Photocatalytic Inactivation of Viruses Using Graphitic Carbon Nitride-Based Photocatalysts: Virucidal Performance and Mechanism

Abstract

The prevalence of lethal viral infections necessitates the innovation of novel disinfection techniques for contaminated surfaces, air, and wastewater as significant transmission media of disease. The instigated research has led to the development of photocatalysis as an effective renewable solar-driven technology relying on the reactive oxidative species, mainly hydroxyl (OH^●) and superoxide (O₂^●−) radicals, for rupturing the capsid shell of the virus and loss of pathogenicity. Metal-free graphitic carbon nitride (g-C₃N₄), which possesses a visible light active bandgap structure, low toxicity, and high thermal stability, has recently attracted attention for viral inactivation. In addition, g-C₃N₄-based photocatalysts have also experienced a renaissance in many domains, including environment, energy conversion, and biomedical applications. Herein, we discuss the three aspects of the antiviral mechanism, intending to highlight the advantages of photocatalysis over traditional viral disinfection techniques. The sole agenda of the review is to summarize the significant research on g-C₃N₄-based photocatalysts for viral inactivation by reactive oxidative species generation. An evaluation of the photocatalysis operational parameters affecting viral inactivation kinetics is presented. An overview of the prevailing challenges and sustainable solutions is presented to fill in the existing knowledge gaps. Given the merits of graphitic carbon nitride and the heterogeneous photocatalytic viral inactivation mechanism, we hope that further research will contribute to preventing the ongoing Coronavirus pandemic and future calamities.

1. Introduction

The outbreak of the Coronavirus infectious disease has drenched the world in devastating health impacts [1]. Globally, the Coronavirus pandemic has caused over 4 million fatalities as of 28 July 2021 at a steady rate [2,3,4]. Hence, a paradigm approach to preparedness and a timely communal response are required to battle against the high contagiousness of viruses.

An emerging treatment strategy against viral outbreaks is the nanotechnology route of designing antiviral therapeutics [5]. Nano-antimicrobials (i.e., nanoparticle-derived antiviral agents) are of nanometer size, which makes them well-suited for biochemical interactions with a nanosized virus. The properties of nanoparticles behind the antiviral activity include the small size with a large surface area and the selective, targeted, and stimulus-responsive action on the virus [6]. The functionalization of carbon-based nanomaterials, including carbon quantum dots, graphene, graphene oxide, fullerenes, and graphitic carbon nitride, has been used in antiviral research under visible light irradiation for human immunodeficiency virus (HIV), avian influenza virus A (H1N1), poliovirus, herpes simplex virus, and human adenovirus [7].

Thereby, rigorous research is required to develop a facile, stable, cost-effective, highly efficient, and sustainable technique for disinfecting virus-contaminated water, air, and surfaces. Fortunately, an advanced oxidation process is a promising technology to alleviate infectious viruses in the environment. AOPs include ozonation, the homogenous photo-Fenton process, photo-electro-oxidation, ultrasound, and photocatalysis and can be used to efficiently treat virus contamination [8,9]. Certainly, photocatalysis is an advanced “green disinfection” strategy that targets wide-scale viruses present on surfaces and in air and wastewater owing to the high redox abilities of the produced reactive oxidative species (ROSs), such as hydroxyl (OH^●), superoxide (O₂^●−), hole (h⁺), and peroxide (O₂⁻²) radicals [10,11]. A broad survey of literature on photocatalytic systems for virus disinfection is presented in Table 1 [12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31], further instigating researchers to exploit photocatalysis against SARS-CoV-2.

Table 1. Summary of potential photocatalysts for virus disinfection.

