In this study, zinc oxide (ZnO) nanorods have been synthesized using a simple template-free precipitation technique and deposited on glass substrate. The meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) has been synthesized and then immobilized on the surface of ZnO nanorods to prepare an organic/inorganic composite. The samples were characterized by various techniques such as X-ray diffraction, diffuse reflectance spectra, Fourier transform-infrared spectroscopy and scanning electron microscopy. In addition, the photobactericidal activity of TPPS/ZnO composite, TPPS and ZnO nanorods was tested against the pathogenic bacterium of Escherichia coli under visible LED lamp irradiation. The results indicate that the photobactericidal activity of TPPS-loaded ZnO nanorods was better than TPPS or ZnO nanorods, separately.

INTRODUCTION

Semiconductors such as titanium dioxide (TiO2) and ZnO (zinc oxide) can act as sensitizers for a light-reduced redox process for organic material mineralization (Hoffmann et al. 1995; Boer et al. 2002; Li & Haneda 2003) and indicate bactericidal activity or degradation on the organic cell wall of bacteria (Lu et al. 2005; Applerot et al. 2009; Panigrahi et al. 2011). ZnO is a non-toxic and biocompatible material with excellent chemical and thermal stability (Özgür et al. 2005; Zhao et al. 2012). It is well known that the shape of crystal accumulation has an important role in its application (Shen et al. 2008). Semiconductors with one-dimensional shapes such as rods or tubes are desirable due to their novel optoelectronic properties (Cui et al. 2001). Pure ZnO, with a large direct band gap energy of 3.37 eV, cannot make efficient use of the solar spectra because the solar spectra only contain approximately 4% UV light (Guillen & Herrero 2006). The utility of porphyrin as sensitizer is considered as an approach to extend the absorption range to the visible region (Gerdes et al. 1997; Sun et al. 2013). In previous studies, promising results from ZnO nanoparticles' antibacterial effect alone and the incorporation of different materials, such as porphyrin compounds, have been investigated under various conditions (Senthilkumar et al. 2013). Conversely, using LED as a visible light source has many advantages such as low voltage electricity, no cooling requirement, high photon efficiency, power stability during long operation times and broader spectral wavelength emission.

In this paper, synthesized ZnO nanorods were immobilized on glass using a simple method without any surfactant and were then modified by meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) to form TPPS/ZnO composite. The antibacterial activity of TPPS, TPPS/ZnO and ZnO nanorods were investigated under visible LED light irradiation.

EXPERIMENTAL

Materials and methods

All the chemicals used in this work were analytical grade reagents and used without further purification. Escherichia coli (PTCC 1330) was used as a Gram-negative bacterium.

The samples were characterized by X-ray diffraction (XRD) using a JEOL X-ray diffractometer (JEOL, Tokyo, Japan) with Cu Kα radiation. The Fourier transform-infrared spectroscopy (FT-IR) analyses were carried out on a Shimadzu FTIR-8400S spectrophotometer (Shimadzu, Kyoto, Japan) using a KBr pellet for sample preparation. Diffuse reflectance spectra (DRS) were prepared via a Shimadzu (MPC-2200) spectrophotometer (Shimadzu, Kyoto, Japan). The particle morphologies of the ZnO powder were observed by an AIS2100 scanning electron microscope (SEM; Seron Technology, Gyeongsu-daero, Uiwang-si, Gyeonggi-do, Korea).

Preparation of TPPS

Ten millimoles of freshly distilled pyrrole, 10 mmol of benzaldehyde, 100 mL of propionic acid and 15 mL of nitrobenzene were added to a 250 mL flask. The mixture was allowed to reflux under stirring at 120 °C for 60 min. After that, the resulting mixture was cooled overnight at room temperature and filtrated under reduced pressure. The crude product was purified by column chromatography (Silica gel, chloroform/ethyl acetate = 20:1 as an eluent) and the desired purple solid of meso-tetrakis(phenyl)porphyrin (TPP) was obtained (30%).

