CuO, CuWO4 and materials of copper and tungsten were prepared through simple wet chemical route. X-ray diffraction (XRD) results showed the formation of two predominant crystalline phases: CuO and CuWO4. Specifically, the materials of copper and tungsten exhibited those crystalline phases in different concentrations for each oxide. A first semi-quantitative analysis allowed determine most of crystalline parameters that characterize the samples. Complementary, Fourier transform infrared spectroscopy (FTIR) analysis allowed to verify the crystalline phases formation because any vibration band associate at other compound was detected. In terms of morphological properties, two different morphologies were observed: polyhedral grains associated to CuO and nanoparticles linked to CuWO4, fact that confirm the heterostructure formation. These properties were supplemented with the estimation of surface parameters and parameters of hydrodynamic stability, which induce the principle of application of the heterostructures obtained in biological applications. Then, the bactericide response was evaluated. 100:1 and 100:2 materials showed an outstanding bactericide response, due to similar structural, morphological and superficial properties, and the fact to Cu2O secondary phase was detected at a lower percentage. In this investigation the capacity of CuO/CuWO4 heterostructures in bactericide activity against Gram negative bacteria was verified.
Los óxidos CuO y CuWO4, y materiales de cobre y tungsteno se prepararon mediante un método de vía húmeda sencillo. Los resultados de difracción de rayos X mostraron la formación de dos fases cristalinas predominantes: CuO y CuWO4. Específicamente, los materiales de cobre y tungsteno exhibieron estas fases cristalinas en diferentes concentraciones para cada óxido. Un primer análisis semicuantitativo permitió determinar la mayoría de los parámetros cristalinos que caracterizan las muestras. Complementariamente, el análisis de espectroscopía infrarroja por transformada de Fourier permitió verificar la formación de las fases cristalinas, ya que no se detectó ninguna banda de vibración asociada a otro compuesto. En cuanto a las propiedades morfológicas, se observaron dos morfologías diferentes: granos poliédricos asociados al CuO y nanopartículas ligadas al CuWO4, lo que confirma la formación de la heteroestructura. Estas propiedades se complementaron con la estimación de parámetros de superficie y de estabilidad hidrodinámica, que inducen el principio de aplicación de las heteroestructuras obtenidas en aplicaciones biológicas. Posteriormente, se evaluó la respuesta bactericida; los materiales 100:1 y 100:2 mostraron una respuesta bactericida destacada, debido a sus propiedades estructurales, morfológicas y superficiales, y al hecho de que la fase secundaria Cu2O fue detectada a un porcentaje más bajo. En esta investigación se verificó la respuesta de las heteroestructuras CuO/CuWO4 en la actividad bactericida frente a bacterias Gran negativas.
Several investigations have shown the presence of microbiological contaminants in drinking water sources [1]. Since, as a consequence of the presence of microbial strains, living beings have developed resistance to various drugs, so that persistent diseases and infections have developed in the environment, becoming an emergency of worldwide interest [2]. In this context, the research and development of new inorganic antimicrobial materials is an efficient alternative to control bacterial growth. Metal oxides with high surface area, low particle size, and multiple morphologies have presented optimal structural, microstructural, optical, and electrical properties to inhibit bacterial growth [3]. Specifically regarding to the particle size, this has a direct function in the permeability of the bacteria, because the surface-to-volume ratio increments when the particle size is reduced toward the -nano-size therefore, size effects related to nanoparticles become more notorious. For example, ZnO [4], TiO2[5], and SnO2[6] nanoparticles, among others, are semiconductors, inexpensive, non-toxic, and abundant, with diverse applications such as gas sensors, photocatalysts, memory storage devices and photovoltaic devices, which due to their nano size have the ability to easily penetrate the cell wall of pathogenic bacteria, becoming an influential factor in elevating bactericidal activity [7].
Copper oxide (CuO) is a p-type semiconductor with narrow bandgap of 1.7eV and monoclinic crystalline phase [8]. CuO nanoparticles are attractive due to their simplicity and stability, which encourages the development of potential applications that directly depend on the synthesis methods and their various morphologies. Metal-based CuO nanostructures have been synthesized from inexpensive methods. Among which is the hydrothermal method, by which anisotropic morphologies similar to rectangular nano-bars with high crystalline quality were obtained improving their properties [9]. Likewise, the presence of surfactants drastically influences the CuO formation process, for example, microwave and sonochemical assisted synthesis form nano-sheets and nano-discs, respectively, while by the combustion method, disordered and porous flakes are obtained; For this reason, the influence of the synthesis method on the behavior of the electrical properties (conductivity and capacitance) and bactericidal activity on bacterial populations, such as Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, among others, is established [10]. Similarly, CuO nanoparticles from green synthesis has been a well-studied option, as it is environmentally friendly, highly efficient, reliable and low cost [8]. However, this alternative has encountered the challenge of manipulating and controlling its nanometric size, high purity, morphology and crystallinity therefore, multiple biological compounds have been used as substitutes for chemical reagents that act as coating and reducing agents; for example: fruits, agricultural waste, plant extracts, algae, coffee powder, among others [11]. In general, these nanoparticles have been of high interest due to their applications in the field of biomedicine, among which are treatments of bacterial and viral diseases [12], besides being promising in the process of – in vitro photothermolysis – of cancer cells in the development of nano-therapeutics against cancer [13].
