Exploration of the antibacterial capacity and ethanol sensing ability of Cu-TiO₂ nanoparticles

Abstract

Titanium oxide (TiO₂) is one of the most scrutinized material because of its in-built fundamental properties and has been developed as an outstanding photo-catalytic material intended for many different industrial applications. In order to further explore the properties of TiO₂, we prepared Copper-loaded TiO₂ (Cu-TiO₂) nanoparticles (NPs) for inhibiting the growth of bacterial cells and also to serve as a chemical sensor. The physico-chemical characteristics of the synthesized Cu-TiO₂ NPs were characterized by many different techniques for the crystallinity, bonding and functionality, morphology, elemental composition, and absorption characteristics. From the results, we confirm for the formation of anatase phase of TiO₂ having a tetragonal crystal system, while the morphology studies indicated that the Cu dope TiO₂ has spherical morphology. The elemental analysis confirmed for the inclusion of Cu into TiO₂ crystal lattice and the absorption spectroscopic analysis helped for the bandgap calculation and visible light absorption property of Cu-TiO₂ NPs. The metal nanoclusters of Cu are observed to be deposited on different phases and sites of TiO₂ resulting in the inter-band transitions. Further, the sensitivity of Cu-TiO₂ as a chemical sensor is determined by fabricating the electrode at the FTO glass substrate where the ethanol sensitivity was found to be little increased/enhanced with Cu loading. Finally, the antibacterial activity of Cu-TiO₂ NPs was confirmed by its activity against various bacterial cultures and are found to be efficient.

Keywords:

• Cu-doped TiO2
• antibacterial activity
• ethanol detection
• chemical sensors
• phase transition
• escherichia coli
• staphylococcus aureus

1. Introduction

The recent increase in the synthesis of monodispersed nanocrystals of any origin (inorganic or organic) having many different shapes and sizes is being motivated by the specific role played by thus formed crystals in various fields including the cosmetic and pharmaceutical, energy and environment, photovoltaics and smart technology probes, construction, and engineering, etc [1–5]. Since the nanoparticles (NPs) carries the self-assembling properties which are supported by their narrow sizes and high surface charges, and that also helps them to form the super-ordered structures having a range of practical applications. Although many different techniques are available for the formation of NPs with desired characteristics specific to the biological application like size, shape, surface charges, morphology, hydrophilicity, thermal stability, and porosity, the persistence of such properties even at the desired targeted site is still a challenge [6,7]. As an example, some of the protein-tagged metal or metal oxide NPs when got released into the biological environment mostly get agglomerated and primarily lost their inbuilt properties. Similarly, the metal-based composites with an active antibacterial metal when used for the antibacterial applications, it is expected to see a decrease in the overall efficiency with some composites at least to a limited level, when making up the metal to have inclusive with other properties like hydrophilicity, prevention of agglomeration, biofunctionalization, etc [7,8]. Hence to overcome such limitations of metal-containing composites applicable to the biological sector, the selection of nanoparticulate probe is based on the unique physicochemical properties and high surface area to volume ratio with stable and sustainable characteristics in all the environments. Among many different biomaterials applied in the biomedical sector, the probes in particular employed with an active antibacterial character should be associated with high surface charges and areas, in order to exert strong oxidative stress against the bacterial cells and some ways to tune such properties includes the doping, composite formation, coating with other metals, etc [9,10].