For the synthesis of TPPS, 0.2 g of TPP was dissolved in 5 mL of concentrated H2SO4. The mixture was heated in a steam bath for 4 h and then allowed to stand for 48 h. The resulting solution was neutralized by NaOH solution (3 N) and then the solvent was removed under vacuum at room temperature. The product was washed with methanol until no TPPS could be detected in the solution. Then, TPPS was immobilized on the glass. The glass slides had previously been immersed in a mixture of ethanol and NaOH for a week to activate their surfaces.

Preparation of ZnO nanorods

In order to obtain the concentration of 0.04 M, 5.94 g of Zn(NO3)2·6 H2O was added to 500 mL distilled water. The ammonia solution (25%) was added dropwise to the solution to achieve pH = 11, then the solution was refluxed under stirring. Finally, the mixture was centrifuged and the white solid was collected and washed with distilled water. The obtained solid was dried at 100 °C in an oven for 3 h, followed by calcination at 450 °C for 3 h. For deposition of ZnO particles on the glass slides, a piece of glass was utilized during the synthesis of ZnO. The glass slides had previously been immersed in a mixture of ethanol and NaOH for a week to activate their surfaces.

Modification of ZnO nanorods by TPPS

First, 0.02 mmol of TPPS was dissolved in 50 mL of H2O and a piece of glass coated with white ZnO nanorods was placed in this solution and refluxed for 5 h. Then, the glass slide coated with ZnO and the glass slide coated with ZnO modified with TPPS were dried at 100 °C for 5°h. Then, the glass slide coated with white ZnO and the glass slide coated with ZnO modified with TPPS were dried at 100 °C for 5 h. Consequently, the product was washed with ethanol until no porphyrin could be detected in the supernatant by UV–visible spectrophotometer (UV–vis).

Antibacterial activity

Escherichia coli was grown aerobically at 37 °C in nutrient broth. Thirty microliters of this broth were aseptically transferred onto nutrient agar plates and spread on the surface with a sterile spreader. The glasse slides (0.5 × 1.5 cm2 each sized) with three samples were placed onto nutrient agar. In addition, uncoated glass was also placed on agar as a negative control. The plates were incubated at 37 °C for 20 min and illuminated with the LED lamp (5 watts) for 60 min. They were then incubated at 37 °C overnight. The zone of inhibition was measured to evaluate the antibacterial effect of three samples. The experiments were carried out in triplicate. Bacterial growth was examined visually by measuring inhibition zones around the glass slides. A diameter larger than 10 mm was considered a positive response.

RESULTS AND DISCUSSION

X-ray powder diffraction

Figure 1(a) shows the XRD pattern of ZnO nanorod powder. It can be seen that all these peaks are in good agreement with hexagonal (wurtzite) ZnO (JCPDS Card, No. 36-1451). Table 1 shows that the calculated d-values are in good agreement with those taken from the JCPDS card file data for ZnO powder. No other ZnO phase or impurities were observed, which indicates the high purity of the obtained ZnO nanorods. On the whole, these diffraction peaks are sharp, narrow and symmetrical with a low and stable baseline, suggesting that the sample is well crystallized.

Table 1

The XRD parameters of (h k l), 2Θ; and d-value of the synthesized ZnO nanorods

h k l Synthesized ZnO nanorods
 
JCPDS 36-1451
 
2Θ (degree) d-Value (Å) I (%) 2Θ (degree) d-Value (Å) I (%) 
1 0 0 31.8194 2.81239 61.23 31.770 2.81430 57 
0 0 2 34.4762 2.6015 54.61 34.422 2.60332 44 
1 0 1 36.2973 2.47505 100 36.253 2.47592 100 
1 0 2 47.5898 1.9108 19.69 47.539 1.91114 23 
1 1 0 56.6653 1.62443 22.6 56.603 1.62472 32 
1 0 3 62.9221 1.47712 21.67 62.864 1.47712 29 
2 0 0 66.4036 1.40787 1.83 66.380 1.40715 
1 1 2 67.9976 1.3787 15.89 67.963 1.37818 23 
2 0 1 69.1258 1.35893 7.95 69.100 1.35825 11 
0 0 4 72.6191 1.30193 1.78 72.562 1.30174 
2 0 2 76.9981 1.23743 2.21 76.955 1.23801 
h k l Synthesized ZnO nanorods
 