Copper tungstate (CuWO4) is an n-type semiconductor with a bandgap around 2.2eV and triclinic crystalline phase, this ternary oxide has excellent optical, catalytic [14], photoluminescent [15], and multiferroic [16] properties, in addition to its stability and low production cost. These properties have generated high interest due to their use in various applications such as photoanode in electrochemical devices [17], gas sensors [18], and in photoelectrochemical and photocatalytic water splitting [19]. Regarding environmental remediation, it presents optimal properties in the degradation of organic pollutants, specifically the elimination of residual drugs in water sources has been evaluated, due to the fact that its presence generates pharmacoresistance greater to various bacterial strains [20]. CuWO4 can be used as inorganic antibacterial material because, under visible light irradiation, it generates electron-hole pairs, which generate redox reactions to produce reactive oxygen species (ROS); these ROS are in charge of attack bacterial cells and achieving bactericide effects. On the other hand, the combination of CuWO4 with polymeric materials has produced alternative systems to evaluate antibacterial treatment and dye degradation. Despite these studies, reports in the literature investigating the bactericide response of this semiconductor in the absence of external light sources are scarce.
A heterostructured system is the union of two or more semiconductors, whose capacity is to improve their properties compared to individual semiconductors [21]. For the CuO/CuWO4 heterostructure, an increase in photocatalytic and photoelectrochemical properties has been presented, which have been employed for the degradation of dyes and colorants under UV/visible light irradiation [22]. Similarly, its bactericidal activity synthesized by the hydrothermal calcination method has been evaluated, where an increase in the inhibition zone on E. coli and Puccinia graminis populations was evidenced for this heterostructure, compared to CuO nanoparticles. This result was attributed to the increase in the surface area of the heterostructure [23].
The interest in obtaining the CuO/CuWO4 heterostructure lies in its enhanced photocatalytic properties, due to it can efficiently absorb UV radiation compared to CuO and CuWO4 oxides. These properties are related to its bactericide performance, leading to an examination of their multifunctional response to environmental problems. Taking into account the multiple properties of CuO and CuWO4 individual semiconductors and the efficiency of the heterostructured systems, in this work the CuO/CuWO4 heterostructure was synthesized from a wet chemical route, where the structural, morphological and surface properties were evaluated. Additionally, its bactericidal applicability was studied in (a) P. aeruginosa, (b) E. coli, and (c) Salmonella enteritidis strains, evidencing slight increase in the inhibition zones for heterostructured systems.
Materials and methodsSynthesis of CuO/CuWO4 heterostructuresThe samples were obtained by a soft chemical route. Copper (II) nitrate 3-hydrate pure (Cu(NO3)2·3H2O 98%,) was entirely solubilized in ethanol (C2H5OH 96%, Merck, Germany) at 1M. Ammonium paratungstate ((NH4)10H2(W2O7)6 99%) (APT) was entirely solubilized in distilled water at 0.02M. Passing 6h, both solutions were mixed, and the pH value was established in 2. This final solution was evaporated for 30min at 50°C until a gel was formed. Afterwards a thermic process (200°C – 4h) in oven was developed to stabilize the gel. Then, the resulting material was ground in an Agata mortar. Finally, the final powders were crystallized at 300°C – 2h. Specifically, in the 100:1, 100:2, 100:3, 100:4, 100:5, and 100:6 materials the moles relationship between copper and tungsten is given by the concentration of cooper precursor – concentration of tungsten precursor. CuO was synthesized following the same methodology without adding ammonium paratungstate. CuWO4 was obtained adding the appropriate stoichiometrically quantity of ammonium paratungstate.
Characterization of CuO/CuWO4 heterostructuresStructural and vibrational characterizationTo determine the crystalline phases, present in each material and to obtain experimental data needed to realize a semi-quantitative structural analysis was used the X-ray diffraction (XRD) technique. For this, an X’pert Pro PANalytical diffractometer with a real-time multiple strip (RTMS) detector in Bragg Brentano mode (Cu Kα2 radiation at λ=1.542Å) was used; the 2θ angle range was set between 10° and 80° with a step of 0.0263°. Additionally, the 3D vision image of the unit cell of each of the phases present in the analysis materials was generated using VESTA software.
