Microorganisms or microbes are described as microscopic organisms which can be survived everywhere. Although some of them are beneficial for the environment, animals, and plants , most of the diseases originate from the microbes via toxins propagation in foods and water sources . Traditional disinfection methods produce harmful wastewater or byproducts and are expensive. On the other hand, the use of antibiotics in food-producing industry causes antibiotic resistance . Recently, new materials have been developed to deal with these problems. Some of the metallic and ceramic semiconductors are of the modern disinfectants which can degrade or prevent growth and proliferation of microbes. Ag-containing composites and ZnO NPs are two famous and popular compounds to defeat cancer cells, bacteria, and infected cells. It is suggested that their toxicity originates from the disruption of ion homeostasis and/or cell wall-damaging [4, 5]. TiO2-containing NPs or nanohybrids are the other worth and mostly applied materials due to their microbicidal activity. This performance is a result of activation of semiconductor compound and generation of charge carriers. TiO2 NPs have two prominent characteristics. (i) These NPs are active against several types of microorganisms, such as Gram-positive and negative-bacteria, fungi, etc. (ii) TiO2-containing nanohybrids are not toxic and their biocidal performance is a non-contact action . The possible performances of UV-activated TiO2-containing compounds are being extensively used to oxide and/or degrade microorganisms [7-9]. However, these systems require UV-irradiation at high intensity; so their applications are restricted. Tackling this problem is possible by (i) decreasing the band gap energy (Eg), and/or (ii) using other activation methods.
TiO2 doping with metallic ions could lead to decrease Eg and is taken into consideration. Some of the most popular dopants that are intended to modify TiO2 NPs include Fe [10, 11], Ag , and Cu .
Recently, the influence of different metallic dopants on TiO2 NPs has been investigated elsewhere. Correspondingly, among Cd, Ag, Fe, Ce, and Cu, only Cd and Ag could improve absorbance of UV-visible light. On the other hand, both of them are induced by sonoirradiation . Considering the interesting results in the previous studies, we have decided to investigate the antimicrobial behavior of Cd-TiO2 compound under different irradiation sources.
Another point that should be considered is the tendency of NPs to be agglomerated and precipitated in a suspension. This phenomenon causes to decrease the performance of NPs. The surface covering of NPs with a polymeric compound (such as PEG) is a way to deal with this problem. Microorganisms need folate species to be alive. This characteristic has been used to design smart materials [15-17].
In this work, Cd-doped TiO2 NPs were synthesized by sol-gel / solvothermal method to achieve NPs with high specific surface area and uniformity. PEG was loaded on the as-synthesized NPs to avoid agglomeration and sedimentation. Then, folic acid was added to Cd-TiO2/PEG compound and subsequently, the microbicidal performance of the Cd-TiO2/PEG/FA nanocomposite was assayed under UV-, visible-, and sonoirradiation. This was investigated to evaluate the possibility of using Cd-TiO2/PEG/FA nanocomposite (Cd-TiO2 NPs to provide sono- or photo-activity, PEG to avoid sedimentation, and FA to increase the vicinity of nanoparticles and bacteria) in an antibacterial application. Although using folic acid promotes the anticancer performance of nanoparticles in photodynamic therapy due to the folate receptors, the influence of using this inorganic compound on the antibacterial performance of a photocatalyst would be studied for the first time.
MATERIALS AND METHODS
Titanium isopropoxide (TTIP, Ti(OCH(CH3)2)4) as Ti-containing precursor, cadmium nitrate tetrahydrate (Cd(NO3)2.4H2O) as Cd–containing precursor, isopropyl alcohol (iPrOH, C3H8O, >99.5%) as solvent, nitric acid (HNO3, 65%) as pH adjusting agent, deionized water as hydrolytic agent, polyethylene glycol (PEG, 6000 g/mol), and folic acid (FA) were purchased from Merck® (Frankfurt, Germany) and used as starting materials.
N,N′-dicyclo hexyl carbodiimide (DCC, >99%) and dimethyl sulfoxide (DMSO, 99%) were utilized for FA activation process.