JCPDS 36-1451
 
2Θ (degree) d-Value (Å) I (%) 2Θ (degree) d-Value (Å) I (%) 
1 0 0 31.8194 2.81239 61.23 31.770 2.81430 57 
0 0 2 34.4762 2.6015 54.61 34.422 2.60332 44 
1 0 1 36.2973 2.47505 100 36.253 2.47592 100 
1 0 2 47.5898 1.9108 19.69 47.539 1.91114 23 
1 1 0 56.6653 1.62443 22.6 56.603 1.62472 32 
1 0 3 62.9221 1.47712 21.67 62.864 1.47712 29 
2 0 0 66.4036 1.40787 1.83 66.380 1.40715 
1 1 2 67.9976 1.3787 15.89 67.963 1.37818 23 
2 0 1 69.1258 1.35893 7.95 69.100 1.35825 11 
0 0 4 72.6191 1.30193 1.78 72.562 1.30174 
2 0 2 76.9981 1.23743 2.21 76.955 1.23801 
Figure 1

(a) The XRD pattern of the synthesized ZnO nanorods. (b) The FT-IR spectrum of the synthesized ZnO, TPPS/ZnO and pure TPPS. (c) The DRS of the synthesized ZnO, TPPS/ZnO and the UV–vis spectrum of pure TPPS (in methanol). (d) The plot for band gap energy (Ebg) of ZnO.

Figure 1

(a) The XRD pattern of the synthesized ZnO nanorods. (b) The FT-IR spectrum of the synthesized ZnO, TPPS/ZnO and pure TPPS. (c) The DRS of the synthesized ZnO, TPPS/ZnO and the UV–vis spectrum of pure TPPS (in methanol). (d) The plot for band gap energy (Ebg) of ZnO.

FT-IR

Figure 1(b) shows the FT-IR spectra of the ZnO nanorod, TPPS and the TPPS/ZnO. The appearance of a sharp band at 401 and 501 cm−1 confirms the synthesis of ZnO because of the characteristic absorption band for the Zn–O stretching vibration. In addition, the broad absorption peaks centered at around 3,446 cm−1 are caused by the O–H stretching of absorbed water molecules.

For the FT-IR of TPPS, the stretching vibration of = C–N and –C = N bands (pyrrole) appeared at 1,373 cm−1 and 1,720 cm−1, respectively. The stretching asymmetric and symmetric vibration bands attributed to the C–H (CH2) bands are discernible at 2,846, 2,918 and 2,939 cm−1. The peaks appearance at 890 cm−1, 1,100 cm−1 and 3,387 cm−1 could be attributed to C6H4 (phenyl), the SO3 group and the N–H band, respectively.

For the FT-IR of TPPS/ZnO, the appearance of peaks corresponding to TPPS confirm that porphyrin has immobilized on the surface of the ZnO nanorods, but it is difficult to give these peaks precise values because of very low intensities due to a low loading of the macrocycle into the inorganic matrix.

Optical properties

The UV–vis DRS of the synthesized ZnO nanorods and TPPS/ZnO and the UV–vis spectrum of pure TPPS (in methanol) are shown in Figure 1(c). The spectrum reveals a characteristic absorption peak of ZnO at a wavelength of 368 nm.

The absorption coefficient for direct transition is given by: 
formula
where Eg (eV) is the energy gap, (eV) is the energy of incident photon and A is constant. The energy band gap was determined as 3.22 eV (Figure 1(d)).

The absorption range of the composite is wider in comparison with those of pure TPPS and ZnO nanorods. The UV–visible spectrum of pure TPPS contains a Soret peak at 413 nm and four Q peaks at 513, 553, 557 and 633 nm. Moreover, the composite porphyrin Soret and Q bands are red-shifted relative to those of the pure TPPS. Furthermore, the intensity ratio of Soret band to Q bands of the TPPS/ZnO is lower than that of the pure TPPS. Based upon these observations, it can be demonstrated that the porphyrin has been assembled on the surface of ZnO nanorods and there exists a strong interaction between ZnO nanorods and porphyrin in the TPPS/ZnO.

