Graphitic Carbon Nitride: A Novel Two-Dimensional Metal-Free Carbon-Based Polymer Material for Electrochemical Detection of Biomarkers

Abstract

Graphitic carbon nitride (g-C₃N₄) has gained significant attention due to its unique physicochemical properties as a metal-free, two-dimensional, carbon-based polymeric fluorescent substance composed of tris-triazine-based patterns with a slight hydrogen content and a carbon-to-nitrogen ratio of 3:4. It forms layered structures like graphite and demonstrates exciting and unusual physicochemical properties, making g-C₃N₄ widely used in nanoelectronic devices, spin electronics, energy storage, thermal conductivity materials, and many others. The biomedical industry has greatly benefited from its excellent optical, electrical, and physicochemical characteristics, such as abundance on Earth, affordability, vast surface area, and fast synthesis. Notably, the heptazine phase of g-C₃N₄ displays stable electronic bands. Another significant quality of this semiconductor material is its excellent fluorescence property, which is also helpful in preparing biosensors. Based on g-C₃N₄, electrochemical biosensors have provided better biocompatibility, higher sensitivity, low detection limits, nontoxicity, excellent selectivity, and surface versatility of functionalization for the delicate identification of target analytes. This review covers the latest studies on using efflorescent graphitic carbon nitride to fabricate electrochemical biosensors for various biomarkers. Carbon nitrides have been reported to possess excellent electroactivity properties, a massive surface-to-volume ratio, and hydrogen-bonding functionality, thus allowing electrochemical-based, highly sensitive, and selective detection platforms for an entire array of analytes. Considering the preceding information, this review addresses the fundamentals and background of g-C₃N₄ and its numerous synthesis pathways. Furthermore, the importance of electrochemical sensing of diverse biomarkers is emphasized in this review article. It also discusses the current status of the challenges and future perspectives of graphitic carbon nitride-based electrochemical sensors, which open paths toward their practical application in aspects of clinical diagnostics.

1. Introduction

Road Map from Carbon to g-C₃N₄

Graphitic carbon nitride, g-C₃N₄, is a novel two-dimensional carbon-based material that has garnered intense interest for constructing such electrochemical sensors due to its excellent properties. Carbon nitride, one of the earliest metal-free synthetic polymeric materials, was first discovered by the scientist Berzelius in 1834 [1]. In the meantime, Lie-big investigated the use of tri-s-triazine and triazine to confirm the fundamental units of melamine, melem, melam, and melon. When polymerizing aluminium chloride with potassium thiocyanate, a yellow powder with C–N bonding is obtained, known as “melon” [2]. Later, Franklin investigated melon derivatives in 1922 and found that by heating a mixture of melon and thiocyanate, carbon nitrides (g-C₃N₄) were produced, which were similar to a graphite-like structure [3]. Scientists Redemann and Lucaset pointed out that CN is a polymer based on tri-amino-tri-s-triazine with properties similar to graphite and melon [4]. The term “graphitic” comes from graphite, also known as sp² hybridized graphene. The materials are separated by weak van der Waals forces between layers [5]. The fundamental structure of carbon nitride (CN) is composed of tri-s-triazine units, as Pauling and Sturdivant established in 1937. Research on g-C₃N₄ was significantly hampered because other compounds cannot dissolve it. Many scientists paid attention to β-C₃N₄ in the early 1990s because of its diamond-like structure, high bulk modulus and hardness, and sp³ structure [6]. Then, theoretically and practically, Liu and Cohen developed C₃N₄ in 1989, creating b-C₃N₄, which becomes as hard as a diamond when Si is substituted with C in b-Si₃N₄ [7].

Graphitic carbon nitride (g-C₃N₄) is a naturally occurring n-type semiconductor with a 2.7 eV bandgap [8]. Its constituent raw materials are carbon, nitrogen, and hydrogen atoms [9]. Owing to its superior structural resemblance to graphitic nanostructure, it is named “g-C₃N₄” [10]. It exhibits good blue photoluminescence properties over a broad visible absorption range of 430 to 550 nm [11]. Its chemical stability strengthens it against weak acids and bases that enable solubility in organic solvents, such as diethyl ether, acetone, ethyl acetate, aqueous alkaline, and acid solutions [12]. It shows up-and-down conversion fluorescence (FL) properties, often closely associated with the s-triazine ring’s π–π* transition [2]. It has more robust atomic frameworks and exhibits unique electrical, mechanical, thermal, and optical properties [13]. Its response to visible light lacks metal attributes and displays optical responsiveness within the visible spectrum. It is a potential candidate for various applications due to several properties. They have a high refractive index, exceptional thermal conductivity, low cost, chemical inertness, fast charge transfer, ease of synthesis, biocompatibility, lightweight, and high activity. These qualities contribute to the tunability of the material and its electronic band structure [14].

2. Structural Properties of g-C₃N₄

2.1. Phases of g-C₃N₄

Initially, Hemley and Teter suggested five different forms of C₃N₄ in 1996, including cubic C₃N₄, pseudocubic C₃N₄, α-C₃N₄, β-C₃N₄, and g-C₃N₄, all of which were shown to be hard materials, as represented in Figure 1. The first four CN materials with crystal structures have unusual valence bond structures that make them as hard as diamond. In contrast, the g-C₃N₄ material is in the most stable phase, the soft phase, and has minimum energy with greater stability, spectacular semiconducting properties, and a simple preparation process [15]. It comprises only bonds between carbon and nitrogen, with no electron localization in the p state. The tri-s-triazine (heptazine)-based structure, s-triazine-based orthorhombic structure, and s-triazine-based hexagonal structure are the three forms of g-C₃N₄ [16]. The band gaps for each of the seven phases are 5.49, 4.85, 4.30, 4.13, 2.97, 0.93, and 2.88 eV, in that order [17]. The two main tectonic units that form g-C₃N₄ among the seven phases are triazine C₃N₃ (melam) and tri-s-triazine C₆N₇ (melem). Interestingly, the building block of g-C₃N₄ is tri-s-triazine-based g-C₃N₄, which is considered to be the most stable phase of the varied g-C₃N₄ structure and possesses evenly distributed three-sided nanopores (3.11 A°) throughout the entire laminar framework [18]. The parts of melem are tri-s-triazine subunits (C₆N₇) joined by nitrogen atoms. There are indirect band gaps in every phase of g-C₃N₄, except the pseudocubic and g-h-triazine phases [19].