Photocatalyst	Virus	Light Source	Wavelength	Virus Level	Major Reactive Species	Inactivation Efficiency/Time	Ref.
Ag doped TiO2	Hepatitis B	UV cut-off filter	420 nm	1g/L	OH●, O2●−	81.7 %/12 h	[12]
TiO2 coated ceramic	Hepatitis B	Hg lamp (125 W)	365 nm	-	OH●, HOO●	97 % /240 min	[13]
TiO2	Human adenovirus 40	UV lamp	254 nm	1 × 108 PFU/mL	OH●	0.49 log/14.3 min	[14]
TiO2	Herpes simplex virus	UV-A lamp (Philips TLD 15 W/05)	365 nm	109–1010 PFU/mL	OH●, O2●−	100 %/360 min	[15]
TiO2	Influenza virus H9N2	UV (Black light 20W)	365 nm	1 × 104 PFU/mL	OH●	4-log/150 min	[16]
TiO2	Influenza virus H1N1	UV-A light (Tubular 20 W black light fluorescent lamps)	352 nm	1 × 108 PFU/mL	OH●, O2●−	5.2-log/480 min	[17]
TiO2	Murine Norovirus	UV lamp (8 W)	385 nm	7.8 PFU/mL	OH●	4 log/10 min	[18]
Cu-doped TiO2	Norovirus	UVA-LED	373 nm	2.89 ± 0.11 log10	OH●, O2●−	5 log/60 min	[19]
TiO2	Bacteriophage MS2, Murine Norovirus, Influenza	UV light Blacklight Blue lamps (BLB,4 W, Philips Co.	360 nm	1 × 106 PFU/mL	OH●	1.5 × 10−5 mg/L/36 min, 1.4 × 10−5 mg/L /32 min, 1.7 ×10-5 mg/L/40 min	[20]
ZnO	Bacteriophage MS2, ΦX174, PR772	UV-B	295 nm	1 × 105 PFU/mL	OH●	0.37, 0.67, 0.59 L/kJ /360 min	[21]
Ag-AgI/Al2O3	Human rotavirus type-II Wa	350-W Xe-arc lamp	450 nm	3.2 × 103 PFU/mL	O2●−, h+	3.2 log/40 min	[22]
Pt/WO3 films	Influenza virus H1N1	Fluorescentlamps (FL20SS W/18) 1000 lx	470 nm	1 × 107 PFU/mL	OH●	>5.5 log/120 min	[23]
C60	Rhabdoviridae	Hg lamp (350 W)	495 nm	10 log TCID 50/mL	1O2	7 log10 /360 min	[24]
C60/SiO2	Bacteriophage MS2	Fluorescent bulbs	470 nm	5 × 104 PFU/mL	1O2	3.55 log/75 min	[25]
C70/SiO2	Bacteriophage MS2	Visible, Sunlight	420 nm	3 × 108 PFU/mL	1O2	4.35 log; 4.4 log/90 min	[26]
g-C3N4	Bacteriophage MS2	Xenon lamp	400 nm	1 × 108 PFU/mL	O2●−	6.58 log/180 min	[27]
g-C3N4	Bacteriophage MS2	300 W Xenon lamp	450 nm	1 × 108 PFU/mL	O2●−	8 log/360 min	[28]
Ag3PO4/g-C3N4	Bacteriophage f2	UVA lamp (8W)	500 nm	3 × 107 PFU/mL	h+	6.5 log/80 min	[29]
g-C3N4/Expanded perlite	Bacteriophage MS2	Xenon lamp (300 W)	300 nm	1 × 108 PFU/mL	OH●, O2●−	8 log/240 min	[30]
O-doped g-C3N4	Human adenovirus	7 W LED lamp	450 nm	-	OH●, O2●−, H2O2	5 log/120 min	[31]

In addition to metal-based semiconductors, carbon-based photocatalysts are potential candidates in this regard. Among all the antiviral photocatalysts, graphitic carbon nitride (g-C₃N₄)-based photocatalysts have emerged recently. With an analogous structure to graphene, g-C₃N₄ has a unique hexagonal framework with C-N bonds in place of C-C bonds and is a sustainable alternative to metal-based materials [32]. The exceptional properties of g-C₃N₄ include exceptionally high thermal stability, versatile physicochemical, optical, and electronic properties, and a wide range of synthesis routes, which promote its application in heterogeneous catalysis, environmental remediation, the conversion of energy into usable fuels, and the biomedical field [33]. Several comprehensive reviews have systematically summarized the synthesis, properties, and applications of g-C₃N₄ photocatalysts [34,35,36]. Additionally, there are reviews on the use of metal oxides [37], graphene [38], and carbon-based nanomaterials [39] for controlling the SARS-CoV-2 virus. This review seeks to provide a complete overview of the g-C₃N₄-based photocatalysts applied for virus disinfection in wastewater. The aim of the review is to make readers understand the potential of photocatalysis phenomena in relevance to virus disinfection. The following section provides an overview of the antiviral performance of g-C₃N₄-based photocatalysts by discussing the relevant reported research. Furthermore, innovative research in the healthcare realm, including pathogenic biofilm control, therapeutics, and bioimaging, using graphitic carbon nitride-based photocatalysts is reviewed. Finally, challenges and possible sustainable solutions for designing g-C₃N₄-based photocatalysts for viral inactivation purposes during the pandemic and beyond are addressed.

