1 Introduction

In the era of rapid development of nanotechnology, continuous progress in the synthesis and self-assembly of nanomaterials in a controlled and repeatable manner has been a problem. Because of its possible potential applications in various research fields, it has attracted much attention [1, 16]. Copper and copper-based nanoparticles (NPs) are of particular interest due to their several applications such as surface properties for low cost electronic devices, conductive films of researchers [20], lubricants, nanofluids, catalysis and antimicrobial activity, against bacteria, fungi, algae and viruses [1, 22, 26]. These characteristics make them particularly attractive for a broader range of multiple applications [13]. However, high oxidation tendency, extreme sensitivity to air creates difficulty in its stability of copper NPs [7]. Various methods have been accepted for the synthesis of copper NPs but most of them have encountered the formation of mixed phases, complex synthetic strategies, and structural formation control is poor [4, 19]. But the laser ablation technique is simpler, with high purity and faster method of NPs synthesis than other methods. It involves ablating a solid target placed in a liquid environment to produce a NP collected as a colloidal dispersion. Laser ablation has the several advantages such as short reaction time, mild temperature conditions, no chemical precursors and by-products, high purity, and one-step synthesis route [14, 18]. In addition, many different types of NPs can be produced from metal, semiconductor and polymer nanostructures. Laser parameters such as wavelength, pulse repetition rate, pulse width and pulse energy affect and control the morphology and properties of the resulting NPs, including shape, size and distribution [8, 24]. Therefore this physical technique can be used for the purpose of environment friendly methods of nanomaterial synthesis. Cuprous oxide (Cu2O) is one of the most stable copper oxides, because of its behavior similar to that of the P-type semiconductor [11], the theoretical direct band gap is 2.2 eV, and so the researchers’ attention can be utilized (theoretical efficiency ~ 18%) [21, 23]. In this work, we have studied the synthesis of copper and its oxide nanomaterials by the pulse laser ablation (PLA) method and their characterization by XRD, UV–VIS and ATR-FTIR spectroscopy. The antibacterial test of prepared Cu2O and CuO NPs were performed against S. aureus, MIC and MBC value was also evaluated.

2 Experimental sections

2.1 Materials

The copper target (99.9% purity) was purchased from Johnson Matthey (spec-pure), analytical grade double distilled water, polyethylene glycol 400 (PEG) from Merck and Mueller–Hinton Agar (MHA) and Muller Hinton broth (Himedia Pvt. Ltd) was used for the antibacterial assay.

2.2 Laser ablation technique for synthesis of NPs

Laser ablation of a metal target immersed in 20 mL of distilled water and other 20 mL (18 mL of distilled water and 2 mL of PEG) was carried out at room temperature and atmospheric pressure (~ 760 mm Hg). The copper target was illuminated with a focused Nd:YAG laser, which provided a 10 ns pulse at a wavelength of 1064 nm with a maximum pulse energy of 40 mJ. The laser beam was focused vertically downward on the surface of the metal target using a dichroic mirror and focused using a convex quartz lens of 30 cm focal length, as setup shown in Fig. 1. The container and target were translated in a horizontal plane to evenly remove material and avoid surface pit formation [15].

Fig. 1
figure 1

Experimental setup of PLA used for the synthesis of copper oxides NPs

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3 Instrumentation

The UV–VIS absorption spectra of the synthesized NPs colloidal solution were recorded between spectral region 300–800 nm using a Perkin Elmer Lambda 35 dual beam spectrophotometer. The Cu2O and CuO NPs as well as PEG capping species were confirmed by infrared (IR) absorption spectra, which were recorded using FTIR spectrometer (ABB, Bomem Inc.) equipped with ATR unit in the spectral region 500–4000 cm−1 at a resolution of 4 cm−1. The X-ray diffraction (XRD) was performed using Proto A-XRD diffractometer equipped with CuKα (λ = 1.54 Å) radiation. However, the crystalline size of copper oxides NPs were calculated as earlier [1, 15]. The samples were scanned over a 2θ range of 200–800 with a step size of 0.060. In order to study PEG interaction on the surface of copper NPs, high-resolution transmission electron microscope (Tecnai G2-20, FEI Company, Netherland) operating at 200 kV was used for the size and shape measurements of the prepared copper NPs. Samples for the transmission electron microscopy were prepared by keeping a drop of colloidal solution on the carbon-coated copper grid and drying under the IR lamp. However the synthesized copper concentration was analyzed using Inductively Coupled Plasma Mass Spectrometer (ICP-MS) (Thermo Fisher Scientific, Germany).