2.2. Electronic Structure of g-C₃N₄

The topology of the two-dimensional polymeric conjugated sp²-hybridized g-C₃N₄ exhibits a zigzag-like layered structure. It comprises planar amino groups joined to aromatic poly-tri-s-triazine (heptazine) units, which exhibit Bronsted basic functions, Lewis basic functions, and hydrogen bonding motifs, as represented in Figure 2a [20]. Weak van der Waals forces hold the layers together through hydrogen bonds, with strong C–N covalent connections maintained at a distance of 3.3 nm between each layer, which allows for facile exfoliation into nanosheets [21]. It comprises carbon and nitrogen atoms with four and five valence electrons and identical atomic sizes, as shown in Figure 2b. These atoms form constant covalent bonds. In g-C₃N₄, lone pair electrons of nitrogen are primarily responsible for forming the valence band and the band structure (Figure 2c) [22]. Generally, it has been determined that the band gap of g-C₃N₄ produced in a laboratory is around 2.7 eV, representing the absorption from the lowest unoccupied molecular orbitals (LUMOs) to the highest occupied molecular orbitals (HOMOs). The valence band (VB) comprises σ and π states, whereas the conduction band (CB) mostly comprises π* states. This arrangement improves electrical characteristics by enabling an efficient charge transfer and separation process. The nitrogen P_z and carbon P_z orbitals give rise to the HOMO and LUMO levels, respectively [23]. Like many semiconductor materials, g-C₃N₄ nanomaterials exhibit unique photoluminescence (PL) characteristics. These features arise from the radiative transitions involving antibonding orbitals (π* or δ*) in the conduction band (CB) associated with sp² C−N bonds, as well as the lone pair (LP) in the valence band (VB) from the edge N 2p orbitals [24]. The electronic structure of g-C₃N₄ allows exciting electrons to higher energy states followed by recombination, resulting in photoluminescence. The fluorescence of g-C₃N₄ can be controlled by changes in its structure or the introduction of defects [25]. Defects (such as vacancies or nitrogen defects) can affect how the material performs in various applications by introducing localized states inside the band gap that can improve the mobility of charge carriers and reactivity [26].

Because of the previously described properties of CNs, g-C₃N₄ material can be used in various prospective fields, including the conversion of solar energy, water purification, carbon dioxide capture and storage, hydrogen storage, catalysis, and sensing [27]. This young carbonaceous nanomaterial has many advantageous characteristics, including a porous framework with electron-rich properties, fundamental surface functionalities, exceptional electrical, thermal, optical, and mechanical qualities, a high specific surface area that provides more active sites and allows for increased substrate adsorption, surface functionality, exchangeable N–H groups, and redox activity. All of these qualities combine to make it an excellent material [28].

3. Construction of g-C₃N₄

The construction of g-C₃N₄ involves choosing the appropriate precursor material and synthesis routes to prepare the different morphologies.

3.1. Precursor Material

Generally, bulk g-C₃N₄ can be synthesized through direct thermal polymerization or thermal polycondensation of carbon and various nitrogen-rich, oxygen-free compounds that do not contain direct C–C bonds [29]. Examples of precursor materials include cyanamide, dicyandiamide, urea, thiourea, melamine, formamide, ammonium thiocyanate, guanidine hydrochloride, cyanuric acid, and mixtures of precursors such as urea-citric acid monohydrate, citric acid-thiourea, melamine-thiourea, melamine-urea, sodium citrate-urea, ammonium citrate-urea, tartaric acid-urea, barbituric acid-dicyanamide and sulphur-mixed melamine [30]. Studies have shown that the preparation protocol and precursors can significantly affect the physicochemical characteristics of g-C₃N₄, such as the C/N ratio, surface area, porosity, absorbance, and photoluminescence. The source materials are processed at temperatures between 400 to 650 °C for different time intervals to achieve a carbon-to-nitrogen ratio as close to the theoretical value of 0.75 as possible, yielding g-C₃N₄, as illustrated in Figure 3. Various factors influence the optical and electrical features of g-C₃N₄, such as its precursor type, heating rate, and reactivity conditions, including time and pyrolysis temperatures [31].

3.2. Morphology of g-C₃N₄:

The bulk form of g-C₃N₄ has a low quantum yield (QY), weak dispersibility, smaller active sites, and low surface area, making it impractical for biosensing applications [32]. The physicochemical characteristics and the sensing abilities have been improved and enhanced through the creation of various nanoarchitectures (Figure 4) of g-C₃N₄, including 2D nanosheets [33,34], 2D nanotubes [35], 0D nanodots [36], 1D nanorods [37,38], 1D nanowires [39], 0D quantum dots [40,41], 0D hollow spheres [42], nanoparticles [43], and 3D g-C₃N₄ mesoporous materials [44], nanofibrous materials, g-C₃N₄ film or self-standing structures, and nanocomposites through various synthesis routes. These nanostructures can be employed as the base material for sensing applications [19].

3.3. Synthesis Route

Generally speaking, there are two categories of methods for synthesizing g-C₃N₄ nanomaterials: the top-down and bottom-up approaches, as visualized in Figure 5.

3.3.1. Top-Down Technique

Top-down synthesis involves cutting down bulk materials into smaller ones by using different driving forces, such as mechanical grinding, liquid exfoliation, chemical-oxidation exfoliation, chemical etching exfoliation, microwave-assisted solvothermal methods, and solvothermal/hydrothermal treatment. Due to its cost-effectiveness, low-temperature demand, simplicity of operation, and variety of design variations, this method tends to be more suitable for large-scale production. While this method is fast, it has some drawbacks, such as time consumption, poor efficiency, laborious handling, and poor surface quality, where defects may be introduced during the process. g-C₃N₄ nanomaterials made from bulk g-C₃N₄ are promising for sensing applications because of their chemical and thermal stability, photosensitivity, adsorptive capacity, and photo-conversion ability [19].

Exfoliation Technique

Exfoliation involves converting bulk layers into thin sheets to increase the number of active sites and improve physicochemical and optical properties. Controlled exfoliation is possible in layered materials by incorporating surfactants and ions and the ability to control parameters such as temperature and shear strength [30]. Top-down exfoliation is most commonly implemented through chemical, thermal, or ultrasonic methods [45]. By treating g-C₃N₄ with a strong acid (Conc. HCl), carboxyl groups are introduced with protonation, resulting in g-C₃N₄ nanosheets (g-C₃N₄NSs) [46]. It is well suited for forming tunable heterostructures for reassembly with various charged materials to produce stable g-C₃N₄NS colloids with amphoteric properties. After sulphuric acid (H₂SO₄) reflux treatment, g-C₃N₄ was soluble in water after using strong oxidative acids. However, g-C₃N₄ typically loses its planar structure after acid treatment. Due to the large electronegativity of oxygen elements, g-C₃N₄ can be isolated using an alkaline medium. Amino groups in g-C₃N₄ can be replaced with stable hydrogen bond groups in alkali [47]. By using hot water for thermal exfoliation, ultrathin g-C₃N₄NSs are obtained. Water molecules may intercalate into carbon nitride interlayer spaces, further hydrolysing links between tri-s-triazine units [48]. According to Dong et al., thermal exfoliation of g-C₃N₄NSs led to significant improvements in performance between 450 °C and 550 °C due to enhanced valence band (VB) max and conduction band (CB) min [49]. It is noteworthy that ultrasonic exfoliation, such as exfoliation in water for 16 h, is an efficient top-down strategy [50]. Liquid-phase exfoliation is becoming more popular among the most commonly used exfoliation methods. The production of g-C₃N₄NSs can be achieved by adding an appropriate liquid to bulk g-C₃N₄ and then ultrasonically exfoliating it. A remarkable dispersing efficiency is achieved by matching the surface energies of bulk g-C₃N₄ and the solvent, respectively, 115 and 102 mJm⁻². Bulk C₃N₄ can also be exfoliated using several solvents, including isopropanol, NMP, water, ethanol, and acetone, where IPA produces free-standing g-C₃N₄NSs due to its reduced mixing enthalpy [51].

