An Update on Graphene Oxide: Applications and Toxicity

Graphene oxide (GO) has attracted much attention in the past few years because of its interesting and promising electrical, thermal, mechanical, and structural properties. These properties can be altered, as GO can be readily functionalized. Brodie synthesized the GO in 1859 by reacting graphite with KClO3 in the presence of fuming HNO3; the reaction took 3–4 days to complete at 333 K. Since then, various schemes have been developed to reduce the reaction time, increase the yield, and minimize the release of toxic byproducts (NO2 and N2O4). The modified Hummers method has been widely accepted to produce GO in bulk. Due to its versatile characteristics, GO has a wide range of applications in different fields like tissue engineering, photocatalysis, catalysis, and biomedical applications. Its porous structure is considered appropriate for tissue and organ regeneration. Various branches of tissue engineering are being extensively explored, such as bone, neural, dentistry, cartilage, and skin tissue engineering. The band gap of GO can be easily tuned, and therefore it has a wide range of photocatalytic applications as well: the degradation of organic contaminants, hydrogen generation, and CO2 reduction, etc. GO could be a potential nanocarrier in drug delivery systems, gene delivery, biological sensing, and antibacterial nanocomposites due to its large surface area and high density, as it is highly functionalized with oxygen-containing functional groups. GO or its composites are found to be toxic to various biological species and as also discussed in this review. It has been observed that superoxide dismutase (SOD) and reactive oxygen species (ROS) levels gradually increase over a period after GO is introduced in the biological systems. Hence, GO at specific concentrations is toxic for various species like earthworms, Chironomus riparius, Zebrafish, etc.


INTRODUCTION
Graphene is a single-layered structure with C-atoms (sp 2 hybridized) forming hexagons. Due to the presence of free electrons, the conductivity of graphene is significantly high. Good electrical and thermal conductivity, high tensile strength (stronger than steel), 1 the largest strength to mass ratio, and high surface area (1168 m 2 g −1 ) 2 are some characteristics which make graphene and graphene-based materials excellent candidates for a large number of applications. Nanoelectronics, 3 drug delivery, 4 catalysis, 5 sensors, 6 energy storage (batteries), and tissue engineering 7 are some areas where graphene/graphene oxide (GO) could play an important role. Lots of π−π stacking between the layers causes graphene to aggregate and create a hydrophobic moiety. To overcome this problem, oxidation of graphene is done via the Hummers method followed by exfoliation. 8 GO is a single-layered oxidized form of graphene. Because of the presence of oxygen-containing groups on the surface, π−π stacking is significantly reduced, which also reduces the conductivity and introduces lattice defects in GO. It can be dispersed in water and a few organic solvents due to the formation of hydrogen bonds between hydroxyl groups present on the surface and in the solvent. It is easy to synthesize and keep stable at room temperature. Owing to this property, thin films of GO and its derivatives are easily fabricated on different substrates. GO is readily functionalized because of the presence of large oxygencontaining functional groups, making it highly receptible to complex with metal ions and different compounds. Through reduction, the electrical conductivity of GO can be enhanced. Reduced graphene oxide (rGO) is the substance formed after reduction. rGO is a near relative of graphene, but it has its own identity within the graphene family due to its unique properties. This substance has numerous applications ( Table  1) and a promising future. The variety of existing and future 3.1. Spectroscopic Characterization. X-ray photoelectron spectroscopy (XPS), UV−Visible, Raman, Fourier   transformation infrared (FTIR), and X-ray diffraction (XRD) are spectroscopic techniques used for the characterization of GO as shown in Figure 4.

UV−Visible.
Kigozi et al. synthesized GO from graphene by the Hummers method (HM) and modified Hummers methods (MHM1 and MHM2) and compared the GO obtained. As shown in Figure 5, the graphite flakes and GO samples were scanned between 200 and 700 nm in wavelength. The spectra's absorption peaks were noticed at two distinct wavelengths. These two distinct types are characteristic features that are utilized to distinguish GO from graphite flakes with a peak in the 320−360 nm region, as seen in Figure 5a, which emerged at a wavelength of 310 nm. In the absorbance spectra, two major peaks were observed: one at 230 nm and the other in the range 280−300 nm. The former one is because of the C−C bond's π−π* transition, and the latter one is due to the C� bond's n−π* transition. 63 3.3. X-ray Diffraction (XRD) Analysis. The presence of a peak at 10.44°in the XRD pattern of GO nanosheets manufactured by Muniyalakshmi and the team supports the formation of GO nanosheets and reveals that the sheets are separated by 0.846 nm after oxidation and exfoliation. As depicted in Figure 6, the diffraction pattern is captured between 5 and 50°. 64

Fourier Transform Infrared (FTIR) Analysis.
The appearance of a broad peak at about 3440 cm −1 (Figure 7) is due to the carboxylic group's O−H, while a sharp signal at 1720 cm −1 indicates the presence of carbonyl in the carboxylic group. The C−OH stretch of the carboxylic and the alcoholic groups attached to the graphene-conjugated system are related to the two signals observed at 1363 and 1226 cm −1 , respectively, while stretching for epoxy groups lies at 1085 cm −1 . Further, a signal at 1630 cm −1 corresponds to sp 2 carbons of arenes. 65 3.5. Raman Spectroscopic Analysis. The typical Raman bands of single-layered GO include the G-band (1585 cm −1 ) created by sp 2 -hybridized carbons, the D-band (1350 cm −1 ) caused by sp 3 -hybridized carbons connected to hydroxyls and epoxides, and the 2D-band caused by GO sheet stacking. It has been observed that upon stacking of multiple layers (bilayer) the positions of the G-and 2D-bands shift to lower and higher values by 6 cm −1 and 19 cm −1 as shown in Figure 8(B). 71 Also, the intensity of the D-band was increased upon stacking.
3.6. X-ray Photoelectron Spectroscopic (XPS) Analysis. The presence of C and O is indicated by two strong peaks in the survey scan spectra of GO, which are designated as C 1s and O 1s peaks and correspond to binding energies of 280−290 eV and 528−536 eV, respectively. The first enlarged peak in Figure 9a corresponds to C−C bonds, whereas the  second signal of stretching nearly at 287 eV suggests the existence of epoxy functionality. Another signal at 289.50 eV corresponds to the carbonyl of carboxylic acid, and the fourth signal at 291.28 eV indicates the presence of an ester functionality. As can be seen in Figure 9b, the C 1s peak of GO is really composed of five smaller peaks, each of which has a unique binding energy that reveals a different functional group. The first expanded peak is centered at a binding energy of approximately 284.76 eV and shows single-bond C bonds. The second peak is at around 287.00 eV and shows singlebond O bonds. The third peak is at 289.50 eV and shows double-bond O bonds. The fifth peak is at 291.28 eV and shows single-bond C�O bonds. 66

Energy Dispersive X-ray (EDX) Analysis.
The wt % of carbon is reduced to 44.59% on oxidation of graphite using the Hummers technique (HM), resulting in a 2.08% rise in the atomic percentage of oxygen. This indicates that the approach increased the amount of oxygen in the graphite, resulting in GO formation. Other atoms like sulfur, chlorine, calcium, potassium, and iron were incorporated during the process. This might be due to unfinished reactions and a GO cleaning process that is not up to par. These elements might have come from the oxidation processes' starting chemicals and reagents. 63 Figure 10 represents the EDX of (a) graphite, (b) GO−HM, (c) GO−MHM1, and (d) GO−MHM2.

Microscopic Characterization.
State-of-the-art microscopy tools are used to observe graphene on substrates directly. Electron microscopy is used to identify qualitative and quantitative details of graphene flakes. The degree of exfoliation, number of layers, lateral size, and atomic-level flaws were characterized by transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), and scanning electron microscopy (SEM). 66 Figure 11 represents some techniques for microscopic characterization of GO.
3.9. Scanning Electron Micropscopic (SEM) Analysis. Figure 12 contains SEM images of the low and high oxygen concentration samples. In both cases, severely wrinkled graphene layers can be seen, indicating that the leftover oxygen has caused a deformation in the graphene layers. 67 3.10. Thermo-gravimetric Analysis (TGA). TGA analysis was used to determine the stability of Go-M-Cu at temperatures ranging from 0 to 550°C. From roughly 50 to 550°C, the TGA graph exhibited three primary weight losses. The first weight loss, which begins around 50°C and continues to 150°C, is credited to elimination of adsorbed water, while the subsequent weight loss that begins at 200°C and extends to 300°C is for the decomposition of functional groups embedded on GO as well as the decomposition of organic ligands that they adsorbed physically on the surface of GO. The final and significant loss of covalently and chemically immobilized organic ligands and copper complexes on the basal plane of GO is attributed to the temperature range between 300 and 550°C. 15 3.11. Density. The density and specific gravity of all the samples were measured using an electronic densimeter MD-300S. In Figure 13, the density reading indicates that E-GO has the highest density (2.260 g/cm 3 ). This is due to agglomeration, as GO in ethanol has been reported to be slightly agglomerated. A-GO, on the other hand, had the lowest density (1.167 g/cm 3 ) because GO does not agglomerate in acetone. As a result, agglomeration can result in the formation of closely packed particles. 68

COMPARISON OF GRAPHENE OXIDE WITH OTHER 2D MATERIALS
Recent research has centered on the creation of semiconductor nanocomposite materials based on 2D reduced GO (rGO) to enhance its catalytic uses (Table 4). Apple juice and zinc acetate were used to produce in situ 2D rGO−ZnO (rGZn) nanocomposites, which is a commonly reported green synthesis technique. A smart cotton material coated with rGZn has been developed, and its photocatalytic self-cleaning property has been proved by the degradation of methylene blue, rhodamine B dyes, and tea stains even under sunlight irradiation, which is rare in the literature. 73 Multilayered laminates of graphite oxide (GtO) and GO are hydrophilic materials that are readily sandwiched by water and other polar solvents. The high adsorption capacity of GO materials makes them useful as sorbents for treating wastewater, elimination of numerous contaminants, humidity sensors, protective semipermeable coatings, gas and liquid mixture separation, nanofiltration, fuel cell and battery membranes, and water   desalination. 74 Due to their favorable features for essential applications, ultrathin 2D MOs beyond graphene and other 2D nanomaterials (TMDs, metal carbides) have evolved into a new class of nanomaterials. Kumbhakar et al. have investigated a number of 2D MOs with varied oxidation states (MO, MO x , and M x O y ), spinel-type MOs, perovskite nanomaterials, and their applicability in a variety of fields. Some ultrathin 2D MOs have shown performance that has the potential to outperform existing commercial technology. 75 To develop a carbon-neutral economy, photocatalysts may gather sunlight to extract H 2 and O 2 from H 2 O, fulfilling the energy demand while minimizing greenhouse gas emissions. Carbonaceous semiconductors are excellent candidates to transfer solar energy into chemical energy due to their structural and chemical modifiability. 76 In another example, it was found that the copper−graphene (Cu−Gr) nanocomposite has antiviral activity. This slows infection development by reducing viral gene expression, replication, and progeny virus particle generation, so it might reduce the spread of respiratory viruses. 77 Due to its advantageous inherent features, GO has been utilized in the construction of numerous biosensors, including electrochemical, optical (fluorescence, colorimetric, and Raman), and mass analysis. 78

