Langmuir 2003, 19, 8765-8768
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Cell Damage Induced by Photocatalysis of TiO2 Thin Films Zhe-Xue Lu,† Lei Zhou,† Zhi-Ling Zhang,† Wan-Liang Shi,‡ Zhi-Xiong Xie,‡ Hai-Yan Xie,† Dai-Wen Pang,*,† and Ping Shen‡ College of Chemistry & Molecular Sciences and College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China Received May 12, 2003. In Final Form: July 25, 2003 When illuminated by near-UV light, titanium dioxide (TiO2) exhibits excellent bactericidal activity. However, there exist some different mechanisms for cell killing via photocatalysis. In the present study, the photocatalytically bactericidal mechanism of TiO2 thin films was investigated by atomic force microscopy (AFM) in conjugation with some other techniques. The decomposition process of the cell wall and the cell membrane was directly observed by AFM for the first time. The resultant change in cell permeability was confirmed by potassium ion (K+) leakage. Quantum dots (QDs) were designed originally as a probe to examine the cell permeability for macromolecules. The corresponding bactericidal activity of TiO2 thin films was examined by cell viability assay. These results suggested that the cell death was caused by the decomposition of the cell wall and the cell membrane and the resultant leakage of intracellular molecules.
Introduction Titanium dioxide photocatalyst has been extensively studied over the last 30 years for removal of organic compounds from polluted water and air. TiO2 photocatalyst in the anatase crystal form possesses strong oxidizing power when illuminated by UV light with wavelength < 385 nm. The photocatalytic process generates reactive oxygen species (ROS) such as hydroxyl radical, hydrogen peroxide and superoxide, and so forth. These reactive oxygen species can damage various cells.1 In 1985, Matsunaga and co-workers2 reported for the first time that TiO2 photocatalyst could kill bacterial cells in water. Since then, much research work related to the bactericidal effect of TiO2 photocatalyst has been reported. However, there were some different opinions on the bactericidal mechanisms of these photocatalysts. A deep understanding of the mechanisms has been quite important. In the early studies, Matsunaga et al.2,3 proposed that the direct oxidation of coenzyme A (CoA) in the TiO2treated cells, which led to the decrease in respiratory activities, was the vital reason for cell death. Lately, some workers4-9 proposed some other explanation of cell death, which resulted from the destruction of cell structure. Saito and co-workers4 used potassium ion leakage as a criterion for measuring membrane damage. Sunada7 found the degradation of endotoxin, which is a lipopolysaccharide * To whom correspondence should be addressed. Phone: 8627-87686759. Fax: 86-27-87647617. E-mail:
[email protected]. † College of Chemistry & Molecular Sciences. ‡ College of Life Sciences. (1) Blake, D. M.; Maness, P.-C.; Huang, Z.; Wolfrum, E. J.; Jacoby, W. A.; Huang, J. Sep. Purif. Methods 1999, 28, 1. (2) Matsunaga, T.; Tomada, R.; Nakajima, T.; Wake, H. FEMS Microbiol. Lett. 1985, 29, 211. (3) Matsunaga, T.; Tomoda, R.; Nakajima, Y.; Nakamura, N.; Komine, T. Appl. Environ. Microbiol. 1988, 54, 1330. (4) Saito, T.; Iwase, T.; Morioka, T. J. Photochem. Photobiol. B: Biol. 1992, 14, 369. (5) Sakai, H.; Cai, R.; Hashimoto, K.; Kato, T.; Hashimoto, K.; Fujishima, A.; Kubota, Y.; Ito, E.; Yoshioka, T. Photomed. Photobiol. 1990, 12, 135. (6) Sakai, H.; Ito, E.; Cai, R.-X.; Yoshioka, T.; Hashimoto, K.; Fujishima, A. Biochim. Biophys. Acta 1994, 1201, 259. (7) Sunada, K.; Kikuchi, Y.; Hashimoto, K.; Fujishima, A. Environ. Sci. Technol. 1998, 32, 726.