Thermal Polymerization

Graphitic carbon nitride is said to be a material with some special features. It is commonly prepared using thermal polymerization by condensing precursors rich in nitrogen, such as melamine, cyanamide, dicyandiamide, and urea, within an inert atmosphere at temperatures ranging from 500 to 600 °C [52]. The organic precursors decay during the process, and gases such as ammonia result in the residual material transforming into the conjugated framework of g-C₃N₄ [53]. The material is heated and then cooled to room temperature. Further optional post-synthesis procedures can be included until it can be synthesized [54]. It is highly valued, especially in terms of ease of operation and economy. It also allows massive scales besides the purity of the produced g-C₃N₄, which has a graphitic, layered structure, making it an ideal candidate for the semiconductor-based process and photocatalysis [55]. Tian Liu et al. report that ultrathin g-C₃N₄ nanosheets vacancy defect modifications exhibit remarkably enhanced photocatalytic activity, while the recordable hydrogen evolution rate is up to 5.12 mmolh⁻¹g⁻¹, 7.64-fold greater than that of standard g-C₃N₄. Meanwhile, ultrathin carbon nitride (UCN) could degrade 85% of pollutants, tetracycline (TC) and ciprofloxacin (CIP), within 30 min with optimal use of visible light and rapid charge transfer [56]. L. Madiededo Florentino et al. prepared g-C₃N_4, especially in an atmosphere of CO₂, and showed higher hydrogen evolution reaction (HER) rates up to 3858 μmolh⁻¹g⁻¹ for g-C₃N₄ derived from urea, whereas those based on melamine reached 2225 μmolh⁻¹g⁻¹ in static air. There was no apparent relationship between HER and the degree of polymerization, but high band gap values corresponding to s-triazine structures led to better HER [57].

Hydrothermal/Solvothermal Synthesis

Hydrothermal and solvothermal processes are the most effective and most accessible methods for generating g-C₃N₄ nanostructures. g-C₃N₄ was synthesized in deionized water using a one-pot green hydrothermal technique by Zhang and coworkers. An appropriate quantity of bulk g-C₃N₄ was dissolved in deionized water using ultrasonication to create a solid–liquid mixture. It was discovered that g-C₃N₄ nanostructures were produced over 12 h at 200 °C using a hydrothermal method. After 24 h of natural precipitation and cooling to room temperature, a clear supernatant was obtained. It was demonstrated that even a tiny quantity of freshly synthesized g-C₃N₄ has a remarkable lubricating effect [58]. By utilizing a strong base and EtOH to thermally condense bulk g-C₃N₄, Zhan and his colleagues discovered a straightforward, one-pot method for producing highly luminous graphitic carbon nitride quantum dots (g-C₃N₄QDs) and g-C₃N₄NSs (KOH). Usually, ethanol was used to dissolve bulk g-C₃N₄ powder before combining it with a concentrated KOH solution. In an autoclave, the mixture was stirred and maintained at 180 °C for 16 h. The generated g-C₃N₄QDs had a mean diameter of about 3.3 nm and a particle size distribution [59]. A hydrothermal method supported by ascorbic acid produced mesoporous g-C₃N₄ with greater crystallinity. A specific quantity of ascorbic acid (AA) powder was dissolved in pure water before ethanol was added to create the ascorbic acid (AA) solution. The AA solution was then thoroughly blended with a large amount of g-C₃N₄ powder. The g-C₃N₄ suspension was put into a Teflon-lined autoclave and kept at 180 °C for 4 h after ultrasonic dispersion. The sample was centrifuged, cleaned with ultrapure water and ethanol, and then dried in a hot oven overnight to obtain the mesoporous g-C₃N₄ samples [60].

3.3.2. Bottom-Up Approach

Bottom-up techniques typically involve assembling and constructing complex structures out of simple components. Highly reliable massive structures can be assembled atom by atom as a consequence of the physical and chemical activities at the nanoscale. The method effectively produces g-C₃N₄ and offers potential nanostructure products with uniform composition and superior long- and short-range ordering. It encompasses a variety of approaches, including templating and template-free techniques, microwave-assisted methods, supramolecular preorganization, hydrothermal/solvothermal, ultrasonication, chemical cleavage, and sol-gel synthesis, etc. [32].

Templating Technique: Hard and Soft

Templating is a promising way to create nanoscale g-C₃N₄ with porous structures, sizable guest-accessible surface areas, and many active sites. There are two techniques: soft templating and hard templating [61]. By changing the type of hard and soft templates, the morphology and pore structure of g-C₃N₄ can be controlled. The modification of g-C₃N₄ nanostructures can be done more sustainably using the soft templating method. To incorporate porosity and even doping elements into the g-C₃N₄ framework, organic templates such as surfactants, amphiphilic block polymers, and ionic liquids are typically utilized. These templates might gather near the interfaces, impacting the development of the inorganic phase close by [62]. Hard templating methods refer to using hard templates during the fabrication of g-C₃N₄, such as silica and alumina oxide. Numerous nanostructured g-C₃N₄ have since been created, including hollow nanospheres and nanospherical g-C₃N₄, nanosheets, and nanorods. First, the mesopores of the templates are infused with the precursor solution. g-C₃N₄ would arise in the defined porosity due to the hard templates’ fixed architecture. It is accessible when the hard template has been eliminated. The last step is the removal of soft and hard templates from the final product by various methods such as etching, calcination, and acid treatments [63]. Groenewolt et al. pioneered the direct polymerization of cyanamide in a hard template, such as a mesoporous silica host matrix, to create g-C₃N₄ mesoporous nanoparticles with a diameter of 5 nm. However, in the case of soft templating, residual carbon could be discovered even after the last step, which can impact the catalytic properties of the resulting g-C₃N₄. The removal of the template in the last step of hard template plating processes typically calls for powerful acids like hydrogen fluoride, which could cause the pore structure to collapse [64]. Additionally, concentrated acids may harm the environment, demanding further treatment. Wang et al. developed porous g-C₃N₄ with calcium carbonate particles as the hard material to avoid the need for concentrated acids [61].