Morphological characterizations

The morphology of TPPS/ZnO and ZnO nanorod particles was exhibited by SEM images, as shown in Figure 2. Figure 2(a) and 2(b) show the SEM images of the pure ZnO and Figure 2(c) and 2(d) are related to TPPS/ZnO nanorods. It was found that all synthesized ZnO nanorods were quite uniform in size.

Figure 2

SEM images of the synthesized ((a), (b)) ZnO and ((c), (d)) TPPS/ZnO nanorods.

Figure 2

SEM images of the synthesized ((a), (b)) ZnO and ((c), (d)) TPPS/ZnO nanorods.

The results of antibacterial activity

The Gram-negative bacterium E. coli selected in this study is a well studied model organism for antibacterial experiments. The effects of ZnO-coated, TPPS-coated and TPPS/ZnO-coated glass slides alongside a control (uncoated glass) were investigated on E. coli bacterium. Figure 3(a) depicts the growth of colonies around the uncoated glass but reveals no bacterial growth adjacent to the glass slides immobilized with porphyrin, ZnO and TPPS/ZnO, which are shown, respectively, in Figure 3(b)3(d). The average of the inhibition zone is shown in Table 2. It is known that even ZnO-coated and TPPS-coated glass is observed to have a significant toxic effect on both the bacterial cultures, but the glass immobilized with TPPS/ZnO was observed to have the broadest inhibition zone (radius of 13.8 mm).

Table 2

The effect of uncoated glass slide and glass slides coated against E. coli

Samples Average diameter of inhibition zone (mm) 
Glass coated with ZnO 8.2 ± 0.83 
Glass coated with porphyrin 7.4 ± 1.14 
Glass coated with TPPS/ZnO 13.8 ± 1.92 
Uncoated glass slide – 
Samples Average diameter of inhibition zone (mm) 
Glass coated with ZnO 8.2 ± 0.83 
Glass coated with porphyrin 7.4 ± 1.14 
Glass coated with TPPS/ZnO 13.8 ± 1.92 
Uncoated glass slide – 
Figure 3

Zone of inhibition measurement of ZnO thin films treated with bacterium E. coli. The samples are (a) control, (b) ZnO film, (c) porphyrin film and (d) TPPS/ZnO film.

Figure 3

Zone of inhibition measurement of ZnO thin films treated with bacterium E. coli. The samples are (a) control, (b) ZnO film, (c) porphyrin film and (d) TPPS/ZnO film.

The photocatalytic mechanism of ZnO sensitized by TPPS is shown in Figure 4. When the visible light irradiates on the surface of the ZnO, porphyrin molecules on the surface of the ZnO nanorods generate photoinduced electrons by absorption of visible light. The photoinduced electrons transfer to the conduction band of ZnO. Meanwhile, the excited porphyrin (TPPS*) changes the electrons from the solution into TPPS. The electrons presents on the conduction band of ZnO reacted with dissolved oxygen to generate peroxyl radicals (·O2), which then become highly oxidative hydroxyl radicals (·OH) through a series of oxidation reactions. The hydroxyl radicals (·OH) can oxidize the organic cell wall of bacteria. The synergistic effect of the porphyrin sensitization makes a photocatalyst that exhibits a better degradation effect in the process of photodegradation of the organic cell wall of bacteria.

Figure 4

Antibacterial activity of ZnO/TPPS against the cell wall of bacteria.

Figure 4

Antibacterial activity of ZnO/TPPS against the cell wall of bacteria.

CONCLUSION

In this study, the photobactericidal activity of ZnO nanorods modified using porphyrins has been investigated. First, ZnO nanorods were successfully synthesized and deposited on glass substrates by a simple technique and then ZnO film was modified with porphyrin. The results of FT-IR and DRS spectroscopy show that porphyrin was successfully immobilized on the surface of ZnO nanorods. After that, its photobactericidal activity was tested against E. coli under visible LED lamp irradiation. In comparison to porphyrin and ZnO nanorods, the glass immobilized with TPPS/ZnO showed excellent antibacterial property against Gram-negative bacterium E. coli.

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