APPLICATION OF GRAPHENE OXIDE AS THE CATALYSIS FOR DIFFERENT TYPES OF REACTIONS
Catalysis with higher efficiency and lower environmental impacts has emerged as a popular option for industrial processes. The oxygen-containing groups on the GO surface have a high degree of hydrophilicity and chemical activity. Furthermore, the functional groups embedded in GO operate as excellent harboring sites for numerous active catalytic species. A significant number of sp 3 -hybridized carbon atoms bond to a variety of oxygen-containing groups, which serves to insulate GO and leads to a resistance of 1012 sq −1 or larger per  sheet of GO (Table 5). The GO's sheet resistance can be reduced significantly, thereby turning it into a semiconductor. The band gap of GO can be monitored by changing the arrangement, coverage, and functional groups. GO's mechanical and conductive qualities make it an attractive material for catalysis. Hamed et al. 9 studied the heterogeneous copper complex immobilized on GO for Beckmann rearrangement to convert aldoximes into primary amides as in Scheme 1. GO− metformin−Cu (1.6 mol %) using water as solvent gives 95% yield in 0.5 h, which is the highest among other heterogeneous catalysts.
Khatun et al. 94 reported the synthesis of carbamates via the reaction of amine (5 mmol), benzyl bromide, or n-butyl (5 mmol) in the presence of Zn(II)DETA@GO (40 mg) as a catalyst. TLC was used to monitor the reaction's development. EtOAc was added to the mixture for dilution, and after the completion of the reaction brine was used for the workup. EtOAc was recovered and dried with sodium sulfate (Scheme 2). Gas chromatography was used to examine the conversion. Column chromatography was used to purify the product, and    11 reported the reaction between aromatic/ aliphatic amines, aromatic aldehydes, and trimethyl phosphite to get α-aminophosphonates at room temperature (Kabachnik−Fields reaction) as shown in Scheme 3. The presence of carboxylic acid and hydroxyl groups in GO may explain its higher catalytic activity in comparison to other catalysts. Thombal et al. 12 investigated the reactions of unprotected sugars like D-glucose with allyl alcohol (glycosylation) in the presence of catalyst (sulfonated GO) in the absence of solvent. They have used 5 wt% of GO to validate the impact of GO (Scheme 4).
According to Anouar et al., 10 liquid organic hydrogen carriers (LOHCs) can manufacture hydrogen as shown in Scheme 5. High rates (turnover frequency) must be achieved at mild circumstances and are one of the most essential requirements for being a catalyst for hydrogen production from LOHCs. The nongaseous physical nature of formic acid and its derivatives as well as its widespread availability via catalytic hydrogenation of CO 2 are the key reasons for this choice. In a 10 mL solution of 1 M NH 4 HCO 2 (ammonium formate), 50 mg of the catalyst (2.5wt % of Pd) was added.
Chyana et al. 95 studied the role of GO as a catalyst for the synthesis of dihydropyrimidine (Biginelli reaction) from cinnamaldehyde. The optimal conditions for this reaction are 7.5% catalyst at 100°C and 30 min to get a product of 83.7% (Scheme 6).
Mittal et al. 13 found that covalently grafting the Cu(II) Schiff base complex on GO resulted in a heterogeneous catalyst, which was characterized by numerous spectroscopic methods. The catalyst presented is easy to use and stable under ambient circumstances due to its ease of separation from the reaction mixture, as shown in Scheme 7. The designed catalyst could be recycled four times without losing its catalytic activity. The yield obtained was satisfactory.
Rana et al. 14 investigated the reaction between nitromethane and p-hydroxybenzaldehyde using a triamine-functionalized GO catalyst (TGO) under a N 2 environment, as shown in Scheme 8. Nitromethane (10 mL), p-hydroxybenzaldehyde (1 mmol), and TGO catalyst (0.05 g) were mixed for 2 h at 50°C to get the product in high yield (Scheme 8).
Chattopadhyay et al. 96 found that GO contains highly mobile electrons, which might help to boost the activity of Ag−GO NPs employed as shown as in Scheme 9. The Ag−GO nanocomposite can serve better as a platform for dispersing silver NPs. As a result, the Ag−GO nanocomposite inhibits Ag NP agglomeration and has a huge surface area to get a larger number of active sites for catalysis. GO may potentially boost the catalytic activity of Ag NPs in the process, rather than only acting as a support.
Zhang et al. 98 used GO−NH 2 −NEt 2 in cooperative catalysis for the conversion of benzaldehyde into β-nitro styrene, as in Scheme 11. In this reaction, tertiary amines activate the nucleophiles, while the primary amines activate the carbonyl compounds by formation of imine intermediates in the nitroaldol reactions. The catalytic synergistic effect can be understood using GO−NH 2 −NEt 2 with an NH 2 :NEt 2 in different ratios. The ratio of 1:1 shows the highest catalytic activity with selectivity toward the trans-β-nitro styrene of 100%.
Epoxidation of various alkenes using H 2 O 2 (oxidant) in the presence of PDA/GO was explored. High yields and low reaction times were obtained for the epoxide products. This novel catalyst was recovered and reused easily multiple times before showing a significant reduction in efficiency. The yield was 98% when CHCl 3 was used as solvent for 2 h (Scheme 12). 99 Trzeciak et al. 100 studied carbonylative Suzuki−Miyaura cross-coupling, which involves the reaction of 4-iodoanisole with phenylboronic acid using Pd/GO or Pd/GO-TiO 2 as promising catalysts to get the products in a smaller span of time. At 1 atm of CO and a modest quantity of catalyst, Pd/ GO showed good efficiency in the reaction, yielding the   The great effectiveness of PTT was demonstrated when mice tumors that had been BP-treated and then exposed to radiation shrank in size in just three days, and the animals continued to live for more than a month as a result of the treatment. Fluorescence bioimaging

DOX
The PEGylated g-CNQDs show improved physiological stability and a 9.3% quantum yield in their fluorescence emission. In contrast to neutral pH, the DOX release from the PEGylated g-CNQDs was higher in acidic circumstances.
necessary diarylketones (0.2 mol %). Pd/GO-TiO 2 was used in four consecutive cycles and had the highest productivity, with almost 95% of the ketone production. The catalytic efficiency was evaluated by reacting lactic acid (50%) and ethanol (esterification) by Vu et al. 101 as shown in Scheme 13. The weight ratios of the GO catalysts and sulfonated biochar to lactic acid were 1% and 5%, respectively. Because it is hard to filter GO, the weight ratio of the GO catalyst was not more than 1%.
For the esterification process, GO was discovered to be an effective and reusable acid catalyst. Many aromatic acids, aliphatic acids, and alcohols reacted well under standard conditions and produced high yields of the desired products. With strong catalytic activity, the heterogeneous catalyst may be readily recovered and recycled in dichloroethane solvent.
To test the effectiveness of the catalysts for nucleophilic substitution processes, Yang et al. 121 explored the synthesis of iodine octane from NaI and chlorobenzene (Scheme 14). Among the GO and GO−Px catalysts, GO-P600 showed the best activity with 95% yield and nearly 100% selectivity. Zarnegaryan et al. 103 studied cross-coupling reactions (Suzuki−Miyaura coupling) in the attendance of the GO− N 2 S 2 using the reactions between phenyl boronic acid with 4halo (Cl, I, and Br) nitrobenzene as shown in Scheme 15. The yield was 99% when GO−N 2 S 2 was used with 4-iodo nitrobenzene and phenyl boronic acid.
In the synthesis of 3,4-dihydropyrimidinones, the clay−GO (10:1) nanocomposite gave good yields in a shorter period of time. Narayanan et al. 104 showed better results with GO as compared to the current reported catalysts (Scheme 16).
Styrene was oxidized according to the findings of Wu et al., who used Mn−salen−GO as a catalyst for the reaction. The yield was good (90%). 105 Acocella et al. 106 investigated the effects of several graphitebased nanofillers in ring-opening reactions of epoxide (Scheme 17) triggered by amines for diglycidyl ether (DGEBA) or bisphenol. Their findings revealed that GO had a catalytic activity on epoxy resin cross-linking by amines as the viscosity of the reaction mixture increased with time, depicting an increase in polymerization/cross-linking ( Figure 14).
In the reduction of 4-nitrobenzene with aqueous NaBH 4 , Zhang et al. 107 studied the catalytic activity of the nanocomposite to get the 4-aminobenzene in high yield (98%) as shown in Scheme 18.
Li et al. 108 established a radical addition approach in an O 2assisted one-pot procedure to synthesize 3-trifluoroalkylated quinoxalin-2(1H)-ones under suitable conditions with 80 wt % of GO (Scheme 19). According to mechanical investigations, GO inhibited the synthesis of superoxide trifluoromethyl radicals, allowing it to execute an autotandem radical addition. In the absence of metal catalysts, this unusual multicomponent tandem process permitted access to CF 3 -substituted quinoxalinones, whereas GO acted as the catalyst. These changes, in a larger sense, highlight the potential of GO as a catalyst in synthetic chemistry rather than solely as a source material to graphene-based products. GO helps CF 3 SO 2 Na to form the electrophilic CF 3 radical, which allows for an addition reaction with alkynes to produce alkyl nucleophilic radicals that react with the quinoxalin-2(1H)-ones' C-3 position. With MeCN and ethyl acetate at 100°C, 85% yield was recorded with 80 wt % of GO.
Eftekhar et al. 15 used a GO-functionalized copper complex for the synthesis of the 1,2,3-triazole derivative. This catalyst can efficiently catalyze the synthesis of 1,2,3-triazoles in H 2 O. At first, the catalyst was dispersed in H 2 O (10 mL) by sonication for 10 min, and then sodium azide, phenyl acetylene, and benzyl bromide were added to the mixture and refluxed to get the product of interest, that is, 1,2,3-triazole derivatives in a high yield of 95%.
Sachdeva et al. 109 reported a huge range of functional groups such as methoxy, methyl, or bromide present in either dicarbonyl compounds or nitroaniline along with aldehyde or ketone, providing promising transformations. GO/rGO can be used as a catalyst in a one-pot synthesis for good yields (83− 95%) (Scheme 20). Catalysts can be recycled without losing any activity for up to four runs.
Ganesan et al. 110 exploited GO as a simple catalyst for the synthesis of β-aminoketones via a three-component Mannich reaction under moderate conditions, as shown in Scheme 21. Without any particular functionalization, native GO acts as a carbonaceous solid Bronsted acid catalyst, generating a range of β-aminoketones under metal-free conditions. The current catalytic technique eliminates the need for hazardous workup, and chromatographic purification produced a high yield of βaminoketones. The catalyst can be used for up to six successive catalytic cycles without losing substantial activity.
Sarvi et al. 111 employed a ZnO-immobilized GO-based catalyst for the anaerobic oxidative conversion reaction of alcohols to nitriles in H 2 O (Scheme 22). Under an oxygen balloon with a ZnO/GO catalyst, aliphatic/heteroaromatic/ aromatic primary alcohols transformed to nitriles. Without considerable reduction in activity, ZnO/GO can be utilized for seven consecutive runs.
Mirheidari et al. 112 covalently attached an amino-functionalized SiO 2 sphere/cobalt combination to a GO surface. In the production of amino naphthoquinones in ethanol solvent, the catalyst demonstrated significant catalytic activity for one-pot synthesis to enhance the yield (96−98%) in a smaller span of time (5−8 min).
Kumar et al. 113 identified GO−MnO 2 as a possible catalyst for the production of chalcones by Claisen−Schmidt condensation (Scheme 23). When compared to pure MnO 2 and GO, the catalytic activity of GO−MnO 2 was better.
Several reaction parameters, such as temperature, solvent polarity, and catalyst weight percent, were changed extensively to vary the reaction conditions. At 110°C, under solventless circumstances, a high yield of chalcones was achieved in a short amount of time. The catalyst was easy to separate and may be reused several times with very slight activity changes. When compared to other catalysts described in the literature, the GO−MnO 2 nanocatalyst showed much higher activity in a shorter length of time.
Ulvan's chemical structure is made up of two disaccharide units, type A 3s glucuronorhamnose and type B 3s iduronorhamnose, which are organized in a regular sequence inside the heteropolymer chain, as shown in Scheme 24. 114 For esterification of fatty acid and trimethylolpropane, Su et al. 115 utilized a SnO@GO catalyst. It can facilitate the stoichiometric esterification of trimethylolpropane and fatty acids (C6−C10). The resulting lubricating oil production might be as high as 98%. Furthermore, the SnO@GO catalyst has a high reusability and minimal residual property in goods, making it an environmentally responsible and cost-effective choice for the manufacturing of lubricating esters. The SnO@ GO composition may be recycled up to six times without deactivating.  (473 K), but on inclusion of GO, the reaction temperature was lowered (373 K) along with increased conversion percentage.
Using GO as a catalyst, the Friedel−Crafts addition of indoles to α,β-unsaturated ketones and nitro styrene was investigated. As shown in Scheme 25, several indole compounds were synthesized with good yields. The manufacture of GO catalyst from easily available and simple raw ingredients makes this process more cost-effective. 117 When the NGO catalyst was used to oxidize benzyl alcohol derivatives, the reaction continued past the aldehyde stage and yielded the corresponding carboxylic acid (Scheme 26). Based on the reaction optimization experiments, the optimal settings were 2.2 equiv of H 2 O 2 , 20% (mass fraction) of NGO, and 80°C as the reaction temperature. The low yield for benzyl alcohol oxidation in the absence of NGO proved that NGO served as a good catalyst. 118 GO is a simple and efficient catalyst for aza-Michael addition of amines and electron-deficient olefins. These reactions proceed under mild circumstances and produce high yields in shorter durations (Scheme 27). The catalyst is easily recoverable and recyclable, with consistent catalytic activity. 119 This procedure imparts GO with a high reactivity with 2,2,6,6-tetramethyl-piperidin-1-oxyl (TEMPO) as a cocatalyst for the selective oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-diformylfuran (DFF) under specific conditions (100% HMF conversion with 99.6% HMF selectivity at 80 wt % GO loading and 1 atm air). According to this study, GO may act as an oxidant in the reduction of the −COOH groups in HMF during its anaerobic oxidation. 120