The recent increase in the usage of inorganic antimicrobial agents such as titanium (Ti), zinc (Zn), cerium (Ce), etc over the organic agents is due to their improved safety, stability, hydrophilicity, and photocatalytic efficiency [11]. Among all other agents, titanium dioxide (TiO₂) is a renowned metal oxide semiconductor and has greater potentials for environmental applications such as dye degradation, inclusion into nano-paints, cosmetics, sensors, solar cells and electronics [12,13]. Also, taking into consideration of the surface plasmon resonance property that has an affect on the photocatalytic ability, a number of formulations with a change of morphology and architecture are being developed including the Ag-TiO₂ nanocermet thin films [1,2], Au–VO₂ nanocomposite thin films [3], MgO:CuO nanocrystals [4], Au − VO₂ thermochromic nanocomposite [5], etc. However, each nanocomposite has its limitations in terms of thermal stability, aqueous dispersibility, complex synthesis procedures, and at least associated with high costs. Since the unique antibacterial properties of TiO₂ makes it an excellent candidate to incorporate in the manufacturing of medical device probes and for the coating of sanitary ware surfaces and so the quest is to produce the TiO₂-based composites at a limited cost without compromising the inbuilt properties. For the TiO₂ NPs in the biomedical sector, the major governing factors are primarily payable to the oxidative stress and formation of reactive oxygen species (ROS) along with the hydroxyl radical generation from hydrogen peroxide (H₂O₂) under UV light illumination [11]. Moreover, it was observed that an improvement in the antimicrobial activity of TiO₂ NPs can further be achieved by forming composites with that of other metals having the inbuilt antimicrobial property like Ag, Cu, Zn, etc [14,15]. Thus formed TiO₂ nanocomposites are observed to be versatile materials among the other semiconductor oxides in their purest form (e.g. ZnO) due to the photostability, Physico-chemical structure, optical, and electrical properties.

By making use of the composite technology, the antibacterial and photocatalytic activity of TiO₂ NPs can be fine-tuned and in one of the study, the TiO₂ nanocomposite was being used for the removal of toxic organic compounds by decomposition, destruction of pollutants from the contaminated water/air, killing of harmful bacteria and cancer cells [16]. Therefore, in the present work, we hypothesized that the formation of Cu-TiO₂ NPs can further improve the photocatalytic and antibacterial properties of TiO₂ by taking advantage of the same inbuilt properties of Cu. Also, the Cu’s addition into TiO₂ can be economical to achieve the potentially higher activity levels at a minimum cost as Cu is cheaper as compared to TiO₂. Following the synthesis of Cu-TiO₂ NPs by a simple chemical route, the product was thoroughly analyzed for the crystallinity and crystal size, surface morphology, elemental composition, functionality, and bondings, etc. Finally, the antibacterial activity test was performed with the Cu-TiO₂ NPs against four different bacterial cell types where the inhibition zones were investigated.

2. Experimental methods

2.1. Synthesis of Cu–TiO₂ NPs

For the synthesis of Cu-TiO₂ NPs, we started with copper acetate and titanium isopropoxide precursors in stoichiometric ratios, where both prepared solutions were dissolved in absolute methanol using a magnetic stirrer to form a homogeneous solution. Now ammonium hydroxide (as precipitation agent) was added slowly to the earlier formed homogeneous solution until the pH reaches 9 and the formed solution was kept for 24 h to obtain the precipitated product. The precipitate was separated, washed with double distilled water until the pH becomes neutral, dried at 60 °C for 16 h, and finally subjected to annellation at 600 °C for 3 h to get the final product of Cu-TiO₂ NPs.

2.2. Instrumental analysis

The powdered X-ray diffraction (XRD) analysis for pure TiO₂ and Cu-TiO₂ NPs was used to investigate the crystal size and purity and for that, the XPERT-PRO diffractometer consisting of Cu K_α radiation operating at a wavelength (λ) of 1.541 Å was used. For the surface bonding and functionalization, Fourier transforms infrared (FTIR) spectroscopy, JASCO FT/IR 4600 instrument in the range of 4000-500 cm⁻¹ and for the optical absorption studies, Shimadzu-1800 UV–vis spectra in the range of 200–800 nm was used. In order to observe the surface morphology, particle shape analysis, and elemental composition of Cu-TiO₂ NPs, the field-emission scanning electron microscopy (FESEM, Supra- 55) connected to an energy dispersive analysis by X-rays (EDAX) detector from BRUKER was used.

2.3. Antibacterial activity test

The antibacterial activity of Cu-TiO₂ NPs was tested against four different pathogenic bacteria, Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Candida albicans (C. albicans), and Bacillus subtilis (B. subtilis). For the assay, about 1 mL of 10³ CFU/mL (CFU-colony forming units) concentration of bacteria was expelled onto the pellets positioned on the petri dish and were then disclosed to the UV radiation for 0, 1, 2, 3, and 4 h periods. Following that, the petri dish was permeated using MacConkey agar and swiftly rotated to evenly spread the agar and permitting to harden it (about 10 min). Further incubated for another 24 h at 37 °C in a 5% CO₂ incubator. Finally, the viable colonies count of bacteria on each agar plate was monitored, and its disinfection efficiency was evaluated. The bacterial reduction fraction was assessed according to the following relation (1). (1) $$\text{Bacterial reduction fraction}=100\mathrm{*}\frac{{\mathrm{N}}_0-\mathrm{N}}{{\mathrm{N}}_0}$$(1) where N₀ and N are the average counts of live bacterial cells per milliliter (control), and agar treated pellets, respectively.