3.1 Bactericidal test

The Kirby–Bauer’s disc diffusion assay of synthesized colloidal oxide of copper NPs (with PEG and without PEG) was performed to determine zone of inhibition. All experimental protocols were performed under adequate ascetic measures. For the disc diffusion method, approximately 5 × 106 CFU ml−1, 100 μl of S. aureus suspension spread using sterilized cotton swabs over Mueller–Hinton agar plate. The disc (20 µL capacity) was first dipped into the colloidal solution of copper NPs, followed by putting disc onto agar plate having uniform lawn of S. aureus suspension. The solution gradually defused spread to adjacent bacterial lawns and inhibited the growth of bacteria around the disc, which was then measured as antibacterial nature of the prepared NPs. The S. aureus culture plate was then incubated to overnight for 37 °C. The circular zone of bacterial growth inhibition/clear zone was expressed in terms of the average diameter of the zone of inhibition in millimetres [2].

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of synthesized Cu2O and CuO were examined using procedure specified by [1]. The effect of copper NPs on S. aureus bacterial and growth kinetics was studied using optical density measurements at 600 nm of inoculated bacteria. The optical absorbance of control (copper NPs absent from inoculated bacteria) and test samples were measured for the period of 0–48 h. The changes in optical density over time were used to produce growth curves at different concentration of CuO and Cu2O NPs against control. All the experiments were carried out in triplicate.

4 Results and discussion

4.1 UV–Vis spectroscopy

The UV–VIS absorption spectra of the synthesized two different colloidal copper oxides NPs in pure water and with 10% PEG were recorded within the wavelength region of 200–800 nm. The recorded spectra are represented in Fig. 2a. This spectrum showed a peak at wavelength of ~ 270 nm followed by another peak with cooperatively low intensity at ~ 538 nm. The peak at 270 nm is attributed to the characteristic Brillouin transition of Cu2O [3, 6] and the surface plasmon resonance (SPR) peak at 538 nm of copper shows that the collective oscillation of the conducting electrons on the surface of the nanosized particle absorbs visible electromagnetic waves which confirms copper particles are nano-sized [5]. Another absorption peak recorded at a wavelength of 640 nm found responsible for CuO-NPs species. The colloidal CuO-NPs are consistent with the peak reported in the 590–640 nm spectral regions [5, 18]. It shows that the formation of a copper oxide nanocolloid produces peak at about 640 nm without a PEG medium. The simultaneous presence of spectral peaks at wavelengths of 270 nm and 640 nm strongly supports the presence of CuO NPs. In case of PEG assisted synthesis, the small absorption humps were seen around 230–270 nm and the same time another absorption SPR peak at 538 nm which confirms the presence of Cu2O and Cu NPs. However, PEG exhibited capability to restrict the oxidation of copper NPs during the PEG-mediated copper target ablation, therefore initially trace amount of Cu2O was observed. It might be possible to form a Cu NPs core shell surrounded by a thin layer of Cu2O NPs whose outer surface continuously interacts with dissolved oxygen in the aqueous medium and after passing few days, thin layer of Cu2O might grow and finally CuNPs become converted into pure Cu2ONPs.

Fig. 2
figure 2

a Recorded UV–VIS Cu2O and CuO and b corresponding dln(αhν)/d(hν) curve of synthesized colloidal oxide of CuO-NPs

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The optical band gap of synthesized CuO-NPs was estimated to employing absorption data, the absorption coefficient, (α) of the colloidal solution of CuO-NPs under the Beer’s law, is related to its band gap energy by:

$$ \upalpha = {\text{A}}\left( {{\text{h}}{\upnu } - {\text{Eg}}} \right)^{\text{n}} /{\text{h}}{\upnu} $$
(1)

where A is a constant, Eg is the band gap of material, and the exponent n may have the values 1/2, 2, 3/2, and 3 corresponding to allowed direct, allowed indirect, forbidden direct, and forbidden indirect semiconductor, respectively.

