The preparation of Co3O4 NPs was carried out in a hexagonal LLC medium. A solution of TX-100 (4.2 g, wt %) and water (5.8 g, wt %) exhibits a hexagonal phase at 25◦C. The hexagonal phase formed using this binary mixture displayed a broken focal-conic texture under POM (Fig.S1a). In this medium, Co3O4 NPs were prepared by reducing the CoCl2.6H2O using NaBH4. Interestingly, the texture of the mesophase was retained even after the formation of the NPs (Fig.S1b). This indicates well dispersion of the NPs in the mesophase. In fact, the NPs were stable in the mesophase for about three months without any aggregation. Fig.S1c shows the NPs in the isotropic phase at 29◦C.
3.1. UV- visible and FT-IR spectroscopy analysis
UV–vis and FT-IR spectrum of Co3O4 NPs is given in Fig.1a and Fig.1b respectively. The CoCl2 solution displayed a broad absorption at 512 nm whereas the spectrum of Co3O4 NPs’ dispersion displayed a broad absorption band in the wavelength range of 591-641 nm (Fig.1a). The higher and the lower wavelength shoulder of this peak corresponds to O2– (2p) → Co2+ (t2) and O2–(2p) → Co3+(eg) charge transfer processes respectively [32, 34].
The FT-IR spectrum of Co3O4 NPs displayed prominent peaks at 660 cm-1 and 559 cm-1. These peaks were assigned to stretching vibration of Co-O and bridging vibration of O-Co-O bond respectively [40]. The band at 660 cm-1 is characteristic of Co2+–O vibration in a tetrahedral site, and the band at 559 cm-1 is attributable to the Co3+–O vibration in an octahedral site of the Co3O4 lattice [36]. These peaks confirm the spinel structure of Co3O4 NPs. A broad absorption band at 3384 cm-1 corresponds to OH stretching which is probably due to moisture from the ambience.
3.2. SEM studies
Representative SEM images of Co3O4 NPs over an ITO substrate are provided in Fig.2a and b. The images showed that NPs having nearly spherical-like morphology having a size of ∼100 nm.
3.3. XPS analysis
A survey scan of the Co3O4 particles is shown in Fig.3a. The survey spectrum indicated the presence of Co, O and C elements. Fig.3b, c and d show the individual spectrum for C, O and Co respectively. The peak at 283.86 eV due to carbon is from the substrate (carbon tape used for sample preparation). The two strong peaks at 779.93 eV and 795.81 eV in Fig.3d correspond to Co 2p3/2 and Co 2p1/2 respectively, characteristic of the Co3O4 phase [41, 42], Co 2p1/2 - Co 2p3/2 energy separation is approximately 15.88 eV. This result confirmed the formation of the Co3O4 NPs.
3.4. Anti-bacterial activity of the Co3O4 NPs
Anti-bacterial behaviour of some of the selected NPs is fairly attractive. Considering Co3O4 NPs, their catalytic activity has been explored in detail compared their biological applications. Few examples in which biological application of Co3O4 NPs is investigated are given below.
Khalil et al. have synthesised the Co3O4 NPs by greener method using aqueous leaf extracts of Sageretiathea as chelating agent [37]. They studied the anti-bacterial potential of Co3O4 NPs (with and without UV illumination) against three gram-positive (S. epidermis, S. aureus and Bacillus subtilis) and three gram-negative [Pseudomonas aeruginosa (P. Aeruginosa), Klebsiella pneumonia, and E. coli] bacterial pathogens. They found that S. aureus and E. coli are the most susceptible strains having MIC (minimum inhibitory concentration) and MICuv as 31.25 and 31.25 µg/mL respectively. Whereas, P. Aeruginosa have been least with MIC and MICuv as 250 and 62.5 µg/mL. Increase in the concentration of the NPs resulted in increase in the antibacterial activity. Also, these researchers have used Co3O4 NPs for cytotoxic, antioxidant and enzyme inhibition assays. Sabir et al. prepared the Co3O4 NPs through biological method in the presence of phytolaccadodecandra leaf extract and through chemical method using co-precipitation technique [38]. Between these two methods, the NPs prepared through biological method have shown better anti-bacterial activity even at low concentration (25 µg/mL) having a zone inhibition of 10.55 mm and 8.3 mm for E. coli and S. aureus due to the enhanced production of reactive oxygen species. On the other hand chemically synthesised NPs showed the zone inhibition of 8.8 mm and 8.11 mm for both gram-negative and gram-positive strains respectively.
In our work, we studied the anti-bacterial activity of Co3O4 NPs towards gram-positive (S. aureus) and gram-negative (E. coli) bacterial pathogens. For a given concentration of the Co3O4 NPs (25, 50 and 100 µg/mL), for the gram-negative bacteria (E. coli), inhibition zones measured were 29, 31 and 33 mm while for gram- positive (S. aureus) bacteria the corresponding inhibition zones were 23, 24 and 25 mm (Table 1). It is important to note that activity of Co3O4 NPs against E. coli was higher than S. aureus as seen from Fig.4. These results indicate the potential of the Co3O4 NPs against human pathogens.
In the current study, it is important to note that activity of Co3O4 NPs against E. coli was higher compared to S. aureus.
3.5 MTT assay of in-vitro MCF-7
We evaluated the cytotoxic efficacy of Co3O4 NPs against breast cancer cell lines by MTT assay (Fig. 5). The physicochemical properties of the NPs are crucial in deciding their biological application and cytotoxicity [43, 44].
