Photocatalytic dye degradation and biological activities of the Fe2O3/Cu2O nanocomposite

The present study reports the synthesis of the Fe2O3/Cu2O nanocomposite via a facile hydrothermal route. The products were characterized using X-ray diffractometry (XRD), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), high-resolution transmission electron microscopy (HR-TEM), energy dispersive spectroscopy (EDS) and Brunauer–Emmett–Teller (BET) techniques. The composition, morphology and structural features of the nanoparticles were found to be size-dependent due to the temperature response in the particular time log during hydrothermal synthesis. HR-TEM confirmed the formation of hexagonal rod-shaped bare Cu2O, rhombohedral-shaped Fe2O3 and composite assembly. Rhodamine-B (RB) and Janus green (JG) were chosen as model dyes for the degradation studies. Photocatalytic degradation of the dyes was deliberated by altering the catalyst and dye concentrations. The results showed that the Rhodamine-B (RB) and Janus green (JG) dyes were degraded within a short time span. The synthesized materials were found to be highly stable in the visible light-driven degradation of the dyes; showed antibacterial activity against E. coli, P. aeruginosa, Staph. aureus and B. subtilis; and exhibited less toxicity against the Musmusculus skin melanoma cells (B16-F10). The fusion of these advantages paves the way for further applications in energy conversion, biological applications as well as in environmental remediation.


Introduction
Photocatalysis is a fascinating tool for energy conversion and environmental decontamination due to its conspicuous nature. 1 Using metal-semiconductors for photocatalysis is an environmentally safe process that simulates the photosynthesis process to speed up chemical reactions that require or involve light. 2,3 The visible light photocatalytic process has been implemented in semiconductor integrated circuits for harvesting solar energy. 4 To overcome the band gap problem, metal oxides can be used as a photocatalyst, which, upon exposure to UV lights, visible lights or both, allows photo-excited electrons to be promoted to the conduction band to speed up the recombination of huge charge carriers. [5][6][7] Broad band gap issues can also be overcome by introducing a hetero or homo barrier made with a combination of different metal oxides, 8 nanowire, 9 core-shells 9 and nanowires, 10 which offers superior charge transfer competence and commotion compared to a sole metal oxide semiconductor. [11][12][13] This allows the competent and viable catalysts to perform under visible light irradiation, which constitutes nearly 46% of the astral band. 14 In recent years, the investigation of semiconductors for harvesting energy in the visible spectrum has led to the progression of the invention of non-titania-based semiconducting photocatalysts for dye degradation. 15,16 In addition, the plasmon-resonance of ne metal oxide nanoparticles (MNP's) also enhances the photocatalytic process by absorbing incident photons much more effectively. Cu 2 O has been used as a prolic p-type photocatalyst with a through band gap in the visible range of 2.0-2.2 eV. [17][18][19] However, Cu 2 O suffers from photo-wavering and photo-corrosion, which can over time cause deactivation in solar irradiation. 20 Iron oxides (Fe 2 O 3 ), on the other hand, are an extremely constant n-type semiconductor under standard ambient conditions. 21 The valence band-edge of Fe 2 O 3 is an outstanding catalyst for the photo-squalor of unrened pollutants and is not only abundant in nature but also massproducible. 22 However, photocatalytic activity in Fe 2 O 3 is oen overwhelmed by its hole diffusion length (approximately, 10 nm) and an extremely short electron-hole recombination time. [23][24][25] Most studies focus on the antibacterial activity of metal nanoparticles and its composites, including those that are against resistant strains of microorganisms. However, there is no exact mechanism for the antimicrobial action of metal oxide nanoparticles. 26 The anti-microbial activity of metal oxide nanoparticles was enhanced with increasing surface area and volume of the nanoparticles and decreasing particle size. 27 Inimitable properties and the assessment of interactions between nanomaterials and the biological system are essential. 28 In recent years, the cell line toxicity of human cell lines using several synthesized metal oxide is a more fascinating topic in understanding the relationship between nanoparticlecell interactions. 29, 30 The size, shape, and morphology of nanoparticles, play a vital role in mammalian cell culture medium, and the nanoparticle are much more toxic than the hydrothermally synthesized nanocomposite semiconductor. 31,32 In this experiment, we focus on exploiting and utilizing some multi-tasking facile materials with high photocatalytic activity and biological applications. To evade these drawbacks, in an attempt to tackle the above issues, we have developed a costeffective method for the synthesis of non-toxic Fe 2 O 3 impurities on the Cu 2 O nanocomposite for various applications ( Fig. 1  and 14). Fe 2 O 3 burdened onto Cu 2 O and the nanocomposite was prepared by a facile method. The (p-n) hetero-junction nanocomposite was used for the effective degradation of organic pollutants; including Rhodamine-B (RB) and Janus green (JG), bacterial degradation and some additional biological screening applications.