Crystallite size (L) was determined through the Debye Scherre's equation, Eq. (1). In this expression, the instrumental parameters used were: Scherrer constant K=0.94, and wavelength λ=0.1540598nm. B corresponds to the Full Width at Half Maximum, FWHM, and θ corresponds to Bragg position. In both cases, CuO and CuWO4, the maximum intensity in the diffractogram was the line selected to calculate L. Additional structural parameters [24] also were determined. To calculate the dislocation density, δ, and the microstrain, ɛ, were used the Eqs. (2) and (3), respectively.
Lattice parameters (a, b, c), and the unitary cell volume, V were calculated through Eqs. (4) and (5)[25] for the monoclinic phase of CuO, and Eqs. (6) and (7)[26] for the triclinic phase of CuWO4, using the Miller indices (h k l) of the three most intense peaks for each oxide. In Eq. (6): S11=b2c2sin2 α, S22=a2c2sin2 β, S33=a2b2sin2 γ, S12=abc2(cos α cos β− cos γ), S23=a2bc(cos β cos γ − cos α), and S13=ab2c(cos γ cos α − cos β). In Eq. (7): x=cos2 α+cos 2β+cos2 γ and y=2cos αcosβ cos γ. α, β, and γ values were taken of powder diffraction standards cards.
The verification of the crystalline phases present in the obtained materials was carried out using the Rietveld refinement method through the GSAS software.
Fourier transform infrared spectroscopy was used to confirm the absence or presence of absorption bands on the materials surface. For these measurements, an FTIR Prestige-21Shimadzu spectrometer was used.
Raman spectroscopy measurements were carried out through a Raman equipment with 514nm of excitation wavelength, 100mW of output power, and 20× lens; additionally, an accumulation time of 45s and five accumulations were used.
Morphological and superficial characterizationTo investigate the morphology of the samples and to estimate their grain sizes a Field emission-scanning electronic microcopy (FE-SEM) was utilized. Images were obtained with a ZEISS equipment working at 5kV and 200nA; the samples were deposited on carbon tape and coated with a Pt layer. Grain size analysis was performed using the ImageJ digital image processing software.
The specific surface area (SBET), pore size, and volume size were determined by means of BET analysis. For this, a Micromeritics ASAP 2020 particle size analyzer was used.
Characterization of hydrodynamic stabilityTo evaluate the hydrodynamic stability of the materials obtained was used the dynamic light scattering (DLS) technique. A Zetasizer Nano Series Malver Instruments 2EN3600 equipment at room temperature was used.
Determination of antibacterial activity by the well-diffusion methodBactericidal activities of the synthesized heterostructures were studied using Gram-negative bacteria (P. aeruginosa, E. coli, and S. enteritidis) through the disk diffusion method or antibiogram method. The bacteria were cultured in a mixture of Mueller Hinton Broth culture medium and AGAR as solidifying agent. Bacterial lawn was obtained by spreading culture broth as solid nutrient agar plates. After solidification, a specific volume of the bacteria solution was poured into each plate. After that, one sample of each material was placed in the plates (in triplicate). Afterwards, the plates were taken brought to the stove at 37°C for 24h. After incubation, the inhibition zone size was measured.
Results and discussionStructural analysisXRD patterns of the bulk CuO, CuWO4, and cooper and tungsten compounds are shown in Fig. 1. CuO XRD pattern exhibits characteristic peaks related to the reflection lines of CuO monoclinic structure – Tenorite phase – PDF No. 01-080-0076 (powder diffraction standards), with a space group C2/c. CuWO4 diffractogram shows peaks associated to the reflection lines of CuWO4 triclinic structure PDF No. 01-088-0269. Regarding cooper and tungsten compounds, two principal phases were determined in all patterns: CuO and CuWO4, with the same indexing cards. Specifically: (a) CuO sample: CuO(100%); (b) 100:1 sample: CuO(69%)/CuWO4(31%); (c) 100:2 sample: CuO(57%)/CuWO4(43%); (d) 100:3 sample: CuO(50%)/CuWO4(50%); (e) 100:4 sample: CuO(42%)/CuWO4(58%); (f) 100:5 sample: CuO(37%)/CuWO4(63%); (g) 100:6 sample: CuO(32%)/CuWO4(68%); (h) CuWO4 sample: CuWO4(100%). Hence, it can be seen that the crystalline phase content of CuWO4 increases with the increases of tungsten precursor concentration.
