Worldwide, use of nanoparticles (NPs) in the biomedical field attracted the attention of researchers. The small surface to volume ratio compared to its bulk material is responsible for their enhanced or unique properties. As it enable the NPs to interact with high degree of specificity and enhanced efficacy to combat infectious diseases1,2. Among the various metal oxides NPs copper oxide nanoparticles (CuONPs) gained significant interest due to their high stability, longer shelf life, high quantum efficiency and antimicrobial activity. Extensive biomedical application of CuONPs as an antioxidant, antimicrobial, anti-inflammatory, antiviral, cytotoxic and anticancer activities has made them strong candidates to be used as therapeutic agents3,4,5,6,7. Longer shelf life and stability also make CuONPs the suitable candidate in environmental biotechnology for purification of water and elimination of pollutants from industrial effluents without the formation of harmful by products8. In human body copper (Cu), is present in trace element that is found in enzymes such as superoxide dismutase, cytochrome oxidase, and tyrosinase6. Moreover it serves as a cofactor for multiple enzymes responsible in neuropeptide production, cell signalling mechanism, oxidative stress, and immune cell function in humans9.

Synthesis of CuONPs can be done by several physical, chemical and biological processes. Physical and chemical synthesis approaches suffers from drawbacks like expensive reagent, hazardous reaction condition, longer time, tedious process to isolate NPs and harm to ecosystems as well as human health10,11. Thus, to overcome these shortfalls, the principle of green chemistry utilizing naturally available resources like viruses, bacteria, cyanobacteria, fungi, algae and plants were utilized for the fabrication of NPs12. Cyanobacteria (blue–green algae) form one of the largest and most primitive ancestral groups with prokaryotic cell structures that have the capability of carbon dioxide-dependent photosynthesis. Cyanobacteria have received a lot of scientific attention for their ability to synthesize NPs, not only due to their high biomass efficiency, but also due to their potential to bioremediate hazardous metals and transform them to more manageable forms13. These are able to synthesize inorganic and metal oxide NPs e.g. selenium, zinc, platinum, palladium, gold, and silver nanoparticles14,15,16,17,18. Cyanobacteria facilitated synthesis of NPs occurs either through extracellularly, that involves the production of reductase enzyme due to electrostatic interactions and intracellularly i.e. within the cells by the activity of enzymes13.

To the best of our knowledge, studies on biomedical applications of CuONPs are limited, prompting us to undertake the present study, to unravel the toxicological evaluation of biologically synthesized CuONPs. In the present study CuONPs were biologically synthesized using cyanobacteria (Phormidium sp.) cell extract and characterized by Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), energy dispersive x-ray spectrum (EDX), X-ray diffraction (XRD), Atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and UV–Vis analysis. As-synthesized CuONPs were studied for antioxidant, antimicrobial, and anti-inflammatory properties. Cytotoxicity of CuONPs was tested against two human lung cancer cell lines A549 and H1299, as well as apoptotic studies were studied. Furthermore, the photocatalytic activity was done to promote the use of Phormidium derived CuONPs as an eco-friendly aspect capable of effectively combating dye degradation.


GC–MS analysis of cyanobacterial cell extract

Based on the retention time, the peaks of mass spectra were identified with the help of NIST/Wiley Library. GC–MS profile of Phormidium sp. cell extract showed 19 compounds—fatty acid esters (94.5%), carboxylic acid (1.42%), esters (0.84%), alkenes (1.75%), phenolics (0.29%), alkanes (1.40%) and others (Fig. S1; Table S1). Three major peaks of fatty acid esters were observed i.e. Peak 1 with area 21.09% for 9, 12-Octadecadienoic acid, methyl ester, Peak 2 with area 17.06% for 9-Octadecenoic acid, methyl ester and peak 3 with area 12.7% for Hexadecanoic acid, methyl ester.

Biosynthesis of CuONPs

Change in color of the solution from dark green to dark brown indicated the synthesis of CuONPs (Fig. S2). Cyanobacteria derived CuONPs exhibited the maximum absorption peak at 265 nm that confirmed the formation of CuONPs in the solution and no absorption band was determined for copper sulphate and the cell extract at the same region (Fig. 1a).

Figure 1
figure 1

Characterization of Phormidium sp. derived CuONPs; (a) UV–visible spectrum; (b) FTIR spectrum; (c) X-ray diffractogram; (d) SEM analysis at a direct magnification of × 10 000; (e) EDX graph and (f) TEM micrograph. The inset in panel (a) depicts the green color of cell extract and powder form of CuONPs.

Physicochemical characterization of CuONPs

FTIR analysis

FTIR analysis of CuONPs and Phormidium sp. cell extract was done (Fig. 1b). The broad peak observed at 3388 cm−1 in the cell extract corresponds to the –OH stretching of phenolic compounds in the cell extract. The fine peak at 2969 cm−1 was attributed to the –CH2and C−H stretching mode in alkanes. The intense peak observed around 1657 cm−1 can be allocated to the stretching vibrations of proteins (amide I) whereas the band at 1536 cm−1is characteristic of amide II. The peak at 1452 cm−1 is characteristic to the C-N stretching of aliphatic amines. The peak around 1402 cm−1 shows the presence of −COO carboxylic acid present in the cell extract. The sharp peak at 1089 cm−1 corresponds to the presence of the stretching vibrations of carboxylic acids and amino groups. The narrow peak observed at 597 cm−1 can be attributed to the bending vibrations of CuO, confirming the formation of highly pure CuONPs.

XRD analysis

XRD analysis showed the prominent peaks at 35.5°, 38.7°, 48.7°, 53.6°, 58.2° and 61.5° that are attributed to the Miller- Bravais indices of (111), (110), (200), (202), (222), and (003), respectively (Fig. 1c). The observed diffraction peaks of the CuONPs comply well with those of the Joint Committee on Powder Diffraction Standards (JCPDS, card No. 89–1461) pattern of CuO which can be indexed to the hexagonal wurtzite crystal structure of the CuONPs. The detailed information of full-width half maxima (FWHM), Miller indices, d-spacing in nanometer, D- granule size of biogenic CuONPs were indexed in (Table S2). According to the most intense diffraction peak at 2θ = 38.7° (110), the average particle size of CuONPs was found to be 22.5 nm using Debye–Scherrer equation19. Moreover, no extra peaks were detected in XRD pattern presenting that CuONPs synthesized by the biogenic method is significantly pure.

SEM, TEM and AFM analysis

SEM analysis showed 28.5 ± 2.5 nm sized CuONPs (Fig. 1d). EDX spectrum gave a strong signal at 8 keV with 96% weight of copper (Fig. 1e), which signified the presence of copper. The average size of CuONPs by TEM was found to be 20.7 ± 2.2 nm with spherical or oval shape (Fig. 1f). Tip-corrected AFM analysis showed spherical/oval shaped CuONPs in the range of 0–22.5 nm. The result showed the 2D and 3D view of the sample surface over a 0.7 × 0.7 µm scan and uniform height distribution (Fig. 2a,b).