APPLICATION OF GRAPHENE OXIDE IN TISSUE ENGINEERING
Tissue engineering, according to Vacanti et al., is the use of life science and engineering ideas for the production of biological substitutes that preserve and improve tissue function. Tissue engineering is a multidisciplinary area that includes mechanical engineering, clinical medicine, genetics, materials science, and other engineering and life science fields. 122 To create the right environment for tissue and organ regeneration, TE depends on the utilization of porous 3D scaffolds. Scaffolds are often embedded with cells and growth factors or are exposed to biophysical stimuli in bioreactors, which is a device or system that applies various forms of chemical or mechanical stimuli to cells. The scaffolds (cell-seeded) are either cultivated in vitro to produce tissues that may later be transplanted into a damaged location, or they are implanted directly into the wounded region, where tissue or organ regeneration is triggered in vivo, using the body's own mechanisms. The tissue engineering trio refers to the combination of cells, signals, and scaffolds. The 3D biomaterial before cells was inserted is referred to as the scaffold (in vitro or in vivo). Scaffold requirements are as follows: (i) Biocompatibility -Tissue engineering scaffolds must be biocompatible, and cells must stick to the surface, operate correctly, move freely in the scaffold, and begin to multiply before laying down a new matrix.
(ii) Biodegradability -In tissue engineering, the body's cells are expected to gradually replace the embedded scaffold over time. Scaffolds are not meant to be permanent. Thus, the scaffold should be biodegradable so that cells can create their own extracellular matrix. Degradation byproducts must be nontoxic and should escape the body without any side effects.
(iii) Mechanical properties -Scaffolds must have mechanical properties similar to the site on which they will be implanted. (iv) Scaffold architecture -To promote penetration into the cellular matrix and appropriate insertions of nutrients into cells within the scaffold and the extracellular matrix generated by these cells, scaffolds should have a connected pore structure with good porosity.
(v) Biomaterials -Scaffolds for tissue engineering are made from synthetic polymers, biomaterials, ceramics, and natural polymers. The bioadaptability and preferential electroconductivity of GO as a biomaterial for tissue regeneration have piqued curiosity. GO has the potential to be used in the controlled growth of stem cells in vivo, the liberation of active biological factors from stem-cell-containing delivery systems, and the intracellular delivery of factors like growth factors, DNA, and synthetic proteins to modulate stem cell differentiation and proliferation. Growth factors (GFs), which play critical roles in migration, maturation, and proliferation, as well as the differentiation of immature precursors into functional tissues can be transported through GO because of properties like surface chemistry and size. 16 Figure 15 represents the general effect of GO-based scaffolds on biological and mechanical properties of tissues, whereas Figure 16 shows tissue engineering for different body parts.
6.1. Bone Tissue Engineering. In bone tissue engineering, GO is commonly employed. The goal of bone tissue engineering (BTE) is to successfully include bone regeneration at the defect location of the host while avoiding problems. BTE   is made up of four main elements: osteogenic cells, a biocompatible framework or scaffold, vascularization, and morphogenetic signals. Bone tissue regeneration requires these following characteristics: (a) osteoinduction, which permits the biomaterial to encourage progenitor cells to differentiate into osteoblasts; (b) osteoconduction, which allows the biomaterial to assist bone tissue growth; and (c) osteointegration, which helps the biomaterial integrate with the surrounding bone tissue by supporting it. In the host environment, biomaterials should be stable mechanically and chemically. For adequate bone tissue regeneration, the scaffold should be biocompatible, osteoconductive, and compatible with cell adhesion as well as proliferation on the surface and inside its pores; should have good mechanical characteristics and compressive strength for better cancellous and cortical bone; and should have pore interconnectivity (the sizes of the pore are essential for BTE so transportation of nutrients and oxygen is fluent) and good biodegradability. Important considerations in developing a scaffold for use in tissue engineering include biocompatibility and cytotoxicity. The  effect of GO and other compounds on MG-63 cell viability is depicted in Figure 17. 43 Alginate microspheres have a porosity of roughly 92%, while alginate−GO microspheres have a porosity of around 87%. The porosity of alginate−GO−dexamethasone was 84%, which was lower than the porosity of alginate and alginate−GO microspheres, indicating that dexamethasone is likely cross-linked with alginate and GO. The unique composite microsphere (alginate−GO−dexamethasone), which stimulates cell migration and enhances bone regeneration, has a porosity of more than 80% ( Figure 18). The observed porosity difference is observed due the hydrophilic functional group. 43 The recent research in BTE has concentrated on the design of porous biomaterials with a superior biocompatible and mechanical reinforcement matrix that replicates the behavior, form, and microstructure of bone (Table 6). GO has the potential to be lighter than air, have good electrical and mechanical conductivity qualities, and have a high capacity for heat isolation and absorption that makes it effective in tissue engineering applications. Lee et al. explained the significant noncovalent binding properties of GO, which make it possible for it to serve as a preconcentration base for osteogenic inducers, causing quicker MSC growth in the direction of the osteogenic lineage. To study the binding properties of GO and rGO toward various growth factors allows researchers to better understand the molecular causes of rapid differentiation. 123 Omid Akhwan synthesized graphene nanogrids and studied the differentiation and proliferation of hMSCs, which were facilitated by the use of GO as selective 2D templates. Especially in comparison to those that flourished on graphene sheets and PDMS, the size of the cytoskeleton fibers on nanogrids was much lower. The rGONR grids demonstrated   the hMSCs' quickest osteogenic development when the chemical inducers were present. 124 The composite scaffolds showed different decomposition behavior with time. Rapid and high decomposition could be deduced with the glycosidic bond cleavage of β-G and bacterial cellulose. 125 6.2. Neural Tissue Engineering. GO may help nerve regeneration by increasing the rate of neural differentiation in embryonic and neural stem cells. Seeding of GO with a bioactive component to increase the biological activity is facilitated by its various functional groups. NSC survival, proliferation, and neuronal differentiation can all be enhanced by a PLGA/GO-TH composite (L-theanine). 133 In an in vivo investigation, GO release and PCL (polycaprolactone) biodegradation are investigated. A 15 mm sciatic nerve deficit might be effectively repaired using the GO/PCL nerve conduit. 134 The shapes of graphene-based nanomaterials might change their firmness and flexibility in addition to affecting how they interact with cells and tissues. 135 Akhavan et al. synthsised selforganized hNSCs with the pulser laser simulation. The prepared sample was tested and found to be more biocompatible, thermally, and electrical conductive. 136 Heo et al. developed a noncontact electric field stimulation procedure and a graphene/PET stimulator that can improve brain cell-to-cell communication in vitro. A modest electric field encourages the formation of new cell-to-cell coupling and strengthens already-existing connection. Using a graphene/ PET stimulator provides good flexibility and transmittance, as well as a weak field operation with a high electric field optical amplifier. These alterations in cell-to-cell interaction were caused by these abnormalities in the regulation of protein synthesis involved in cell mobility in conjunction with the cytoskeleton. 137 The research revealed that rGO/TiO 2 works as a biocompatible stimulator for efficient development of hNSCs into neurons. On rGO/TiO 2 , the differentiation of cell nuclei grew 1.5 times in response to the stimulation, but on TiO 2 and GO/TiO 2 , it was enhanced by just 24% and 48%, respectively. 138 The amount of deoxygenation and electrical conductivity for GO sheets was the same with ginseng and hydrazine. In an aqueous solution, the ginseng−rGO showed better stability against agglomeration as compared to hydrazine−rGO, as observed from XPS and shown in Figure 19. Despite that the aquaphobic film of hydrazine−rGO has little toxicity against hNSCs, no substantial cell proliferation was seen in these films. 139 6.3. Scaffolds in PC12 Cells. From 3 to 60 days, the biocompatibility of the silk fibroin−raffinose trisaccharide−GO toward PC12 (rat pheochromocytoma) cells improves gradually ( Figure 20). 140 6.4. Cartilage Tissue Engineering. Cartilage damage can occur as a result of an accident or as a result of illnesses like osteoarthritis. Tissue engineering utilizing mesenchymal stem cells (MSCs) is a regenerative therapeutic technique that includes three fundamental components: (1) stem cells, (2)   (3) growth factors (GFs) ( Table 7). 21 After 14 days, ATDC5 cell viability was greater in the chitosan/PVA/GO than in the chitosan/PVA, but the viability of ATDC5 cells was lower in chitosan/PVA nanofibers with a higher level of GO. Differential cell proliferation was observed between chitosan/PVA and chitosan/PVA/4 wt % GO on days 4, 7, and 14 but only between chitosan/PVA/4 wt % GO and chitosan/PVA/4 wt % GO on days 4 and 7. These results show that cell division is increased over time ( Figure  21). 21 Internal implantation of the CSMA/PECA/GO scaffold was done by varying the concentrations and period of exposure (1, 2, 4, and 8 weeks). It has been observed that there is a significant increase in cell viability as the period of exposure increases. The implanted composite on the mice slowly degraded over a period of time, as shown in Figure 22.
Liao et al. studied the subcutaneous implantation of CSMA/ PECA/GO scaffolds in a rat animal model. The scaffold's morphology varied over time after it was implanted ( Table 8). The deterioration of the hybrid scaffold progressed in tandem    with the implantation. It took two months for the scaffold to totally decay. 148 6.5. Skin Tissue Engineering. New skin tissue engineering methodologies have been created that have the ability to imitate the biological features of natural tissue with a high degree of intricacy, flexibility, and repeatability. Biocompatibility, morphology, pore size, porosity, mechanical strength, and water absorption capability of hybrid scaffolds are all improved by GO concentration (Figure 23).
The biological performance of a GO-modified chitosan/PVP nanocomposite was examined by Mahmoudi et al. In the rat, electronic pictures demonstrate that nanofibrous membranes and GO have a significant influence on wound closure, as shown in Figure 24 and Table 9. The inclusion of GO nanosheets provides additional benefits in terms of strength, permeability, and cell attachment. There were no signs of scarring or inflammation in the region that was evaluated. 153 6.6. Tissue Engineering in Dentistry. Antimicrobial activity, regenerative dentistry, oral cancer therapy, drug delivery, improvement of dental biomaterials, and BTE are all possible using GO in dentistry. Because of its biocompatibility, GO scaffolds can be used in bone tissue regeneration, osteointegration, and cell growth. Researchers have also developed GO for biofilm, which takes preventions and changes on the surface for better antibiofilm and antiadhesion capabilities. 156 The bioactivity of periodontal stem cells on Na 2 TiO 3 coated with GO was investigated by Zhou et al. ALP activity has long been employed as a marker for osteoblast-like cells. On days 7 and 10, PDLSCs on GO−Ti substrates displayed higher ALP activity than those on Na−Ti substrates ( Figure 25 and Table 10), showing that the presence of GO promoted an early stage of bone formation. 27