Since, this semi-quantitative structural analysis it is possible to infer that the average width of the peak of greatest intensity of each crystalline phase presents changes in its broadening when changing the concentration of oxides present in the heterostructure. These changes are more noticeable in the 100:3 sample. In particular, in the 100:4, 100:5, and 100:6 heterostructured systems, broadening of the diffraction peak of the CuWO4 phase is identified; this broadening may be evidenced in the reduction of the crystallite size. Therefore, this broadening could be attributed to the possible fluctuations of the atoms in the lattice with respect to the ideal position of the crystalline lattice. These fluctuations are dislocations and/or microdeformations possible of the lattice [27]. In terms of the CuO phase, significant changes in the broadening are not observed, so significant changes should not be found in the crystallite size of these systems. On the contrary, for the 100:1 and 100:2 systems the CuWO4 phase presents phase percentages lower than 50%, the width of the diffraction peak does not present variations so, the crystallite size would remain almost invariant. Hence, a broadening of the CuO phase peak is evident when its concentration is greater than 50%. Then, increasing the concentration of the tungsten precursor for the heterostructured systems, the percentage of CuWO4 crystalline phase increases proportionally, such that when it is higher than 50% the broadening of the predominant diffraction peak is evident, together with its intensity, a trend that can confirm a lower estimate of the crystallite size and a greater crystallinity in the heterostructures [28]. On the other hand, a minimal shift of the most intense diffraction peaks of the CuO phase diffractogram for the 100:5 and 100:6 samples are observed. This behavior can be attributed to the fact that the W ions exchange with the Cu ions in the structure of this oxide, as reported in [25]. However, this behavior does not noticeably affect the heterostructure formation. Finally, the most intense diffraction peak of the pattern obtained for the CuWO4 sample presents a shift that may be due to the formation of this “pure” oxide, thus avoiding the formation of the heterostructure.
Results of structural parameters obtained since semi-quantitative analysis, such as: crystallite size, dislocation density, and microstrain are presented in Fig. 2. Changes in the CuO crystallite size in the presence of CuWO4 are observed (Fig. 2 (a)). An inhibition in the growth of this crystal is detected when the CuO phase percentage decreases in the heterostructured systems. On the other hand, the CuWO4 crystallite size tends to decrease in samples with percentages greater than 50% of CuWO4, promoting thus the increase of the lattice microdeformations; for lower percentages, significant changes in the crystallite size do not occur. Additionally, the microdeformation values indicate the presence of tensile stresses in the presence of the additional phase.
The obtained results for the lattice parameters are presented in Table 1. b parameter of the CuO decreases in the presence of CuWO4 for crystalline phase percentages lower than 50% (100:1 and 100:2 samples), which is attributed to the preferential growth along a crystalline plane which coincides with the inhibition of tensile stress and the crystallite size variation for this oxide (Fig. 2(c)). However, the CuO unit cell volume of does not exhibit considerable changes, so there is not lattice distortion due to the presence of the CuWO4 phase. On the other hand, a and c parameters present strong changes in 100:5 and CuWO4 samples. The first promoted the increase in the unit cell volume, while the second considerably decreased its unit cell volume, as a consequence of changes in the dislocation density of the crystalline lattice (Fig. 2(b)). The lattice parameters determined by this semi-quantitative method are in agreement with the values reported previously [29].
Semi-quantitative structural analysis parameters: inter-planar spacing (d), lattice parameters (a, b, c), and volume of the unit cell (V).
| Sample | Phase | d (Å) | a (Å) | b (Å) | c (Å) | V (Å3) | ||
|---|---|---|---|---|---|---|---|---|
| CuO | CuO | (11−1)2.522 | (200)2.309 | (20−2)1.862 | 4.681 | 3.482 | 5.128 | 81.20 |
| 100:1 | CuO | (002)2.526 | (111)2.322 | (−202)1.866 | 4.688 | 2.425 | 5.123 | 81.11 |
| CuWO4 | (−1−11)3.101 | (111)2.299 | (130)1.864 | 4.663 | 5.832 | 4.902 | 132.09 | |
| 100:2 | CuO | (−111)2.523 | (200)2.310 | (111)2.322 | 4.685 | 2.424 | 5.126 | 81.09 |
| CuWO4 | (−1−11)3.106 | (−111)2.823 | (1−11)2.784 | 4.698 | 5.825 | 4.889 | 132.58 | |
| 100:3 | CuO | (200)2.309 | (−111)2.522 | (111)2.321 | 4.681 | 3.421 | 5.127 | 80.98 |
| CuWO4 | (100)4.667 | (021)2.517 | (002)2.438 | 4.708 | 6.011 | 4.883 | 136.91 | |
| 100:4 | CuO | (−111)2.523 | (111)2.322 | (200)2.310 | 4.685 | 3.424 | 5.126 | 81.09 |
| CuWO4 | (010)5.795 | (100)4.667 | (002)2.439 | 4.708 | 5.843 | 4.883 | 133.09 | |
| 100:5 | CuO | (−111)2.523 | (111)2.322 | (200)2.310 | 4.685 | 3.424 | 5.126 | 81.09 |