2. Photocatalytic Virus Inactivation: Mechanism

The use of photocatalysis technology in virus inactivation was first reported over a TiO₂ photocatalyst for MS2 phage inactivation by destroying the protein capsids upon the oxidative action of OH^● radicals [40]. Since then, the development of photocatalytic antiviral agents has been ever-increasing, with a paradigm shift in research from metal-oxide-based photocatalysts to metal-free visible light active photocatalysts [41]. It is worth clarifying that the photocatalytic viral inactivation process targets the structure of the virus, involving genetic core (RNA or DNA) damage, protein oxidation, and shape rupturing/distortion. Viral inactivation by the photocatalysis process is proposed [42] to occur by three routes, as shown in Figure 1, which are: (i) physical damage to capsid protein shells of viruses that causes their inactivation; (ii) metal ion toxicity in synergism with photocatalysis that induces the release of metal ions to increase the speed of viral inactivation kinetics; and (iii) chemical oxidation, which is the most effective photocatalytic virus disinfection process due to the participation of reactive oxidative species with strong oxidizing power that target the degradation of the cell wall and cytoplasmic membrane of the virus.

Figure 1. Proposed mechanisms of viral inactivation induced by photocatalysts via different routes (a) physical damage induced by rupturing of cell wall, (b) attack of metal ions, and (c) action of reactive oxidation species (ROS). Reproduced with permission from [42] with license number 5191840171112. Copyright Elsevier 2019.

In brief, photocatalytic viral inactivation utilizes renewable solar energy and an abundance of oxygen to generate reactive oxidative species. The photocatalytic virus inactivation has the following step-wise mechanism: (i) upon irradiation with light, semiconductor-based photocatalysts induce the production of electron–hole pairs with the simultaneous migration of charges to the reactive surface; (ii) the holes in the valence band react with the H₂O molecules or surface-adsorbed OH species to produce OH^●, which targets the chemical composition of the shells and capsids of viruses; and (iii) the electrons in the conduction band react with the molecular oxygen (O₂) for the production of highly reactive radicals, including O₂^●−, OH^●, and O₂⁻², which attack the virus adsorbed on the photocatalytic surface [43,44]. Figure 1c illustrates the sequential heterogenous photocatalytic viral inactivation mechanism, wherein the released reactive species oxidize viruses by damaging coenzyme A on the cell membrane, causing inhibition of cellular respiration activity and eventual cell death [45,46]. The mechanistic action of ROSs for enveloped viruses (influenza, Hepatitis B [13], Rhabdoviridae [24], etc.) follows a different route of inactivation due to the assembly of the structural proteins (i.e., the spike, nucleocapsid, membrane, and envelope proteins). The plausible attack of ROS on enveloped viruses, as summarized by Costa et al. [47], includes: (i) alteration of protein cross-linkage; (ii) destruction of the protein structure; and (iii) variation in the charge and mass. For instance, Korneev et al. [48] investigated the photodynamic inactivation performance of the photosensitizer octacationic octakis(cholinyl) zinc phthalocyanine on influenza virus and observed an attack of singlet oxygen on spike glycoproteins. The TEM images indicated baldness of virions at higher concentrations of photosensitizer (4 µM) upon light exposure for more prolonged durations, leading to baldness of the virion and its infectivity. Regarding photocatalysis, as an alternative route for combatting enveloped SARS-CoV-2, an understanding of the actual mechanism of virus inactivation is vital. To date, diverse photocatalysts have been explored for photocatalytic viral inactivation; however, the research has yet to yield large scale application.