PHOTOCATALYTIC BEHAVIOR OF GRAPHENE OXIDE
The basic mechanism behind the photocatalytic reactions involves irradiation of the photocatalyst surface with light energy equal to more than the band gap of photocatalysts. After absorption of energy containing light photons, the electrons get excited. The transfer of electrons from the valence band (VB) to the conduction band (CB) of holes is generated in the VB. These electrons and holes in the CB and VB eventually migrate to the photocatalyst's surface for photocatalytic reactions. GO attracts researchers' attention toward photocatalytic reactions because its band gap is easily tunable. Moreover, it paved its way for being an electron-trapping layer. In the mechanism of photocatalysis, there are possibilities that the photogenerated charge carriers might recombine and stop the photocatalytic activity. GO serves as a support to prevent this charge recombination by forming heterojunctions or composites with other materials. The formation of the heterojunction enables transfer of electrons from one material to another; i.e., the photogenerated charge carriers lie on the surface of different materials, and hence their probability to recombine is prohibited. Three major photocatalytic applications of GO are, i.e., photocatalytic hydrogen generation, photocatalytic CO 2 reduction, and photocatalytic degradation of organic contaminants.

Photocatalytic Degradation of Organic Contaminants.
Industrialization has been increasing on a very fast pace which ultimately leads to a scarcity of energy resources and a release of toxic organic contaminants into the environment. An increasing population results in increasing energy demands. These contaminants have dangerous effects on the environment as well as on human health. Looking at freshwater scarcity, it has become an essential need to provide fresh water. Photocatalysis has been an easy and useful technique to deal with these environmental concerns. The primary source being sunlight, present abundantly in this method, makes it superior over other alternative methods present to deal with environmental concerns.
GO-based semiconductor composites have been most appropriately utilized in the photocatalysis process for environmental pollutants. The chemically active surface of GO causes different organic entities to get attached to its surface, which ultimately modify its electronic properties. The binding of different pollutants to the GO surface is due to π−π interaction between aromatic pollutants and sp 2 -hybridized graphene. 159 This property of GO is of utmost use in photocatalysis, as GO can improve the photocatalytic behavior of various semiconducting materials by forming composites or heterojunctions with them. GO has been known to enhance the photoactive response of metal oxides such as ZnO. A combination of ZnO with GO exhibited remarkable photocatalytic performance due to the ability of GO to decrease the aggregation of ZnO particles, acting as an electron acceptor and inhibiting charge recombination. ZnO−GO hybrid material has been reported to degrade methylene blue (MB) dye with a photocatalytic efficiency of 80%. The oxygen functional groups of GO interact with those of ZnO, facilitating electron transfer from ZnO to GO upon light  irradiation. In this way, GO contributes to an enhancement of photoactivity of ZnO by reducing charge recombination. 160 Mohanta et al. fabricated a ternary heterojunction, i.e., Au− SnO 2 −rGO, for photocatalytic degradation of clothianidin. rGO by serving as an electron sink prohibits an electron−hole recombination process. 161 The mechanism of the electron transfer in Au−SnO 2 −rGO has been shown in Figure 26. Akhavan et al. synthesized GO platelets and deposited them on anatase TiO 2 thin films. These GO platelets were reduced at different irradiation times, and the reduced platelets were utilized for E. coli's degradation photocatalytically. Under solar light irradiation, the photoactivity for bacterial degradation was enhanced by a factor of 7.5. 162 Also, Akhavan et al. explored graphene-tungsten oxide-based composite film for photoinactivation and photodegradation of viruses. 163 Another graphene-based nanocomposite, i.e., the sulfur-doped GO/ Ag 3 VO 4 nanocomposite, was found to exhibit excellent photocatalytic degradation of cationic and anionic dyes. Also, the nanocomposite photocatalytically degraded dithiocarbamate fungicide thiram within 1 h to yield thiourea as a product. The nanocomposite was reported to show complete mineralization with more than 90% organic content removal. 164 Graphene/TiO 2 composite films with sheet-like surface morphology exhibited excellent photocatalytic performance by inducing cytotoxicity on the C. elegans nematode. This photoinactivation of the nematodes was attributed to the high-level reactive oxygen species (ROS) generation under solar light irradiation. 165 Tuan et al. synthesized SnO 2 −rGO nanocomposites via a one-step simple hydrothermal method for photoassisted degradation of MB. The 90% photocatalytic degradation of MB using SnO 2 −rGO nanocomposites over 30% photocatalytic degradation by SnO 2 signifies the importance of GO. Band gap narrowing was observed in SnO 2 on doping with rGO. The band gap changes from 3.93 to 3.13 eV for SnO 2 to 2% SnO 2 −rGO. rGO has been playing the following major roles in the photodegradation of MB using this composite: increasing the adsorption capacity of MB over the composite's surface, reducing the band gap along with the formation of a large no. of electrons and holes, and reducing the recombination of carriers, etc. The basic mechanism of the work is displayed in Figure 27. 166 A metal−organic framework (MOF) composite with graphene, i.e., MIL-68(In)-NH 2 /GO has been reported to exhibit increased photocatalytic activity for amoxicillin (AMX) degradation. The enhanced activity (93% degradation) for the composite has been attributed to GO which acts as an efficient electron transporter. 30 Concerning the photocatalytic behavior of GO, one of the key points is band gap opening as well as engineering of GO-based materials. One of the methods for band gap engineering of GO is fabrication of a graphene nanomesh. The GO sheets were vertically immobilized at the surface of ZnO NRs having a diameter of 140 nm and less than 1 μm of average length to achieve graphene nanomeshes via local photodegradation of GO sheets. The graphene nanomeshes have shorter oxygen-containing carbon bonds and higher carbon defects. Moreover, the valence band of GO sheets was found to be at different energies than Fermi energy levels. For graphene nanomeshes, binding energies of 1.6 were discovered to be the closest to the Fermi energy level. These materials can be utilized to stimulate hNSC using NIR photocatalysis. The hNSC's proliferation on the GO nanomeshes was correlated to the presence of excess oxygen functional groups formed on the edge of the GO nanomeshes that leads to superhydrophilicity of the surface. The graphene layers revealed cell differentiations, higher differentiation of neurons than glia, and more elongations of the cells under NIR laser stimulation. 167 These semiconductor nanomeshes have been used to laser-stimulate human brain stem cells. With a band gap energy of 1 eV, GO nanomeshes were created by Akhavan et al. and successfully used in NIR laser stimulation of hNSC differentiation into nerve cells. A few other GO-based composites for removal of certain organic pollutants, such as gaseous benzene, oxytetracycline, heavy metals, etc., in the environment have been reported in Table 11. 7.2. Photocatalytic Approach to Generate Hydrogen via Splitting of Water. Coupling GO with semiconductors has been a fascinating approach for photocatalytic H 2 generation during water-splitting reactions. This coupling should be appropriate, keeping in mind the band position of both GO and the semiconductor. GO-supported semiconductors have been reported to show higher yields of hydrogen in photocatalytic water-splitting reactions. This has been attributed to properties of GO, such as expanding light absorption tendency and acting as a supporting material to   Figure 28 provides a basic mechanism of water splitting by a photocatalytic approach. 171 Wang et al. prepared a CuS-ZnO/rGO/CdS composite for visible-light-induced H 2 production ( Figure 29). The heterostructure showed a good photocatalytic hydrogen generation rate of 1073 μmol/h/g. The role of rGO in this heterostructure has been justified, as it prevents the agglomeration of Zn particles during synthesis, serves as a migration channel to provide efficient separation of photogenerated charge carriers, and hence helps in improvement of the migration rate. The role of rGO as a carrier transport channel has also been depicted by Chen et al. in photocatalytic H 2 generation using the NiO@Ni-ZnO/rGO/CdS heterostructure. The higher hydrogen rate using this heterostructure has been attributed to the synergistic effect between ZnO and CdS supported by rGO which behaves as a transport channel for the flow of photoexcited carriers. 172 Wang et al. synthesized the rGO/Pt-TiO 2 nanocomposite and found that the composite exhibited a photocatalytic hydrogen generation efficiency of 1075.68 μmol/h/g. rGO in the composite developed synergetic interactions with TiO 2 to improve the absorption of light tendency and efficient charge separation, which results in excellent hydrogen generation rate, found to be 5-fold and 81fold greater than Pt/TiO 2 and pure TiO 2 , respectively. 173 There are various studies on the hydrogen generation through water-splitting reactions using GO-based photocatalysts tabulated in Table 12. 7.3. Photocatalytic CO 2 Reduction. The significant rise in global temperature due to an increase in greenhouse gases is the most challenging concern of this century. Graphene, being a narrow band gap photocatalyst, has shown immense results with respect to photocatalytic CO 2 reduction in Table 13. The rational design of graphene with wide band gap materials to develop graphene-based photocatalysts has been currently drawing researchers' attention. The Cu 2 O/rGO composite has been reported for photoreduction of CO 2 . In the microwaveassisted fabrication of this composite, rGO not only prevents the charge recombination by electron trapping but also improves the stability of Cu 2 O by acting as a stabilizer. The stability of Cu 2 O after fabrication of rGO was analyzed through inductively coupled plasma optical emission spectrometry (ICP-OES). The results showed that Cu 2 O under-   This higher surface area facilitates enhanced light absorption tendency, and graphene sheets accelerate electron transfer. Moreover, the composite formed has a 2.9 eV band gap which is found to be lower compared to pure TiO 2 (3.2 eV), resulting in a red shift and hence contributing to higher photocatalytic visible-light-driven CO 2 reduction with an efficiency of 21.38 μmol/g. 36 The mechanism of charge transfer in TiO 2 -rGO for photocatalytic CO 2 conversion has been shown in Figure 30.