Figure 2
figure 2

Atomic force microscopy (AFM) results of Phormidium derived CuONPs (a) Unfiltered AFM image showing topographical 2D image; (b) 3D image. Particle size analysis of CuONPs (c) Size distribution through Zeta size analyzer; (d) Zeta potential distribution.

Particle size distribution and zeta potential analysis

DLS analysis suggested the CuONPs size distribution in the range of 68.7 ± 3.4 nm (Fig. 2c). The substances adsorbed on the surface of the nanoparticles (e.g., stabilizers) and the thickness of the electrical double layer (solvation shell), moving along with the particle makes the size bigger in comparison with SEM and TEM microscopic techniques20. The polydispersity index (PDI) of CuONPs was 0.220 which pointed out that these particles are moderately dispersed. As per the manual Malvern Zeta analyzer the PDI value less than 0.05 represent highly mono-dispersed distributions of NPs and the PDI value more than 0.7 indicate poly-dispersed distributions of NPs. The calculations used for determination of size and PDI parameters are defined in the ISO standard documents 13,321:1996 E and ISO 22,412:2008 (Manual—Malvern Zeta Size Analyzer)21. The zeta potential of CuONPs was found to be − 21.1 mV (Fig. 2d).

Biological characterization of Phormidium sp. derived CuONPs

Antioxidant activities of Phormidium derived CuONPs

In the present study the antioxidant activity of aqueous cell free extract, CuONPs and standard ascorbic acid was compared by ABTS, DPPH, SOR and H2O2 assays and calculated the concentrations at which 50% scavenging (IC50) of free radicals was done. Their free radical scavenging activities increased in dose dependent manner (Fig. 3 and Table 1). The order of antioxidant activity (% inhibition of scavenging) was: SOR > H2O2 > DPPH > ABTS. The IC50 value calculated from the percentage inhibition of free radical scavenging revealed that CuONPs showed higher IC50 values than standard ascorbic acid but lower than cell free extract indicating their superior antioxidant nature.

Figure 3
figure 3

Antioxidant activity of cell free extract, biogenic CuONPs and ascorbic acid by different scavenging assays; (a) ABTS; (b) DPPH; (c) Hydrogen peroxide; (d) SOR. Experiments were performed in triplicates; bars represent the mean of values and error bars represent mean ± SD (*P < 0.05).

Table 1 Antioxidant activity of biosynthesized CuONPs.

Steady inhibition of ABTS·+ free radical (3.9% ± 0.5 to 86.33% ± 0.8) by CuONPs was observed in the concentration ranging from 25 to 125 µg ml−1 and corresponding IC50 values for CuONPs, extract and ascorbic acid (Std.) were calculated (Fig. 3a and Table 1). DPPH radical scavenging activity at concentration range 25–125 µg ml−1 showed scavenging percentage ranging from 23.5% ± 1.08 to 79.6% ± 0.4 which directly depends on hydrogen donating tendency of sample to DPPH radical and corresponding IC50 values were calculated (Fig. 3b and Table 1).

Antimicrobial activity of CuONPs

Biogenic CuONPs exhibited significant dose dependent inhibition for all microbial pathogens (Fig. 4a–d). Higher inhibition was observed against Gram positive (Bacillus cereus, Staphylococcus aureus) than gram negative (Escherichia coli, Klebsiella pneumoniae) bacteria with lowest MIC value of 62.5 µg ml−1 against B. cereus (Table 2). Similar results were observed against fungal pathogens as percentage inhibition with maximum inhibition against C. albicans having MIC value of 125 µg ml−1 (Fig. 4e,f; Table 3).

Figure 4
figure 4

The inhibitory effect of Phormidium sp. derived CuONPs and cell extract on; (a) E. coli; (b) K. pneumonia; (c) B. cereus; (d) S. aureus; (e) C. albicans and (f) C. glabrata. Experiments were performed in triplicates; bars represent the mean of values and error bars represent mean ± SD (*P < 0.05).

Table 2 Minimum inhibitory concentration (MIC) against pathogenic bacteria alone and in combination with antibiotic (streptomycin) along with interaction index.
Table 3 Minimum inhibitory concentration (MIC) against pathogenic fungi alone and in combination with antifungal (fluconazole) along with interaction index.

Furthermore, synergistic action was determined to describe the interaction of antimicrobial agents, in which the effect produced by the drugs in combination was greater than their individual effects22. The interaction indexes (synergistic/antagonistic) for each combination was determined by checkerboard methods. The fractional inhibitory concentration (FIC) indexes against pathogenic bacteria in combination with CuONPs and streptomycin were calculated (Table 2). The degree of synergy was higher for Gram negative species than Gram-positive species. Similar results were observed against C. albicans and C. glabrata with CuONPs and fluconazole (Table 3). The possible mechanism that lies behind the enhanced antimicrobial action of CuONPs were summarized (Fig. 5).

Figure 5
figure 5

The proposed antimicrobial mechanisms induced by CuONPs for inhibition in bacterial and fungal cells.

Anti-inflammatory activity of biogenic CuONPs

The maximum inhibition of protein denaturation observed was 86.3% ± 0.33 with 150 µg ml−1 CuONPs (Fig. S3). Aspirin (acetylsalicylic acid), a standard inflammatory drug showed maximum inhibition 93.8% ± 1.11 at the concentration of 100 µg ml−1. The IC50 of aspirin and CuONPs were found to be 34.9 ± 0.87 and 89.9 ± 0.21 µg ml−1, respectively.

Biocompatibility assay of Phormidium derived CuONPs

Biocompatibility of CuONPs against the non-small cell lung cancer cell lines (H1299 and A549) was assessed by MTT assay. CuONPs reduced A549 and H1299 cancer cell line viability in a dose-dependent manner after 24 h exposure (Fig. 6c).

Figure 6
figure 6

Cytotoxic effect of Phormidium derived CuONPs at IC50 concentration against lung cancer cell line (a) H1299 (control & treated); (b) A549 (control & treated); (c) cytotoxic effect of CuONPs against A549 & H1299 lung cancer cell lines at different concentration. Experiments were performed in triplicates; bars represent the mean of values and error bars represent mean ± SD (*P < 0.05).

The IC50 values calculated for biogenic CuONPs was 100.8 ± 1.24 µg ml−1 for H1299 and 88.3 ± 0.74 µg ml−1 for A549 cell line. The IC50 values for standard drug (doxorubicin) was 0.94 ± 0.55 μg ml−1 against H1299 and 1.80 ± 0.05 μg ml−1 for A549 cells, respectively. CuONPs treatment induced apoptosis leads to the conformational changes visualized by DAPI staining as nuclei blebbing, condensation and cracking constitutes the characteristic features of apoptosis (Fig. 6a,b). Cells without treatment (control) had shown intact nuclei with smooth edges having uniform shape and size in both the cell lines.