Fig.5 shows the dose-dependent (6.5, 12.5,25, 50 and 100 µg/mL) cytotoxic effect against
invitro MCF-7 cell lines. Following 24 h incubation, there is a significant cytotoxic activity in vitro cells. The percentage of cytotoxic activity of Co3O4 NPs was observed as 9.88%, 21.73%, 44.07%, 54.15% and 59.88% for MCF-7 cells (Graph 1). The cell incubation was increased with increasing concentrations of Co3O4 NPs. In line with the earlier reports, the cytotoxicity of Co3O4 NPs was dose dependent [45-48]. The half maximal inhibitory concentration (IC50 value) was observed as 30.8 µg/mL of Co3O4 NPs against breast cancer cell lines by MTT assay. This result revealed the excellent cytotoxic effect of Co3O4 NPs against the MCF-7 breast cancer cells.
Comparative study
Table 2 shows a comparative study of Co3O4 NPs with similar other studies for the application of MCF-7 anti-cancer activity. The comparison indicates that the anti-cancer activity of the Co3O4 NPs in the current study is comparable with the other Co3O4 NPs reported. Many of the studies reported a decrease in the cell viability with increasing the concentration of NPs.
3.6. Catalytic activities of the Co3O4 NPs for the degradation of MB and reduction of 4-nitrophenol
The degradation of organic dye MB at room temperature using NaBH4 (2 ml, 2´10-2 M) was studied using Co3O4 NPs as catalysts. The UV–vis spectrum of aqueous solution of MB (2 ml, 2´10-5 M) in presence of Co3O4 NPs (2 mg) showed a broad absorption peak at around 665 nm. The reduction of MB by NaBH4 in the absence of catalyst is used as a control. The degradation process was found to be accelerated in the presence of Co3O4 NPs. The absorption peak at 665 nm for MB was gradually decreased with increase in the reaction time and blue colour of the mixture vanished in 8-10 min indicating that the dye has been degraded slowly (Fig.6a).
Since the concentration of the NaBH4 is considerably higher than the MB, the reaction can be assumed to follow a pseudo first order kinetics. A graph of logarithm of absorbance versus time is shown in Fig. 6b. The apparent rate constant is calculated from the slope of this graph and is found to be 3.1´10-2 min-1 (Fig. 6b). Additionally the Co3O4 NPs showed good recyclability upto 5th cycle. However, the time required for the complete reduction increased with increasing cycles (using 2mg Co3O4 NPs; I cycle 8 min, II cycle 10 min, III cycle 12 min, IV cycle 22 min, V cycle 30 min ). The UV-vis spectra showing the recyclability of Co3O4 NPs (II to V cycles) for the reduction of MB are provided in Fig.S2.
Similarly, the catalytic activity of Co3O4 NPs in the reduction of 4-nitrophenol is investigated. In a typical experiment, NaBH4 (300 µL, 0.1M) is used for the reduction of 4-nitrophenol (300 µL, 1 mM) in presence of Co3O4 NPs (300 µL, 80 mM) as catalyst. In the absence of Co3O4 NPs, a mixture of 4-nitrophenol and NaBH4 showed an absorption band having λmax at 400nm due to 4-nitrophenolate anion. We observed no change in the peak at least for a period of more than 3 h which indicated that the reduction did not take place in the absence of the catalyst. However, addition of a small amount of Co3O4 NPs (300µL, 80 mM) to the reaction mixture triggered the reduction process that was visualized as a change in colour from yellow to black. Time-dependent absorption spectra for the reaction mixture showed the appearance of a new peak at 276 nm corresponding to the formation of 4-aminophenol followed by a gradual disappearance of the peak at 400 nm. The absorption spectra are given in Fig.6c.
The concentration of borohydride in this reduction is higher than that of the reactant, 4-nitrophenol, hence; it will not adversely affect the kinetics of the reaction. Therefore, it may be anticipated that the rate of this reduction reaction will follow pseudo-first order kinetics. Fig. 6d shows a plot of the logarithm of the 4-nitrophenolate absorbance at 400 nm (ln A) versus reaction time which yielded a straight line. An apparent rate constant Kapp of 1.8 ×10-2 min -1 was obtained from the slope of this linear progression.
Moreover, the NPs exhibited good recyclability. Up to the fifth cycle, particles can be collected and recycled without significant loss in their catalytic activity (using 300 µl of 80 mM particles; I cycle 6 min, II cycle 12 min, III cycle 12 min, IV cycle 12 min, V cycle 16 min). The UV-vis spectra corresponding to the recyclability of the NPs are provided in Fig.S3.
Mondal et al. [49] reported the photocatalytic application of cobalt NPs for the reduction of MB (2´10-5 M) using NaBH4 (2´10-2 M). Cobalt NPs were prepared from cobalt sulphate using tetrabutylammonium bromide and NaBH4 as reducing agent. The prepared spherical cobalt NPs (size-86.97 nm) displayed good activity in the degradation of MB along with a recyclability even after 7 cycles. Same researchers also reported the catalytic reduction of 4-nitrophenol (1´10-6 M) using NaBH4 (2´10-5 M) [50]. The estimated Kapp at 29°C was 0.21 min-1 and particles exhibited recyclability till 5th cycle.