Template-Free Technique

Unlike procedures that use templates, template-free techniques do not require dangerous chemicals like hydrofluoric acid to wash away the template (e.g., silica matrices). The benefit of the template-free method is that the pore structure can be created by treating precursors beforehand. This can greatly reduce the number of raw materials used, and toxic and hazardous substances can be avoided. Generally, the decomposition of the aforementioned precursors might provide g-C₃N₄ without using any other substances. The obtained g-C₃N₄ could undergo additional treatment that would alter its morphology. For example, the morphology of g-C₃N₄ could be changed from nanoplates to nanorods using a straightforward reflux procedure that included exfoliation, regrowth, and rolling. Additionally, g-C₃N₄ might be produced by infrared heating of dicyandiamide precursors without the inclusion of any additive chemicals [65]. Han et al. tried combining freeze-drying, assembly, hydrothermal treatment, and thermal calcination to create g-C₃N₄ that resembles seaweed [66]. To prepare nanoporous g-C₃N₄, Gu et al. created a solvothermal treatment that was followed by a post-calcining technique in an argon environment [67]. Xie and colleagues designed a solvothermal method for producing g-C₃N₄ nanofibers and wires using melamine and cyanuric chloride as the starting materials [68]. To make phosphorus-doped carbon nitride, Wang and colleagues employed melamine as a precursor and ammonium dihydrogen orthophosphate as a phosphorus source. Phosphorus halted the growth of the g-C₃N₄ crystal, increased the unique surface area, increased the effectiveness of electron and hole separation, and reduced the energy band gap [69].

Supramolecular Preorganization

In the supramolecular preorganization approach, there is no requirement for an external template [70]. It may employ molecular self-assembly, in which non-covalent bonding can generate a well-defined structure and a strong aggregate morphology [71]. Hydrogen bonds must shape the supramolecular aggregate topologies produced by non-covalent interactions. The generated stable aggregate structures defined the ultimate structure of the compounds. The supramolecular preorganization resulting from combining the melamine precursor and triazine compounds was further calcined to produce g-C₃N₄ [72]. The supramolecular arrangement can be more specially designed by attaching it to a molecular structure. For instance, melamine, 2,4-diamino-6-phenyl-1,3,5-triazine, and cyanuric acid could combine to form a supramolecular compound. The stoichiometric mass of its predecessors may control its morphology. Similarly, barbituric acid, urea, and caffeine could also be used to generate supramolecular complexes with the cyanuric acid-melamine complex [73].

Microwave-Assisted Process

The microwave method has several advantages, including a low processing temperature, time savings, and convenience. The polymerization of the g-C₃N₄ precursors that could help produce g-C₃N₄ can be accelerated by microwave. Additionally, microwave-based p-electron incorporation into g-C₃N₄ is simple [30]. Recent research has demonstrated that g-C₃N₄ with distinct melem and its partially condensed form may be prepared using a microwave, opening up the possibility of examining the contributions of each component to the photocatalytic performance [2]. Urea and sodium citrate were used as precursors in a microwave-assisted method for producing g-C₃N₄QDs, which has already been described. The author obtained a suspension by mixing the precursors in deionized water and whisking them together. For five minutes, the mixture was microwaved in a household microwave. The resultant yellowish material was refined by ethanol treatment and centrifugation after cooling to room temperature. A dialysis membrane was then used to dialyze the solution against deionized water. The resulting solution was then heated to 50 °C and evaporated. Finally, deionized water was combined with the g-C₃N₄QDs. The creation of a g-C₃N₄ layer, which could be uniformly formed on the substrate, can be aided by thermal vapour condensation. Additionally, the vapour–solid deposition approach might modify the form and architecture of g-C₃N₄ [74].

Ultrasonication

The ultrasonication process utilizes the acoustic vibrations of ultrasonic waves to separate the layers. Since the layers of g-C₃N₄ exhibit weak van der Waals forces in the solvent, water, ethanol, isopropanol, acetone, and N-methyl-pyrrolidone are examples of typical solvents [51,75]. Because water can be processed more easily than other solvents after usage, it is the solvent that offers the most promise among them. Zhang’s group was the first to disclose liquid exfoliation of g-C₃N₄ using water as a solvent by ultrasonication. They discovered that liquid exfoliation of g-C₃N₄ might be utilized to modify the physicochemical properties [76,77].

Sol-gel

The preparation of g-C₃N₄ via the sol-gel method is another option. Here, the precursor of g-C₃N₄ is combined with the precursor of silica or sol of silica. The sol-gel approach requires a step to remove the template, just like the hard template method. The silica template is created during the preparation of g-C₃N₄ instead of being pre-synthesized, as is the case with the hard template approach [78].

Chemical Cleavage

Besides the aforementioned widely employed techniques, there are additional ways to prepare g-C₃N₄. Diverse architectures of g-C₃N₄, which typically proceed via hydrolysis under acid or alkaline conditions, could be prepared using chemical cleavage. During the acid or chemical treatment process, the molecule of g-C₃N₄ was partly broken. Different morphologies of g-C₃N₄ could be produced by varying the hydrolysis conditions, including carbon nitride nano-leaves, nanotubes, 3D network carbon nitride, and carbon nitride nanobelts [14,79].

Hydrothermal/Solvothermal

Hard and soft template removal requires laborious procedures and potentially dangerous substances, such as ammonium bifluoride and hydrogen fluoride, both of which are energy-intensive and hazardous to the environment. Therefore, creating an effective process for producing g-C₃N₄ nanomaterials that do not require hard or soft templates is imperative. Melamine and sodium citrate were used as precursors in a simple, one-step hydrothermal method developed by Zhuang and his colleagues to produce g-C₃N₄NSs. Before the treatment with melamine, water was mixed with sodium citrate. This mixture underwent ultrasonication and was heated for 4 h at 200 °C in an autoclave reactor. The resulting g-C₃N₄NSs exhibited bright blue fluorescence with a quantum yield of 48.3% [80]. Lu and colleagues created a facile, one-step method for creating OS-g-C₃N₄QDs from thiourea and citric acid. In the context of cell imaging, the as-synthesized OS-g-C₃N₄QDs displayed bright blue fluorescence with high quantum yield, increased biocompatibility, and reduced cytotoxicity [81]. Zhao and colleagues created three distinct water-soluble g-C₃N₄QDs using a straightforward and environmentally friendly single-step hydrothermal procedure, using urea as the nitrogen precursor and citric acid, as well as its salts, as the carbon precursor [82]. According to the molar proportions of urea to citric acid, sodium, and ammonium citrate, the mean diameters of g-C₃N₄QDs were 4.6, 4.1, and 6.3 nm, respectively [83].