Dye degradation by CuONPs

To investigate the photocatalytic activity of CuONPs the organic dye MB was subjected to photodegradation using biogenic CuONPs (5, 10, 15, 20 and 25 mg/L). The control kept in dark, showed no visual color change. But the reaction mixtures supplemented with CuONPs in sunlight exhibited change in color from dark blue to pale blue. UV–Vis scanning of the reaction suspension for MB at 665 nm showed that the peak height decreased with increase in time that suggested increased dye degradation with time (Fig. 7a). Maximum degradation of MB dye (94%) dye was observed after 90 min (Fig. 7b).

Figure 7
figure 7

Photodegradation of methylene blue (MB) dye, (a) Effect of solar irradiation using CuONPs on photocatalytic degradation of MB dye; (b) Percent photocatalytic degradation of MB dye as effect of time with varying photocatalyst (CuONPs) load; (c) Photodegradation of dye versus irradiation time at varying photocatalyst (CuONPs) measured by optical absorption; (d) Plot of ln(C0/C) versus irradiation time for dye degradation kinetics.

For the identification of CuONPs induced free radical during the photodegradation of dye, elimination studies were performed by the addition of ammonium oxalate (AO) as h+ scavenger, p-benzoquinone (p-BQ) as O2 scavenger and tert-butanol (t-BuOH) as OH scavenger in the reaction suspensions. The addition of AO exhibited the minimal change in the photocatalytic degradation of dyes indicating that h+ radicals played very little role in degradation (Fig. S4). The addition of p-BQ (·O2 scavenger) exhibited higher reduction than h+ radicals indicates their relatively higher role in dye degradation. However, the degradation rate of MB dye reduced maximally upto 65% with the addition of t-BuOH (OH scavenger) and suggested that hydroxyl radicals were mainly involved in dye degradation. The decrease in the removal rate in the presence of scavengers presents the following trend: t-BuOH > p-BQ > AO. To quantitatively study the photocatalytic activity of CuONPs, we performed the plots of ln(C/C0) versus irradiation time, assuming that the degradation reaction of dye by CuONPs under visible light irradiation followed the pseudo-first-order kinetics. It can be modeled as:


where Kabs is the photodegradation rate constant (min−1), C0 is the initial concentration of MB and dye and Ct is the concentration of dye at time t23. Using MB dye initial concentration of 25 mg/L and varying photocatalyst load, plots of best-fit lines of ln(C0/Ct) versus time were plotted (Fig. 7c,d). The linear nature of our data confirmed that the photodegradation of MB dye using photocatalysts fits well with the kinetic model. It is worth noting that dye concentration in these experiment remains in the optical regime where the Beer–Lambert law holds. All correlation coefficient (R2) values were higher than 0.95 with an average value of 0.96 for photocatalytic degradation of MB dye. Our results confirmed the applicability of the pseudo first-order reaction kinetic model for photocatalytic degradation of MB dye. Assuming the reaction kinetics, highest rate constant (k) determined from the slope of ln(C0/Ct) versus time linear plots, corresponding to optimum catalyst load (CuONPs) of 10 mg/L, was 0.026 min−1 for MB dye.


Biogenic CuONPs synthesis got worldwide attention due to their wide applications in biomedicine. During the present investigation, CuONPs had been synthesized using the crude cyanobacterial (Phormidium) cell free aqueous extract. Extra-cellular biosynthesis of CuONPs is much economical than intra-cellular synthesis over the use of chemicals, energy and time required for cell disruption and purification. GC–MS analysis of Phormidium cell extract showed the presence of several active principle compounds that may help in the synthesis of CuONPs. These compounds were also previously reported in the GC–MS profile of Nostoc sp. EA0324, and Synechocystis sp. PCC 733825, and suggested their reducing nature. Fatty acid esters, viz., Octadecanoic acid, methyl ester and 13-Docosenoic acid, methyl ester were found in lesser quantity (1.74% and 0.64% respectively). The derivatives of these fatty acid esters with reducing potential have been also found in Allium saralicum26 and Citrus wax27. Esters like heptadecanoic acid- methyl ester and Octadecenoic acid (Z)—methyl ester were also found in cyanobacterial extract. Heptadecanoic acid, methyl ester from Mentha spicata28 and derivative of octadecenoic acid (Z)-, methyl ester from Thesium humile29 were also reported for their good reducing potential. Apart from the esters, few carboxylic acids i.e. Methacrylic acid (0.13%) and 1, 2-benzenedicarboxylic acid (0.22%) were also identified in extract. Previously, the reducing nature of these two carboxylic acids was reported in Calothrix brevisima30 and Punica species31. Compounds of other classes like alkane (Nonadecane), alkene (Neophytadiene, 1, 3, 12-Nonadecatriene), cycloalkene derivatives (cyclododecyne, tricycle [,16)] triacontan and 1,1':3',1"-tercyclopentane, 2'-dodecyl), derivative of acetanilide (acetamide, N-(3,5-diaminophenyl) and phenol (Phytol) have been also obtained in the present study using Phormidium sp. cell extract. During previous studies some related compounds were also reported with good reducing ability like, Neophytadiene from Plectranthus amboinicus32, 5-(1,5-dimethyl-4-hexenyl from Zingiber officinal33, Phenol, 3,5-bis(1,1-dimethylethyl from Calothrix brevisimma30. The phyto-chemicals present in cyanobacterial extract such as chlorophyll, carotenoids, phycobilliproteins, flavonoids, phenols and proteins act as both reducing as well as stabilizing (capping) agents for NPs synthesis34. The flavonoids have the ability to donate the hydrogen or electron, and the phenolics exhibit a chelating effect on the metal ions, which is responsible for the reduction of Cu2+ from CuSO4.5H2O to copper oxide nanoparticles35. Proteins present in the extract bind to nanoparticles through free amine groups or cysteine residues or negatively charged carboxylate groups of enzymes that help in synthesis as well as stabilization of nanoparticles. Exact chemical nature of reducing and capping agent is not yet explored for CuONPs. But with regard to CdS nanoparticles derived from Phormidium tenue NTDM05, fluorescent phycobilin protein are reported to prevent agglomeration of nanoparticles36. CuONPs synthesized from cyanobacteria exhibited different λ max, e.g. at 259 nm in Spirulina platensis37, 600 nm in Phormidium sp.38, 220 nm in Cylindrospermum stagnale39 and 580 nm in Spirulina platensis40. Furthermore, CuONPs from other organisms have showed slightly different λ max, e.g. at 282 nm in Punica granatum41, 250 nm in Achillea millefolium3, 260 nm in Aloe vera42, 265 nm in Aloe barbadensis Miller43, 250 nm in Caesalpinia bonducella44, 245 nm in Momordica charantia5 and 277 nm in Citrofortunella microcarpa45.

