2D nanomaterials has attracted research attention. g-C 3 N 4 nanosheets supply an iterating choice for bioimaging and bioprobes applications [187,188]. Additionally, the N-contain structure for the g-C 3 N 4 nanosheet provides the potency for coordination with proton or metal ions [189]. The mentioned unique characteristics of g-C 3 N 4 nanosheets make this useful for developing fluorescent biosensors or bioprobes. Table 3 shows some of the developed fluorescent biosensors for detecting different biomarkers. Hatamie et al. applied g-C 3 N 4 nanosheets to develop a label-free bioassay system for diagnosing metronidazole in biological fluids. The switch-off green fluorescence biosensor provided rapid sensing with a linear detection range from 0.01 to 0.10 µg mL −1 [62]. Dopamine is a neurotransmitter with substantial biological functions in neuroendocrine regulations, and its abnormal content in the human serum leads to Parkinson's and Alzheimer's disease. Lv et al. investigated the g-C 3 N 4 nanofibers in the fluorescent probe for dopamine sensing. It provided a sensitive detection platform with a limit of detection (LOD) lower than 17 nM [199].

g-C 3 N 4 -Based Electrochemiluminescent (ECL) Biosensors
Over the past several decades, many studies on electrochemiluminescence (ECL) biosensors have been conducted in various fields, such as chemical analysis and clinical diagnostics or food analysis. Electrochemiluminescence, or electrochemical chemiluminescence, is the light emission produced from molecular types by an electron transfer process. Additionally, ECL is triggered by an electrochemical reaction of the luminophores on an electrode surface. Moreover, the significant advantages of ECL are its high sensitivity and selectivity. In ECL biosensors, electrochemically generated intermediates endure an extremely exergonic reaction to turn out into an electronically excited state. ECL-based biosensors utilize specific biological diagnosis elements, such as enzymes, antibodies, aptamers, peptides, and proteins to selectively recognize a particular analyte and generate an ECL signal [208]. The basis of the method is on diagnosis interaction among biological cognizance elements and the corresponding targets by ECL release alterations. Accordingly, two main components are needed in standard ECL detection: ECL active types and biological cognizance elements.
Depending on the reaction that induces the ECL signal emission, there are several sensing systems for medical applications.
In systems that are based on the chemical reactions of the luminophores and coreactants, the chemical reaction between the luminophore and the co-reactant and is used for detecting diverse biomarkers.
The second type is systems that involve the co-reaction accelerator-involved reactions. In these systems, the reaction mixture is mixed with co-reaction accelerators. These accelerators are involved in generating electrochemiluminescent reactions in terms of facilitating the ECL reaction rate of co-reactant to produce several intermediates.
In systems that incorporate resonance energy transfer (RET) reactions, instead of using only one luminophore, the signal is emitted via two different emitters by incorporating a RET.
For systems that incorporate an enzyme reaction-based signal amplification, binding events between target analytes and probe DNAs initiate. High sensitivity and extension of the dynamic range of the modulation are some of the benefits of these systems [209]. Figure 4 represents the metioned types of ECL biosensors based on the reactions leading to ECL signal emission. of the dynamic range of the modulation are some of the benefits of these systems [209]. Figure 4 represents the metioned types of ECL biosensors based on the reactions leading to ECL signal emission. g-C3N4 has a large surface area, and this carbon-based material can enable more sites to sequester charge carriers. Additionally, g-C3N4 has high electron conductivity, and they can successfully separate and then transfer charge carriers [208]. Some of the electrochemiluminescent biosensors are represented in Table 4.  g-C 3 N 4 has a large surface area, and this carbon-based material can enable more sites to sequester charge carriers. Additionally, g-C 3 N 4 has high electron conductivity, and they can successfully separate and then transfer charge carriers [208]. Some of the electrochemiluminescent biosensors are represented in Table 4. Wu et al. developed an ECL immunosensor to detect the cancer biomarker CA125; nevertheless, its relatively low concentration in human body fluids limits the conventional methods. The disposable and label-free biosensor provided a sensitive detection via ECL emission when multifunctional g-C 3 N 4 captures the CA125 tumor marker in the range from 0.001 to 5 U/mL, with a LOD of 0.4 mU/mL [213]. Wang et al. proposed a novel ECL bioassay system for detecting the HL-60 cancer cells based on g-C 3 N 4 nanosheets and Ag-PAMAM-luminol nanocomposites (Ag-PAMAM-luminol NCs), where g-C 3 N 4 nanosheets were applied as a reductive-oxidative ECL emitter. The overlapping of the ECL spectrum of g-C 3 N 4 nanosheets and the adsorption spectrum of Ag nanoparticles as well as luminol oxidative-reductive ECL emissions simultaneously contributing to the sensitive detection of the HL-60 cancer cells, with 150 cells as the limit of detection [222].

Conclusions and Future Perspectives
The early diagnosis of diseases is the best way to improve the treatment prognosis and decrease the side effects of illnesses. Biosensors based on nanomaterials are efficient for this approach due to the high and rapid sensitivity in diagnosing the target molecules that arises from the specific properties of nanomaterials. In recent years, the nanosheets of g-C 3 N 4 and their derivatives have attracted a lot of interest owed to their outstanding optical properties (high photoluminescence yield), high surface area, electrical conductivity, antimicrobial activity, and good thermal and chemical stability. Several simple and high-yield methods have been used to synthesize g-C 3 N 4 -based materials, such as the pyrolysis of low-cost materials, including melamine and urea. C 3 N 4 -based materials have also been used in various biosensors (SPR, EC, PCL), which demonstrates that they are promising candidates in this field. Moreover, g-C 3 N 4 -based biosensors show high and rapid sensitivity for detecting diseases, such as cancer; other targets in biological samples; or even the detection of pollutants. Thus, g-C 3 N 4 is a new carbon-based 2D nanomaterial for biosensing, and it is expected that in the near future, g-C 3 N 4 -based biosensors will be improved in order to be more sensitive in diagnosis and functionalized in order to have more selectivity to attach the receptors. We anticipate that further research will be conducted on addressing the intrinsic shortcomings attributed to g-C 3 N 4 , including poor specific surface area, limited light absorption range, and poor dispersibility in organic and aqueous media.

Conflicts of Interest:
The authors declare that there is no conflict of interest regarding the publication of this article.