Preparation of Cd-doped TiO2 NPs
The Cd-doped TiO2 NPs were synthesized by solvothermal assisted sol-gel method. Preparation parameters have been optimized to reach the highest sonocatalytic performance. For this reason, TTIP, iPrOH, distilled water, and nitric acid were mixed with the molar ratio of 2.8, 9.2, 166.7, and 1.0, respectively. The obtained solution was relaxed for 24 h to achieve a white sediment. Then, 1 g of the dried sediment powder, 0.063 mmol of Cd–containing precursor, 7.5 mL of water, and 59 mL of iPrOH were mixed and stirred for 24 h to achieve a homogenous solution. The obtained solution was then treated via a solvothermal process at 200 oC for 8 h to precipitate some colored particles. This sample is named as Cd-TiO2.
Functionalization of Cd-TiO2 NPs by PEG
PEG was used as the functionalization agent and conjugated to Cd-TiO2 NPs. For this aim, adequate amounts of the as-synthesized NPs and distilled water were mixed to reach the concentration of 1 g/L and sonicated for 2 h to deagglomerate the NPs before PEG addition. Then, 0.2 g of PEG was added and mixed with 100 mL of the Cd-TiO2 solution and sonicated for 3 h. This sample is named as Cd-TiO2/PEG nanocomposite.
Conjugation of FA to Cd-TiO2/PEG
In this step, Cd-TiO2/PEG nanocomposite and FA should be conjugated. Generally, covalent bonding between FA molecules and NPs could not form directly. FA needs an activation process to form isourea via carboxyl group activation. This procedure has been reported in detail elsewhere . For this reason, FA was solved in DMSO with equal weight ratio. Then DCC was added to the solution under N2 gas flow and stirred for 2 h. Subsequently, Cd-TiO2/PEG nanocomposite powder was added to the obtained solution and stirred under N2 atmosphere for 2 h. The as-modified NPs were separated from the solution by a freeze–dryer (Christ, Germany). This sample is named as Cd-TiO2/PEG/FA nanocomposite. The weight ratio of FA to Cd-TiO2 NPs should be optimized; therefore, four weight ratios (0.2, 0.4, 0.6, and 0.8) were incorporated. The schematic flowchart of the mentioned synthesis procedures is shown in Fig. 1.
The as-synthesized samples were characterized using X-ray diffractometry (XRD, D8 Advance, Bruker) with a Co X-ray tube, transmission electron microscopy (TEM, LEO 912 AB), diffuse reflectance spectroscopy (DRS, UV–visible scanning spectrophotometer,), and Fourier transform infrared spectroscopy (FTIR, PerkinElmer).
Tauc plot was drawn between (αhυ)2 vs. hυ (where α and hυ are absorption coefficient and photon energy, respectively) to measure the optical band gap energy (Eg) .
The concentration of the Cd in TiO2 structure was determined by atomic absorption spectroscopy (AAS) using an Analyst 300 spectrophotometer (Perkin-Elmer, USA).
The antibacterial activity of the samples under ultrasonic, UV or visible light irradiation was determined against facultative Gram-negative Escherichia coli (E. coli - ATCC 43886). Preparation of the bacteria-containing solution has been explained elsewhere . For this reason, E. coli was precultured in nutrient agar (NA) medium at ~ 37 °C for 24 h. The treated cells were then diluted and re-suspended with phosphate buffer solution (PBS) to 2.5–10 × 105 colony forming units per mL (CFU/mL). Using the surface plate method, 200 µL of the bacterial solutions and 1 µL of the samples suspension with a concentration of 10 g/L were mixed and then exposed to irradiation for 5 h. Three types of irradiation sources were used:
• UV irradiation by two UV lamps (365 nm, 15 W),
• Visible light by two ﬂuorescent lamps (18 W)
• Ultrasonic irradiation by an ultrasonic bath at 37 kHz frequency and 90 W output power
After irradiation, two techniques were applied to evaluate the bactericidal responses of the samples:
• Counting the numbers of surviving bacteria for the samples irradiated with UV or visible light.
• Measuring the non-growth halo diameter for the samples irradiated with ultrasonic waves.