| CuWO4 | (1−11)3.111 | (1−11)2.965 | (021)2.517 | 5.138 | 6.054 | 4.459 | 137.47 | |
| 100:6 | CuO | (−111)2.523 | (111)2.322 | (200)2.310 | 4.685 | 3.424 | 5.126 | 81.09 |
| CuWO4 | (100)4.667 | (101)3.306 | (021)2.517 | 4.708 | 6.013 | 4.877 | 136.80 | |
| CuWO4 | CuWO4 | (100)4.667 | (020)2.898 | (111)2.786 | 4.708 | 5.843 | 4.215 | 114.89 |
Complementary, the Rietveld refinement analysis of all obtained diffractograms was realized. The formation of CuO, CuWO4 and CuO/CuWO4 heterostructures was confirmed from this analysis. In addition, this analysis showed the existence of the Cu2O additional secondary phase in all materials with exception of the CuO sample; Fig. 3(a) shows the percentage of the crystalline phases for each sample. Cu2O phase crystallized in the presence of the CuWO4 phase and increased in percentage with the tungsten addition to the heterostructure. This trend can be attributed to the crystallization temperature, which is low for the formation of CuO (inhibiting its crystal growth), and low for the elimination of the secondary copper oxide phases. This trend could also be related to the CuO thermal stability or instability in the absence or presence of metal ions such as the tungsten. Fig. 3(b) shows the monoclinic crystalline structure for the CuO phase, Fig. 3(c) the triclinic crystalline structure of the CuWO4 phase, and Fig. 3(d) the cubic crystalline structure of the Cu2O phase.
Table 2 presents the results for lattice parameters, cell volume, and reliability parameters obtained in the Rietveld refinement analysis. A slight change in the lattice parameters of the CuO phase is evident, which confirms that the presence of CuWO4 promotes preferential crystal growth of CuO in the heterostructures. W ion caused a distortion of the lattice and promoted the growth of the heterostructure in the specific crystallographic direction associated with parameters a and b. On the other hand, the CuWO4 phase exhibits variations in the lattice parameters as the concentration of W ions increases in the heterostructure. These changes confirm the increase of the tensile strain in the lattice. Additionally, χ2<5 indicates reasonable refinements of the CuO, CuWO4, and Cu2O phases. Finally, the fact to the Cu2O phase does not appear in the CuO indicates that the Cu2O is a secondary phase of CuWO4.
Structural parameters and reliability parameters obtained from Rietveld refinement.
| Sample | Phase | a (Å) | b (Å) | c (Å) | V (Å3) | χ2 | R(F2) |
|---|---|---|---|---|---|---|---|
| CuO | CuO | 4.677 | 3.421 | 5.125 | 80.907 | 2.588 | 0.0360 |
| 100:1 | CuO | 4.704 | 3.441 | 5.154 | 82.266 | 2.528 | 0.0354 |
| CuWO4 | 4.691 | 5.823 | 4.866 | 131.730 | |||
| Cu2O | 4.293 | 4.293 | 4.293 | 79.123 | |||
| 100:2 | CuO | 4.685 | 3.429 | 5.132 | 81.324 | 4.564 | 0.0344 |
| CuWO4 | 4.705 | 5.843 | 4.884 | 133.030 | |||
| Cu2O | 4.297 | 4.297 | 4.297 | 79.379 | |||
| 100:3 | CuO | 4.677 | 3.420 | 5.127 | 80.881 | 4.054 | 0.0429 |
| CuWO4 | 4.701 | 5.837 | 4.879 | 132.66 | |||
| Cu2O | 4.297 | 4.297 | 4.297 | 79.323 | |||
| 100:4 | CuO | 4.680 | 3.427 | 5.128 | 81.103 | 4.528 | 0.0419 |
| CuWO4 | 4.701 | 5.836 | 4.879 | 132.620 | |||
| Cu2O | 4.293 | 4.293 | 4.293 | 79.133 | |||
| 100:5 | CuO | 4.667 | 3.414 | 5.116 | 80.395 | 4.530 | 0.0408 |
| CuWO4 | 4.702 | 5.839 | 4.881 | 132.790 | |||
| Cu2O | 4.295 | 4.295 | 4.295 | 79.215 | |||
| 100:6 | CuO | 4.748 | 3.396 | 5.114 | 81.212 | 3.295 | 0.0427 |
| CuWO4 | 4.701 | 5.838 | 4.881 | 132.710 | |||
| Cu2O | 4.286 | 4.286 | 4.286 | 79.087 | |||
| CuWO4 | CuWO4 | 4.700 | 5.838 | 4.881 | 132.69 | 3.121 | 0.0321 |
| Cu2O | 4.236 | 4.236 | 4.236 | 75.996 | |||
Fig. 4(a) shows the FTIR spectra of the as-prepared samples. The FTIR spectrum of CuO sample shows vibrational bands located at 419cm−1, 478cm−1, 555cm−1, and 596cm−1 attributed to CuO vibration attributed to 3d9 configuration [25], CuO bond of CuO6, CuO stretching vibrations along (−202), and CuO bond along (−101) [30], respectively. Regarding FTIR spectrum of CuWO4, the band located at 442cm−1 was assigned to the stretching vibrations own of Cu-O bond of the copper tungstate [31] and the band found at 675cm−1 was related to an infrared active mode presents in the oxide [30]. Additionally, three bands were identified in 793cm−1, 827cm−1, and 905cm−1, which are attributed to the stretching mode of the WOW bond [32], the [WO4]2− tetrahedral mode, and the OWO stretching mode, respectively. Finally, three bands associated to organic groups were detected: 1342cm−1 linked to CO2 presence, 1645cm−1 attributed to OH flexion combined with copper own of hydroxyl groups [33], and 1786cm−1 related to H2O and CO2 chemisorbed or physisorbed at surface level on the oxide [34]. Table 3 presents the FTIR band allocation of all the obtained materials. In general, the bands of heterostructure systems correspond to CuO or CuWO4. Some slight shift to red or shift to blue were detected, attributed to surface defects or morphological changes that induce lattice variations and crystal defects. Other crystalline phases and other impurities were not found detected since FTIR analysis.