Hydrothermal preparation of Fe 2 O 3
The hydrothermal technique has been found to be one of the best techniques to prepare Fe 2 O 3 nanoparticles of the desired size with homogeneity in composition and an extreme degree of crystalline particles. The primary nanoparticles were synthesized by taking advantage of a hydrothermally enabled reaction between FeCl 3 and NaOH. Ammonia (Aldrich, India), 10.14 g (37.5 mol) of FeCl 3 $6H 2 O and 7.45 g (37.5 mmol) of FeCl 2 $4H 2 O were dissolved into 25 ml of distilled water. 25 ml of 25% ammonia was added to the salt solution under stirring at 700 rpm for 2 min. Next, 15 ml of the mixture was put into a Teon-lined stainless steel Morey autoclave. The autoclave was heated to 160 C in an oven and maintained with a 12 hour reaction time. The effects of the reaction temperature on the product were investigated. Temperature plays a crucial role in the formation of a well-dened spherical product. The autoclave containing these chemicals was naturally cooled to room temperature, and the precipitates were then washed with distilled water and isolated under a magnet. The nal products were dried at 60 C and characterized. In the hydrothermal process, the following reaction takes place.

Photocatalytic experiments of the removal of dyes
The photocatalytic behaviour of the synthesized nanomaterials was evaluated by the removal of Rhodamine-B (RB) and Janus green (JG) dyes (200 ml of aqueous solution of dyes (1 Â 10 À5 mol l À1 )) under UV light radiation. The light source used was a 150 W Xe (Xenon) lamp, and the distance between the UV source and the photo-reaction vessel was 10 cm. Prior to irradiation, the suspensions were magnetically stirred in the dark for 30 min. Then, the photoreaction vessel was exposed to UV irradiation under standard ambient conditions. The selected dyes were used in conjunction with 10 mg of catalyst nanoparticles in the photo-removal experiment. At regular time intervals, 3 ml of the suspension was taken for centrifugation to separate the photocatalyst and for further evaluation using a UV-Vis absorption spectrometer. The photo-removal efficiency percentage was calculated from the equation given below. Aerward, recycling experiments were carried out for ve repeated cycles to inspect the permanence of the photocatalytic Fe 2 O 3 /Cu 2 O nanocomposite. The composite catalyst was centrifuged, washed with ethanol and deionised water, and was dried before reuse for the next trial. The photo-removal efficiency percentage was calculated from the equation given below; % Photo-removal efficiency where C 0 is the initial concentration of dye and C is the concentration of dye aer photo-irradiation (nal).