3. Antiviral Characteristics of Graphitic Carbon Nitride

Graphitic carbon nitride (g-C₃N₄) is emerging in the field of antiviral photoactivity mainly due to its metal-free composition, non-toxic nature, biocompatibility, photo-corrosion resistance, and chemically stable features [49]. The facile fabrication of g-C₃N₄ by nitrogen-rich organic precursors (e.g., urea, melamine, thiourea, cyanamide, and dicyanamide) enables low-cost and bulk fabrication [50]. Bulk g-C₃N₄ possesses a suitable bandgap of 2.7 eV, promoting visible light absorption up to 460 nm. According to the bandgap structure of g-C₃N₄, the valence band potential (+1.4 eV vs. NHE) is thermodynamically unfavorable for the production of OH^● (H₂O/OH^● = 2.38 eV vs. NHE; OH⁻/OH^● = +1.99 eV vs. NHE) [51]. Hence, many reports claim that viral inactivation in the g-C₃N₄ disinfection system is due to the photogenerated-h⁺-dominant oxidation process [52,53]. Benefitting from the appropriate positioning of the conduction band (−1.1 eV vs. NHE), the facile production of O₂^●− occurs by favorably reducing O₂ into O₂^●− (O₂/O₂^●− = −0.33 eV vs. NHE). The subsequent formation of OH^● via multistep reduction of O₂ at the reductive site of g-C₃N₄ was identified in MS2 bacteriophage inactivation [27]. Overall, the photocatalytic electron transfer and ROS generation are described by Equations (1)–(7) [54].

$$\mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4+\mathrm{h}\mathsf{\nu }\to \mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4\left({\mathrm{e}}_{\mathrm{CB}}^{-}+{\mathrm{h}}_{\mathrm{VB}}^{+}\right)$$

(1)

$$\mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4\left({\mathrm{h}}_{\mathrm{VB}}^{+}\right)+{\mathrm{H}}_2\mathrm{O}\to \mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4+{\mathrm{OH}}^{●}+{\mathrm{h}}^{+}$$

(2)

$$\mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4\left({\mathrm{h}}_{\mathrm{VB}}^{+}\right)+{\mathrm{OH}}^{-}\to \mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4+{\mathrm{OH}}^{●}$$

(3)

$$\mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4\left({\mathrm{e}}_{\mathrm{CB}}^{-}\right)+{\mathrm{O}}_2\to \mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4+{\mathrm{O}}_2^{●-}$$

(4)

$${\mathrm{O}}_2^{●-}+{\mathrm{HO}}_2^{●}+{\mathrm{H}}^{+}\to {\mathrm{OH}}^{●}+{\mathrm{O}}_2+{\mathrm{H}}_2{\mathrm{O}}_2$$

(5)

$$2{\mathrm{HO}}_2^{●-}+2{\mathrm{H}}^{+}\to {\mathrm{O}}_2+{\mathrm{H}}_2{\mathrm{O}}_2$$

(6)

$$\mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4\left({\mathrm{e}}_{\mathrm{CB}}^{-}\right)+{\mathrm{H}}_2{\mathrm{O}}_2\to \mathrm{g}-{\mathrm{C}}_3{\mathrm{N}}_4+{\mathrm{OH}}^{-}+{\mathrm{OH}}^{●}$$

(7)

In addition, g-C₃N₄ possesses high thermal stability (i.e., resistance to oxidation in air at a temperature of up to 600 °C) and is insoluble in water and organic solvents, contributing to the facile disinfection of viruses prevailing in wastewater and air. The unique biocompatible and non-toxic nature of g-C₃N₄ minimizes its interference during the virus inactivation process. These merits of g-C₃N₄ contribute to its pioneering performance in a wide range of applications, including wastewater treatment, microbial disinfection, environmental remediation, health care, and energy conversion. The dilemma of appropriately tailoring (lowering) the bandgap of g-C₃N₄ to promote the solar spectrum’s utilization while simultaneously reducing the charge carrier recombination remains a significant drawback [55]. The other issues that could arise during the photocatalytic viral inactivation mechanism include: (i) the limited surface area of the g-C₃N₄ photocatalyst inhibiting ROS generation; (ii) agglomeration of g-C₃N₄ nanoparticles in water; (iii) controlled diffusion of surface-bound radicals; and (iv) the counterattack of released radicals with oxidizable substrates of the virus cell wall. As a solution, strategies such as heterojunction formation, doping, dye sensitization, and co-catalyst loading are introduced in bare g-C₃N₄ to increase the photoactivity [56,57]. To date, g-C₃N₄ has been well explored for bacterial disinfection via photocatalysis. In contrast, the photocatalytic disinfection of viruses is difficult to accomplish due to their vast range of classifications, varying composition, complex structure, and higher resistance to oxidative stress [58].