FTIR band assignment of all materials obtained.
| Vibration | CuO | 100:1 | 100:2 | 100:3 | 100:4 | 100:5 | 100:6 | CuWO4 |
|---|---|---|---|---|---|---|---|---|
| Wavenumber (cm−1) | ||||||||
| CuO metaloxygen band | 419 | 415 | – | 419 | 417 | 413 | – | – |
| CuO stretching vibration (CuWO4) | – | 445 | 451 | 438 | 446 | 446 | – | 442 |
| CuO stretching mode along (002) | 478 | 476 | – | – | 476 | 482 | – | – |
| CuO stretching mode along (−202) | 555 | 545 | 555 | 547 | 548 | 550 | 546 | – |
| CuO stretching mode along (−101) | 596 | 596 | 596 | 594 | 596 | 596 | 594 | – |
| Infrared active mode Cu2O | – | 682 | 681 | 679 | 679 | 679 | 677 | 675 |
| WOW stretch mode | – | 794 | 793 | 791 | 795 | 789 | 787 | 793 |
| WO stretching vibrations in tetrahedral [WO4]2− | – | – | – | – | – | 826 | 826 | 827 |
| OWO stretching vibrations in the octahedron WO3 | – | 912 | 910 | 910 | 910 | 910 | 908 | 905 |
| Infrared active mode H2O and CO2 in CuWO4 | – | 1342 | 1342 | 1342 | 1342 | 1342 | 1340 | 1342 |
| Bending and stretching vibration of the OH group | – | 1643 | 1643 | 1643 | 1645 | 1645 | 1645 | 1645 |
| Infrared active mode H2O and CO2 on CuO | – | – | 1786 | 1786 | 1786 | 1786 | 1786 | 1786 |
Fig. 4(b) shows the Raman spectra of the as-prepared samples. The Raman spectrum of the CuO sample shows the vibrational modes characteristic of the monoclinic crystal structure of the oxide. The characteristic signals are found in 286cm−1, 339cm−1 and 622cm−1 consistent with the Raman active modes. These bands are attributed to the bulk CuO [35]. The Raman vibrational modes obtained for the CuWO4 sample show 10 characteristic signals of triclinic CuWO4[36]. The signals with the highest intensity are found at 219cm−1, 277cm−1, 354cm−1, 397cm−1, 477cm−1, 546cm−1, 774cm−1 and 903cm−1, which are associated with the symmetry of Ag[37]. In the heterostructured samples, the coexistence of the vibrational modes of CuO and CuWO4 were observed, confirming the results obtained in the structural analysis by XRD. However, the presence of a vibrational mode at 1062cm−1, corresponding to the Raman active mode of an additional Cu2O phase, was observed [38]. The low intensity of the Raman active mode characteristic of Cu2O in the spectra of samples 100:1 and 100:2 indicates a minimal presence of the Cu+1 ion, which is according to results of Rietveld refinement analysis. In contrast, in the spectrum of 100:3 a significant increase in such intensity is observed, suggesting a higher presence of this phase and, in addition, a high crystallinity. This combination could generate modifications in the properties of the heterostructured system.