4. Biosensing Application of g-C₃N₄

4.1. Electrochemical Biosensor (ECB)

An electrochemical biosensor is a chemical sensor that can detect a biochemical signal produced due to the interaction between the targeted biological analytes and the sensing surface and convert it into electrochemical signals. It rapidly detects small targeted biological compounds with high selectivity and sensitivity as it combines electrochemistry with biological reactions. It works on the principle of a redox reaction, which takes part in the interface between the biological sample and the electrode surface. Here, the electrode plays an important role in converting biological changes into electrochemical signals like voltage and current [30]. It can be measured from the electrochemical workstation setup through electrochemical techniques (ECT), namely cyclic voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry (DPV), amperometry, conductometry, potentiometry, and electrochemical impendence spectroscopy (EIS). It has some significant features, such as being cost-effective, highly efficient, having a faster response time, less operating power, and reliable and onsite detection [84].

4.2. Significance of g-C₃N₄ in Electrochemical Biosensing

Graphitic carbon nitride is more appealing in electrochemical biosensors than other materials due to the high surface-to-volume ratio, tunable electrical characteristics, and broad active edges. The reaction between the molecules/ions of the analyte and the g-C₃N₄ nanosheets modifies the attributes of both original components. These interactions occur by non-covalent contacts or covalent bonding of reactive species which have undergone chemical reactions with g-C₃N₄. Non-covalent interactions are generally beneficial for real-time monitoring that requires rapid reaction and recovery, whereas covalent bonds are required when analytes remain stable and immobile on the surface. Additionally, by combining the atomic-scale characteristics of different materials, the heterostructures of g-C₃N₄ exhibit novel physicochemical interactions that lead to outstanding performance of the device [85]. It acts as a promising aspirant in the bioanalytical platform as the structure involves non-covalent functionalization through electrostatic and hydrophobic interactions and p-stacking [86]. It achieves tremendous attention in the biosensing field as it has numerous significances, which include flexible structural design, abundant in amine functional groups, readily available precursor material, facile synthesis strategy, rapid detection, easy handling, absolute determination, cost-effective, good selectivity, stability, biocompatibility, low toxicity, lower detection volume, outstanding fluorescence quantum yield, and unique electroluminescent and photoelectrochemical properties [87].

The electrochemical mechanism is based on the electron transfer and adsorption mechanism. An enhanced electron transfer rate is observed in g-C₃N₄ when a high surface area and desirable defects exist. This is essential for redox reactions, where swift electron transfer is required for effective sensing. In the adsorption mechanism, surface properties of g-C₃N₄ may influence interaction with the target analytes. Additionally, defects would enhance the adsorption energy of the analytes, leading to improved detection sensitivity [88].

The thickness of efflorescent graphitic carbon nitride significantly influences its physicochemical and electrochemical properties [76]. Typically, increased layers of g-C₃N₄ exhibit decreased porosity and surface area. This limitation in the accessibility of the reactants may hinder chemical reactions and electrochemical activities [89]. In the aspects of the dynamics of charge carriers, the thinner layers of g-C₃N₄ have short diffusion paths, leading to better mobility for charge carriers; thus, catalytic and electrochemical responses will improve [90]. The thicker layers would trap the charge carrier, leading to loss through recombination. The electronic band structure is modified through the thickness of g-C₃N₄, followed by any impact on optical absorption properties. Generally, thinner films result in a smaller band gap that gives greater light absorption efficiency. The bulkier g-C₃N₄ may be more structurally stable throughout electrochemical reactions, but thinner layers lead to reduced electroactivity due to the decreased number of active sites [91]. Additionally, the thickness of g-C₃N₄ affects its catalytic properties. The thinner layers show more accessible sites for reactions to occur; this should boost catalytic efficiency, especially in photochemical applications [92].

The performance of g-C₃N₄ in electrochemical sensors is intricately linked to its morphology, structure, and defects. With an understanding and optimization of these factors, highly sensitive and selective sensors for various applications can be designed. The electrochemical properties depend directly on morphology: The structure of g-C₃N₄ includes three forms, namely, porous, nanosheet, and bulk forms. The thinner nanosheets help improve the sensitivity of the sensors and electron transfer rates by providing a larger surface area with more active sites. This makes it easier for analytes to interact with them, thus improving the detection limits. In the case of porous structures, porosity makes electrolyte and analyte access easier and, therefore, enhances the overall electrochemical response. Furthermore, the linked pores can enhance ion transfer [93,94].

The electronic characteristics are influenced by the crystalline structure of g-C₃N₄. When compared to amorphous forms, graphitic g-C₃N₄ usually shows superior electrical conductivity. This conductivity is essential for the quick movement of electrons during electrochemical reactions, which improves the performance of sensors [95]. The layering effect of the interlayer spacing of g-C₃N₄ may impact ion diffusion and charge transfer kinetics. By optimizing the layer thickness, it is possible to enhance response times [96].

A defect can introduce localized states into the band structure, thus enhancing the mobility and reactivity of charge carriers. These defects are vacancies, interstitials, and edge defects. These defects can improve the adsorption of analytes. In addition, defects can be introduced in g-C₃N₄ by doping or thermal treatments to alter its electrochemical properties. For example, the conductivity and catalytic activity may be increased by doping the molecule with nitrogen or metals [97,98].

4.3. Sensing of Biomarkers

Nanostructured g-C₃N₄-based biosensors have gained much attention in the healthcare sector for identifying numerous biological molecules, including glucose, cholesterol, uric acid, tryptophan, 8-HDG, troponin-I, dopamine, serotonin, noscapine, epinephrine, procalcitonin, riboflavin, and pramipexole, as depicted in Figure 6.

4.3.1. Glucose

Diabetes is a serious metabolic disorder that affects a large number of individuals and has prompted the development of reliable, portable glucose monitors [99]. The research community is focused on using g-C₃N₄-based detection methods to measure glucose, a specialized diabetes biomarker, and use fewer enzymes that become unstable after a few days; nonenzymatic glucose detection provides advantages [84]. According to Javad et al., a carbon paste electrode underwent one such alteration with Co(OH)₂ coated on polymeric g-C₃N₄. To create the g-C₃N₄ material, a furnace was used to heat guanidine hydrochloride to 550 °C for two hours. As a result, g-C₃N₄ powder was created, and TEM confirmed the nanolayered structure it produced [100]. The 2D material prepared on g-C₃N₄ in the current work enhanced the sensitivity and offered a linear range of 25–420 mM impedimetrically and 6.6–9800 mM amperometrically. The Nyquist plots of the multi-depth modified electrodes with g-C₃N₄ on CPE, with and without Co(OH)₂, revealed the reduction of charge transfer over each other, which evidences that cobalt hydroxide has been exfoliated from the g-C₃N₄ matrix [101]. Studies for amperometry found that interfering compounds were not present. The sensor was then tested with real-time blood samples, with a linear range of 0.5 to 10 M and a detection limit of 0.5 M [102]. Through one-step pyrolysis of Cu₃[Fe(CN)₆]₂ and melamine, an efficient technique for the fabrication of graphitic carbon nitride (g-C₃N₄)/iron oxide-copper nanostructures was developed. Iron and copper nanoparticles were used to adorn g-C₃N₄ nanosheets, resulting in Fe₂O₃ and Cu, which increased the redox performance of nonenzymatic glucose sensors. With a detection limit of 0.3 mM, the improved working electrodes made of g-C₃N₄/Fe₂O₃-Cu nanocomposites could monitor glucose in the range of 0.6 mM–2.0 mM [103].