ELECTROCHEMICAL NATURE OF GRAPHENE OXIDE
GO consists of numerous oxygen groups like carboxyl, epoxide, hydroxyl, and carbonyl, which are covalently bonded to sp 3hybridized C networks. 192 However, the exact structure of GO is a much-debated topic and requires further in-depth studies.
Due to its unique structure, a variety of intriguing properties emerge, including electrical, optical, thermal, mechanical, and electrochemical. Recent investigations in the realm of GO's electrochemical characteristics are the most popular. This is evident due to the advantageous electron mobility and the unique surface characteristics of GO. 193 Some of these surface features include a thickness of one atom and a large surface area, which aid in tolerating active species and enable electron transport at the electrode surface. 193,194 In addition to this, GO also exhibits a surmountable amount of electrocatalytic activity and high electrochemical capacitance with good cycle performance. 195 Different methods can be used for the preparation of GObased electrodes, such as simple dispersion of GO-based materials, on an electrode or by confining GO on a functionalized electrode substrate. Alternatively, the method of spin-coating is also used for the preparation of GO electrodes. Self-assembling is also deemed an important technique which boosts the potential applications of GO electrodes in sensor fabrications because it can adjust the electrode dimensions efficiently to form a nanoelectrode assembly. 192 The fundamental process which is directly linked to electrochemical reactions is termed as heterogeneous electron transfer (HET). It denotes the process of electron transfer into or out of the graphene sheets from its surrounding environment. The edge plane and the basal plane are the most common sites for HET studies. However, studies have shown that the basal plane is electrochemically inert, while the edge plane shows efficient HET kinetics and is supposed to contain defects. 196 Further reduction of GOs through a chemical or electrochemical process can increase their efficiency. For instance, the cyclic voltammetry of GO sheets has been determined to be reduction waves ranging from −0.60 V (vs Ag/AgCl reference electrodes) to a maximum of −0.87 V. This process is pH dependent, wherein the reduced GOs were observed to have higher conductivity, and the mechanism for this reduction through electrochemical means is suggested to be as follows:  Because of the large surface-to-volume ratio, its dispersibility in water and organic solvents, and the large reactive functionalized surface, GOs are widely studied for electrochemical applications. In addition to moderate conductivity and good chemical stability, GO is known to demonstrate direct electron transfer to proteins and enzymes as well. Therefore, GOs have applications in the field of electroanalysis, electrochemical luminescence, electrochemical sensors, etc. 196 Akhavan et al. have explored rGO nanowalls (rGONWs) synthesized using the modified Hummers method. The electrochemical activity of free nucleotides, ssDNA, and dsDNA was studied using the synthesized rGO nanowalls. Based on the observed results, they concluded that single nucleotide polymorphism (SNP) can be detected for up to 10 DNA/mL for a specific sequence, which was effective as label-free detection. Therefore, the electrochemical biosensor developed exhibits potential for the detection of nucleotides up to resolution of single DNA. 197 Later, the same researchers fabricated spongy graphene electrodes (SGEs) that are charged with Mg 2+ ions via electrochemical deposition. They used the synthesized SGE for electrochemical oxidation of guanine for ultrasensitive detection of leukemia. Based on their results, they exhibited an improved detection limit of up to 0.02 cells/mL. 198 They further investigated the point of care diagnosis of leukemia using functionalized GO nanoplatelets (GONPs). The GONPs were synthesized using the modified Hummers method. They exhibited guanine oxidation in leukemic cells. Also, they compared the GONPs with rGONWs and observed that guanine oxidation is five times higher in GONPs over rGONWs. However, it is based on the polymerase chain reaction and, therefore, is expensive, and the results are obtained in a couple of days. Yet, it is an effective technology for leukemia diagnosis. 199

GRAPHENE OXIDE AS AN ELECTROCATALYST
GO possesses excellent electrocatalytic behavior as seen in many reactions (Table 14). This behavior of GO can be attributed to its superistic properties such as high surface-tovolume ratio, active functional groups bounded to its surface, high chemical stability, good conductivity, good electron transfer capability, and desirability in water and organic solvents. Zhu et al. constructed rGO-based heterostructures for enhanced electrocatalytic hydrogen production. The heterostructure CoSe 2 −MoSe 2 (1−1)/rGO containing a 1:1 proportion of Co/Mo exhibits a good hydrogen evolution reaction (HER) in both acidic and alkali media. The appreciating performance in both media is due to the excellent electron transfer facilitated by rGO. Also, the CoSe 2 −MoSe 2 interface provides several active sites for the adsorption of hydrogen. 200 The heterostructure exhibits an overpotential of 107 mV and 182 mV at 10 mA/cm 2 with a slope of 56 mV/dec and 89 mV/dec in acidic and alkaline conditions, respectively. This study elaborates the role of GO in constructing energy storage conversion electrodes. Li et al. integrated polyoxometalates (POMs) and pyrrole (Py) on graphene sheets with uniform distribution to obtain ternary nanohybrids, i.e., POMs−polypyrrole/rGO. The GO-synthesized ternary nanohybrid exhibited excellent electrocatalytic HER activity with 0 mV overpotential and a small slope of 33.6 mV/dec The nanohybrid exhibited good stability in an acidic medium. 201

BIOMEDICAL APPLICATIONS
GO has gained considerable interest as a potential nanocarrier platform including biomedical applications. 206 GO has a large surface area, low toxicity, biodegradability, high drug holding capacity, and targeted drug delivery system. 207 Graphite/ graphene/GO (Table 15) is an allotrope of carbon nanomaterials and a relatively new field of major implications for biomedical use. In 2011, Feng et al. published a paper on graphene for biomedical applications. 208 In 2012, Shen et al. were given the great idea of interesting studies to investigate the uses of graphene and its composites in diverse areas like drug delivery, sensors, gene delivery, and GO-based antibacterial nanocomposites. The first stage used GO as a drug carrier, and it was remarkably useful in anticancer drugs with doxorubicin. Notably, the GO could interact with the  doxorubicin by overlapping π-orbitals, and the β-cyclodextrin unit may identify receptors of folic acid in cancer cells. 209 Electrostatic interactions between GO and ionized pharmaceuticals (H-bonding) are the main mechanisms for drug loading, and the aromatic structures may be essential (such as DOX). The in situ drug-loaded nanocomposites are desirable candidates for upcoming nanomedicine therapeutic techniques in malignancies and acidic organs like the stomach. 210 In 2021, aptamers that are smaller, more chemically stable, and less toxic may be able to attach to their targets with more specificity and affinity. However, GO may still connect with single-stranded DNA/RNA (aptamers) efficiently through hydrophobic or π−π stacking interactions. GO-derived novel 2D materials with aptamer functionality can be used to build extremely sensitive biosensors for cancer detection. Materials that are GO−aptamer-conjugated are superior to cancer screening and diagnosis methods based on antibodies. They are quite effective at assisting with the identification and diagnosis of several types of cancer. 211 It was suggested that the nanostructure Au@AF−GO could be a useful medication. It was successfully applied to SPECT imaging and in vivo targeted therapy. An intriguing nanomaterial for upcoming diagnostic techniques is the Au@AF−GO nanostructure due to its exceptional qualities, which include  quick body clearance, effective tumor targeting/imaging, and short Au radioisotope half-lives. 212 For instance, employing the rGO nanomess, one of the most recent graphene constructions with very high near-infrared absorption, allows for extremely successful photothermal treatment. 213 GO has also been studied in the biomedical field due to its surface area. 214 Due to its large surface area, it may also be considered a good adsorbent material such as in drug encapsulation. Additionally, the presence of unsaturation bonds could conjugate with several drug molecules through covalent cross-linking π−π stacking interactions. 215 Through  hydrophobic interactions and π−π stacking, graphene's 2D structure with large surface area may be exploited for efficient drug loading. Furthermore, because of its large surface area, GO may be biofunctionalized at a high density using both covalent and noncovalent surface modification techniques. However, pure GO loses a bioactive site to support cell growth, which is limited to use in biomedicine. The application of graphene in gene delivery is a nonviral graphene-based gene vehicle for transfecting pDNA in mammalian cells. An innovative mixture of ethidium bromide (EtBr) and carboxylated graphene (G-COOH) is used for efficient gene delivery into AGS cells. 216 For simultaneous gene and photothermal cancer treatment, the conjugated histone methyltransferase enzyme SET1 (hSET1) on decreased polydopamine-loaded GO nanosheets was employed (rGO-PDA). Higher nearinfrared absorption is provided by the rGO-PDA nanocarriers, which also better integrate with hSET1 antisense. hSET1 antisense is delivered to breast cancer cells by nanocarriers. 217 Polyethylene glycol is used to stabilize and target gold nanorods or nanospheres coated with GO (P-L-Arg). P-L-Arg raised cellular uptake and gene-retarding effects of coated substances.
Currently, researchers developed the modified GO with carboxymethyl cellulose (GO−CMC) composites as a drug carrier, which bonded small molecules of the doxorubicin hydrochloride (DOX) drug through π−π interaction or hydrogen bonding. 218 A lot of research has been done in recent years to include nanotechnology for drug delivery, and as a result, several drug delivery systems (DDS) have been designed. 218−221 In initially designed DDS, anticancer drugs could not differentiate between cancerous and healthy cells, which caused several side effects and adverse implications for the human body. 218 The DDS should deliver the accurate dose of medicine only at accurate sites. Thus, finding a carrier for the drug is one of the major problems. A good amount of potential drug carriers like nanoparticles, micelles, 222 dendrimers, 223 biopolymers, 224 as well as synthetic polymers have been designed for targeted drug delivery. Nanocarriers have the potential to greatly improve the efficiency and accuracy of drug delivery while also reducing adverse effects, particularly when the drug is insoluble in water. 225 In DDS, GO and GO functionalized with nanoparticles and polymers were frequently employed 226,227 because of their suitable properties, low cost, simple synthesis,  and π-conjugated structure. 228 As a result, a significant amount of research is done on GO and its derivatives for DDS. 229,230 In 2022, polymer-grafted magnetic GO (GO-PVP-Fe 3 O 4 ) was successfully developed and employed in anticancer drug delivery. 231 GO was first functionalized with polyvinylpyrrolidone (PVP) before being implanted with magnetic nanoparticles (Fe 3 O 4 ) using a simple and efficient chemical process. Noncovalent interactions were used to load an anticancer drug quercetin (QSR) on GO-PVP-Fe 3 O 4 , as shown in Figure 31. The drug-carrying capacity was found to be 1.69 mg g −1 .
Comparison of the cytotoxicity of the nanocarrier in human breast cancer cells and nontumorigenic epithelial cells was done with and without drugs. The results demonstrate that the drug-embedded GO-PVP-Fe 3 O 4 nanohybrid is more harmful than the free drug to cancerous cells while being biocompatible to epithelial cells. A smart DDS that includes polymer-grafted magnetic GO (potential nanocarrier) which is pH-responsive for cancer treatment should be useful for drug delivery influenced by an externally applied magnetic field.
In 2021, the new nanoscale GO polymer composite DDS was developed and studied as a potential drug (doxorubicin) for oral drug delivery. 232 A novel doxorubicin-loaded nanocomposite composed of GO/copolymer was formed by reversible addition−fragmentation chain transfer (RAFT) and ring-opening polymerization (ROP), which is given in Figure 32. Doxorubicin was incorporated into the nanocomposite and had a size of 51 nm with a satisfactory encapsulation of 82% ± 1.12%. DOX was bonded to the surface of graphene by π−π stacking and aquaphobic interactions. The anticancer effect of the DOX@GO nanocomposite was revealed in cytotoxicity studies on breast cancer cells named 4T1 murine and could be useful in breast cancer therapy.
In 2021, the global aging population will exponentially increase the rate of Parkinson's disease (PD) incidence. 233 PD is a neurodegenerative disorder and is caused by defaced dopamine neurons in the substantia nigra pars compacta (SNpc), abnormal synuclein (-Syn) deposition, and progressive neurodegeneration in striatal regions. Even after a lot of research into the pathophysiology of PD and the development of therapies to control its progression, no substantial cure has been found to date. Puerarin (Pue) is a naturally occurring substance that possesses outstanding anti-PD capabilities. Due to its poor pharmacological properties, including low water solubility, weak bioavailability, and insufficient blood−brain barrier (BBB) penetration, its use   for the treatment of Parkinson's disease (PD) has been restricted. However, nanotechnology advancements have revealed potential advantages of targeted drug delivery into the brain for PD treatment. Xiong et al. developed for the first time the Pue-loaded GO nanosheets with better drug-carrying capacity, changeable functional groups on the surface, and high biocompatibility. Pue was then moved across the BBB into the brain with the help of lactoferrin (Lf) (targeting ligand) which is capable of binding to the BBB's vascular endothelial receptor. Studies demonstrated that Lf-GO-Pue could be a safe as well as effective treatment for Parkinson's disease (PD), 233 which is represented in Figure 33.
In 2020, Jun et al. synthesized chitosan-functionalized GO nanosheets coupled with folic acid for photothermal cancer therapy guided by near-infrared fluorescence: cancerous cells   were fully killed in vitro following laser irradiation. 234 Additionally, in vivo experiments demonstrated that tumors were entirely destroyed within 20 days after the deployment of this targeted nanosystem under laser irradiation, which is given in Figure 34. After 24 h of injection of FA-CS-GO, a photoacoustic signal was observed around the tumor area in a mouse. FA-CS-GO showed a high tumor-targeting efficiency, outstanding PAI, and powerful photothermal effect. This is the first work to demonstrate the use of nanomaterials with multifunctionalities for highly efficient FL/PAI-guided PTT, which represents an amazing new direction for nanomedicine ( Figure 35). In 2020, this work promised to enhance doxorubicin's (DOX) therapeutic efficacy as an anticancer agent by embedding it into a nanostructure. 235 A nanocomposite was prepared by loading a stimuli-responsive copolymer in magnetically active GO, and its evaluation as a stimuliresponsive DDS was done. Initially, the magnetic GO nanohybrid (MG) was successfully created in this regard. Polymerization of acrylic monomers occurred on addition of vinylic groups to the MG surface to produce N-isopropylacrylamide (NIPAM) and acrylate cyclodextrin (Ac-CD) copolymer brushes, as shown in Figure 36. The MG−PB hybrid nanosystem could be presented as a potential candidate for drug delivery in breast cancer treatment, as it is nontoxic and effective.