Biogenic CuONPs were further purified for the removal of unwanted biomolecules by centrifugation after 3 h. Then physical characterization was done by XRD, SEM, EDX, TEM, AFM and DLS analysis. In FTIR analysis peaks obtained in Phormidium sp. derived CuONPs showed functional groups that may have served as the reducing and capping agents during its synthesis (Fig. 1b). Similar observations were also reported by other researchers e.g. peak at 574 cm−1 in Millettia pinnata flower extract4, at 540 cm−1 in aqueous black bean extract46, peak at 576 cm−1 in brown alga Cystoseira trinodis11, 590 cm−1 in Momordica charantia aqueous extract5 and 529 cm−1 in Aloe vera leaves extract42. The peak observed at 875 cm−1 may be attributed to the aromatic C-H bending. In Citrofortunella microcarpa leaves extract identical peak at 880 cm−1 was reported45. This indicates that the biological molecule present in the cell extract has a dual function of stabilization and formation of CuONPs. XRD analysis of CuONPs from Phormidium sp. cell extract showed well-defined peaks at 2θ value that indicated their crystalline nature (Fig. 1c). When the crystalline size decreases from bulk to nanoscale dimensions, the XRD peaks broaden19. XRD result showed that CuONPs formed are crystalline in nature with average size 22.5 nm close to particle size measured by TEM. Several biologically synthesized CuONPs also showed the crystalline nature viz., from cyanobacteria Phormidium47, Spirulina platensis37, brown alga Cystoseira trinodis11 and Bifurcaria bifurcate48. Higher plants also exhibited crystalline nature of biosynthesized CuONPs e.g. Aloe barbadensis Miller leaves extract43, Caesalpinia bonducella seed extract44 and Achillea millefolium leaf extracts3. Scanning electron microscopy with energy dispersive X-ray (SEM–EDX) was done to confirm the presence of elemental copper in biologically synthesized CuONPs. The presence of copper (96%) and oxygen in EDX analysis confirmed the purity of synthesized CuONPs (Fig. 1d,e).

TEM and AFM analysis were also done for size and surface topology (Figs. 1f and 2b). CuONPs of different shapes and sizes had been reported from cyanobacteria e.g. Phormidium, Spirulina platensis and Bifurcaria bifurcate synthesized quasi-spherical shaped CuONPs in the range of 40–45 nm37,47,48. Other studies on higher plant mediated synthesis have reported lesser purity e.g. CuONPs synthesized from Aloe vera extract exhibited 54% of elemental copper having size of 30 nm with some larger particles (100 nm)42. CuONPs synthesized from Momordica charantia showed an average size of 61.4 nm with 54.5% of copper5. Additionally, large sized spherical CuONPs have been reported from Citrofortunella microcarpa (68–75 nm)45 and from actinomycetes (61.7 nm)49. Dynamic Light Scattering (DLS) was used to determine hydrodynamic sizes, polydispersities and aggregation effects of colloidal sample based on the Brownian movement20. It is the particle diffusion behavior within any fluid, measured by the fluctuations in light intensity that passes through a colloidal solution as a function of time50. The CuONPs size distribution in the range of 68.7 ± 3.4 nm (Fig. 2c). The obtained PDI value of 0.2 for CuONPs suggested their intermediate or moderate nature. As per the manual Malvern Zeta analyzer the PDI value less than 0.05 represent highly mono-dispersed distributions of NPs and the PDI value more than 0.7 indicate poly-dispersed distributions of NPs21. Further, zeta potential measurement was done to check the stability of synthesized CuONPs (Fig. 2d). It is considered as a measure of charges on the surface of nanoparticles. Large positive (> + 30 mV) or negative (≥ 30 mV) charges tends to repel each other whereas low zeta potential value causes the aggregation of NPs due to absence of repulsive force and provide stability to the nanoparticles17. Qamar et al.5 synthesized CuONPs from Momordica charantia leaf extract with zeta potential of − 7.23 mV only. The more negative value of zeta potential of the Phormidium derived CuONPs suggested their higher stability.

Cyanobacteria and many medicinal plants possess free radical scavenging moieties such as phenolic compounds, terpenoids, flavinoids, vitamins and other endogenous metabolites, that are rich in antioxidant activity51. Phormidium derived CuONPs exhibited higher percentage of free radicals scavenging. Cellular respiration leads to the production of reactive oxygen species (ROS) and several other free radicals having unpaired valence shell electrons. These free radicals play vital role in cell signaling but in excess leads to oxidative damage to the cell by reacting with the cellular components that cause cancer, aging, cataract, cardiovascular diseases and dysfunction of organs. Thus, antioxidants play a crucial role to down regulate and eliminate free radicals before they damage the cell52. The present study showed the antioxidant activity of CuONPs by ABTS, DPPH, SOR and H2O2 assays. ABTS·+ is a pre generated free radical and the interaction between antioxidant and ABTS·+ causes bleaching of ABTS·+17. CuONPs exhibited percentage inhibition from 3.9 to 86.33% and corresponding IC50 values for CuONPs, extract and ascorbic acid (Std.) were calculated (Fig. 3a and Table 1). Kumar and Shanmugam53 also reported 88% ± 1.12 inhibition of ABTS·+ free radicals with 500 µg ml−1 of CuONPs synthesized by using Magnolia champaca floral extract.

In DPPH radical scavenging, CuONPs exhibited percentage inhibition ranging from 23.5 to 79.6% and corresponding IC50 values were calculated (Fig. 3b and Table 1). In previous reports, Cystoseira trinodis (brown algae), Solanum nigrum and Mussaenda frondosa derived CuONPs exhibited 50% scavenging inhibition with an IC50 value of 543 µg ml−1, 131.5 µg ml−1 and 1570 µg ml−1, respectively8,11,54.