RESULTS AND DISCUSSION
XRD, DRS, AAS, and TEM results
Fig. 2-A shows XRD patterns of the as-synthesized Cd-TiO2 and TiO2-P25 NPs. The obtained results approved the formation of anatase [PDF No.: 4-477] in pristine TiO2 and also Cd-TiO2 samples. The intensity of the main peak of anatase which is located at 2θ=30° is considered as an index to compare the as-synthesized powder and commercial TiO2-P25 powder . It is clear that the height of this peak was about 56 and 63 for Cd-TiO2 and TiO2, respectively, which are less than that of TiO2-P25. This can be related to the smaller NPs and/or lower degree of crystallinity in both synthesized samples in comparison to the commercial TiO2-P25 powder. On the other hand, dopants cause a structural change in their matrix. The type and intensity of the changes are related to three items; amounts, ionic radii, and electrical charge of the dopants. Referring to the AAS results, the amount of Cd which is doped into TiO2 structure has been measured about 0.59 wt. %. Since the measured value is very close to the theoretical value (i.e. 0.710 wt.%), Cd could be considered as the source of the mentioned changes in TiO2 structure .
Based on Fig. 2-B, Eg value of the as-synthesized sample was ~ 3.11 eV, which is less than that of TiO2-P25, 3.45 eV. It means that Cd-doping led to decrease band gap of TiO2 structure and its photocatalytic activity needs less energy. TEM images of the Cd-TiO2 and TiO2-P25 samples are illustrated in Fig. 2-C and 2-D, respectively. It is clear that the as-synthesized NPs have a uniform shape and particle size about 10–20 nm. This observation is in good agreement with XRD peaks intensity.
The next step in this research was to consider the dispersion state and sedimentation behavior of the as-synthesized NPs. To improve the performance of the Cd-TiO2 NPs, they should be modified by organic molecules . For this aim, PEG was selected as a modification agent. After that, FA was conjugated to the as-prepared compound to enhance its antibacterial performance . FTIR analysis was utilized to evaluate bonding formation between Cd-TiO2 NPs, PEG, and FA molecules. The resulted spectra are illustrated in Fig. 3 and assignments of the bands are presented in Table 1. According to the first row of Table 1 (Cd-TiO2), the vibration at 400-800 cm-1 was assigned to metal oxides (Ti─O and/or Cd─O) bands . The vibrations at the range of 1000-2000 cm-1 may be related to residual raw materials in the synthesis procedure of Cd-TiO2. The vibration at ~ 3500 cm-1 was assigned to O─H groups . According to the second row of Table 1 (Cd-TiO2/PEG), the vibration at ~ 700 cm-1 may be related to Ti─O and/or Cd─O band. The vibrations at 1325, 1455, and 2910 cm-1 were related to the existence of PEG (see 6th raw in Table 1). Moreover, some peaks were observed in Cd-TiO2/PEG pattern which is seen neither in the Cd-TiO2 sample nor in the PEG spectrum. One of these vibrations was located at 1777 cm-1. This new band confirmed the formation of conjugation between Cd-TiO2 NPs and PEG molecules. The mentioned vibration in functional group region ascribes a functional group containing a carbonyl (C=O). According to the third to sixth rows of Table 1 (Cd-TiO2/PEG/FA), in addition to the vibrations which are related to Cd-TiO2 NPs (400-800 cm-1) and PEG (960, 1480 and 2900 cm-1), some new bands appeared in these spectra at 1150, 1225, and 1360 cm -1, which are related to C─O─C, C─O, and C─H vibrations, respectively. These are not basically matched with FA vibrations (see 8th row). It seems that the nonexistence of the FA vibrations in these spectra is related to the low intensity of FA vibrations in comparison to other peaks. On the other hand, the as-formed bands confirmed the formation of bonding between Cd-TiO2/PEG and FA. Accordingly, an acceptable synthesis method was utilized to loading of FA and PEG on the Cd-TiO2 NPs.
Improvement in the bactericidal response of Cd-TiO2/PEG/FA compound under visible light, UV, or ultrasonic irradiations
Bactericidal assays were performed to compare the effects of PEG and FA loading on the Cd-TiO2 NPs under different irradiations. Table 2 represents the results of antibacterial screening under visible light, UV, or ultrasonic irradiations, showing a decrease in E. coli survivability due to the modification process. In the blank and Cd-TiO2 samples under different irradiations, the viabilities of bacteria decreased due to the existence of the photocatalyst NPs; therefore, the main role of bacterial-killing under UV light, visible light, or ultrasonic waves accomplished by photo/sono-activation of the NPs (no irradiation). As can be seen, Cd-TiO2/PEG powder exhibited relatively higher bactericidal effect compared to Cd-TiO2 powder when exposed to three sources of irradiations. Under UV irradiation, the synthesized Cd-TiO2 powder destroyed 2.0 log concentrations of bacteria, while only 1.4 log were killed under visible light. This event is possibly relates to:
1. UV is known as a disinfection agent. It can destroy the nucleic acid of a bacterium and consequently leads to not perform vital cellular tasks and to lose its reproductive capability.