4. Antiviral Performance of Graphitic Carbon Nitride-Based Photocatalysts

To exploit the photocatalytic efficiency of g-C₃N₄ for virus inactivation under visible light irradiation, Li et al., for the first time, conducted an antiviral experiment using the bacteriophage MS2 as a model virus. As illustrated in Figure 2a, the major reactive oxidative species responsible for the 1 × 10⁸ PFU/mL inactivation of bacteriophage MS2 were photogenerated electrons and O₂^●− radicals. The photocatalytic degradation mechanism of MS2 involved oxidative damage to the surface protein, resulting in a shape distortion of the virus followed by disruption of the viral genome RNA, ultimately causing viral death with no regrowth for up to 72 h. The action of reactive oxidative species on the protein coating of the virus, which is shown in Figure 2b, was confirmed by the decrease in protein concentration from 9.8 mg/mL to 0 mg/mL for 1 × 10⁸ PFU/mL of virus treated by 5 mL of g-C₃N₄ after 360 min of irradiation. A comparative study was performed to evaluate the efficacy of g-C₃N₄ compared with other photocatalysts, as shown in Figure 2c, which revealed more than 7-log, 1-log, and 4-log of MS2 deactivation by g-C₃N₄, N-doped TiO₂, and Bi₂WO₆, respectively. Ag@AgCl displayed higher inactivation of viruses of up to 8 log within 300 min, which was attributed to the biocidal and electron sink properties of Ag nanoparticles [28]. Because of the merits of Ag nanoparticles, a Ag₃PO₄/g-C₃N₄ Z-scheme heterojunction revealed the complete inactivation of the bacteriophage f2 virus by the action of released OH^● > h⁺ > O₂^●− radicals at a concentration of 3 × 10⁶ PFU/mL within 80 min of irradiation. The photocatalytic inactivation efficiency had the following trend: g-C₃N₄, 3.6 log; Ag₃PO₄, 4.5 log; and Ag₃PO₄/g-C₃N₄, 6.5 log, which was attributed to the higher electron–hole pair separation and the more comprehensive visible light response (up to 500 nm) in Ag₃PO₄/g-C₃N₄. The higher photo-oxidative ability of Ag₃PO₄/g-C₃N₄ was evident from the photoluminescence spectra, which showed a 48% decrease in PL emission intensity compared with bare g-C₃N₄ [29].

Figure 2. (a) Schematic representation of the viral inactivation mechanism of g-C₃N₄ under visible-light exposure; (b) protein concentration measurements in 100 μL of a solution of viral particle lysates from 5 mL of a virus-related suspension (1 × 10⁸ PFU/mL) and the amount of protein carbonyls during the inactivation process; (c) photocatalytic activity of Ag@AgCl, Bi₂WO₆, N-TiO₂, and g-C₃N₄ samples with 150 mg/L dosages for MS2 inactivation upon exposure to visible light. Reproduced with permission from [28] with license number 5136360326508. Copyright Elsevier 2016.

The dual photoinactivation of an antibacterial (E. coli) and an antiviral (MS2) was investigated for the first time over a g-C₃N₄/expanded perlite 520 floating photocatalyst under visible light irradiation. The scavenging experiment showed that OH^● radicals were dominant in the MS2’s inactivation, whereas O₂^●− radicals participated in both E. coli and MS2 inactivation. The different behavior of the photoinactivation mechanism was accredited to the different sizes and surface morphologies of E. coli bacteria and the MS2 virus. More importantly, the denaturation of the rigid capsid cell wall of the MS2 virus requires highly oxidizing OH^● radicals. The viral photocatalytic efficiency of the g-C₃N₄/expanded perlite 520 photocatalyst was practically assessed in deionized water and natural water, wherein a decrease in removal efficiency from 8 log to 3.7 log was obtained. This was attributed to the effect of natural organic matter constituents in natural water, which tend to absorb the floating g-C₃N₄/expanded perlite 520 photocatalyst, thereby blocking reactive oxidative species generation [30].