4.3.2. Cholesterol

Massive strategies have been advocated for the prevention, mitigation, and treatment of cardiovascular illnesses, such as hypertension, atherosclerosis, and cardiopathy, as well as life-threatening dyslipidemia [104]. Rapid cholesterol detection was performed with a platinum- and phosphorus-codoped carbon nitride smartphone-assisted colorimetric biosensor, which provided a wide linear range of 0.5–600 μgmL⁻¹ and a low detection limit of 59 ngmL⁻¹. It was further applied to real samples, such as serum and food, using colour recognition software and fluorescence signal variation [105]. The primary cause of this disease is an unfavourable buildup of cholesterol, which is brought on by excessive consumption of foods high in cholesterol, a lack of regular exercise, and tobacco use [106]. There is currently a significant increase in the number of individuals with heart- and stroke-related illnesses, which has drawn increased attention and created concerns for global health in the coming decades. Therefore, ongoing blood cholesterol monitoring is necessary for early clinical diagnosis and treatment [107]. The cylindrical spongy-shaped polypyrrole (CSPPy-g-C₃N₄H⁺) nanohybrid composite was utilized to fabricate cholesterol biosensors for the first time. The as-fabricated biosensor electrode (ChOx-CSPPy-g-C₃N₄H⁺/GCE) had great sensitivity (645.7 µAmM⁻¹ cm⁻²) in a broad linear range of 0.02–5.0 mM, a low detection limit (8.0 µM), a quick reaction time (3 s), long-term stability, and good selectivity for cholesterol detection. This method offers a fresh perspective on using the CSPPy-g-C₃N₄H⁺ composite as a superior electrocatalytic and electroconductive material that is inexpensive, biocompatible, environmentally friendly, and electroconductive with great application potential. This approach may open the door to the development of numerous new sensors and biomedical applications [108].

4.3.3. Uric Acid

Graphitic carbon nitride-modified screen-printed carbon (SPC) electrodes were designed for real-time serum uric acid (UA) measurements in gouty patients subjected to different exercise regimens. The sensor responded linearly from 5 to 40 µM of UA and was compared with standard laboratory clinical procedures. The results showed that serum UA rises after exercise, which is contrary to the expectation that it is supposed to decrease, hence supporting the application of the sensor in real life [109]. A Pt@g-C₃N₄/N-CNTs-modified GC electrode was developed for the highly sensitive simultaneous detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA) at low detection limits. Good recovery rates in serum samples demonstrate that the proposed method has potential applications in clinical diagnosis and health monitoring of diseases [110]. Electrochemical methods are promising for uric acid (UA) detection because of quick and cost-effective direct sensing capabilities. This review focuses on nanocomposite-based electrodes and different electrochemical techniques for efficient UA sensing in clinical, biomedical, and food hygiene applications [111]. In this research, a molecularly imprinted polymer (MIP)-based non-enzymatic electrochemical sensor incorporating a modified pencil graphite electrode for the detection of uric acid was prepared. A high sensitivity, good selectivity, stability over 19 days of operation, and the ability to detect uric acid in the presence of interfering molecules were demonstrated [112]. A tungsten oxide/MO-derivatives-incorporated graphene nanocomposite was synthesized for the simultaneous detection of dopamine (DA) and uric acid (UA) using differential pulse voltammetry (DPV). The concurrent low detection limits, large linear ranges for dopamine and uric acid, and successful application in human blood and urine samples established its reliability and stability [113]. In this study, a uric acid-detecting 3D-printed flow-through biosensor that employs a modified screen-printed electrode and immobilized uricase has been developed with a high sensitivity in the range of 4 nM. The biosensor demonstrated excellent selectivity and accuracy in spiked artificial urine samples with considerable routine clinical applications [114]. Blood serum and urine contain uric acid, which plays an important biological role in the body. Abnormal changes in uric acid concentration can cause kidney failure and metabolic problems [115]. Murugan et al. fabricated and deposited g-C₃N₄ nanosheets over glassy carbon electrodes, and the electrochemical technique was employed for the selective and sensitive detection of uric acid in urine samples [116].

4.3.4. Tryptophan

Tryptophan (TRP) is a member of the amino acid family and is a crucial component of many plants and nutritional supplements. Hallucinations and delusions can occur when the human brain is exposed to TRP in improper amounts [117]. The study reports the successful development of an electrochemical sensor based on methionine/graphitic carbon nitride-modified screen-printed electrodes (g-C₃N₄.Meth|SPE) for the rapid detection of tryptophan in food and supplements. The composite associated with the synergistic interaction incorporated into the sensor enhanced high sensitivity, selectivity, and a low detection limit for reliable analysis of actual samples [118]. This study presents a voltammetric sensor based on phosphorus-doped graphitic carbon nitride (P-g-C₃N₄) electrodeposited on a glassy carbon electrode for sensitive and selective TRP detection. The sensor showed a sensitivity as low as 7.1 nM and was successfully applied to real samples, including food, supplements, and clinical specimens [119]. This paper reports the development of a nitrogen defect-rich graphitic carbon nitride-modified glassy carbon electrode (ND-CN/GCE) that can be used for sensitive electrochemical detection of TRP at the very low detection limit of 0.0034 μM, with excellent selectivity. The practical applicability of the sensor in detecting the content of TRP in milk and multivitamin samples also yielded a good recovery rate and reproducibility [120]. This study synthesized a graphitic carbon nitride-modified carbon paste electrode (g-C₃N₄–CPE) for the electrochemical detection of TRP, with a limit of detection as low as 0.085 μM and a linear range between 0.1 and 120 μM. Excellent selectivity, repeatability, and reproducibility were achieved, and successful application was demonstrated in detecting TRP in milk samples with recovery rates of 98–105.2% [121]. Liu et.al and coworkers revealed that g-C₃N₄ nanosheets, produced using the thermal polycondensation technique, were used as a unique stand-alone matrix for the sensing of TRP [122]. The CV and DPV results implied that the strong p–p interaction and fast electron transfer with increased conductivity feasibly enabled the electrocatalytic recognition of TRP through adsorption onto g-C₃N₄ nanosheets [123]. For the detection of TRP, the LoD was found to be 0.024 µM, with a linear range of 0.1–110 µM. TRP was accurately detected in rat blood serum and amino acid administration needles with satisfactory recovery values [124].