COMPUTATIONAL STUDIES
The computational work revealed the conformational information of the nonbonding interaction of GO. Fark et al studied the effects of the interaction of GO with NO 2 , CO 2 , SO 3 , and SO 2 using DFT-based Raman spectroscopy and VCD. They also confirmed stable conformers by MD simulation. Their work notes that VCD spectroscopy can be an alternative for analyzing the interaction between GO and molecules. 236 The physiochemical features of GO are intimately connected to its biological/toxicological activities. 237 It is tough to precisely control the physical and chemical characteristics of GO as they change as soon as it interacts with the environment. Adsorption−desorption interactions between colloids and GO have been observed in a variety of environments (water, soil, and sediment). The agglomeration of GO with organic colloids or other nanoparticles is known as heteroaggregation. 238 Sharifi et al. worked on the interaction of polydiallyl methyl ammonium chloride nanocomposites with nano GO (NGO/PDADMAC). Thermodynamic studies based on B3LYP/6-31+G** revealed that the configuration of the complex produced in the carboxy (−COOH) and hydroxy (−OH) positions has better structural integrity than the epoxy group and that when comparing the groups in experimental and analytical IR spectra the hydroxy agent has a higher probability of overlap. 239 GO showing electronic absorption spectra in solvents potentially gives a different characterization of the GO−solvent interaction, which is significant in GO-based photocatalyst and optoelectronic applications. Meng et al. in their work explained the effect of the polarity of the solvent and Hbonding using DFT by comparing two solvents (NMP and water). The transfer of electrons over GO surfaces dominates the hole−electron pair transition according to the natural transition orbital (NTO) study. 240 A DFT computational study was used to investigate alkali and alkali earth metal interactions with graphene sheets, and the B3LYP theory with a LanL2DZ basis set was used to investigate transition metal interactions. The measured results were compared to the calculated complexation energies (E Adsorption ). These experiments revealed a substantial relationship between the binding energies and charge density of metal ions, implying that there is some charge transfer occurring between the metal ion and  graphene. 241 With the use of MD simulation, the influence of varying size and shape of GO and SiO 2 nanoparticles on the high viscosity of fluids was investigated. Tersoff and Lenard-Jones (LJ) interatomic force fields were used to evaluate the viscosity of fluids containing C, O, Si, and H. MD simulations revealed that on addition of GO and SiO 2 nanoparticles to the virgin fluid the viscosity increased. The viscosity of the pure fluid and GO-incorporated fluid was statistically resolved to 88 Pa s and 94 Pa s, respectively. 242 Hasem et al. created biocompatible, antibacterial, and antiviral nanocomposites in their research. To make dialdehyde cellulose, the initial step was to oxidize cellulose with periodate (DAC). In the second phase, DAC was combined with S-containing amino acids in the vicinity of GO. FTIR, SEM, TEM, and TGA were used to characterize the produced nanocomposites. Furthermore, computational techniques and molecular docking revealed the reactivity and stability of compounds with biological action against gram-positive, -negative, and HSV-1 bacteria. From the experimental and computational data, the interaction of DAC with amino acids improved their reactivity and interaction. 243 MD simulations were used to look at how the presence of an electric field helped separate H 2 O/O 2 gas molecules over a double-layered nanoporous GO membrane. In an external electric field of 10 −4 V, the rate of gas permeation through the membrane for H 2 O molecules was found to be 3.26 × 10 −3 mol m −2 s −1 . Examining the change in interaction energy with electric field intensity has also shed light on the process of improved H 2 O/O 2 separation. Hydrogen bond interactions between H 2 O molecules and H 2 O membranes are inhibited by the electric field. Accelerating desorption in the presence of electric fields would allow for an additional adsorbent surface on the membrane, enabling the passage of H 2 O molecules. 241 Orekhav et al. reported a computational and experimental evaluation for the reduction of GO using nanosecond infrared laser irradiation (Figures 37, 38, and 39). Researchers reported that rapid aerobic heating to 3800 K results in a unique regime of high-quality GO reduction. This surprising outcome is the consequence of two different processes: (i) combustion on extremely defective regions of GO and (ii) defective reduction in the rest of the material. Under certain pulse regimes, GO transforms into rGO. 244 The application of B-and N-codoped rGO (BN-GN) as an electrolyte electrochemical degradation of paracetamol was described in this study. DFT calculations, characterization, quenching tests, and electron paramagnetic resonance analyses were utilized to investigate the reaction process, which focused on the catalyst surface at the atomic level and dominating radical species created by the reaction. The inclusion of N and B functionalities into GN increased the catalytic activity by generating new surface defects, active sites, and improving conductivity, according to the characterization data. Experimental and theoretical results revealed that codoped BN-GN boosted the catalytic activity significantly, and the B elements in C−N−B groupings were considered as the major reactive sites. 245 Computational and experimental methodologies were used to investigate tetracycline adsorption on magnetic GOFe 3 O 4 in the work. The structural and electrical character-  istics of magnetic nanoadsorbent and tetracycline are revealed by combining ab initio and DFT, demonstrating chemical adsorption between tetracycline and GOFe 3 O 4 . The reaction was spontaneous, exothermic, and chemical, according to the thermodynamic characteristics. Theoretical and experimental analyses were in agreement, demonstrating that tetracycline adsorption on GOFe 3 O 4 is mediated by a chemisorption process. 246 A complex of Cu with N,N′-bis(4-hydroxysalicylaldehyde)ethylenediamine (Salen) was incorporated on Cl-modified GO, resulting in a heterogeneous catalyst (Cu-f-GO). The structural and electrical characteristics as well as the system that determines SOCl 2 with each accessible functional group of GO were investigated using first-principles-based DFT. Other thermodynamic parameters were calculated like HOMO, LUMO, chemical potential, electronegativity, energy band gap, adsorption energy, and global electrophilicity, as shown in Figures 40 and 41. According to the data observed, the prepared catalyst performed poorly. 247 The reduction of U(VI), Se(VI), Se(VI), Re(VII), and Se(IV) in a homogeneous Fe(II) solution is not thermodynamically viable. Surface-mediated Fe(II) reduction, on the other hand, has long been thought to represent a primary avenue for the immobilization of these radionuclides. In this article, a study using DFT calculation and spectroscopic Figure 49. (a) Eyeball exposed to double-distilled water for 7 days (control group). (b) Eyeball exposed to 25 μg/mL of RGO for 7 days. (c) Eyeball exposed to 50 μg/mL of RGO for 7 days. (d) Eyeball exposed to 100 μg/mL of RGO for 7 days. (e) Eyeball exposed to 25 μg/mL of GO for 7 days. (f) Eyeball exposed to 50 μg/mL of GO for 7 days. (g) Eyeball exposed to 100 μg/mL of GO for 7 days. (h) Eyeball exposed to 25 μg/ mL of GO for 10 days. (i) Eye exposed to 100 μg/mL of GO for 7 days in vivo. 261 Copyright@2018, Elsevier, Ltd., Experimental Eye Research.  examination demonstrated that GO mediates the reduction of U(VI), Se(VI), Se(VI), Re(VII), and Se(IV) by aqueous Fe(II). The pseudo-second-order model was very well explained by the dynamics of all adsorption systems, indicating a chemical interaction. The Freundlich model might better represent the isotherms for all reaction systems than the Langmuir model. Spectrophotometry analyses revealed that the implementation of GO was due to the graphitic surface's easier transfer of electrons and, in particular, the lowered redox potential caused by Fe 2+ surface adsorption on GO. Furthermore, the inclusion of fulvic acid (FA) may increase the rate by boosting the electron transport capacity. 248 Researchers developed a design that uses the aluminum atom to boost the optoelectronics, optical properties, and absorption capacity of the GO. In this paper, they provide results from DFT approaches that enabled us to comprehend the consequences of aluminum atom doping on GO nanosheets in the gas phase. 249 The interface architectures and interaction mechanisms between cellulose derivatives and GO were determined using first-principles calculations. It has been reported that H-bonds and weak surface forces play a significant role in the synthesis of composite systems. The amount of hydrogen bonds is also influenced by steric hindrance: the lower the steric hindrance, the more hydrogen bonds are formed. 250 Alkaline earth metal functionalized GO and montmorillonite (MMT) aerogels were produced by Xin Hao and colleagues for effective Cu(II) removal of wastewater. As revealed by systematic adsorption studies, Sr-G/M possesses denser slitshaped pores, causing an effective 97.1% Cu(II) removal efficiency. In Figure 42(a), the peaks of 1421 and 1600 cm −1 shift to 1423 and 1602 cm −1 , suggesting that the −COOH group is coordinated with Cu 2+ , while the characteristic peak of −OH moves from 3430 to 3422 cm −1 , indicating that the −OH group is involved in adsorbing Cu 2+ . 251 Ionic liquids (ILs) and GO membranes have been proposed for the CO 2 /CH 4 gas separation method. CO 2 /CH 4 dynamical properties and interactions in 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF 4 ]), 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Bmim][TF 2 N]), and 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]-[PF 6 ]) have been reported. Because of the strong bond between GO cations, the interaction between cations and anions is reduced, making CO 2 adsorption simpler. CO 2 /CH 4 is mostly distributed in the IL part of the IL/GO membrane, allowing for gas selectivity. The CO 2 /CH 4 IL RDFs reveal that the limited IL/GO system performs better than bulk ILs at collecting gases. 252 Energy that can be harvested from commonplace moisture is gaining popularity as a means of directly powering electrical equipment. Fabricating high-performing moisture-electric generators (MEGs) with high and steady electrical output remains a challenge. Relying on the instrument fabrication of GO/PVA MEGs, we present a straightforward technique for modifying the oxygen-based groups of GO using HCl treatment, which enhances the electric output. The MEG that results has a constant voltage of 0.85 V and a current of 9.28 A (92.8 A cm −2 ), which are some of the highest values ever documented. Much better, by simply connecting four MEG units in series or parallel, electric output may be increased even further. 253   SOD activity increases in the first two weeks but decreases on the 21st and 28th day ( Figure 43). 255 12.3. Ceriodaphnia dubia. Ceriodaphnia dubia is a type of water flea in the class Branchiopoda. GO showed toxicity toward Ceriodaphnia dubia, and after 48 h, the EC 50 value was found to be 1.25 mg/L. After 24 h exposure to a sublethal     concentration, the C. dubia scientifically increases the ROS level (Table 16). 256 12.4. Drosophila melanogaster. Drosophila melanogaster is a species of fly belong to family Drosophilidae. Different concentrations of GO caused nearly to 100% mortality on day 16. After exposure to GO, larval crawling was first decreased to 50 μg/mL and increased to 200 μg/mL. An amount of 300 μg/ mL causes fatal damage to the neuromuscular coordination of larvae ( Figure 44). 257 12.5. Artemia salina. Artemia salina is a species of brine shrimp. LC 50  12.7. A549 Cells. A549 cells are lung carcinoma cells that constitute a cell line. To find the toxicity of GO on A549 cells, they (cells) were exposed to different concentrations of GO: m-GO (430 ± 300 nm), l-GO (780−410 nm), and s-GO (160 ± 90 nm). s-GO has more cell viability loss than m-GO and l-GO (concentration-dependent). After 24 h of post-exposure, cell viability is 67% at 200 μg/m of Lof GO. It was observed that s-GO causes severe oxidative stress among all GO samples compared to others: s-GO caused 3.9 times, m-GO caused 2.1 times, while l-GO caused 2.6 times more ROS levels than the controlled group ( Figure 48). 260 12.8. Mouse Eye. Mouse is the most common species found widely all over the world. The eyes are an essential part of any animal to see the world. It was reported that after exposure of 50 μg/mL and 100 μg/mL of GO for one week, corneal opacity was developed ( Figure 49). After 24 h of exposure, GO can significantly induce cell viability loss ( Figure  50). 261 12.9. Bacteria Biofilm. GO shows the concentrationdependent enhancement of cell growth in 12 h for all concentrations of bacteria biofilm (Figure 51). A concentration of 50 to 500 mg/L of GO enhances biofilm formation ( Figure  52). It was observed that GO significantly enhances cell growth and biofilm formation up to 500 mg/L concentration. 262