A significant dose dependent inhibition was observed with biogenic CuONPs against bacterial strains (Fig. 4a–d). Karuppannan et al.55 synthesized CuONPs from Cardiospermum halicacabum extract exhibited the antibacterial action with MIC value of 1 mg ml−1 for S. aureus and E.coli. Manasa et al.54 synthesized CuONPs from Mussaenda frondosa L that exhibited antibacterial potential with MIC value of 305 µg ml−1 against E. coli. Furthermore, CuONPs synthesized from brown algal extract of Bifurcaria bifurcate48 and cyanobacteria Spirulina platensis extract37 showed antibacterial potential against S. aureus, B. cereus, E. coli and K. pneumonia by disc diffusion. CuONPs synthesized from actinomycetes49, Achillea millefolium leaf extracts3 and Momordica charantia5 exhibited the antibacterial activity with maximum zones of inhibition. However mechanically synthesized CuONPs also showed the antibacterial activity but with higher MIC value of 2.5 mg ml−1 for S. aureus and 3.75 mg ml−1 for E.coli56. CuONPs synthesized using Psidium guajava leaf extract exhibited enhanced antibacterial activity against gram negative (E. coli, P. aeruginosa) and gram positive (S. pneumoniae, S. epidermidis) bacterial strains using agar well diffusion, and CuONPs interaction with bacterial membrane was shown by confocal laser scanning microscopic (CLSM)57. CuONPs synthesized using Abutilon indicum leaf extract showed antibacterial activity against E. coli, S. typhi, B. subtilis and S. aureus bacteria by using agar well diffusion method58. Limited studies were known in the literature on the antifungal activity of CuONPs with reference to Candida species. CuONPs synthesized from Achillea millefolium leaf extracts3, Brassica oleracea var. extract59 and chemically synthesized CuONPs60 exhibited the potent antifungal activity against C. albicans and C. glabrata. However, commercially synthesized CuONPs showed the antifungal activity with MIC value of 1 mg ml−1 for C. albicans and C. glabrata61. Our results highlight the presence of synergistic interactions between CuONPs and antibiotic/fungicides combinations (Tables 2 and 3). The possible mechanism behind the enhanced synergistic antimicrobial action of CuONPs may be due to the involvement of active functional groups such as hydroxyl and amino groups present in the antibiotics/fungicides that can be chelated by CuONPs22,62. Since CuONPs has potential antimicrobial properties with synergistic potential they may be used in food preservation and packaging. The antimicrobial mechanism of CuONPs may be due to the generation of reactive oxygen species (ROS) generation that disrupts the cell membrane causing direct cellular toxicity63. The possible mechanisms involved by the CuONPs induced antimicrobial activity can be summarized as (Fig. 5). The carboxylic and amines group present in cell membrane facilitates the penetration of CuONPs by the intracellular dissolution of soluble copper ions and accumulation of superoxides or hydroxyl radicals that leads to oxidative stress. CuONPs generates potent cytotoxicity by DNA damage, mitochondrial degradation, ribosomes disruption and dysfunction of different proteins channels present in cell membrane that leads to cell death64,65.

Inflammation is a complex process, associated with pain and involves increased protein denaturation, vascular permeability and membrane alteration. During denaturation, proteins lose their secondary and tertiary structure due to stress or heat that causes inflammation. Limited reports are available on the anti inflammatory activity of CuONPs. Maximum protein denaturation observed was 86.3% with 150 µg ml−1 CuONPs (Fig. S3) and the IC50 of aspirin and CuONPs were found to be 34.9 ± 0.87 and 89.9 ± 0.21 µg ml−1, respectively. Thiruvengadam et al.4 observed 80% of albumin denaturation by the CuONPs synthesized using Millettia pinnata flower extract. Biosynthesized CuONPs using Triumfetta rotundifolia exhibited 57.4% of human RBCs membrane stabilization at 1 mg ml−1 and 58% at 100 mg ml−1 using Mussaenda frondosa L. extract54,66. CuONPs synthesized using Bacopa monnieri leaf extract exhibited the in-vivo anti-inflammatory activity by 74% inhibition of edema in comparison to diclofinac sodium as standard by 24% after 48 h67. CuONPs synthesized using Cissus quadrangularis leaf extract showed the enhanced anti-inflammatory activity in membrane stabilization and anti-proteinase activity in comparison to standard drug68. Chemically synthesized CuONPs were also tested for their anti-inflammatory activity both in-vitro and in-vitro on albino mice. Results showed that BSA denaturation was highly inhibited by CuONPs in the in-vitro anti-inflammatory activity and in-vivo activity revealed that low doses (5 mg/kg) of CuONPs were more potent in inhibiting inflammation than high doses (10 and 20 mg/kg) of free drugs69.

Cytotoxicity of CuONPs against the non-small cell lung cancer cell lines (H1299 and A549) was assessed by MTT assay. Appreciable anticancer activity was observed against A549 cells with lower IC50 value than H1299 cells (Fig. 6c). DAPI staining was done to determine the apoptosis caused by CuONPs on cell lines that showed the nuclei blebbing and condensation in treated cells (Fig. 6a,b). Manasa et al.54 showed an IC50 value of 218.18 µg ml−1 from the biosynthesized CuONPs using Mussaenda frondosa L. extracts against A549 cancer cells. The CuONPs synthesized from Ficus religiosa extract evaluated against A549 cell line by MTT assay showed an IC50 value of 200 μg ml−170. Green synthesized CuONPs from Ficus religiosa leaf extract inhibited histone deacetylases (HDAC) level and exhibited apoptosis mediated anticancer activity against A549 lung cancer cell line with an IC50 value of 200 μg ml−171. CuONPs synthesized using Abutilon indicum leaf extract exhibited the cytotoxicity against human lung A549 and breast MDA-MB-231cancer cell lines through MTT assay58. CuONPs-mediated anticancer activity involves oxidative stress, ROS accumulation, chromosomal aberration, genetic material fragmentation, caspase production, and enhancement of intrinsic and extrinsic apoptotic pathways64. CuONPs caused DNA damage, halted cell cycle, and inhibited cell proliferation in cervical cancer HeLa cells72. CuONPs synthesized using marine entophytic actinomycetes showed the cytotoxicity against A549 lung cancer cells at concentration of 500 µg ml−1 with 54% inhibition and an irregular morphology of cancer cells73. Furthermore, Singh et al.74 and Elsayed et al.75 observed green synthesized CuONPs as an efficient antiproliferative activity in the treatment of targeted breast cancer. The lower IC50 values of Phormidium derived CuONPs against cancer cell lines (significant at P ≤ 0.05) clearly put forward the fact that biosynthesized CuONPs have potent cytotoxicity.

The photocatalytic activity of CuONPs was done using MB dye at different concentrations (5, 10, 15, 20 and 25 mg/L) showed change in color from dark blue to pale blue. UV–Vis scanning exhibited the reaction peak for MB at 665 nm (Fig. 7a). The percent degradation under visible light irradiation observed was 94% after 90 min (Fig. 7b) and was calculated by the corresponding percent adsorption value using the equation given by Kwon et al.76. Sinha and Ahmaruzzaman.77 reported that CuONPs (10 mg/L) synthesized from aqueous extract of L. rohita scales degraded 96% of MB dye in 135 min and CuONPs (50 mg/L) synthesized from brown algae Cystoseira trinodis degraded 89% of MB dye in 210 min11. Biogenic CuONPs (20 mg/L) synthesized from plant extracts of Mussaenda frondosa leaf extract exhibited 97% of MB degradation in 140 min54 and CuONPs (10 mg/L) from cell extract of Cardiospermum halicacabum exhibited 93% degradation in 210 min55. CuONPs (0.2 g/L) synthesized from plant extract of Achillea millefolium3, degraded 96% of MB dye in 120 min. It is necessary to determine which reactive species plays greater role in the photocatalytic degradation process. Biosynthesized CuONPs (20 mg) using Psidium guajava leaf extract showed 89% degradation of MB dye in 150 min57.