To further comprehend the photocatalytic viral inactivation mechanism in g-C₃N₄, Zhang et al. studied the effect of various operating parameters, including temperature and photocatalyst loading, within 180 min of visible light irradiation via 3D response surface and 2D contour plots. Figure 3a illustrates a variation in reaction temperature from 1.48 ℃ to 43.5 ℃, where the maximum MS2 viral inactivation of 6.5 log PFU/mL was obtained at 24.05 ℃ compared with the g-C₃N₄ photocatalyst. The inhibition of viral inactivation at higher temperatures was due to the decrease in dissolved oxygen levels, which further lowered the concentration of O₂^●− radicals. Further, investigation of the effect of a photocatalyst loading from 15.91 to 135 mg /L resulted in an increase in MS2 inactivation efficiency due to the increased specific surface area of the g-C₃N₄ photocatalyst with a large number of active sites. The vital factor of light intensity required to induce electron–hole pair transitions was optimized at 199.80 mW/cm² concerning varying photocatalyst loadings (Figure 3b) and temperatures (Figure 3c) [27]. With the integrated merits of doping and the Z-scheme charge transfer mechanism, Zhang et al. evaluated the performance of O-doped g-C₃N₄ coupled with hydrothermal carbonation carbon on human adenovirus type 2. Figure 4a shows the virucidal mechanism, which depicts direct Z-scheme electron migration from the conduction band (−0.27 eV) of the hydrothermal carbonation carbon to the −0.86 eV conduction band of the O-doped g-C₃N₄. The enhanced electron–hole pair separation resulted in the generation of higher amounts of O₂^●− and OH^● radicals for the distortion and rupture of, and formation of holes in, human adenovirus type 2 viral capsids, as confirmed by TEM images, under 120 min of visible light irradiation, causing a loss of pathogenicity (Figure 4b). As shown in Figure 4c, the toxicity assessment of photocatalysts through an XTT proliferation colorimetric assay with human A549 lung carcinoma cells displayed negligible cell viability even at a higher concentration of 150 µg/mL of photocatalyst, indicating the excellent biocompatibility of the synthesized photocatalysts [31].

Figure 3. The plots demonstrate the photocatalytic viral inactivation efficacy by g-C₃N₄ of (a) photocatalytic loading X₂ vs. intensity of light X₁; (b) intensity of light X₁ vs. reaction temperature X₃; and (c) reaction temperature X₃ vs. photocatalytic loading X₂. Reproduced with permission from [27] with license number 5136341150728. Copyright Elsevier 2018.

Figure 4. (a) The plausible charge transfer route in photocatalysis over the O-g-C₃N₄/HTCC Z-scheme heterojunction; (b) cytotoxic effects of O-g-C₃N₄/HTCC, and O-g-C₃N₄; and (c) TEM images of HAdV-2 mixed with O-g-C₃N₄/HTCC taken during different intervals of time. Reproduced with permission from [31] with license number 5136341407889. Copyright Elsevier 2021.

Based on the above-described literature, it is evident that the virucidal potential of g-C₃N₄-based photocatalysts has been well-explored for non-enveloped viruses.

5. Promising Role of Graphitic Carbon Nitride-Based Photocatalysts in Healthcare

The development of modified g-C₃N₄-based photocatalysts with high water solubility and biodegradability, a small nanoscale size, and enhanced light absorption is important to the achievement of target-specific applications [59]. Various strategies and synthesis routes have been investigated in this respect. Subsequently, bare g-C₃N₄ and its derivatives were used in healthcare applications, particularly pathogenic biofilm control, diagnostic imaging, and therapeutics [60]. g-C₃N₄ possesses superior stability, good biocompatibility, low toxicity, a unique fluorescence emission, and high ROS production potential; hence, it may play a vital role in diagnosis and therapy. The following discussion summarizes the healthcare applications of graphitic carbon nitride-based photocatalysts based on ROS generated upon light irradiation.