Pseudomonas putida.
Pseudomonas putida is a soil bacterium. It is an uncommon cause of skin and soft tissue infections. For the toxicity test of GO on P. putida, 10 g/L of aqueous GO solution was used. It was found that the presence of GO has a negative effect on the bacterial growth and viability of P. putida. The growth of P. putida was inhibited in 0.05 mg/mL of GO. Higher concentrations than 0.5 mg/mL and 1.0 mg/mL of GO have a negative impact on the viability of bacteria. 263 12.11. Chironomus riparius. Chironomus riparius is a harlequin fly, a species of a non-biting midge. C. riperius fourth instar larvae were exposed to 0, 50, 500, and 3000 μg/L of sGO (500 nm), lGO (∼10 μm), and mlGO (∼9 μm) (monolayer) for 24 h and 96 h. After 24 h of exposure to the concentration of 3000 μg/L, GO accumulation was mainly found in the digestive tract (Figures 53, 54, and 55). 264 In the experiment, it was observed that after 24 h of exposure to GO there was significant activation of SOD levels.

Microcystis aeruginosa.
Microcystis aeruginosa is a type of cyanobacteria that lives in fresh water. It can cause harmful algal blooms that are important for the economy and the environment. After 96 h of exposure to GO, the EC 50 value was 49.32 mg/L. It was reported that the fluorescence intensity of GO was 15.0−58.5%, which is higher than the controlled group. It was concluded that after exposure to 96 h, chlorophyll was reduced by 7.4% at 0.1 mg/L of GO. 265

Adult Zebrafish.
Zebrafish is a freshwater fish belonging to the minnow family of the order Cypriniformes. Zebrafish was exposed to GO for ∼14 days at concentrations of 0.1 and 1 ppm, a high ROS level is observed at a concentration of 1 ppm. 266 12.14. Lemna minor. The common duckweed, also known as duckweed, is Lemna minor. It is a freshwater aquatic plant species. Three distinct synthetic approaches were used to test         but it dropped to 0.78 on days 5 and 7. The number of leaves were tripled from the original (Figure 56). 267 12.15. Earthworm (Eisenia fetida). Two methods were used to determine the toxicity of GO on earthworm. One is a filter paper contact test, and another is a soil contact test. The author reported that after 24 and 48 h of exposure the EC 50 values were 2.52 and 2.36 mg/mL, respectively. GO has a negative effect on earthworm growth, and earthworms had a significant weight loss of >30% at 10−30 mg/mL ( Figure  57). 268 12.16. Zebrafish Embryo (Danio rerio). The embryonic stage of zebrafish was investigated to find the toxic effects of GO. It was found that GO significantly affects the mortality rate at higher concentrations (0.4−1 mg/mL) 269 (Figures 58  and 59).
12.17. Microcystis aeruginosa. Microcystis aeruginosa is a species of freshwater cyanobacteria, and its EC 50 value after exposure to GO for 96 h was found to be 11.1 μg/mL. 270 12.18. Zebrafish Embryo. The toxicity of GO on zebrafish embryos has been reported. Solutions of GO having different concentrations of 1, 5, 10, 50, and 100 mg/L were prepared, and embryos were exposed to these solutions for 96 h (Figure 60). 271 In the case of heart rate, it was reported that after exposure to GO (conc. of 100 mg/L) for 48 h the heart rate of zebrafish embryos was significantly decreased (Figure 61). 271 12.19. Arabidopsis thaliana. Arabidopsis thaliana is a small flowering plant native to Eurasia and Africa. The toxic     effect of GO on this species within the range of μg/L was investigated. No significant change in germination within the range ( Figure 62). 272 At a concentration of 1 μg/L, considerable accumulation of GO was found. It was reported that there was no significant change in seeding and root length from day 4 to day 8 ( Figure  63). 272 12.20. Zebrafish Embryo. The toxicity of different sized GO on zebrafish embryos was investigated. Three different sizes of GO, 50−200 nm (s-GO), <500 nm (m-GO), and >500 nm (l-GO), with an exposure time of 4−124 h, were investigated. Zebrafish embryos showed the sized-based side effects of GO. After exposure of 120 h, the survival rate was significantly reduced. It was concluded that after 48 h hatching rates are inhibited in 100 mg/L of s-GO, 0.1 mg/L of m-GO, and 10 mg/L of l-GO. It was observed that body length was also inhibited at a high concentration of GO (100 mg/L) ( Figure 64). 273 12.21. Algae. The toxicity of GO on three classes of algae (cyanobacteria, green algae, and diatom) was examined. After 96 h of exposure to GO, algae growth was significantly inhibited with a 10 mg/L concentration. All species have resistance power to oppose the effect of any chemical, so different algae showed different side effects against GO, as shown in Figure 64. A significant difference in chlorophyll-a was observed with the concentration of 10 mg/L as compared to a control, as shown in Figure 65. 274 12.22. miR-21 and miR-29a in Human Cell Lines. miR-21 and miR-29a are from the family of micro RNA. Micro RNA is a small single-stranded noncoding RNA. A noncytotoxic dose of 15 μg/mL of GO (100 nm) was selected to examine the toxicity of GO. A fluctuation of miR-21 in MCF-7, KMBC/71, and HUVEC cells was observed, while the expression of miR-29a was only changed in MCF-7 and KMBC/71. No significant change was observed in HUVEC cells of miR-29a ( Figure 66). 275 12.23. Bacteria. Gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) models of bacteria were used to assess the toxicity of GO against bacteria. Cell damage was discovered when bacteria came into direct contact with the razor-sharp edges of the nanowalls. Gram-negative bacteria were less susceptible to the damage that nanowalls induced to their outer meninges than gram-positive bacteria. Nanowalls made of GO also have antibacterial properties. After 1 h, 26.5%   of S. aureus bacteria and 41.8% of E. coli bacteria could survive 276 (Figures 67 and 68).