CuONPs induced free radical during the photodegradation of dye, showed in the presence of t-BuOH (OH- scavenger) the degradation rate of MB dye reduced maximally upto 65% (Fig. S4). Sharma and Dutta78 also reported that hydroxyl radicals were the most important reactive oxygen species in the dye degradation by specific ROS scavenger test using isopropyl alcohol as scavengers for ·OH radicals. After establishing photocatalytic role of CuONPs by measuring percentage degradation of MB dye, attention was paid for kinetic study. The plots of best-fit lines of ln(C0/Ct) versus time were plotted using MB dye initial concentration (25 mg/L) and varying photocatalyst load, (Fig. 7c,d) that exhibited the linear nature of our data and confirmed the photodegradation of MB dye fits well with the pseudo first-order reaction kinetic model. The higher photocatalytic degradation rate of biosynthesized CuONPs may be due to its smaller particle size (20.7 nm, TEM analysis). Moreover, this can be attributed to the higher density of electron-deficient sites generated during synthesis that can trap photogenerated electrons and reduce recombinations, thereby improving the photocatalytic activity77. The probable mechanism for the photocatalytic activity of CuONPs based on the interaction with light source i.e. associated with photon absorption and connectivity among the surface reactivity and, surface radical formation among (H2O, O2)3. The photocatalytic mechanism was initiated when the photons were absorbed by CuONPs, get photo excited and undergoes plasmonic decay. According to Ghareib et al.79 complete reaction occurred during photocatalytic degradation of MB dye by CuONPs can be summarized as below:

$${\text{Cu}} + {\text{h}}v \to {\text{h}}^{ + } \left( {{\text{Cu}}} \right) + {\text{e}}^{ - } \left( {{\text{Cu}}} \right)$$
$${\text{H}}_{{2}} {\text{O}}\left( {{\text{ads}}} \right) + {\text{ h}}^{ + } \to {\text{OH}} + {\text{ H}}^{ + } ({\text{ads}})$$
$${\text{O}}_{{2}} + {\text{e}}^{ - } \to {\text{O}}_{{2}}^{ - } \left( {{\text{ads}}} \right)$$
$${\text{O}}_{{2}}^{ - } \left( {{\text{ads}}} \right) + {\text{ H}}^{ + } \leftrightarrow {\text{HOO}}^{.} \left( {{\text{ads}}} \right)$$
$${\text{2HOO}}^{.} \left( {{\text{ads}}} \right) \to {\text{H}}_{{2}} {\text{O}}_{{2}} \left( {{\text{ads}}} \right) + {\text{ O}}_{{2}}$$
$${\text{H}}_{{2}} {\text{O}}_{{2}} \left( {{\text{ads}}} \right) \to {\text{2OH}}^{.} \left( {{\text{ads}}} \right)$$
$${\text{Dye}} + {\text{OH}}^{.} \to {\text{CO}}_{{2}} + {\text{ H}}_{{2}} {\text{O }}\left( {\text{dye intermediates}} \right)$$
$${\text{Dye}} + {\text{h}}^{ + } \to {\text{Oxidation products}}$$
$${\text{Dye}} + {\text{ e}}^{ - } \to {\text{Reduction product}}$$

The electron or holes generated due to plasmonic decay react with O2 and, H2O molecules to generate active species; anionic super oxide radical (O2.) and hydroxyl radical (OH.), respectively. Furthermore, the hydro peroxyl radical (HO2) obtained by the protonation of superoxide ion (O2) radical converts to H2O2, that inevitably dissociates into highly reactive hydroxyl radicals (OH). Here, in this process CuONPs play an important role as an electron carrier. The greater degradation efficiency of CuONPs, may be attributed to the reducing agents presents in the algal extract that promotes the formation of oxygen species (e.g. superoxide and hydroxyl radical) upon irradiation 11. Such an efficient photodegradation provide a convenient path for the treatment of dyes under visible light irradiation.

Conclusion and future prospects

In summary the present study emphasizes a cost effective, environmentally benign and bio-inspired protocol for the synthesis of CuONPs followed by its biomedical ad photocatalytic applications. The phytochemicals present in cyanobacteria Phormidium cell extract act as a bio-reductant of Cu2+ ions as well as a stabilizing agent for biosynthesis of CuONPs. Dark brown coloured, small (20.7 nm), spherical shaped CuONPs with high purity (96%) were successfully synthesized. Towards its biomedical applications, biogenic CuONPs proved its excellent efficiency in the free radical scavenging and exhibited the pronounced toxicity against pathogenic microbial strains with high degree of synergistic interactions. Results of cytotoxicity suggested the therapeutic use of CuONPs in the treatment of inflammatory diseases and human lung cancer. In addition CuONPs exhibited its convenient utilization for the efficient photocatalytic degradation of the carcinogenic methylene blue dye that poses serious threat to human health. However, for the advanced CuONPs biomedical applications, more extensive research is still required to reduce its toxicity while improving its biological efficiency. The applications of CuONPs in the field of sensors, food packaging and antimicrobial action will give its future applications by taking in consideration the safety aspects as a serious concern. The findings of the present study may expand the prospects for the biogenic synthesis of CuONPs and their use in a variety of biomedical and biotechnological applications.

Materials and methods


Analytical grade chemicals used were purchased from Sigma-Aldrich (India) or Merck (India). All culture media were purchased from Hi Media (India). All the buffers and reagents used were prepared in double-distilled water (DDW). Glassware were thoroughly washed and air-dried before use.

Test organism and growth conditions

Cyanobacterial strain Phormidium sp. (NCCU-104) was procured from IARI, New Delhi, India. Axenic culture was routinely grown in BG-11 liquid medium in culture room at 30 ± 2 °C and illuminated with cool white Sylvania 40W T12 fluorescent lamps having a lux intensity of 2000 ± 200 for 12 h light/dark cycles80. Cyanobacteria were harvested in the mid-exponential growth phase for extract preparation. For antibacterial study, Klebsiella pneumoniae (KJ 938546), Staphylococcus aureus (MCC 2708), Bacillus cereus (MCC 2243), and Escherichia coli (MCC 2052) were received from the microbial culture collection of the National Centre for Cell Science (NCCS), Pune, India. Clinical isolates of Candida albicans (MCC 1151) and Candida glabrata (MCC 1432) were obtained from National Fungal Culture Collection of India (NFCCI), Pune, India. For anticancer study A549 and H1299 (Human NSCLC cell lines) were obtained from the National Centre for Cell Science (NCCS) Pune, India and maintained in DMEM (Cat # 10569010, Gibco, Waltham, MA, USA) and incubated at 37 °C and an atmosphere of 5% CO2.

