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Abstract
Adhesion of bacteria to a surface followed by biofilm formation causes many problems in human health care and, in some cases, can even cause human death. Therefore, reducing bacterial attachment to surfaces and antibacterial surface fabrication are two of the most important issues in many applications, including healthcare, medical, food packaging, etc. Polycarbonate (PC) is one of the most widely used polymers in medicine. However, it does not have antibacterial properties. On the other hand, laser treatment is used as a standard method for surface modification of different materials. In this paper, excimer laser irradiation at a fluence below the ablation threshold was used for surface patterning and modification of the polycarbonate sample, aiming to improve its antibacterial properties. The results show that super-hydrophilic nanostructured polycarbonate surfaces have antibacterial properties compared to non-treated PC, which has no antibacterial properties.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Bacterial adhesion to a surface is a physico-chemical process. Therefore, surface chemical changes or physical modifications are two possible methods to resist bacterial attachment to the surface [1]. In the last decade, physical modifications of the surfaces have attracted the attention of many researchers due to the advantages of the physical modified surfaces in comparison with the chemical modifications [1].
Although many parameters such as surface energy and surface charge influence the bacterial adhesion to the surface [2], wettability and roughness are supposed to be the most important and effective factors in controlling the bacterial attachment to the surface [3]. Changes in surface wettability and topographical modifications as a results of laser irradiation [4,5], have great potential to bacterial adhesion rate modification [6]. On the other hand, nano-scale surface roughness [7] and nanoparticles [8–10] greatly influence on different properties of a surface such as the bacterial adhesion rate. According to the laser irradiation parameters such as the laser fluence, the number of pulses and the pulse repetition rate, different micro/nano structures are formed on the surface [11,12]. Based on the Wenzel [13] or Cassie-Baxter [14] model, nano/micro scale structures change the surface morphology and influence on the surface properties such as wettability [15]. On the other hand, laser irradiation of a surface may cause chemical modifications. Since chemical modifications also affect the wettability behavior [1], by tailoring specific functional groups on the surface, bacterial adhesion rates can be altered by chemical modification, following laser irradiation.
A lot of research has been done on fabricating antibacterial polycarbonate with different approaches including chemical coatings [16], phase separation and selective solvent/non-solvent methods [17]; however, since polycarbonate is a widely used polymer in many medical applications, it is important to fabricate an antibacterial surface in a simple, high efficiency and one-step approach. Recently, some studies have investigated the fabrication of antibacterial surfaces by ultrashort lasers as a single step and high efficiency method [18,19], however, nanosecond laser treatment has advantage over ultrashort laser treatment due to lower maintenance costs.
Polycarbonate (PC) has been reported to be one of the most widely used polymers in many industrial applications such as agriculture, electronics and medicine [20,21]. This polymer offers unique benefits in healthcare devices and is irreplaceable for many medical applications. Polycarbonate is used in hemodialyzers, blood oxygenators and many other healthcare devices [22]. Therefore, antibacterial polycarbonate fabrication with modern methods seems to be essential for different uses. It is worth noting that polycarbonate has high absorption at 248 nm excimer laser, therefore the dominant mechanisms for the laser interaction with PC is photochemical.
In our previous works [4,23] we investigated the effect of an excimer laser irradiation at fluences above and very close to the ablation threshold on the surface contact angle and bacterial adhesion rate of the polycarbonate samples. Our previous results showed that the formation of the hierarchical structures following surface treatment at the ablation threshold fluence (∼40 mJ/cm2) and above the ablation threshold (80 mJ/cm2) with a high number of pulses leads to a superhydrophobic behavior and antibacterial properties of the surfaces [23].
Here, the effect of the sub-threshold fluence excimer laser irradiation of PC on the morphology, chemistry, wettability and antibacterial properties is investigated. The results show a single-step approach, no need of adding chemicals, for fabrication of antibacterial polycarbonate samples.
2. Materials and method
2.1 Sample preparation
Commercial polycarbonate samples with the thickness of 1 mm, were cut into 1 cm × 1 cm dimensions. They were washed with water and ethanol before the laser treatment to remove any contaminations from their surfaces.