2.4. Fabrication of Cu-TiO₂ NPs-based ethanol (chemical) sensor

The synthesized Cu-TiO₂ NPs along with ethyl acetate (EA) and butyl carbitol acetate (BCA) were mixed thoroughly and coated onto the surface of FTO (Fluorine-doped tin oxide) glass possessing at an active surface area of 0.25 cm². In order to obtain a consistent coating, the electrode was placed in a muffle furnace maintained at 50–60 °C for about 12 h. To form the sensor, the FTO glass plate coated with Cu-TiO₂ serves as the working electrode along with the copper (Cu) wire serving as a counter electrode. The phosphate buffer solution (PBS, 0.1 M, pH 7.0) was prepared by incorporating 0.2 M Na₂HPO₄ and 0.2 M NaH₂PO₄ in 100 mL of de-ionized water. To be used as a target chemical, ethanol (1.0 M, stock solution) was diluted at various concentrations using the double-distilled water, while the PBS (0.1 M, pH 7) amount was kept constant to 10 mL during the measurement. The analyte solution equipped for various ratios of ethanol ranging from 0.625 M to 10 M. With the use of an electrometer, the I–V characteristics were deliberated using a straight forward two-electrode technique and likewise, the graph was plotted for the current versus concentration (i.e. the slope of calibration curve) and this graph can be used for estimating the ethanol sensitivity towards the electrode.

3. Results and discussion

3.1. Structural study

Figure 1 shows the comparison of powdered XRD patterns of pure TiO₂ and Cu-TiO₂ NPs, where the 2θ peaks for the pure TiO₂ are observed to the diffraction (h k l) planes of (101), (044), (200), (105), (211), (204), (116), (220) and (215) respectively. From the analysis of peaks data, it can be confirmed that the peaks are in a good agreement with the standard pattern (JCPDS card no: 21 − 1272). It confirms the presence of anatase phase of TiO₂ in the sample and belonging to the tetragonal crystal structure of TiO₂ [17,18]. Similarly, the Cu-TiO₂ NPs also confirm the presence of anatase phase as indicated by the 2θ peaks changing to sharper and the intensity of individual planes, (1 0 1) seems to be modified when compared with the pure TiO₂ peaks. The maximum intensity of 2θ peak (1 0 1) of pure TiO₂ was found to be increased in the Cu-TiO₂ sample and this might be due to the occurrence of modifications in the atomic planes of TiO₂ with the incorporation of Cu ions into the Ti structure. Also, the changes occurring to the 2θ peaks of TiO₂ suggest a change in the overall morphology following the occupation of Cu atoms at the substitutional sites of Ti without making any alteration to the host crystal lattice. The line width of the maximum peak used for estimating the particle’s size and for that, the Debye’s Scherrer formula was used [19]. The average crystalline size was found to be ∼27.32 nm and ∼25.26 nm for pure TiO₂ and Cu-TiO₂, respectively [20]. Since the Cu ions substitute and compensate the anion vacancy in the TiO₂ lattice and that helped to reduce the concentration of free electrons, which is also confirmed by the UV–visible analysis.

Figure 1. Comparison of the powdered XRD patterns of pure TiO₂ and Cu-TiO₂.

3.2. Vibrational studies

Figure 2 shows the FTIR spectral comparison of pure TiO₂ and Cu-TiO₂ NPs and from the figure, the pure TiO₂ sample peaks observed at 3400 cm⁻¹ and 1878 cm⁻¹ corresponding to the hydroxyl group’s (from the surface water/moisture) stretching and bending vibrations. The same bands are observed as broad for the Cu-TiO₂ NPs sample too at 3445 cm⁻¹ and 1620 cm⁻¹. The pure TiO₂ sample confirmed a peak around 760 cm⁻¹, which exposes the existence of stretching vibrations of Ti–O bond and for the Cu-TiO₂ sample, the absorption band of this peak appeared in the range of 630–830 cm⁻¹ [22]. Also, for the Cu-TiO₂ sample, the broad absorption band in the range of 550–1000 cm⁻¹ is associated with the Cu-O vibrations. Thus, from the FTIR examination, it can be confirmed for the formation of Cu-TiO_2, and also, a lower number of OH groups for the Cu-TiO₂ sample is maintained as against the pure ones.