Preparation of cell extract and its characterization

Thoroughly washed biomass pellet were suspended in 100 ml of sterile water and sonicated in a Branson Sonifier 450 (Branson Ultrasonics Corp., USA) for 3–5 min at maximum output and duty cycles to ensure complete breakage of the cells. If the extract showed the presence of filaments/cells, steps of sonication was repeated. The resulting extracts were centrifuged at 10,000 g for 30 min, filtered through Whatman No. 1 filter paper and stored in refrigerator at 4 °C for further use as cell extract18. The chemical profile of cyanobacterial extract was analysed using GC–MS to find out the probable compounds that have reducing potential and aided the synthesis of nanoparticles. Samples for GC–MS analysis were prepared by dissolving the dried extract in methanol. The GC–MS analysis was done by Shimadzu GC–MS QP 2010 Plus equipment in electron ionization (EI) mode fitted with a RTX-5 capillary column (60 m × 0.25 mm × 0.25 μm). Helium was used as carrier gas with 0.7 ml min–1of flow rate. The temperature of the injector was fixed at 260 °C. The initial and final temperature of the column was 80 °C and to 280 °C at the rate of 10 °C min–1 and 15 °C min–1 respectively. A 3.5 min solvent delay was used. Mass spectra were recorded under scan mode in the range of 40–650 m/z. Compounds were identified by comparing with NIST11/ WILEY library81.

Biosynthesis of copper oxide nanoparticles

For the extracellular synthesis of CuONPs, in 10 ml of algal extract, added 90 ml of copper sulphate solution (0.1 M). Together with this, algal extract (without copper sulphate) and copper sulphate solution alone were taken separately in two flasks to serve as positive and negative controls. The reaction mixture was kept under vigorous stirring at 90 °C, with gradual addition of 5 ml of 0.1 M (NaOH) the deep blue solution gradually turned to brick red coloration which changed to dark after vigorous stirring for 3 h. The black solid product obtained was centrifuged thrice at 15,000 rpm for 20 min after thoroughly washed with distilled water and 90% ethanol successively to remove any impurities. The resulting solid precipitate was oven-dried, crushed into powder, and stored in airtight container for further analysis48,49. Biosynthesized CuONPs were analyzed by UV–Vis spectrophotometer in 200–500 nm range (Labtronics LT 2900).

Physiochemical characterization of Phormidium derived CuONPs

Biosynthesized CuONPs were characterized for their physicochemical properties with the help of different physical techniques like UV–Vis Spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), and Dynamic light scattering (DLS), Scanning Electron Microscopy (SEM) with EDX, Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and Zeta potential analysis. Biosynthesized CuONPs were analyzed using a Double-beam UV–Vis spectrophotometer (Labtronics LT 2900). The CuONPs suspension was centrifuged at 10,000 rpm/15 min and air dried sample analysis was recorded on the FT-IR spectrophotometer (Perkin Elmer, USA) in the range 500–4000 cm−1 at a resolution of 4 cm−182. XRD (Rigaku X-ray diffractometer, Ultima IV, Japan) of CuONPs was recorded at 2θ in the range from 20° to 80° at 40 kV/30 mA with CuKa radiation (k ¼ 1.54056°A), and the crystalline domain diameter of CuONPs was calculated from the XRD peaks by the Debye–Scherrer equation:

$$D=\frac{0.94\lambda }{\beta cos\theta }$$

where, D is the crystalline domain diameter of ZnONPs, “λ” is the wavelength of the X-ray source used (0.1541), and “β” is the full width at half maximum (FWHM) of the diffraction peak and “θ” is the Bragg’s angle. Dynamic Light Scattering (DLS) (Malvern, UK) was used to determine hydrodynamic size and polydispersity of nanoparticles. Size distributions of synthesized CuONPs were measured using a Nano Zetasizer system (Zeta sizer, Malvern, UK). Before measuring the sample were passed through a 0.2 μm polyvinylidene fluoride (PVDF) membrane with the parameters used such as measurement temperature (25 °C), medium viscosity, and material refractive index (1.59)18. The size of CuONPs was determined by SEM (Carl Zeiss, USA) equipped with EDS, and TEM. For SEM analysis (Sigma version 5.05 Zeiss, USA), thin films of the CuONPs were prepared by dropping an exceedingly small amount of the sample on the silicon wafer at an accelerating voltage of 20 kV with time ranging from 60 to 100 s82,83. The scientific instrument Sirius SD model (the EDX spectrometer-equipped SEM) was used to determine the elemental analysis of the CuONPs. TEM analysis was done on (Philips EM-410LS, JEOL, Japan) using 10 μl of the CuONPs solution kept on the carbon-coated copper grids, making a thin film on the grid and kept in a grid box, sequentially82. Selected portion of the images were observed using Image J and evaluated to obtain the size distribution histogram. The surface topology of synthesized ZnO NPs was obtained by AFM analysis. A thin film of CuONPs deposited on silica glass plate by dropping few drops of the CuONPs solution on the plate and then allowed to dry at 30 °C for overnight. Images were taken, with AFM (Model Ntegra Prima AFM, NT-MDT, Russia).

Biological characterization of Phormidium sp. derived CuONPs

In vitro antioxidant potential of CuONPs

The standard protocols were adopted for determining antioxidant activity like; 2, 2′ -azino-bis 3-ethyl benzthiazoline-6-sulfonic acid (ABTS)84, 2,2′-diphenyl-1-picrylhydrazyl (DPPH)85, hydrogen peroxide (H2O2)86 and superoxide radical (SOR) scavenging assays87. These methods are based on inhibition of free radical, which vary greatly according to the generation of free radical, its reproducibility, and the endpoint. Further, their potential was compared with standard ascorbic acid. Suspensions of CuONPs were sonicated to avoid NPs agglomeration (details given in ESI Section S1). The absorbance was measured spectrophotometrically against the corresponding blank solutions and the percentage inhibition of free radical scavenging was calculated using following equation18.

$${\text{\% Inhibition}}/{\text{Scavenging}} = \frac{Ac - At}{{Ac}} \times 100$$

where where Ac = Absorbance of control, At = Absorbance of test. The IC50 values of CuONPs were calculated and compared with the standard ascorbic acid.

Determination of antimicrobial and synergistic activity of CuONPs

To evaluate the antibacterial efficacy of biologically synthesized CuONPs, broth microdilution method as recommended by CLSI88 was performed against Gram-negative (Escherichia coli, Klebsiella pneumonia) as well as Gram-positive (Bacillus cereus, Staphylococcus aureus) bacteria. Antifungal activity was also determined against Candida albicans and Candida glabrata following standard guidelines of CLSI89. Different concentration of CuONPs (0.24–1000 µg ml−1), streptomycin and fluconazole as positive control, were placed into 96-well plate in a final volume of 100 µl. The test pathogens were harvested and their turbidity was assessed according to the McFarland 0.5 standard. Then, 100 µl of cell cultures (approximately 2.5 103 cells per ml) were placed into the 96-well microtitre plate (Tarson) and incubated at 37 °C for 24 h. After incubation, the growth/turbidity was recorded at 600 nm using a spectrophotometer18. The lowest concentration of CuONPs at which no visible growth occurred represented its MIC (Minimum Inhibitory Concentration) value. All experiments were concurrently performed in triplicates.