Determination of antibacterial activity and live and dead cell analysis
The

Cell culture and treatment
Musmelanoma cells (B16-F10) were seeded in tissue culture asks and full-grown in Dulbecco's modied Eagle's medium (DMEM; Thermo Fisher, USA Gibco), balanced with 5% fetal bovine serum (FBS, Thermo Fisher; Gibco) and 1% penicillin/ streptomycin blend (Santa Cruz Biotechnology, USA). A detailed experiment procedure was clearly provided in S2 in ESI. †

Statistical analysis
Systematic data replicates (three) were analyzed for every attempt and for every analysis of discrepancy (ANOVA) using SPSS-Inc. 16    The nanoparticles synthesized by hydrothermal method resulting in the formation of nano/mesopore structure show excellent photocatalytic performance. 43,44 HR-TEM of the synthesized nanoparticles was shown in (Fig. 4); the highquality images of the hexagonal rod-shaped Cu 2 O, rhombohedral-shaped Fe 2 O 3 and Fe 2 O 3 /Cu 2 O composite nanoparticles with partial agglomeration are shown. Additionally, the nanocomposite results in the formation of mesopores between the particles, which were clearly conrmed by BET surface area and pore volume measurements. The sizes of both individual nanoparticles were between 30-60 nm, which are in accordance with the results of XRD. It was also observed that the d-spacing measurements of the twin domains were measured to be approximately 0.2Å to 0.4Å nm and corresponded to the (111) and (220)  The carbon peak appears due to the carbon tape used sample holder.
The particle size distribution was analyzed using a dynamic light scattering instrument. The Fe 2 O 3 /Cu 2 O particles were dispersed in a solvent. The distribution of particle sizes when immersed in the solvent ranged from 42-593 nm, with approximately 266 nm being the mean particle size. Histogram of the DLS analysis for the particle size distribution of Fe 2 O 3 / Cu 2 O composites is depicted in (Fig. S1 in ESI †). Zeta potential measurements by DLS were considered as an authentic technique for evaluating the particle size and zeta potentials of nanoparticles in a suspension and also plays a crucial role during interaction with other biological systems as well as environmental degradation. The particles examined presently possessed zeta potentials of À0.1 mV for Cu 2 O, 13.9 mV for Fe 2 O 3 , and À46.2 mV for the Fe 2 O 3 /Cu 2 O composite. Nanoparticles with a zeta potentials between À10 and +10 mV have a neutral charge, while if it is greater than +30 mV or less than À30 mV, it is considered to be strongly cationic or anionic.
Surface area is an important parameter to determine the photocatalytic activity of the nanoparticles. A photocatalyst with a high surface area is likely to absorb more dye molecules and react faster (Table 1); the BET surface area as a function of the pore volume of the prepared samples undoubtedly demonstrates a similar type II curve. The BET surface area of Fe 2 O 3 , Cu 2 O and Fe 2 O 3 /Cu 2 O were found to be 5.676, 9.90, and 10.401 m 2 g À1 , respectively (Fig. 6). According to the hysteresis loop in the relative pressure region around 0.4-0.9, the nitrogen adsorption/ desorption isotherms showed that the Fe 2     O were found to be 6.9 and 7.2 respectively. It was observed that RB and JG in an acidic environment can advantageously increase the electrostatic attraction between the protons from the catalysts, including the dyes, RB and JG. Thus, photo-removal activity is high. At low pH (below 5), the chances for agglomeration are high, thus reducing the active surface area available for dye adsorption and photon absorption. At an optimum pH, the predominant iron site, namely Fe(OH) 2+ , not only forms Fe(II), the major catalytic species in the photoremoval reactions, but also produces additional À OH responsible for dye removal. When the pH value was greater than pzc, the surface of Fe 2 O 3 /Cu 2 O became negatively charged. Therefore, the negatively charged dye molecules were repelled by the catalyst surface, leading to a decrease in the effective photocatalytic activity. Highly alkaline conditions are favourable for the generation of a large number of less reactive high-valence composite iron species (Fig. 7).
The Langmuir-Hinshelwood model was engaged to investigate the kinetics of RB and JG photo-removal. The photocatalytic experiments were carried out under optimal reaction conditions [Fe 2 O 3 ¼ 0.75 g l À1 , both RB and JG ¼ 9 mM and pH 5]. Fig. 8 shows the logarithmic plot of RB and JG concentration as a function of irradiation time. The photocatalytic removal of   This journal is © The Royal Society of Chemistry 2019 RB and JG follows pseudo-rst-order kinetics; the pragmatic rate constant for Fe 2 O 3 /Cu 2 O of 1.21 Â 10 À2 s À1 is signicantly higher than those of Fe 2 O 3 (4.36 Â 10 À3 s À1 ) and CuO 2 (6.45 Â 10 À3 s À1 ). Hence, the activity of the Fe 2 O 3 nano-porous material is about approximately 2.6 times higher than that of other demonstrated materials in a systematic manner. DRS was used to investigate the light-harvesting nature of the Fe 2 O 3 /Cu 2 O photocatalyst. Estimating the conduction-band minimum (CBM) and the valence-band maximum (VBM) is vital to understanding the mechanism of the photocatalytic degradation of the photocatalyst. To investigate the CBM and VBM of Fe 2 O 3 , Cu 2 O, or Fe 2 O 3 /Cu 2 O, the UV-DRS spectra were used to record the spectrum. As shown, the associated band gap values were calculated using the following eqn: 48 The calculated band gaps of bare Fe 2 O 3 and Cu 2 O were found to be 1.96 and 1.89 eV, (Fig. 9) respectively, which are consistent with similar results obtained from related work. 49 The observed small red-shi in the band gap value (1.85 eV) of the Fe 2 O 3 /Cu 2 O photocatalyst is possibly due to the formation of a p-n heterojunction between the p-type Cu 2 O and the n-type Fe 2 O 3 . 50 In the exploration of the photocatalytic dye removal activity, it was found that the 1.5% loading of Fe 2 O 3 on Cu 2 O exhibits sophisticated performance compared to those of 0.8 and 2.4% loading. Fig. 7  was a much more effective photocatalyst that enhances the removal rate of Rhodamine-B and Janus green.