4.3.5. 8-HDG

Among cancer biomarkers, endogenous electrophiles, and oxidants, 8-hydroxy-2′-deoxyguanosine (8-HDG) is particularly important. Apart from the role that DNA (deoxyribonucleic acid) plays in various biological and chemical processes, 8-HDG is an extremely significant biomolecule [125]. Excessive concentrations indicate DNA damage in the body. Therefore, analytical researchers are more concerned about controlling the 8-HDG concentration. Rajaji and his team developed an electrochemical sensor for detecting the oxidative stress biomarker 8-hydroxy-2′-deoxyguanosine (8-HDG) using a Cu₂O/g-C₃N₄/GCE-modified working electrode. It showed a LoD of 4.5 nM for the linear range of 8-HDG from 25 nM to 0.91 mM in 0.1 M PBS. As a result of the modified sensor, 8-HDG could be detected in biological samples with high selectivity and sensitivity [126].

4.3.6. Troponin-I

In this study, a novel dual-mode electrochemiluminescence (ECL) and photoelectrochemistry (PEC) immunosensor was fabricated on hollow graphitic carbon nitride (H-g-C₃N₄) through careful optimization of the shell thickness with the addition of TiO₂ for the ultra-sensitive detection of cardiac troponin I (cTnI). The signal cross-checking ensures highly reliable bioanalysis [127]. Troponin I (cTnI), a biomarker of acute myocardial infarction (AMI), is released into the bloodstream within 1–4 h after heart muscle injury occurs [128]. Using porous graphitic carbon nitride (P-g-C₃N₄) nanomaterial-integrated biosensors, Khushaim et al. detected picogram concentrations of cardiac troponin with sensitivity and specificity. Furthermore, they developed a low-cost and easily accessible aptasensor that uses gold nanoparticle-functionalized P-g-C₃N₄ (P-g-C₃N₄-AuNPs) to detect real blood samples. P-g-C₃N₄-AuNPs are practical in the blood (cTnI-spiked), and the LoD was 0.01 pgmL⁻¹ for a 20 μL sample with a fast detection time (2 min) [129].

4.3.7. Dopamine

Here, AuNPs/graphitic-C₃N₄ nanocomposites have been employed to synthesize a susceptible electrochemical sensor for dopamine detection with a detection limit of 0.24 nM. The sensor detected dopamine in human urine samples, providing broad linear ranges and brilliant performance [130]. The paper also functionalized a carbon-metal oxide composite (g-C₃N₄/MnO₂)-coated screen-printed electrode (SPE) for improved sensitivity toward dopamine detection. The determined detection limit was 10 nM, and its sensitivity was 44-fold better. A modified SPE was used to determine the dopamine content in pharmaceutical formulations, foodstuffs, and clinical samples. Its potential application lies in disposable and low-cost sensors [131]. The synthesis of a three-dimensional mesoporous graphitic carbon nitride composite decorated with gold nanoparticles (Au/3Dim CN) was achieved and found to display exceptional electrochemical performance towards dopamine (DA) detection with a limit of detection of 0.02 µM and high selectivity. The sensitivity of the sensor is improved by combining Au nanoparticles with mesoporous g-C₃N₄ and anti-interference capability [132]. This study developed a g-C₃N₄/Gd₂S₃ nanocomposite for dual applications in electrochemical dopamine sensing and photocatalytic water treatment. The material demonstrated high sensitivity for dopamine detection (LOD of 1.2 nM) and effective visible light degradation of contaminants, showcasing its potential for real-time environmental and analytical use [133]. This paper designed a portable electrochemical sensor based on 3D phosphorous- and sulphur-doped g-C₃N₄ (3D-PS-doped CNHN). They obtained a detection limit of 7.8 nM for highly sensitive and selective detection of dopamine (DA). It exhibited excellent stability and reproducibility in human fluids and, thus, might be viable for clinical monitoring of neuronal disorders [134]. Silver nanoparticle-decorated graphitic carbon nitride (g-C₃N₄@Ag) nanocomposites were designed to generate multicolour electrochemiluminescence (ECL) emissions tunable from blue to yellow and for the detection of dopamine. A multicolour ECL detection array supported by machine learning improved its linear detection range from 0.1 nM to 1 mM, with a detection limit of 44 pM, enhancing the quantification through a deep neural network algorithm [135]. An important function of DA is to regulate the central nervous system and maintain hormone balance. It has been shown that DA levels are linked to various nerve disorders, including Alzheimer’s and Parkinson’s. In this study, high-sensitivity graphitic carbon nitride nanosheets were synthesized by direct thermal oxidation and used to detect ascorbic acid (AA), dopamine (DA), and uric acid (UA) simultaneously. Because of its high specific surface area, hierarchical pore structure, and excellent signal response, the electrochemical sensor can effectively detect AA, DA, and UA. The detection limits for AA, DA, and UA are 3.7 μM, 0.07 μM, and 0.43 μM, respectively [136].

4.3.8. Serotonin

The central nervous system of mammals is primarily controlled by monoamine neurotransmitters such as serotonin (5-hydroxytryptamine, 5-HT) [137]. Multiple sclerosis, neuroblastoma, and Parkinson’s disease are just a few of the neurological conditions that have been related to changes in 5-HT transmission [138]. This study investigated engineered graphitic sensors in combination with fast-scan cyclic voltammetry (FSCV) for the optimization of in vivo detection of serotonin; thus, it was possible to shed light on complex electrochemical side reactions, including water-driven redox events. The results show the requirement for more refined protocols that deploy FSCV to improve monitoring reliability with minimal side reactions and water oxidation effects [139]. For the purpose of this research, a highly stable composite of Nb₂CTx/protonated carbon nitride (Nb₂CTx/PCN) was developed for electrochemical detection of serotonin with a proposed limit of detection of 63.24 nM. The developed sensor had excellent sensitivity and selectivity, thus significantly improving the potential of analysing human serum for neurotransmitters [140]. In this research, a binary electrochemical sensor for serotonin and melatonin detection was prepared and coated on reduced graphene oxide–carbon nanotube composite film using vertically oriented mesoporous silica–nanochannel films. The sensor provided outstanding sensitivity and anti-fouling properties and was appropriate for conducting analyses in complex biological fluids such as human blood and cerebrospinal fluids [141]. Recently, scientists reported an electrochemically exfoliated graphitic carbon nitride nanosheet-based highly sensitive serotonin sensor developed at neutral pH with a detection limit of 150 pM. The sensor demonstrated excellent selectivity, electrocatalytic activity, and stability, with good dynamic range detections from 500 pM to 1000 nM. The g-C₃N₄ nanosheets have considerable electrochemical performance for detecting 5-HT in a continuous standard range between 500 pM and 1000 nM. The developed 5-HT sensor’s detection threshold and sensitivity were determined to be 150 pM and 1.03 µAµM⁻¹cm⁻², respectively. High sensitivity, exceptional selectivity, repeatability, and stability are great benefits of the developed g-C₃N₄ nanosheet-based sensor, ehich can detect concentrations in the picomolar/nanomolar range [142].