The antimicrobial synergistic activities of CuONPs in combination with the standard antibiotic/fungicides were evaluated by the checkerboard assay90. A microtitre plate was inoculated with 50 µl of CuONPs (0.48–125 µg ml−1) and standard antibiotic/fungicides (0.24–31.2 µg ml−1). Each well was inoculated with 100 µl of microbial suspension to make up the final volume 200 µl and checkerboard plates were incubated overnight at 37 °C. The fractional inhibitory indexes (FIC) were calculated according to the Eq. (11).

$${\text{FIC}}_{{{\text{index}}}} = \frac{{\text{MIC of test sample in combination}}}{{\text{MIC of test sample alone}}}{ } + \frac{{{\text{MIC of antibiotic}}/{\text{antifungal in combination}}}}{{{\text{MIC of antibiotic}}/{\text{antifungal alone}}}}$$

where synergy and antagonism were defined by FICI ≤ and > 4, respectively. Synergy was defined by FICI < 0.5, partially synergistic were defined by 0.5 < FICI < 1, whereas indifferent was defined by FICI ≤ 491.

Anti-inflammatory activity of biogenic CuONPs

Inhibition of albumin denaturation is used to analyze the anti-inflammatory activity of CuONPs by the modified method of Lavanya et al.92. The control solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (1% w/v aqueous solution) and 0.05 ml of distilled water. The test sample (0.5 ml) consists of 0.45 ml of distilled water and 0.05 ml of CuONPs (25, 50, 75, 100, 125, 150 µg ml−1). The standard solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (1% w/v aqueous solution) and 0.05 ml of various concentrations of diclofenac sodium. All the above solutions were adjusted to pH 6.3 using 1N HCl. Firstly, the samples were incubated at 37 °C for 20 min followed by the additional incubation at 57 °C for 5 min. After being cooled, 2.5 ml of phosphate buffer was added to the above prepared solutions. The absorbance was measured using a UV–Visible spectrophotometer at 416 nm. The control represents 100% protein denaturation and the results were compared with diclofenac sodium and percentage inhibition of protein denaturation was calculated using Eq. (12).

$${\text{\% Inhibition}} = 100 - \frac{{{\text{Optical density of control}} - {\text{optical density of test sample}}}}{{\text{Optical density of control}}} \times 100$$

Biocompatibility assay of CuONPs

Biocompatibility of CuONPs derived from Phormidium sp. was carried out quantitatively by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and qualitatively by 4′,6-diamidino-2-phenylindole (DAPI) staining using the method of Mossman93 and Liu et al.94. Briefly, 1 × 104 ml−1 cell in their exponential growth phase were seeded in 96 well plates with Dulbecco’s modified eagle’s media (DMEM) along with 5% (v/v) Fetal Bovine Serum (FBS) and 1% antibiotics and antimycotic solution (Gibco). Incubated for 24 h at 37 °C in a humidification incubator with 5% CO2. After incubation the cells were treated with different conc. of CuONPs (20, 40 60, 80, 100, 120 and 140 µg ml-1), incubated for 24 h and washed with PBS thrice. After 24 h of incubation, in each well added MTT dye (5 mg/ml) was mixed with DMEM complete media in 9:1 ratio and again incubated at 37 °C for 3–4 h. Then, media was removed and 100 µl of DMSO was added in each well. The plates were analyzed in a microplate reader (BIO-RAD microplate reader-550) at 595 nm and percentage viability was calculated using Eq. (13).

$${\text{\% of viable cells}} = { }\frac{{{\text{A}}_{{{\text{T }} - }} {\text{ A}}_{{\text{B}}} }}{{{\text{A}}_{{\text{C}}} { } - {\text{ A}}_{{\text{B}}} }}{ } \times 100$$

where AC = absorbance of the control: AT = absorbance of the treated cells, AB = absorbance of the blank. IC50 values of CuONPs were determined for each cell line.

Cells were stained with fluorescent 4',6-diamidino-2-phenylindole (DAPI) to detect nuclear condensation and blebbing. After 48 h of treatment, the cells in a 12 well plate were washed with PBS thrice. Cells were then fixed with 4% paraformaldehyde for 8 min and then washed with PBS thrice. After that, the cells were permeabilized by treating with 0.1% Triton X-100 for 2 min. Again washed 3 times with PBS and treated with fluorescent dye DAPI 1 μg/ml. After 5 min PBS was added to the well to keep the cells hydrated while imaging under fluorescent microscope ZOE Fluorescent Cell Imager, (Bio-Rad)17.

Photocatalytic degradation by CuONPs

The CuONPs derived from Phormidium sp. were also evaluated for degradation of methylene blue (MB) dye. For this 5, 10, 15, 20 and 25 mg/L of CuONPs were added to 100 ml of aqueous solution containing 25 mg/L of MB dye. Prior to the irradiation (500 W xenon lamp), the suspensions were magnetically stirred for 1 h in the dark to reach an adsorption/desorption equilibrium between CuONPs and the dye solution. The suspension mixtures were withdrawn at regular interval and centrifuged. In order to determine the remaining dye concentration the absorption spectra were recorded at 665 nm for (MB) using UV–Vis spectrophotometer77. Another set (control) was allowed to react under identical condition without CuONPs (photocatalyst). The percentage degradation efficiency for photocatalytic reaction was calculated using the Eq. (14).

$${\text{\% Degradation}} = \frac{{{\text{A}}_{{0}} - {\text{A}}}}{{{\text{A}}_{{0}} }} \times 100$$

where A0 and A are the initial and final absorbance of MB dye at λmax, respectively.

Identification of CuONPs induced free radical in dye degredation

Specific free radical scavenger protocols were adopted to identify their role in degradation of methylene blue by CuONPs. Different scavenger were added in the reaction mixture, viz. ammonium oxalate (h+ scavenger), p-benzoquinone (·O2 scavenger) and tert-butanol (·OH scavenger)95. CuONPs (10 mg/L) and radical scavengers (10 mM) were placed into 100 ml of 25 mg/L dye solution along with respective scavengers. The reaction mixtures were placed under sunlight for 1 h. Finally the degradation rate of the dye was calculated to determine the active role of scavenger species.

Statistical analysis

Data analysis was carried out using standard statistical software Origin 8.1. Descriptive statistical measures, especially the mean and standard deviation, were used to summarize the collection of data for each measurement. Two-way analysis of variance was used to evaluate the influence of independent variables as well as possible interactions between them in the antibacterial and antioxidant activity study. Values of *p < 0.05 were considered as statistically significant. Each test was performed in triplicates and results were presented as mean ± SD.