Total organic carbon (TOC) and chemical oxygen demand (COD) studies of Rhodamine-B (RB-B) and Janus green (JG)
This analysis is necessary because the disappearance of dye colour alone cannot be used as a measure to determine the complete mineralization of the dyes. [51][52][53][54] Noxious and longlasting reaction intermediates were formed during the photoremoval of the dyes. Simultaneously, it was essential to calculate the degree of degradation of Rhodamine-B (RB) and Janus green (JG) during photo-removal using the Fe 2 O 3 /Cu 2 O photocatalyst. Aer the degradation process, it was a necessity to measure the chemical oxygen demand (COD) and total organic carbon (TOC) to assess the purity of degraded dyes before the discharging process. Total organic carbon (TOC) analysis was performed in order to determine the extent of mineralization of Rhodamine-B (RB-B) and Janus green (JG) during the photocatalytic degradation process. The present work shows the signicantly declining performance in the particular functioned period (Fig. 10). Furthermore, the photocatalytic degradation process can result in the formation of colourless dye intermediates resulting in the disappearance of colour, which may actually be more toxic than the dye itself. The present study revealed that the colour disappearance of the dye was faster than the degree of mineralization with maximum TOC removal (Table S3 in ESI †). The rapid loss of colour might arise from the cleavage of the azo bond, while the high TOC value may be due to difficulty in converting the N-atom of the dye into oxidized nitrogen compounds. This mechanism clearly explained that the dye molecules were converted to other intermediates and that the dye was systematically decolourized in 120 min, which may lead to complete mineralization. Similarly, the reduction of  COD reects the extent of removal or mineralization of an organic species (Table S4 in ESI †), the percentage change in COD and TOC during photo-removal was measured under optimum reaction conditions [Rhodamine-B (RB) and Janus green (JG) concentration 9 mM, catalyst concentration 0.75 g l À1 , pH ¼ 5 and irradiation time of up to 120 min]. The solutions obtained aer 120 min of photo-removal showed a signicant decrease in COD and TOC concentration. It has been observed that Rhodamine-B (RB) and Janus green (JG) molecules were partially degraded to intermediates, and only a small fraction was subjected to complete mineralization; the COD showed a related emergent action similar to TOC.
To evaluate the stability and re-usability of the Fe 2 O 3 /Cu 2 O photo-catalyst, ve additional cycles of dye RB and JG removal were performed with it. Fig. 13 shows the good recyclability of the Fe 2 O 3 /Cu 2 O photo-catalyst for ve consecutive cycles. A slight decrease in activity for the h cycle of RB and JG may be due to the loss of catalyst due to its recyclability. This result   Paper clearly indicates that both dyes degraded into carbon dioxide and water as the nal products. The experiments were carried out under optimal reaction conditions (RB and JG ¼ 9 mM, Fe 2 O 3 , Cu 2 O and Fe 2 O 3 /Cu 2 O ¼ 0.75 g l À1 and irradiation time ¼ 120 min) in the presence of scavengers (2 mM for 200 ml of respective dye solution) such as t-BuOH for $OH, 58 benzoquinone (BQ) for O 2 c, 59 and potassium iodide (KI) for holes and $OH. 60 The effect of t-BuOH, BQ and KI on the photo-removal percentage of RB and JG is shown in (Fig. 6). It was clearly observed that the photo removal percentage of RB was reduced to 35.79%, 39.78%, 43.11% and 91.25% aer the addition of KI, t-BuOH, BQ and blank, respectively. Then again, the Janus green (JG) is as follows: t-BuOH-43.65%, BQ-44.78%, KI-38.65%, and blank-89.14%. The photocatalytic activity of the nanomaterial surprisingly concealed the scavenge ring effect, indicating that both O 2 c and the $OH are actively implicated in the photo-removal process.  (Fig. 15). The result of antibacterial activity showed that there exists a signicant zone of inhibition against test pathogens (Table 3). It should be noted that, among the test samples, due to its large surface area, Fe 2 O 3 /Cu 2 O composites respectively yields the maximum inhibition zones of 20.13, 21.09, 08.23 and 20.60 for Staph. aureus, P. aeruginosa, B. subtilis and E. coli. In the case of B. subtilis, whilst only a slight response to Fe 2 O 3 /Cu 2 O nanocomposite is observed, we have not observed any zone of inhibition in SDW (Sterile Distilled Water) diffused discs against test pathogens. This study clearly suggests that the Fe 2 O 3 /Cu 2 O nanocomposite inhibits bacterial pathogens by rupturing the outer and inner walls of the cell, which leads to disorganization and leakage of the cell membrane (Fig. 16).