(LPS) cell wall constituent of Gram-negative bacteria. Maness and co-workers8 suggested that TiO2 photocatalysis promoted peroxidation of the lipid membrane, thus inducing cell death. However, the decomposition process of the cell wall and the cell membrane and its contribution to cell death has not been deeply investigated. In this study, the bactericidal mechanism of TiO2 thin film photocatalyst was investigated by AFM in conjugation with some other techniques. The dependence of the permeability of the cell membrane and cell viability on the stepwise cell wall and cell membrane damages was examined. The decomposition of cell walls and cell membranes caused by an illuminated TiO2 thin film may be the main reason for cell death. Experimental Section Cultures. Escherichia coli JM-109 was grown in Luria-Bertani (LB) medium, cultured at 37 °C with shaking, and harvested in the late exponential phase of growth. The harvested bacteria were centrifuged at 5000 rpm for 3 min, and the wet pellets were resuspended in ultrapure water and recentrifuged at 5000 rpm for 3 min to remove the growth medium. And the final pellets were resuspended in ultrapure water for analysis. Preparation of TiO2 Thin Films. The preparation of TiO2 thin films was as described in the literature.10 Ammonium hexafluorotitanate ((NH4)2TiF6; 0.1 mol/L) in 0.3 mol/L boric acid (H3BO3) was used as the treatment solution. Microscope slides were used as substrates. The preparation procedure for thin films was as follows: the substrate was degreased and washed ultrasonically in dilute nitric acid, ethanol, and ultrapure water. Then it was immersed in the treatment solution and vertically suspended. The temperature of the treatment solution was kept at 35 °C. After 9 h, the sample was removed from the treatment solution, washed with ultrapure water, and dried at room temperature. To obtain the maximum photocatalytic activity, the samples were calcined at 300 °C in air for 2 h. Photocatalytic Reaction. A TiO2 thin film coated glass slide (TiO2, 0.24 mg/cm2; glass slide size, 2 × 2 cm2) and a glass slide without any film for control were immersed in the bacterial suspension (about 108 cells/mL), respectively. The photocatalytic reaction was carried out by overhead illumination of the slide (8) Maness, P. C.; Smolinski, S.; Blake, D. M.; Huang, Z.; Wolfrum, E. J.; Jacoby, W. A. Appl. Environ. Microbiol. 1999, 65, 4094. (9) Huang, Z.; Maness, P. C.; Blake, D. M.; Wolfrum, E. J.; Smolinski, S.; Jacoby, W. A. J. Photochem. Photobiol. A: Chem. 2000, 130, 163. (10) Zhou, L.; Zhao, W.; Fang, Y. Chin. J. Appl. Chem. 2002, 19, 919.
10.1021/la034807r CCC: $25.00 © 2003 American Chemical Society Published on Web 09/17/2003
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immersed in the bacterial suspension with a 125 W high-pressure mercury lamp (Shanghai YaMing Electric Bulb Holding Co. Ltd.). The light with wavelengths < 300 nm was filtered. The light intensity reaching the surface of the bacterial suspension was 0.7 mw/cm2. At time intervals of 0, 5, 10, 20, 40, and 60 min, 300 µL of the bacterial suspension was transferred and used immediately for various assays described below. Atomic Force Microscopy (AFM) Imaging. Samples were analyzed using a Picoscan atomic force microscope (Molecular Imaging, Tempe, AZ, USA) in contact mode with commercial MAClever II tips (Molecular Imaging, USA), with a spring constant of 0.95 N/m. At every time interval, 10 µL of the illuminated bacterial suspension was dropped on a 2 × 2 cm2 glass slide and then air-dried for AFM imaging. Inductively Coupled Plasma-Mass Spectrometry (ICPMS) Experiments. At every time interval, 200 µL of the illuminated bacterial suspension was centrifuged at 12 000 rpm for 10 min, and 150 µL of the supernatant was diluted to 2 mL for ICP-MS analysis on Agilent 7500a. Fluorescence Measurements. Quantum dots (QDs) are colloidal nanocrystalline semiconductors with unique light emitting properties. CdSe/ZnS quantum dots in hexane were prepared according to the method developed in our laboratory. Nearly monodisperse CdSe quantum dots were first synthesized according to the scheme reported by Peng,11 and then a capping process was carried out by using hexamethyldisilathiane ((TMS)2S) and Zn(Ac)2 as precursors and trioctylphosphine oxide (TOPO)/hexadecylamine (HAD) as solvents. Modification of QDs with 3-mercaptopropionic acid was performed as described elsewhere.12 Fifty microliters of E. coli treated respectively by illumination for 0 and 20 min was mixed with 50 µL of mercaptomodified QDs and incubated at 37 °C for 40 min, washed twice, and resuspended in ultrapure water for fluorescence imaging on an Aviovert 200M inverted microscope (Zeiss, Germany). Cell Viability Assay. Loss of viability was examined according to the viable count procedure. Forty microliters of serially diluted cell suspensions was respectively spread onto Luria-Bertani agar plates. All plates were incubated at 37 °C for 24 h, and the numbers of colonies on the plates were counted.