• Pathogenic biofilm control: The issue of the unwanted growth of pathogenic biofilms on surfaces raises crucial health concerns since these bacteria can persist for a prolonged time. The most commonly contaminated surfaces are those of hospital equipment, packaging materials, and food-processing facilities, which can become vulnerable routes for disease transmission. The conventional disinfection strategies include UV-light irradiation, mechanical cleaning, and chemical sprays, which need to be frequently applied to maintain the efficacy of biofilm control. The photocatalysis process holds promise in biofilm control due to its broad-spectrum effectiveness under room conditions (ambient light/sunlight), simplicity, good recyclability, and low cost. For instance, the biofilm-dispersing ability of a Ag/g-C₃N₄ photocatalyst was assessed, wherein the attack of O₂^●− radicals resulted in complete degradation of the protein coating, exopolysaccharides, and nucleic acid of S. aureus biofilms [61]. The promising role of a g-C₃N₄/chitosan photocatalyst was revealed by the biofilm inhibition of S. epidermidis, P. aeruginosa, and E. coli in urine samples. The ¹O₂ radicals released under visible light irradiation were dominant in the biofilms’ eradication [62].
• Photodynamic therapy: The property of ROS generation under light illumination makes g-C₃N₄ a suitable candidate for destroying tumor cells by damaging the gene structure. Principally, the phenomena of photodynamic therapy involve the production of ROSs (O₂^●− and OH^● radicals), which block the electron transport chain in mitochondria, resulting in a lack of supply of energy and nutrients to tumor cells, leading to cell death. The photosensitization effect of Ru coordinated to g-C₃N₄ has been exploited for the photodynamic therapy of hypoxic tumor cells as explained in the mechanism shown in Figure 5a. The invasion of hypoxic tumor cells by Ru⁺²-g-C₃N₄ occurs under visible light irradiation involving photoinduced reduction of the Ru⁺³ center to the Ru⁺² center to generate multiple cytotoxic ROSs (OH^●, O₂^●−, and ¹O₂), which causes mitochondrial dysfunction leading to apoptosis [63].
  

  Figure 5. (a) The photocatalytic ¹O₂ generation mechanism of Ru-g-C₃N₄ in the presence of H₂O₂ or H₂O. The superscripts on the Ru ions denote the ground state Ru²⁺, the triplet state Ru²⁺, and the radical, cationic species Ru³⁺; (b) schematic representation of ZnO/CDots/g-C₃N₄ Z-schemes for photo-assisted bacteria inactivation; (c) diagrammatic illustration of the growth of neural PC12 cells on g-C₃N₄/graphene composite fibers on exposure to visible light. Reproduced with permission from [63,64,65] with license numbers 5161471258951 and 5161480119676. Copyright American Chemical Society 2017, Elsevier 2020, 2021.
• Wound healing: The ROSs generated under light irradiation tend to inhibit the growth of bacteria and act as wound healers. The accelerated wound healing performance was evaluated for a Z-scheme ZnO/C-dots/g-C₃N₄ ternary heterojunction (Figure 5b), wherein C-dots serve as a bridge to reduce electron–hole pair recombination for the migration of electrons from the conduction band of ZnO to the valence band of g-C₃N₄. The subsequent production of OH^● and ¹O₂ radicals resulted in 99.97% and 99.8% disinfection of Streptococcus aureus and Escherichia coli, respectively. The released Zn⁺² intruded into the bacteria and exhibited a hyperthermia effect, triggering the growth of fibroblasts for a rapid wound healing process [64].
• Bioimaging: The quantum confinement and edge effects of g-C₃N₄ photocatalysts limit the electron mobility, contributing to fluorescence emissions. In addition, g-C₃N₄ emits wavelengths of blue and green light that can be optically tuned to enhance the fluorescence quantum efficiency for cell imaging to display a contrasting image. Zhang et al. performed neuronal differentiation of PC12 cells using the phenomena of non-invasive light stimulation. Under visible light irradiation (450 nm), the biocompatible interface of g-C₃N₄/graphene photocatalysts dispersed on polycaprolactone/gelatin fibers exhibited distinguished outgrowth of neurite while simultaneously improving the electron–hole pair charge carrier separation as demonstrated in Figure 5c [65].

6. Challenges and Sustainable Solutions

Despite the performance of g-C₃N₄-based photocatalysts in diverse applications, the research, perception, and interpretation remain in their infancy with respect to the virus inactivation mechanism and large-scale practical efficiency. In the race to fully explore and utilize g-C₃N₄-based photocatalysts for virus disinfection purposes, we need to put in effort to identify possible solutions to overcome the prevailing challenges. In this respect, it can be roughly divided into the following aspects:

• Insufficient validation of the reaction mechanism: our understanding of the characterization techniques for validating the photocatalytic virus disinfection mechanism is insufficient to support: (i) the extent of ROS generation; (ii) the co-existing action of O₂^●− and OH^● radicals in attacking the virus cell wall; (iii) the pathway selectivity during viral mutation; and (iv) the offensive response of the virus upon exposure to light irradiation. Another significant challenge involves the stronger resistance of certain viruses to UVC (200–280 nm), UVB (280–320 nm), and UVA (320–400 nm) light, which requires the development of visible-light-active or near-infrared-responsive photocatalysts [66]. It is thus necessary to precisely identify and characterize the role of participating ROSs to obtain a reasonable photocatalytic virus disinfection mechanism for the amendment of a practical photocatalysis process.
• Photostability, recyclability, and scalable production are the most crucial factors in the enhancement of the efficacy and robustness of g-C₃N₄-based photocatalysts that target virus eradication [67]. Considering the extended time during which viruses persist in the environment (air, water, and infected surfaces), the stability of g-C₃N₄ photocatalysts needs to be enhanced to overcome the economic barriers. The improvement of the current separation techniques for recovery of g-C₃N₄ photocatalysts from wastewater and photoreactors is recommended to solve the challenging issue of photocatalysts’ recyclability [68]. In accordance, magnetic isolation via the incorporation of nanosized suspended particles into the contaminated reaction mixture has been explored [69]. In terms of the large-scale production and applicability of g-C₃N₄ photocatalysts, cost-effective engineering strategies should be practiced, and photoreactor designs need to be optimized for extended solar energy utilization. Hence, the commercialization of metal-free g-C₃N₄ material is vital at the industrial scale for the future mass utilization of solar energy with a minimal environmental impact. The photoreactor design needs to be optimized for an increased light-capturing rate with the maximum use of renewable sunlight to achieve the sustainable disinfection of viruses. The most suitable photoreactor is the compound parabolic collector (CPC), which effectively utilizes diffuse and direct solar irradiation. Hence, the commercialization of photoreactors is vital at the industrial scale for the future mass utilization of solar energy with a minimal environmental impact [42].
• Conflicting band edge modulation achieves broad-spectrum solar energy absorption while maintaining a high redox potential with a reduced electron–hole pair recombination rate. The type II and Z-scheme charge transfer routes are two cutting-edge strategies for solving these predicaments [70,71]. Based on our critical assessment, it is not surprising that g-C₃N₄-based photocatalysts have benefitted a diverse range of photocatalytic fields. As a matter of course, it is expected that the above challenges will be resolved with advances in research. Even though various disinfection technologies have been developed to mitigate the spread of viruses, photocatalytic virus disinfection is a sustainable alternative that may lessen the environmental impact.

7. Conclusions

This article provides an overview of virus disinfection by the semiconductor photocatalysis process to address the problems associated with virus pandemics. The unique merits of g-C₃N₄-based photocatalysts and their utilization in diverse fields, including energy, environment, and biomedicine-related applications, have instigated researchers to provide perspectives on g-C₃N₄-driven virus disinfection. Their development is emerging and rapidly progressing owing to their metal-free, visible-light-active, non-toxic, and biocompatible nature for the next generation of sustainable virus eradication techniques. It was discovered that the critical photocatalytic virus inactivation mechanism for g-C₃N₄ involves the action of reactive oxidative species (ROSs), mainly O₂^●− and OH^● radicals, which rupture the capsid shell, deactivate proteins, damage genes, and eventually inhibit virus regrowth. This article reviewed and assessed the latest advancements in g-C₃N₄-based photocatalysts for virus inactivation. The photocatalysis operating parameters, such as temperature, light intensity, photocatalyst concentration, pH, and the effect of organic matter, affecting the antiviral performance of g-C₃N₄ have been well-explored to enhance the reactive oxidative species production rate and maximize the visible light spectrum’s utilization. The contributions of g-C₃N₄-based photocatalysts in healthcare applications, including bioimaging, therapeutics, and bacterial biofilm control, exemplify the prominent role of the ROSs released under visible light irradiation. However, particular long-term challenges remain regarding both g-C₃N₄ and the photocatalysis process, which restrict the large-scale applicability of g-C₃N₄-based photocatalysts in virus disinfection. Future studies should undertake pilot-scale photoreactor research and fill the remaining knowledge gaps on the exact photocatalytic viral inactivation mechanism. We hope that this review helps to realize the potential of photocatalysis as an alternative sustainable technology to prevent the regrowth of viruses.