Figure 2. Comparison of the FTIR spectrums of pure TiO₂ and Cu-TiO₂.

3.3. FESEM analysis

The FESEM images of Cu-TiO₂ NPs showed in Figure 3 indicate that the particles are of spherical morphology and the observed spherical shape was a little bit distorted due to the addition of Cu making the TiO₂ particles to be shrinken. Also, we see the particles in their agglomerated phase and are expected because of the absence of suitable surfactant at the surface of Cu-TiO₂ particles which usually protect the particles, making them appear in their monodispersed form.

Figure 3. FESEM images of Cu-TiO₂ NPs at different magnifications in the range of 1–10 µm.

3.4. EDAX analysis

Figure 4 shows the comparison of EDAX data for the pure TiO₂ (a-c) and Cu-TiO₂ NPs (d-f) respectively. From the analysis, the data confirmed for the presence of ∼40 weight percentage of Ti in the pure TiO₂ sample and ∼76 weight percentages in the Cu-TiO₂ sample. The increase of weight observed in the Cu-TiO₂ sample shows that ∼20 weight percentage of Cu is getting added to the TiO₂ sample. Also, the appearance of Cu peak at the respective position (Figure 4(f)) also provides further evidence for the successful inclusion of Cu to the TiO₂ lattice.

Figure 4. Comparison of the EDAX data for the pure TiO₂ (a-c), and Cu-TiO₂ (d-f) samples.

3.5. UV-vis analysis

Figure 5 shows the UV-visible spectral comparison for the pure TiO₂ and Cu-TiO₂ samples and it can be observed from the graphs that the absorbance spectra for pure TiO₂ have been modified following Cu’s incorporation onto it. The wide absorption band in the region of 200–350 nm was observed and the presence of a strong absorption band indicates that the absorbed energy in the form of photons can be utilized for the promotion of electrons from the valence band to the conduction band. The shift is observed as an optical absorption edge and moves from UV to visible range and this shift may perhaps be due to the inter-band transitions in the Cu clusters which easily get deposited on different phases and sites of TiO₂ nanocrystals. A similar effect was reported on the doping of TiO₂ nanocrystals with Pt- and Ag [21] showing the close association among the light absorption properties and the photocatalytic activity. With an enhancement of absorbance in the UV–vis region in the Cu-TiO₂ NPs, the generation of more number of photoelectrons and holes also increase and actively get engaged in the photocatalytic reaction, and thereby supporting for an enhancement in the photocatalytic activity of TiO₂. Further, the observation of a peak at 350 nm for the Cu-TiO₂ NPs (Figure 5) could be due to the formation of metal nanoclusters and exhibit a plasmon band. A similar effect has been observed in the Au-TiO₂ nanoclusters too [22].

Figure 5. UV-visible spectrum for the pure TiO₂ and Cu-TiO₂ samples.

3.6. Antibacterial activity of Cu-TiO₂

The antibacterial activity of Cu-TiO₂ NPs was tested against the pathogenic bacteria (E.coli, S. aureus, C. albicans, and B. subtilis) and for that, the agar disk diffusion method was used. The experimental condition requires no light or dark room where the gram-positive bacterium such as S. aureus and gram-negative bacterium such as E. coli species were incubated with Cu-TiO₂ NPs. The bacterial growth of both gram-positive and gram-negative bacteriums was found to be the same at the beginning of the experiment and we observed no significant decrease in the bacterial cells with simple Cu-TiO₂ NPs incubation under the dark condition, as the particles are not active under these dark conditions. However, under the same conditions of Cu-TiO₂ NPs treatment along with the exposure of UV light, we observed a significant decrease in the number of viable bacterial cells, and also, we have investigated the survival rate of NPs treated cells by CFU counting after 24 h of the incubation period. A similar procedure was repeated for the other two bacterial cell lines of C. albicans, and B. subtilis and the effect of Cu-TiO₂ NPs inactivation of bacteria is depicted in Figure 6(A). Similarly, the zone of inhibition (ZoI) for the sample treated cells were measured and are shown in Figure 6(B), where from the analysis, we observed a maximum ZoI for S. aureus to be 20 mm, followed by E. coli of 12 mm, then C. albicans 11 mm, and B. subtilis 10 mm, respectively. Thus, from the antibacterial activity test, it is confirmed that the Cu-TiO₂ NPs are maintaining the activity only in the UV light presence and no activity in the dark and thereby confirming for the photocatalytic activity of the synthesized particles in an externally controlled manner.