2. The lower concentration of the as-formed OH radicals by the semiconductor under visible light in comparison to the UV irradiation.
However, similar different results were obtained in other samples when the targets were irradiated with UV or visible light. Measurement of bactericidal effect under ultrasonic irradiation was accomplished via a different method; therefore, the comparison between these results and UV/visible light irradiated samples cannot be correct.
Antibacterial performance of Cd-TiO2 NPs was improved by PEG addition under UV, visible or ultrasonic irradiation. There is an energy gap in a semiconductor material. A source of energy is required to overcome this gap. When a semiconductor is irradiated, if the provided energy is equal to or higher than that of the band gap, the excitation process could occur and superoxide and peroxide radicals would form. These radicals are effective in destroying bacteria . In this process, the free surface of NPs is important and determinative. The NPs tend to form aggregates, decreasing their free surface and deteriorating the surface-depended properties. Addition of PEG to the NPs led to decrease agglomeration by covering their surfaces . This phenomenon led to improving photo and sono-activity of the Cd-TiO2/PEG compound in comparison to the Cd-TiO2 NPs.
Folic acid is essential for microorganisms to secrete the nucleic acid that makes up their DNA. This characteristic can be a reason for the accumulation of bacteria around folate derivatives biomaterials .
To evaluate this supposition, Cd-TiO2/PEG/FA compounds were prepared and their antibacterial performances with different amounts of FA were compared under UV, visible light and ultrasonic irradiation.
Table 2 indicates that the bacteria survivability decreases with more folate conjugation and minimizes in the Cd-TiO2/PEG/FA (0.6) sample. This approach is probably related to the tendency of microorganisms to accumulate near the folate sources; therefore as-formed free radicals could easily damage their walls and antibacterial performance of the as-synthesized Cd-TiO2/PEG/FA compounds improved by increasing the FA to 0.6. Afterward, the antibacterial performance decreased at higher folate content. High amounts of folate may promote its connection to the NPs and cover their surface, as well as intensifying the accumulation of microorganisms around NPs. These events lead to reduce the exposure of light beams (UV or visible light) to the surface of the NPs; therefore the photoexcitation process would be limited.
It should be noted that the antibacterial behavior under ultrasonic irradiation is somewhat different. In this case, the existence of FA led to more decrease in viability (in comparison to the Cd-TiO2/PEG sample), means that FA molecules cannot prevent ultrasonic waves from reaching NPs. However, the antibacterial performance of the as-synthesized Cd-TiO2/PEG/FA compound improved via the addition of FA, probably due to the accumulation of bacteria around NPs and facilitating interaction between bacteria and as-formed free radicals.
Cd-doped TiO2 NPs were synthesized by a solvothermal assisted sol-gel method. PEG and folic acid as the anti-sedimentation and bait agents were added to the as-synthesized NPs. According to the FTIR results, chemical bonds have been generated between the NPs and PEG molecules and also between Cd-TiO2/PEG nanocomposite and folic acid molecules. The results of the bactericidal assay pointed out that the performance of PEG and FA on the antibacterial characteristic was conﬁrmed. PEG plays its role by avoiding agglomeration of the as-synthesized NPs. FA acts as a bait agent for bacteria and led to accumulating them around the NPs. Its efficacy is dose-dependent and maximized at 0.6 weight ratio of FA to Cd-TiO2. More concentration of FA led to decrease in the antibacterial efficiency under UV or visible light, whereas; it didn’t show considerable effect under ultrasonic waves. In comparison to pure Cd-TiO2 NPs, as-optimized Cd-TiO2/PEG/FA nanocomposite was more efficacious in bacterial killing especially under ultrasonic irradiation, increasing the diameter of the non-growth halo from 4 to 10 mm.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.