Morphological, superficial and colloidal propertiesFE-SEM images of the CuO, CuWO4 and CuO/CuWO4 heterostructures are showed in Fig. 5. In the case of CuO, Fig. 5(a) allows to identify large grains (around of 350nm (Fig. 6)) with irregular geometries. For the CuWO4Fig. 5(h), a predominant type of morphology was identified – nanoparticle agglomerates. Fig. 5(b)–(g) exhibits two different morphologies: polyhedral with well-defined faces and nanograins of irregular geometries. These tendencies allow to infer that the grains greater correspond to CuO and the nanoparticle agglomerates correspond to CuWO4. The morphological results indicate that the CuO/CuWO4 heterostructure was produced in the six cases analyzed, despite the formation of a secondary phase, which was confirmed since Rietveld refinement analysis. Each crystalline phase is lodged in one grain that have one specific morphology. Finally, there is not a tendency in the size of two grains. Greater grains were obtained for the CuO and minor grains were formed for the CuWO4.
Specific surface area BET, SBET, pore and volume size, hydrodynamic dimension, and Z potential are tabulated in Table 4. 100:4 sample is the heterostructure with higher SBET, showing an optimal configuration to increase that property in the individual oxides. SBET results and FE-SEM images show concordance between values, because greater SBET measurements are related to small grain size. Similarly, an increase in the porosity is associated to an increase in the stress, fact to is verified with obtained structural results (Fig. 2). Pore size values of the samples with higher CuWO4 phase concentration exhibit pores between 13nm and 23nm, which makes them promising candidates for bactericide applications. Hydrodynamic dimension was estimated (Table 4) with the purpose to predict the behavior of materials in aqueous solutions keeping in mind their possible applications as bactericide agent. In fist time, the distribution of this parameter was monomodal, which indicates that the nanostructure agglomerates are stables and they are compound by the two oxides. This fact is a verification of the heterostructure formation. On the other hand, the heterostructure formation promotes the agglomerates consolidation, which directly benefit the application of these materials in biological applications. Finally, the Z potential results (Table 4) demonstrate that all materials are stable in aqueous suspension because these are between −30mV and +30mV, meaning they have less possibility of diffusion in bacterial culture media.
Superficial and colloidal parameters of the obtained materials.
| Sample | Oxide | SBETa (m2/g) | Pore sizea (Å) | Pore volumea (10−3cm3/g) | Hydrodynamic dimensionb (nm) | Z potentialb (mV) |
|---|---|---|---|---|---|---|
| CuO | CuO | 1.096 | 74.906 | 2.052 | 2248.33 | -16.8 |
| 100:1 | CuO | 1.619 | 112.515 | 4.553 | 1240.5 | -17.8 |
| CuWO4 | ||||||
| 100:2 | CuO | 1.623 | 130.325 | 5.289 | 851.7 | 19.8 |
| CuWO4 | ||||||
| 100:3 | CuO | 0.990 | 95.828 | 2.372 | 866.4 | -24.7 |
| CuWO4 | ||||||
| 100:4 | CuO | 6.604 | 231.264 | 38.182 | 623.4 | 24.3 |
| CuWO4 | ||||||
| 100:5 | CuO | 0.628 | 131.333 | 2.062 | 443.1 | 24.6 |
| CuWO4 | ||||||
| 100:6 | CuO | 0.283 | 132.235 | 0.937 | 411.1 | 18.5 |
| CuWO4 | ||||||
| CuWO4 | CuWO4 | 2.876 | 159.745 | 11.487 | 563.3 | 20.7 |
CuO, CuO/CuWO4, and CuWO4 inhibition zones against gram-negative bacteria (a) P. aeruginosa, (b) E. coli, and (c) S. enteritidis are presented in Fig. 7. In the (a) P. aeruginosa case, the 100:3 sample exhibited a slight coloration additional; this behavior was associated to a general diffusion process of metals presents in the sample. In the (b) E. coli case, all samples showed inhibition zone with well-defined sizes between 2mm and 3mm. In the (c) S. enteritidis case, the inhibition halo was most noticeable in the CuWO4 sample. The synthesized heterostructures in this investigation could cross the cell membranes through bacterial pores due to they have dimensions in the nanometer scale [39]. Morphological description showed that the CuWO4 oxide presents lower grain size than the CuO oxide in the heterostructures, which leads to the surface-to-volume ratio increments when the particle size is reduced toward the -nano- size then, size effects related to nanostructures become more prominent. Other important aspect lies in the fact to the CuO crystalline phase has minor crystallite size in the heterostructure which is achieved by the in situ crystallization of both oxides in the heterostructured system. Specifically, 100:1 and 100:2 samples showed inhibition halos slightly larger which can be attributed to their similar surface properties and hydrodynamic stability characteristics, besides the secondary crystalline phase percentage (Cu2O) has the lowest level in theses samples (around 3.8%) hence, it can be infer that the Cu2O secondary crystalline phase inhibits the bactericide response in the others heterostructures which that phase is between 13 and 18%.