Results and Discussion Gradual Destruction of Cell Structure Distinguished by AFM. E. coli, a species of Gram-negative bacteria, possesses an outer membrane beyond the peptidoglycan layer which is missing in Gram-positive bacteria. It is estimated that there are approximately 3.5 million molecules of LPS covering three-fourths of the outer membrane of E. coli.13,14 AFM has proved to be a suitable method for investigation of cell morphology and structures. Significant and even very subtle changes in the cell membrane could be observed by AFM.15-20 Figure 1 illustrates the AFM images of E. coli after treatment for different periods of illumination time in the presence and absence of TiO2 thin films. In the case of no illumination (Figure 1A and B), many protrusions were observed on the surfaces of rodlike bacteria, which were proven to be patches of LPS by Amro (11) Qu, L. H.; Peng, X. G. J. Am. Chem. Soc. 2002, 124, 2049. (12) Mitchell, G. P.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1999, 121, 8122. (13) Raetz, C. R. H. Annu. Rev. Biochem. 1990, 59, 129. (14) Rietschel, E. T.; Kirikae, T.; Schade, F. U.; Mamat, U.; Schmidt, G.; Loppnow, H.; Ulmer, A. J.; Zahringer, U.; Seydel, U.; Di-Padova, F.; Schreir, M.; Brade, H. FASEB J. 1994, 8, 217. (15) Kasas, S.; Felay, B.; Carganello, R. Surf. Interface Anal. 1994, 21, 400. (16) Johansen, C.; Gill, T.; Gram, L. Appl. Environ. Microbiol. 1996, 62, 1058. (17) Braga, P.; Ricci, D. Antimicrob. Agents Chemother. 1998, 42, 18. (18) Camesano, T. A.; Natan, M. J.; Logan, B. E. Langmuir 2000, 16, 4563. (19) Amro, N.; Kotra, L.; Wadu-Mesthrige, K.; Bulychev, A.; Mobashery, S.; Liu, G. Langmuir 2000, 16, 2789. (20) Stoimenov, P. K.; Klinger, R. L.; Marchin, G. L.; Klabunde, K. J. Langmuir 2002, 18, 6679.
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and co-workers.19 After illumination of 40 min in the absence of TiO2 thin film, the cell morphology and its surface structure changed little (Figure 1C and D). Only a few of the LPS protrusions disappeared. However, things were quite different in the case of illumination in the presence of TiO2 thin film. The morphology of E. coli illuminated for 60 min changed from rodlike to ellipselike, and a large hole appeared in the cell membrane (Figure 1M), which was also observed by Stoimenov.20 The corresponding surface structure also changed greatly. After illumination of 5 min, a groovelike rift (shown with an arrow in Figure 1F) was observed in the outer membrane of the cell wall, which suggests the beginning of decomposition of the cell wall. After illumination of 10 min, large numbers of the protrusions disappeared, indicating that the cell wall was decomposed, with the cell membrane being exposed (shown with an arrow in Figure 1H). Then, there appeared some rumples in the cell membrane after illumination of respectively 20 and 40 min, suggesting that the cell membrane was damaged (Figure 1J and L). For a cell illuminated for 60 min, its membrane appeared to have been considerably damaged, and its integrity was largely destroyed (Figure 1N). From the above AFM studies, it is very obvious that the damage of cell walls can take place immediately after illumination in the presence of TiO2 thin films. The cell wall will be severely decomposed via photocatalytic reaction 10 min after illumination, followed by a further damage of the cell membrane. The results confirmed the previous report by Maness.8 TiO2 photocatalysis would induce major disorder in the E. coli cell membrane. However, the current results show that ROS produced by TiO2 photocatalysts have no significant selectivity in attacking cellular components. Photocatalysis-Related Change of Cell Permeability Determined by K+ Leakage and QDs Entry. The LPS layer of the outer membrane plays an essential role in providing a barrier of selective permeability for E. coli and other Gram-negative bacteria.19 From the above AFM studies, TiO2 thin film photocatalysts could decompose the LPS layer and peptidoglycan layer, and subsequently destroy the cell membrane, leading to a change in the cell membrane permeability and resultant leakage of intracellular substances. K+ exists universally in bacteria.21-23 It plays roles in the regulation of polysome content and protein synthesis.23 Therefore, K+ leakage from E. coli was used, in the present work, to examine the permeability of the cell membrane. Figure 2 shows the dependence of K+ concentration on illumination time. In the case of illumination in the absence of TiO2 thin film, there was nearly no K+ leakage from E. coli cells. However, in the presence of TiO2 thin film, K+ would leak out from the bacterial cells immediately after illumination. The K+ concentration remarkably increased 10 min after near UV illumination, which should be attributed to a notable change in the structure of the cell wall (see Figure 1H). The K+ level reached to relatively steady values with increasing illumination time, and then it further increased notably when illumination time was increased to 60 min, due to the appearance of holes in the cell membrane, as observed by AFM (Figure 1M and N). The results demonstrate that the K+ leakage was consistent with the decomposition of the cell wall and the cell membrane. With the destruction of cell structure by photocatalytic reaction, macromolecules in bacteria, such as proteins (21) Heefner, D. L. Mol. Cell. Biochem. 1982, 44, 81. (22) Willis, D. B.; Ennis, H. L. J. Bacteriol. 1968, 96, 2035. (23) Ennis, H. L. Arch. Biochem. Biophys. 1971, 143, 190.