Escherichia coli.
Suspensions of GO of 0.05, 0.1, 0.5, 1, and 5 mg/mL concentrations were used to investigate the toxicity of GO toward Escherichia coli. The suspension of GOS−melatonin with a functional group containing oxygen might capture the bacteria. Bacterial activity in the bacterial suspension decreased as GOS concentration was increased. No active bacteria were found in the GOS−melatonin−bacterial suspension at a concentration of 5 mg/mL ( Figure 69). 277 12.25. Mice. GO was produced in concentrations of 1, 10, 100, and 400 g/mL, and exposure times of 24 h were used to determine the cyto-genotoxicity of GO on mouse spermatogonial stem cells. The number of spermatogonia colonies and viable cells dropped at concentrations of 100 and 400 g/mL, and there was considerable cell death ( Figure 70). The ROS levels significantly increased at concentrations of 100 and 400 g/mL as well ( Figure 71). 278 12.26. Pichia pastoris. P. pastoris was exposed for 24 h to various GO concentrations (0−4000 ppm). Twenty-four hours of exposure to a higher concentration of 500 ppm greatly reduced the cell development ( Figure 72). The IC 50 value for this study was found to be 1125 ± 40 ppm. Concentrations ≥1000 ppm of GO led to an increase of intercellular ROS level ( Figure 73). A concentration greater than 1000 ppm caused membrane damage and oxidative stress together in P. pastoris. 279 12.27. Escherichia coli. In this study, E. coli was exposed to GO concentrations for 0−48 h. The oxygen-containing functional groups of the GO were reported to have dropped by about 60%, indicating a relative chemical reduction of the sheets as a result of the interaction with the bacteria. After exposure, bacteria were shown to have reduced GO concentrations due to their metabolic activity, namely, their glycolysis process. 280 12.28. Escherichia coli. In this study, GO was found to have antibacterial properties against E. coli at various concentrations (0−400 g/mL). GO has stronger antibacterial properties when compared to rGO, graphite, and gold oxide. They further stated that both the membrane and oxidative stress may be responsible for bacterial cytotoxicity. After being exposed to E. coli at 40 g/mL for 4 h, the viability gradually increased ( Figure 74). 281 12.29. Human Stem Cells. The building blocks of the body are stem cells. The authors evaluated the toxicity of rGONPs on human stem cells. rGONPs with average leteral dimensions (ALDs) of 114 nm showed strong potential in cell wall destruction at concentrations of 1 g/mL and could enter the hMSCs' nuclei and exhibit some genotoxicity due to DNA fragmentation and chromosomal aberrations at low concentrations of 0.1 and 1.0 mg/mL after 1 h. After 1 h, the cytotoxicity of rGO sheets with ALDs of 3.804 nm emerged at high concentrations of 100 g/mL. 282 12.30. Mice. The findings of this study demonstrated that mice treated with a dose of 2000 g/mL had high levels of GO absorption in their testicles. Additionally, a 45% decrease in sperm viability and motility was discovered (Figure 75). After being exposed to GO, the mice's semen likewise produced ROS. 283

Graphene oxide and Nafion Polymers on
Zebrafish Embryos. In this study, the toxicity of nanocomposite membranes made using a Nafion polymer and GO has been investigated. The zebrafish is thought to be an  effective animal model for understanding developmental toxicity pathways. The author reported that the composite of GO and the nafion polymer did not show a significant effect on HO-1 and iNOS. They concluded that GO causes more toxicity to zebrafish embryos (Figures 76 and 77). 284 13.2. GO−TiO 2 Composite on A549 Cells. In the present experiment, the cytotoxic effect of the GO−TiO 2 composite on A549 cells has been evaluated. The GO−TiO 2 composite showed a significant decrease in cell viability after an exposure of 4 h. It has been reported that a high concentration led to a low level of cell viability. 285 13.3. GO/Alginate/Silk Fibroin Composite. Herein, modified GO with natural Alg and added SF were used to get a hybrid material, that is, GO/Alg/SF. The cell viability of this nanostructure was found to be 89.2%, and the hemolytic effect was found to be less than 6% at high concentrations (1000 μg/mL) ( Figure 78). 286 13.4. Magnetic Chitosan/GO (MCGO) Composite on A549 Cells. The toxicity of GO and its composite was evaluated on the A549 cells. Cells were exposed for 24 h in the concentration range of 50−250 μg, and the toxicity was investigated. It has been reported that viable cell percentages at 50 and 100 μg concentrations were 53.7% and 44.8%, respectively. 287 13.6. Human Breast Cancer Cells. Human breast cancer cell lines (MDA-MB-231 and SKBR3 cell lines) were chosen to find the potential of curcumin/rGO. At a higher concentration of 100 μg/mL, ∼15−25%, the cell destruction has been observed ( Figure 79). 288

CARBON-FIBER-REINFORCED COMPOSITES (CFCS) AND GRAPHENE OXIDE COMPOSITE
The authors have reported that the CFC + GO residue did not show a pro-inflammatory response, while soot CFC + GO induced a significant difference to the control. There was no significant toxic effect on cytotoxicity due to CFC + GO. The authors have also reported that CFC + GO soot induced ROS production in a concentration-dependent manner (Figures 80  and 81). 289

GRAPHENE OXIDE AND AG NANOPARTICLE COMPOSITES
Huge growth inhibition has been seen with GO−Ag composites after 24 h. After direct incubation of 24 h on fibroblasts and HUVECs (human umbilical vein endothelial

GRAPHENE OXIDE + AG NANOCOMPOSITES
J774 is a cell line isolated from ascites of a patient with reticulum cell sarcoma. On exposing the composite to J774 for 24 and 48 h, IC50 values were found to be 2.9 and 3.8 μg/mL, respectively. Ag can be up taken by J774 tumoral macrophages after 24 and 48 h of exposure with a concentration of 1000 μg/ L. After exposure to GO−Ag, 124 and 124.2 μg/L (12%) of Ag were internalized by J774 ( Figure 84). 291 GO−Ag NPs interact with the cell in multiple stages, which start from macrophage endocytosis along with vesicle maturation. After that the nanocomposite degrades, releasing the Ag ions into the cell/cytoplasm. Due to this release, mitochondria stop working properly, and their function imparted causes oxidative stress, as shown in Figure 85.

PROTEIN/DNA INTERACTION WITH GRAPHENE OXIDE
Protein−GO complex formation involves various interactions such as electrostatic, H-bonding, hydrophobic, and π−π interactions and van der Waals forces. 292 Such interactions are surface-dependent; hence, the formation of a complex is dependent on the functional groups situated at the protein surface and GO, as shown in Figure 86. 293 17.1. Graphene Oxide with Lysozyme. Fluorescence spectroscopy of GO interaction with lysozyme reflects changes in the structure of our protein. Due to conformational changes in lysozyme, there is some red shift. 294 The fluorescence intensity of our protein is observed to decrease when the pH is increased above 7.4, at which the intensity was the highest. Lower pH values did not show much change as shown in Figure 87. This shows that at higher pH lysozymes may undergo some conformational changes. 295 17.2. Graphene Oxide with BSA. The fluorescence spectra ( Figure 88) show some decrease in fluorescence with the increase in the GO concentration. This reduction in intensity is attributed to the strong interactions between GO and BSA. 296 These interactions altered the environment, and fluorescence intensities of fluorophores were lowered due to fluorescence quenching. 297 17.3. Graphene Oxide with Trypsin. Fluorescence spectroscopy was done on the 1:1 construct of GO−trypsin. It can be seen in Figure 89 that there is a decrease in the intensity of the spectra which shows that GO quenches the protein. Also, there is a red shift on the interaction with protein, suggesting opening of the β-sheet structure of protein.
Trypsin has a characteristic signal at 336 nm, but when it interacts with GO, the signal shifts to 342 nm, indicating that the sheet structure opens up and quenching occurs. 298 17.4. Graphene Oxide−Human Serum Albumin (HSA). The UV spectra of GO−HSA overlap, which makes it difficult to differentiate the effect of their interaction. Singular value decomposition (SVD) of the spectra allowed us to separate the bands. On increasing the [GO]:[HSA] ratios, there is a blue shift as the weight is increased, as shown by the shape of the curve (Figure 90). 299 17.5. Graphene Oxide with Bovine Hemoglobin (BHb). Each α and β chain of BHb contains three tryptophan units. Three-dimensional fluorescence spectra showed quenching of BHb spectra when it interacts with GO. The quenching can also be identified through a decrease in the intensity of the fluorescence at different pH, which implies there are hydrophobic interactions possible. 300 Fluorescence emission spectra of BHb with different concentrations of GO at various pH levels are shown in Figures 91 and 92. 17.6. Corona-Coated Graphene Oxide. The author employed GO to observe the biological reaction to corona derived from various diseases. The author of this study determined the impact of corona-coated GO. With GO sheets, human plasma that had been exposed to various diseases was cultured. The results revealed that varied corona decorations on GO sheets affected the cellular toxicity, ROS generation, and lipid peroxidation in diverse ways. 301 17.7. Human Epithelial Cells. The prevention of cell growth and activation of cell death were examined at a dosage of 100 μg/mL. The results also showed that a concentration  ≤50 μg/mL caused no significant toxicity to the cells. After 24 h post exposure at a concentration of 100 μg/mL, viability loss was observed ( Figure 93). 302 17.8. Human Mesenchymal Stem Cells. The toxicity of rGO nanoribbons (rGONRs) and rGO nanosheets (rGOSs) on human mesenchymal stem cells was investigated in this work. The author claimed that 10 g/mL of rGONRs produced cytotoxicity, including cell viability, after exposure for 1 h. The results demonstrated that 96 h after exposure the same cytotoxity was still present at a concentration of 100 g/mL. The findings also demonstrated that rGONRs, even at a concentration of 1.0 g/mL, produced DNA fragmentation following exposure for 1 h. 303 17.9. Corona-Coated Spinal-Graphene Nanomaterial. In this study, the author assessed the therapeutic benefits of spinal-graphene nanoparticles coated with corona against cancer. The outcome demonstrated a correlation between the amount of protein corona absorbed on spinal graphene and local and global heating brought on by laser irradiation. The study's findings indicated that the effectiveness of graphenebased photothermal therapy in the treatment of cancer is correlated with the quantity of corona generated by laser irradiation. A reduction in the quantity of corona during laser irradiation had an impact on the therapeutic and harmful effects of NPs. 304

OPTOELECTRONIC APPLICATIONS OF GO
GO can function as a fluorescence quencher by adsorbing dye molecules on its surface, followed by fluorescence resonance energy transfer (FRET), to quench the fluorescence signal. 305 GO absorbs laser light and transfers it to surface molecules. GO increases the Raman signal via a chemical process on its surface and possesses peroxidase-like enzymatic activity. 306 Electrochemical, optical (fluorescent, colorimetric, and Raman), and mass analysis biosensors have been developed using GO because of its useful inherent characteristics. 307 Choi et al. utilized intrinsic properties of GO and synthesized a GO sheet−Pt composite for dye-sensitized solar cells, and it was observed that GO sheets increased the surface area, number of active sites, and crystallinity of Pt. 307 In another example, GO nanosheets were synthesized via a simple method, and the results exhibited comparatively better characteristics, indicating that the synthesized GNSs are a credible potential option for optoelectronic devices such as solar cells, supercapacitors, electrochemical and bio sensors, and biomarkers and also appropriate for low-temperature fuel cells. 308 In a recent work of Kant et al., the MgO−rGO nanocomposite was synthesize,d in which carbon networks with conductive properties were established and caused enhanced dielectric performance and better optical transmissions. 309 Biosensors using field effect transistor (FET) technology may detect BNP significantly faster than with traditional clinical trials. The usability of GO was found in another class of sensitive biosensors know as field effect transistors (FETs). A number of review articles have been published summarizing the advantages of using GO or GO derivatives for the preparation of biosensors. 310−312

CONCLUSION
In this article, the authors present a variety of approaches for the synthesis and characterization of graphene oxide (GO) and its composites. At the moment, the Hummers method is utilized quite frequently for the synthesis of GO but with a few modifications. Due to the layered structure of GO, which also contains groups like carbonyls, hydroxyls, and epoxides that contain oxygen, the active surface area is quite large, as after exfoliation GO sheets are separated out. The results of several chemical reactions point to GO as a promising candidate for the role of catalyst. The regeneration of tissues and organs on GO scaffolds is an important field that needs additional research. This review also discusses certain other applications, such as photocatalytic activity and biological applications like drug delivery. Some of GO's applications are reported here. GO is not one of the best electrical conductors, but its conductivity can be altered by reducing the functional groups present on it to obtain rGO. rGO has quite a lot of applications in different fields as well. One feature that needs more attention is toxicity; it was discovered that the concentration of GO affects whether or not it is physiologically harmful, which restricts its use in the field of medicine. It has been found that there are a lot of animal species for which GO showed some cytotoxic nature. This is one of the aspects that needs more study. GO can be functionalized quite easily. Therefore, there is a need for more research to find ways to reduce its toxicity.