Figure 6. (A) Antibacterial activity of Cu-TiO₂ NPs, and (B) ZoI comparison against various microorganisms of E.coli, S. aureus, C. albicans, and B. subtilis.

Based on the literature reports with similar samples containing 5% Cu-TiO₂ nanofilms [23], ZnO NPs [24], and NiO NPs [25], we indicate that the observation of antibacterial effect for our synthesized Cu-TiO₂ NPs in the presence of UV light is due to the shattering of cell membranes by the oxidative stress [26]. Since, the treatment of Cu-TiO₂ NPs along with the UV light exposure to the bacterial cells acts in two different mechanisms, i.e. (1) the toxicity produced by the Cu ions (Cu⁰, Cu⁺, and Cu²⁺) against the cell walls, and (2) the absorbed energy by the TiO₂ above its bandgap initiates the formation of oxidative species that further attacks the cell’s organic matter [23]. The similar other studies with that of ZnO and NiO NPs also supported for the involvement of oxidative species formed by any other means towards the reduction of bacterial cell number and thereby responsible for the maintenance of antibacterial activity [24,25]. Also, some of the other experiments on Staphylococcus haemolyticus demonstrated a swift aggregation of Cu ions equally on both dry and damp metallic Cu surfaces, with no considerable toxic effects, showing non-lethal DNA damage, whereas fatally damage to the cell membrane due to the rapid increase of Cu ions in the cell within a scale of minutes. Besides, to support this pathway of mechanism, Lemire et al. [27] and Macomber et al. [28] verified that the reduction of Cu²⁺ ions is linked with the fast growth of reactive oxygen group, and this encouraged the burning up of antioxidants and thereby plummeting the ability of cell walls to cause damage from these species.

3.7. Ethanol sensing ability of Cu-TiO₂ NPs

Figure 7(a) shows the electrical and ethanol sensing behavior of Cu-TiO₂ NPs where the I–V characteristics of this chemical sensor are related to various concentrations of ethanol in the range of 0.625 to 10 M for different voltages from 0 to 2.0 volts. Also, the figure depicts a noteworthy non-linear intensification in the current to the applied voltage and this non-linear behavior can be accounted for due the existence of Schottky contact sandwiched between the FTO (fluorine-doped tin oxide) electrode and the TiO₂ layer [29,30]. Besides, the response current was also found to be enhanced with an increase in the ethanol concentration. Thus, the conductivity of TiO₂ film increases due to an increase in the ion concentration demonstrating the superior sensing characteristics of the as-prepared Cu-TiO₂ NPs. The rise in conductivity of the system [31] depends on the redox response drawn at the surface of TiO₂ nanostructure manifested by the equations (2)(2) $${\text{TiO}}_2\left({\mathrm{e}}^{-}\right)\mathrm{ }+{\mathrm{ }\mathrm{O}}_2\to {\mathrm{ }\mathrm{O}}_2{}^{-}$$(2) and (3)(3) $${\text{TiO}}_2\left({\mathrm{e}}^{-}\right)\mathrm{ }+{\mathrm{ }\mathrm{O}}_2{}^{-}\to {\mathrm{ }2\mathrm{ }\mathrm{O}}_2{}^{-}$$(3) respectively. (2) $${\text{TiO}}_2\left({\mathrm{e}}^{-}\right)\mathrm{ }+{\mathrm{ }\mathrm{O}}_2\to {\mathrm{ }\mathrm{O}}_2{}^{-}$$(2) (3) $${\text{TiO}}_2\left({\mathrm{e}}^{-}\right)\mathrm{ }+{\mathrm{ }\mathrm{O}}_2{}^{-}\to {\mathrm{ }2\mathrm{ }\mathrm{O}}_2{}^{-}$$(3)

Figure 7. (a) I–V characteristics, and (b) the calibration curve of Cu-TiO₂ NPs.