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Figure 1. AFM images, in topograph (A, C, E, G, I, K, M) and deflection mode (B, D, F, H, J, L, N), of E. coli illuminated by UV light in the presence or absence of TiO2 thin films at different time intervals. Images of individual bacterium were labeled with A, C, E, G, I, K, and M, and their corresponding zoom-in surface structures were shown respectively in B, D, F, H, J, L, and N. E. coli illuminated in the presence of TiO2 thin film at time intervals of 0 (A), 5 (E), 10 (G), 20 (I), 40 (K), and 60 min (M); E. coli illuminated for 40 min in the absence of TiO2 thin film (C). Image sizes: (A, C, E, G) 3.5 × 3.5 µm2; (I, K, M) 2.6 × 2.6 µm2; (B, D, F, H, J, L, N) 800 × 800 nm2.
Figure 2. Leakage of K+ ion from E. coli cells illuminated by UV light in the presence (A) and absence (B) of TiO2 thin films at different time intervals.
and RNAs, should also be released besides the leakage of small molecules. If this is the case, particles with similar size to that of intracellular macromolecules should be able to enter the bacterial cells. Highly luminescent semiconductor quantum dots have been used widely in biological
fields. Mercapto-modified QDs were found not to get into cultured HeLa cells if they were not conjugated with transferrin.24 So mercapto-modified QDs of about 5 nm in diameter were exploited to study cell membrane permeability in this research. The fluorescence and transmission images of the same vision field of a sample can be obtained by changing the mode of a microscope for collecting light. By comparing the images in the two different modes, it is easy to judge whether QDs have gotten into the cells. Figure 3 shows the fluorescence and transmission images of E. coli having been mixed with mercapto-modified QDs obtained from the same area. When E. coli cells were illuminated for 20 min in the presence of TiO2 thin film, QDs were found to have entered the bacterial cells (Figure 3 and B). When the bacterial cells were not illuminated (Figure 3C and D) or illuminated for 20 min in the absence of TiO2 thin film (Figure 3E and F), no fluorescence was observed. The results demonstrate that during 20 min of illumination in the presence of TiO2 a thin film could greatly change the permeability of the cell membrane, which facilitated (24) Warren C. W.; Nie, S. Science 1998, 281, 2016.
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Figure 4. Effect of photocatalytic reaction by TiO2 thin films on cell viability. The survival curves were obtained by colony counting of E. coli cells illuminated in the absence (A) and presence (B) of TiO2 thin films at different time intervals.
still viable after illumination for 60 min in the absence of TiO2 thin films. However, in the presence of TiO2 thin films (Figure 4B) 13.5% of the cells lost their viability after illumination for 5 min. Ten minutes of illumination made ∼70% of the cells lose their viability. Sixty minutes of illumination made about 90% of the cells die. These results imply that TiO2 thin film photocatalysts have excellent bactericidal activity. Conclusions
Figure 3. Fluorescence (A, C, E) and transmission (B, D, F) images of E. coli cells illuminated in the presence (A, B) or absence (C-F) of TiO2 thin film at different intervals: 0 min (C, D); 20 min (A, B, E, F). Image sizes: 24 × 24 µm2.
QDs to enter bacterial cells. On the contrary, the intracellular macromolecules besides the small intracellular molecules could also leak out from the cells. Loss of Cell Viability. With the decomposition of the cell wall and the cell membrane, the leakage of intracellular molecules will result in a change in the cell viability. Colony counting experiments were utilized to determine the viability of illuminated cells. The survival curve in Figure 4A shows that nearly 90% of the E. coli cells were
From our above studies, it has been found that the loss of cell viability is consistent with the decomposition of the cell wall and the cell membrane and the leakage of intracellular species. A possible bactericidal mechanism was proposed. When cells are illuminated in the presence of TiO2 thin films, the cell wall will be first decomposed, and subsequently the cell membrane will be destroyed, resulting in the increase in the permeability of cells, the leakage of intracellular molecules, and the final death of cells. Acknowledgment. The authors wish to thank Mr. Jie Chen for ICP-MS and Wei Cheng for fluorescence microscopy. This work was supported by the National Science Fund for Distinguished Young Scholars (Grant No. 20025311) and by the National Natural Science Foundation of China (Grant Nos. 20299034; 20207005). LA034807R