The region of fundamental absorption, which corresponds to the electronic transition from top of the valance band to the bottom of conduction band, can be utilized to determine the band gap energy of the material using above relation. The hν derivative of ln(αhν) = ln nA(hν − Eg) makes following expression

$$ \frac{{{\text{D}}\left\{ {\ln \left( {{\upalpha }{\text{h}}{\upnu }} \right)} \right\}}}{{d\left( {{\text{h}}{\upnu }} \right)}} = \frac{\text{n}}{{\left( {{\text{h}}{\upnu} - {\text{Eg}}} \right)}} $$
(2)

The plot of d{ln(αhν)}/d(hν) versus hν shows a divergence at energy equal to the band gap Eg, corresponding to the electronic transition as displayed in Fig. 2b. This plot suffers comparatively less error in the band gap determination as compared to the Tauc plot. Band gap of the CuO-NPs obtained by LA-PLA of copper in distilled water has band gap energy of 3.22 eV.

4.2 X-ray diffraction

The X-ray diffraction pattern of the PEG mediated laser ablated samples were recorded and analyzed after 15 days of sample preparation. The peak positions with 2 theta values of 12.0°, 14.7°, 17.03°, 24.1° and 28.34° were indexed as (110), (111), (200), (220) and (222) planes, which are in good agreement with those of powder Cu2O NPs obtained from the International Center of Diffraction Data card (JCPDS File No. 03-0898) confirming the formation of a crystalline cubic phase of Cu2O with a cuprite structure shown in Fig. 3 [10]. There was no observed any additional diffraction peaks showing its high purity. The average crystalline size of 15 nm and cubic cuprous oxides (Cu2O) were calculated from XRD peaks using Debye–Scherrer’s formula. It was clearly observed that the initially prepared copper NPs slowly oxidized and were converted in the form of cuprous oxide.

Fig. 3
figure 3

X-ray diffraction of Cu2O NPs prepared in the presence of PEG

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4.3 Transmission electron microscopy

TEM is well known technique for imaging solid materials at the level of atomic resolution. This technique was used visualize the size and shape of the oxides of copper NPs. The small size clusters of nanorods of PEG assisted copper NPs are shown in Fig. 4a, b. The TEM micrograph showed most of the copper NPs are rod shaped with the length ranges between 30 and 50 nm and width in range of 8–15 nm. As prepared CuO NPs were visualized round shape particles through TEM micrograph, with average size range of 22 nm It can be attributed that the small widths of nanorods could directly be penetrated to the cell wall of bacteria, which may disrupt the function of bacterial cell that leads toxic effect to the bacteria, whereas, larger size of CuO may be less effective due to slower penetration into the bacterial cells.

Fig. 4
figure 4

TEM micrograph of a Cu2O NPs prepared in the presence of PEG b CuO NPs prepared absence of PEG

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4.4 ATR-FTIR

ATR-FTIR spectrum of colloidal sample of copper oxides were recorded in the spectral region 400–4000 cm−1 and it has been represented in Fig. 5. The three characteristic bands were observed at 432 cm−1, 497 cm−1, and 613 cm−1 which were assigned to the Au mode, Bu mode, and other Bu mode of CuO. Similar observation were also in accordance to the findings of Kliche and Popovic [9]. In case of without PEG the IR absorptions at around 440 cm−1, 497 cm−1 and 530 cm−1 can be attributed to the lattice vibration of the oxide of copper, confirming the formation of pure CuO-NPs present as thin layer [25]. The similar study done by Dang et al. [5] states that the possibility of electrostatic interaction through ester bond of PEG to copper leads to very slow oxidation of copper NPs. The ester bond characteristics were found at wave-number around 1090 cm−1 with of PEG assisted Cu2O synthesis which is in favor of the observations reported by Dang et al. [5].