In view of the above equations, the sensing characteristics of ethanol-based sensors are primarily attributed to the existence of scarcity in oxygen ions that get spontaneously adsorbed onto the surface of TiO₂ nanostructures. The extent of oxidation relies on its oxidizing potential and the negatively charged adsorbed oxygen ion promotes the oxidation of ethanol content into insignificant toxic component CO₂ and H₂O and thus radiating the free electrons (6 e⁻) onto the surface of TiO₂ nanostructure as in equation (3)(3) $${\text{TiO}}_2\left({\mathrm{e}}^{-}\right)\mathrm{ }+{\mathrm{ }\mathrm{O}}_2{}^{-}\to {\mathrm{ }2\mathrm{ }\mathrm{O}}_2{}^{-}$$(3) . Hence, mutual conductivity and sensitivity get magnified. (4) $$C{H}_{3}C{H}_{2}OH\left(\mathit{ads}\right)\mathrm{ }+\mathrm{ }6O\left(-\mathit{ads}\right)\mathrm{ }⟶2C{O}_2+\mathrm{ }3H_{2}O+6e-(C.B.)$$(4)

(Ethanol) (Adsorbed oxygen) (Byproduct) (Free-electron)

A calibration graph between the concentration (M) and current (mA) showed in Figure 7(b) helps to monitor the sensitivity of as-prepared Cu-TiO₂ NPs surface. The calculated sensitivity of Cu-TiO₂ was determined to be ∼0.07212 mAM⁻¹cm^−2. The calculated ethanol sensitivity of TiO₂ is observed to be enhanced with an already reported sensitivity ∼0.052 mAM⁻¹cm⁻² [32] on Cu doping. It uncovered through a linear dynamic range from 0.625 to 10 M, proportional to the linear regression line of r = 0.9384, analogous with a detection limit around ∼0.46 M. Therefore, the calculated sensitivity of the sensor can be designated as a result of appropriate catalytic behavior, perhaps with an excellent biocompatibility and appropriate correlation amongst the active sites on the surface of Cu-TiO₂ and the FTO glass electrode.

4. Conclusion

In conclusion, the structural, optical, and antibacterial activity of Cu-TiO₂ nanocrystals synthesized by the chemical route is described in this work. The crystallinity of synthesized samples was analyzed by powder XRD, where the average crystalline size was found to be ∼27.32 nm and ∼25.26 nm for pure TiO₂ and Cu-TiO₂, respectively. The morphology analysis confirmed the formation of spherical Cu-TiO₂ NPs and the EDAX confirmed for the presence of Cu in the TiO₂ NPs. As indicated by the FTIR analysis, the surface functional groups of Ti-O seemed to be affected by the Cu loading and also the pure TiO₂ particles are found to have maintained a higher amount of surface OH groups than the Cu-TiO₂ NPs. Optical studies indicates that the Cu-loading onto the TiO₂ NPs led to a shift in its optical absorption edge from UV into the visible range. From the antibacterial assay of Cu-TiO₂ NPs, the maximum ZoI of 20 mm was found for S. aureus, followed by E. coli, then C. albicans, and B. subtilis. Further, the ethanol sensing ability of the Cu-TiO₂ NPs was tested and the sensitivity was estimated to be ∼0.07212 mAM⁻¹cm⁻². Overall, this practice can be imposed on the development of exceptional TiO₂ NPs intended for the monitoring of hazardous chemicals in the laboratories, environment, and also in the healthcare sector.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

The authors gratefully acknowledge the support from Universiti Putra Malaysia research grants of 9457700 and 9456800. We also appreciate the Ministry of Education Malaysia (under Fundamental Research Grant Scheme) of 5524429, 5540050, Exploratory Research Grant Scheme (ERGS) 5527188 and MyBrain 15 for providing financial support. King Saud University authors acknowledge the funding from Researchers Supporting Project number (RSP-2020/54), King Saud University, Riyadh, Saudi Arabia.