4.3.9. Noscapine

Noscapine (NOS) is a chemical that is frequently given as a cough suppressant and is present in opium made from poppies in concentrations ranging from 2% to 8%. A composite of graphitic carbon nitride–graphene nanoplatelets (g-C₃N₄/GNP) was used to detect NOS selectively and sensitively using electrochemical voltammetric methods. The findings confirmed that the working electrode developed for DPV technology yields a sensitive NOS detection method (LOD, 10 nM) in the phosphate buffer (pH 6.0) solution. The peak current variation as a function of NOS concentration was linear in the range of 0.05–27.0 µM under ideal circumstances. Using g-C₃N₄/GNP-GCE to monitor NOS in actual samples demonstrated the technology’s potential [143].

4.3.10. Epinephrine

Epinephrine (EPI), often referred to as adrenaline, is a neurotransmitter in the body’s fluids as a massive organic cation. It is a key component in the development of various chronic disorders, including hypertension and myocardial infarction, which are linked to EPI levels in body fluids. The existence of EPI in biological fluids is an important issue. Yola et al. introduced a molecularly imprinted polymer-based electrochemical recognition system for epinephrine detection using a graphitic carbon nitride/N-doped carbon dot composite (g-C₃N₄/NCDs). An electrode imprinted with epinephrine demonstrated good sensitivity for epinephrine recognition throughout a broad linear range of 1.0 × 10⁻¹²–1.0 × 10⁻⁹ M and a detection limit (LOD) of 3.0 × 10⁻¹³ M. A sensor imprinted with epinephrine was used to analyse epinephrine in urine samples [144].

4.3.11. Procalcitonin

Procalcitonin (PCT) is entirely generated in C-cells and cannot enter the bloodstream. Therefore, its amount in healthy patients is below the detection level. The PCT levels will increase after patients develop bacterial sepsis [145]. This electrochemical biosensor has been realized using PCT detection with NiCoP/g-C₃N₄ nanocomposites, displaying sensitivity from 1 µgmL⁻¹ to 10 ngmL⁻¹ and a detection limit of 0.6 µgmL⁻¹. The sensor successfully detects PCT in diluted human serum, holding good potential for clinical testing applications [146]. Because of its distinctiveness and superior sensitivity and selectivity, PCT is usually regarded as the most promising biomarker [147]. An ultrasensitive label-free electrochemical sensor was designed by utilizing graphitic carbon nitride nanosheets for procalcitonin detection. A broad detection range was achieved to be 0.15–11.7 fgmL⁻¹, and a low detection limit was 0.11 fgmL⁻¹. The developed sensor offered great application potential for PCT detection in human serum, which might be applied practically in clinical environments [148]. Liu and his workers designed a label-free PCT detection using the gC₃N₄ nanostructure. By using differential pulse voltammetry (DPV), the sensing capability of this sensor was assessed. The results revealed detection accuracy with a dynamic range of 0.15 to 11.7 fgmL⁻¹ with a LoD of 0.11 fgmL⁻¹.

Additionally, the developed biosensor was successfully employed to identify PCT in human serum samples, and the outcomes point to its possible application in clinical settings. This review discusses recent advances in electrochemical immunosensors for detecting procalcitonin with sandwich-type assays and nanointerfaces to enhance sensitivity. It then outlines current bottlenecks and emerging directions toward flexible point-of-care PCT detection devices [149]. A highly sensitive electrochemical biosensor lacking labelling, based on ultrathin graphitic carbon nitride nanosheet probes, could detect procalcitonin at 0.11 fgmL⁻¹ in the response medium. For the first time, it was applied to detect ProCT in human serum samples, and it can potentially be used as a sepsis diagnostic tool [150]. Using a platform consisting of sulphur-doped MXene and gold nanoparticles and carboxylated graphitic carbon nitride to amplify the signal, we designed an ultra-sensitive sandwich-type electrochemical immunosensor for the detection of procalcitonin. The fabricated sensor had a detection range of 0.01–1.0 pgmL⁻¹ and offered a sensitivity limit of 2.0 fgmL⁻¹ with superb stability and accuracy in detecting plasma samples [151].

4.3.12. Riboflavin

Riboflavin (RF), a crucial water-soluble vitamin, must be determined in pharmaceutical samples at ultrasensitive and minuscule levels due to its importance [152]. This research study proposed the development of a CuO nanoflake-modified graphitic carbon nitride (g-C₃N₄.CuNF|GCE) electrochemical sensor for the highly sensitive detection of riboflavin (RF) with a detection limit of 6 nM. The sensor offered selectivity, reproducibility, and applicability to detect RF in food and pharmaceutical samples [153]. A Dy₂O₃/g-C₃N₄ nanocomposite made by co-precipitation followed by ultrasonication exhibited electrocatalytic performance for the detection of RF, which was notably high; this would include a detection limit at 48 nM and sensitivity for 2.5261 μAμM⁻¹cm⁻². The sensor is characterized by excellent selectivity, stability, and reproducibility and can detect RF in honey and milk samples for favourable recovery rates [154]. Rajkumar et al. prepared the biosensor to efficiently determine RF Ruthenium-Sulphur doped (Ru/S-g-C₃N₄) g-C₃N₄ composite with the LoD of 54.3 pM (3 sm⁻¹). The development of RF sensor systems for analytical substances, such as food supplements and pharmaceuticals, is now possible thanks to this research [155].

4.3.13. Pramipexole

Pramipexole (PMXL), a benzothiazole compound used to treat Parkinson’s disease, corresponds to the undesirable consequences that can result from overdosing. Therefore, a sensitive analytical technique is required to detect trace levels. Shanbhag et al. constructed the biosensor using a g-C₃N₄-modified working electrode to detect PMXL effectively. A lower detection limit (LD) of 0.012 µM was attained for the chosen concentration range (0.5 to 30 µM) for the manufactured g-C₃N₄-CPE, and a high linearity range was observed from 0.05 to 500 µM. Interference research was conducted to explore the selectivity of electrodes in PMXL detection, and tablet sample analysis showed the electrode’s sensitivity and real-time use. The effective use of the electrode for PMXL measurement is demonstrated by the good recovery values from the analysis [156].

5. Summary and Outlook

To summarize, this review demonstrated the significance of metal-free polymeric carbon g-C₃N₄ nanoarchitectures toward the development of electrochemical biosensors. This aspect includes background, history, various phases, and electronic structures concerning the first synthesized carbon nitride polymer. Additionally, the diverse methods for producing g-C₃N₄ nanostructures, generally categorized into top-down and bottom-up approaches, were explained elaborately. Its significance emerges from the nature of the precursor and synthesis approach used, and the nature of the morphology of g-C₃N₄ nanostructures. Further, it has also been simplified to quickly identify the value of g-C₃N₄ in the electrochemical biosensing of diverse biomolecules. As a result of its impressive salient features and design flexibility, 2D g-C₃N₄NSs have found the most use as an exceptional material within the electrochemical biosensing platform. This makes g-C₃N₄ a potential material for developing a biosensing device that, ideally, would be reliable, sensitive, and fast.