Fig. 5
figure 5

Recorded ATR-FTIR spectra of the synthesized colloidal oxide of copper NPs

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5 Antibacterial

5.1 Disc diffusion

The synthesized Cu2O and CuO were further tested against S. aureus Gram-positive bacteria as the research object using Kirby–Bauer’s standard disc diffusion method. As the, Gram positive bacteria composed of thick peptidoglycan layer with rigid structure leads slower penetration of NPs in comparison to Gram-negative bacteria having thin layer of peptidoglycan, which allows comparatively faster penetration of NPs [12]. The discs containing 20 μl of Cu2O and CuO NPs with concentration 35 µg/mL were placed over the bacterial lawn and incubated for overnight. Discs soaked with both Cu2O and CuO types of NPs 35 ± 0.86 µg/mL remarkably inhibited the growth of test bacteria. The highest zone of inhibition 10 mm was recorded with Cu2O NPs, which was followed by 7 mm with CuO NPs after 24 h of incubation. The diffusion of both NPs was clearly visible with light blackish colour making inhibitory zone. The significant zone of inhibition was observed ~ 7 mm and 10 mm around the disc containing CuO and Cu2O respectively (Fig. 6).

Fig. 6
figure 6

Disc diffusion essay of a control, b Cu2O NPs and c CuO NPs synthesized by LAPLA

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5.2 Bacterial growth curve

Dose dependent growth kinetics of S. aureus was variably influenced by the treatments with variable concentrations of CuO and Cu2O NPs which has been illustrated in Fig. 7a, b. Bacterial growth was reduced with increasing concentration of both types of copper oxide NPs. The introduction of Cu2O NPs remarkably affected the growth kinetics of S. aureus strain as compared to the negative control (culture grown in absence of copper NPs). At their respective MBC values, there was no visible bacterial growth observed up to the time of 28 h, that represents the bactericidal concentration for the S. aureus strain. The initial concentration 20, 40 and 80 μl/mL Cu2O NPs were responsible for the 12%, 25% and 85% reduction of S. aureus density as compared to control sets respectively. Further, increasing concentration of Cu2O NPs at the level of 120 μl/mL and 140 μl/mL caused complete absence of bacterial growth as these concentrations represent MIC and MBC values respectively. Similarly, the initial concentration of CuO 20 40 and 80 μl/mL leads to reduce the growth of same bacteria up to 10%, 18% and 70% respectively. Whenever, these CuO concentrations increased up to the level of 130 μl/mL and 150 μl/mL resulted as the MIC and MBC values respectively. The above result is the good agreement with Surapaneni et al. [17]. They suggested that the intracellular protein have high affinity towards Cu2O in compare to CuO. In other hand, the function of inhibitory and bactericidal nature of both copper oxides NPs might be due to smaller width of rod shaped NPs. The unique rod shaped Cu2O NPs have influenced and the facilitated the penetration of NPs into the bacterial cell wall. Such phenomenon of rapid penetration of rod shaped NPs leads to disrupt cellular integrity might have to contribute in ROS generation and protein binding. These actions can cause improper metabolism and replication of their genetic materials consequently, cell death.

Fig. 7
figure 7

Growth profile of S. aureus bacterial strain in presence of varying concentrations of a CuO NPs and b Cu2O NPs

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6 Conclusion

The UV–VIS absorption spectra carried out without PEG and PEG have clearly indicated the formation of Cu2O NPs and CuO NPs. Initially copper NPs slowly oxidize and convert into stable cuprous oxide NPs have high purity, smaller in size than synthetic oxides due to the continuous capping of ablated copper in presence of PEG. On the other hand, in the absence of PEG CuO-NPs was formed due to the natural oxidation of the dissolved oxygen in medium. The comparable growth kinetics of S. aureus bacteria were also studied in the presence of CuO and Cu2O NPs. The small size, rod shape Cu2O NPs have remarkable bactericidal capacity (MIC 120 μl/mL and MBC 140 μl/mL) against S. aureus bacteria. These remarkable antibacterial properties of synthesized stable Cu2O NPs can be employed in the field of clinical, medical and environment.