CuO individual nanostructures are larger, which difficult its performance bactericide. CuO alone has excellent bactericidal properties [40] however, in this research, its size limited the reactive species penetration necessary to promote bacterial inhibition. In addition, the CuO nanoparticles obtained possibly did not succeed to attach to the bacteria surface therefore, they did not interact directly with the cell membrane to penetrate it, and then interfere with the bacterial cells metabolic processes causing damage to them. Similarly, it is inferred that bactericidal inhibition mechanisms such as the production of reactive oxygen species (ROS) and the release of Cu+2 ions occurs at a moderate level. In the first case, the ROS production occurs directly at the defect sites on the surface of nanocrystalline CuO then, it was concluded that the CuO obtained material in this work presents deficiencies in those sites. In the second case, the reduction of the Cu+2 ion to the Cu+1 ion, which is responsible for generating chemical species with the ability to react with reactive species to generate hydroxyl radicals, was not promoted because, the release of Cu+2 ions in the obtained CuO nanoparticles was not completely favored [41]. Thus, responsible cellular components responsible of the inactivation of bacteria, such as E. coli, were not produced. On the other hand, the bactericidal activity of the CuWO4 nanoparticles against Staphylococcus aureus strains has been reported as promising, because this material promotes the ROS formation due to factors such as the culture medium, its pH, and the concentration and the composition of nutrients [42]. However, this CuWO4 property has not been widely studied against other microorganisms, which is due to a bactericidal inhibition mechanism has not been fully established.
It is known that the bactericide properties of nanoparticles are depending on its grain size and specific surface area, hence it is expected that the nanoparticles with minor grain size and greater SBET have greater interaction with bacteria, which is a special case of 100:4 sample. In this sample, a low bactericide response can be attribute at the capacity low of each oxide to release metallic ions, besides the obtained copper and tungsten materials allowed successful the W ion release, which has been reported as highly toxic [43]. Additionally, in the 100:4 sample the Cu2O secondary phase (around 17%) may have affected its performance bactericide.
A synergistic interaction between the two semiconductors in the 100:1 and 100:2 heterostructured materials occurred, which contributed to the heterostructure adhering to the cell membrane allowing three possible phenomena (Fig. 8). (1) The CuWO4 nanoparticles in the heterostructure, specifically the tungsten ions, may interact with the cell membrane promoting its perforation. (2) The charge migration between the two semiconductors may promote the ROS species production, possibly OH· and O2− radicals [44], which penetrated the cell membrane likely reacting with the existing phosphate and sulfate groups. This process could cause the cell death or could prevent the cell replication by stopping the production of essential proteins [44]. (3) The heterostructure may have absorbed photons in the visible spectrum thus, enabling the free radical's production essential for the ROS formation. These radicals diffused through the bacterial membranes, which interact with the unsaturated fatty acids present in the membrane. This chain reaction continued until the membrane ruptured due to oxidative stress, which may have led to the protons release across the membrane and then increased its permeability. The increased permeability allowed protons to promote the breakdown of the proton gradient (difference in proton concentration inside and outside the mitochondrial membrane) thus, inhibiting Adenosine triphosphate (ATP) synthesis, ultimately leading to cell death [45].
Conclusions
CuO and CuWO4 oxides, and six CuO/CuWO4 heterostructures were successfully obtained. Cu2O secondary phase was established since Rietveld refinement analysis realized to XRD pattern; this phase does not appear in the CuO sample but appears in the CuWO4 sample, which allowed to infer that the Cu2O was a secondary phase to samples with CuWO4 contents. This discovery was verified since Raman results. Additionally, two different morphologies were observed since FE-SEM image, demonstrating that each principal crystalline phase of the heterostructure is lodged in each different grain, and the Cu2O secondary phase is lodged in the CuWO4 grain. Specifically, the 100:4 heterostructure exhibit superficial properties improved. All materials showed appropriate colloidal stability properties adequate for biological applications. In the case of bactericidal activity, all material exhibited some response. In particular, 100:1 and 100:2 samples exhibit the better bactericide response. This behavior was attribute to the similar tendency of their structural, vibrational and morphological properties. In general, the change in the concentration of the metallic precursors will be reflected in a change in the population density of the oxides, which was first evidenced in the percentage of crystalline phases determined. The synergistic contribution of semiconductors in those heterostructures promote interaction with gram-negative bacteria, promoting bacterial inhibition. Migration of reactive species on the surface of the heterostructure and adhesion of the nanostructures with CuWO4 induces perforation of the cell membrane, increasing its permeability and inhibiting ATP production, resulting in cell death.
Authors acknowledge financial support from the Universidad Francisco de Paula Santander, Cúcuta, Colombia, FINU Project No. 0.10-2022. This study was also supported by Universidad Pedagógica y Tecnológica de Colombia, SGI Project No. 3649, and Universidad Nacional de Colombia.