Probable photocatalytic mechanism with Fe 2 O 3 /Cu 2 O as the photocatalyst
This section clearly explains how the movement of the photoinduced charge carriers occurring between Fe 2 O 3 and Cu 2 O have been systematically estimated using Mullikenelectronegativity theory. 61 This theory helps explain how a p-n heterojunction functions, according to Anderson's model. 62,63 The Mulliken-electronegativity of the semiconductor, E CB and E VB , are respectively the conduction and valence band edge potential, where the band gap of the semiconductor E g and E e is the free energy of electrons on the H 2 (4.5 eV), for Cu 2 O and     Table 2. Both the conduction band edge of Cu 2 O and Fe 2 O 3 are respectively negative and positive with respect to the hydrogen reduction potential on the normalized hydrogen scale (Fig. 17). Likewise, the valence band edge of Fe 2 O 3 is more positive than that of Cu 2 O. Both semiconductors with different electronegativities and bands positions in an internal electric eld at either side of the junction will build up, being directed from the Fe 2 O 3 surface to the Cu 2 O surface and become exposed to visible spectrum (400 nm). The photo-generated electrons will, under the inuence of an inner electric eld, shi from p-type Cu 2 O to n-type Fe 2 O 3 , and the photo-created holes force to transfer from the valence bands of Fe 2 O 3 to the valence band of Cu 2 O. These processes in effect separate and mobilize the photo-generated electron and holes, thereby enhancing the photocatalytic activity of the Fe 2 O 3 /Cu 2 O photocatalysts.

Photo-removal mechanism of dyes
Hydrogen generation from photo-induced water break-down is increasingly seen as a feasible choice to concurrently solve energy and environmental problems. Various photo-induced H 2 generation techniques and photocatalytic water splitting was demonstrated in the visible light spectrum. [64][65][66][67][68] The low cost and high sustainability features of the reaction system in the photo-removal mechanism of dyes are explained in S1 in ESI. † Electron-hole pairs in the excited Fe 2 O 3 could be efficiently separated to facilitate an efficient shi into photo-induced electrons between Fe 2 O 3 and Cu 2 O. This radical splitting process makes a crucial task in the removal of dyes. The effectiveness of the photo-reaction very much depends on the efficiency of the adsorption of untreated organic contaminants on the photocatalysts and the process of splitting photo-created electron-hole pairs. The holes can either react or be adsorbed through surface hydroxyl to form hydroxyl radicals. As a result, the adsorption equilibrium was destroyed, permitting dye molecules to move from single elucidation to the interface and to consequently decompose into CO 2 , H 2 O and other raw materials through redox reactions.

Re-usability of photocatalysts
The sustainability of a photocatalyst is the most important concern for the booming industry. In order to investigate the stability and durability of Fe 2 O 3 /Cu 2 O, nanomaterial recycling experiments were conducted for the photo-removal of RB and JG. Aer the completion of each cycle, the photocatalyst was collected using an external magnet, washed with double    distilled water, dried overnight, and reused. 66,67 The photoremoval percentages of RB and JG for ve successive cycles were found to be 79.15%, 74.65%, 75.45%, 74.98% and 73.24%, respectively, and are shown in Fig. 18. The Fe 2 O 3 /Cu 2 O nanomaterial exhibits an average of 76.24% for photocatalytic activity aer ve successive cycles. The reduction in activity aer two cycles is due to the loss of catalyst during the washing process. 69 In addition, there is no obvious change observed in the XRD pattern of Fe 2 O 3 in (Fig. 18) aer ve cycles. These results indicated that the Fe 2 O 3 /Cu 2 O materials could be used as a stable photo-catalyst for the removal of organic pollutants in the form of dyes in industrial wastewater.

Toxicity tests of Fe 2 O 3 , Cu 2 O and Fe 2 O 3 /Cu 2 O
To further illustrate the bio-compatible advantage of the proposed photocatalyst, up-conversion materials for emitting visible light were used as candidates for both in vivo and in vitro bio-imaging as well as in photodynamic therapy. We measured the toxicity of Fe 2 O 3 , Cu 2 O and Fe 2 O 3 /Cu 2 O using Musmusculus skin melanoma cells of various concentrations (5-500 g ml À1 ). The results suggested that the viability of (B16-F10) cells decreased in a dose-dependent manner in each sample (Fig. 19), clearly showing that, at a 5 g ml À1 concentration, the lowest concentration of the three respective samples used in the assay did not reduce the cell viability noticeably as compared to other increased concentrations, among which Fe 2 O 3 /Cu 2 O was found to be low in a systematic manner. Therefore, Fe 2 O 3 /Cu 2 O is the safest and the superlative alternative in terms of toxicity in biological application.

Conclusions
In this experimental work, a unique and visible light-driven Fe 2 O 3 /Cu 2 O composite photo-catalyst was prepared by a simple, eco-friendly and cost-effective hydrothermal method for multi-purpose application. Rhodamine-B (RB) and Janus green (JG) could be easily decolorized by Fe 2 O 3 /Cu 2 O under visible light irradiation. Furthermore, the photocatalyst was recycled several times without any observable loss of photocatalytic activity, making it suitable for use in dye removal in wastewater. Moreover, the synthesized materials were found to be highly stable in the photocatalytic process; displayed antibacterial properties against E. coli, P. aeruginosa, Staph. aureus and B. subtilis; and anti-cancer properties against Musmusculus skin melanoma cells (B16-F10). These results suggest that these composite materials can be utilized in the biomedical eld and for catalysis and energy conversion systems.

Conflicts of interest
The authors declare no competing nancial interests.