2.2 Laser treatment
The samples were irradiated with a pulsed KrF laser (248 nm and 25 ns) and the pulse repetition rate of 10 Hz. A circular metal hole with the diameter of 1 cm was used to select the central part of the laser spot. The samples were irradiated at the fluence of 15 mJ/cm2 with different number of pulses.
2.3 Sample characterization
Sessile drop method was used for water contact angle measurement of the pristine and irradiated samples. A droplet was deposited on the sample by a syringe which is positioned above the sample surface. Then a high-resolution camera captured the image from the profile or side view. The image was measured using image-J software. Each experimental condition was repeated for 3 times and the average value of wettability values was considered. For each value of the reported wettability, there is an error of about ±2 degrees.
A scanning electron microscopy (SEM, Quanta 450) was used for investigation of the surface morphology and bacteria adhesion. The roughness of the samples before and after irradiation was examined by Atomic Force Microscopy (AFM, Iran Arapajohesh, Brisk).
The chemical changes induced on the PC surfaces upon irradiation were investigated with ATR-FTIR spectrometer (Perkin Elmer, Spectrum-100).
2.4 Bacterial adhesion
E-Coli bacteria culture was inoculated into Muller-Hinton broth and then incubated at 37◦ C to an OD600 of approximately 0.5. The culture was diluted 1:3 in sterile phosphate-buffered saline (PBS) for testing the adherence of bacteria to the polycarbonate samples. For each PC sample, discs were cut to fill sequential wells of a 96-well plate. Diluted bacteria cultures (500 microlitres) were added to each well and PBS (500 microliters) was added as a negative control. The plate was incubated at 33◦ C for 1 hour. Before fixation, bacterial cells were washed in PBS and fixed with glutaraldehyde.
Experimental fixation solution was used including 2.5% glutaraldehyde (extra pure grade) in PBS for 20 minutes. Immediately afterwards, an alcohol gradient was used. The samples were placed into 20%, 40%, 60%, 80% and 100% alcohol for 10 minutes each, respectively. After that, they were placed into 100% ethanol and sent for SEM analysis.
3. Results
3.1 Laser irradiation
The fluence for the single pulse ablation threshold of PC samples in 248 nm was obtained experimentally to be about 40 mJ/cm2 [4]. The samples were irradiated at the fluence of 15 mJ/cm2, below the ablation threshold, with 5000, 10000 and 15000 pulses
3.2 Surface wettability
Surface wettability is a key feature in the bactericidal activity of a surface. The findings of the researches about the effect of the surface wettability on the bacteria- surface interaction indicate that super-hydrophilic nanopillared surfaces are superior choice for mechano-bactericidal activity, whereas superhydrophobic surfaces, although not bactericidal, may exhibit antibiofouling properties through their self-cleaning effect [24]. On the other hand, laser processing of a surface, affects the wettability of the sample. The water droplet contact angles on the irradiated surfaces, versus the number of laser pulses at the fluence of 15 mJ/cm2 and the pulse repetition rates of 10 Hz is shown in Fig. 1.
Fig. 1. Water contact angle of the samples irradiated at the fluence of 15 mJ/cm2 and the pulse repetition rate of 10 Hz with different number of pulses.
As it is clear from the figure, the value of the water droplet contact angle decreases drastically with increasing the number of laser pulses and a super-hydrophilic surface is formed with 15000 pulses.
3.2 Scanning electron microscopy and bacterial adhesion analysis
Modification of the surface roughness and morphology can reduce the contact area between the bacteria and the surface to weaken the initial adhesion force. On the other hand, fabrication of a superhydrophobic surface or modification of hydrophilic functional groups may hinder the bacterial adhesion. However, it has been shown the E-Coli bacteria prefer to attach to the hydrophobic surfaces [25].
Fig. 2. SEM images of the polycarbonate samples irradiated at the fluence of 15mJ/cm2 with a)5000, b)10000, c)15000 laser pulses at a repetition rate of 10 H. Some adhered E-Coli bacteria on the surface are determined by red circles.
The non-irradiated and laser treated samples were investigated by scanning electron microscope to check E-Coli bacterial adhesion to the surface. E-Coli is a typical gram-negative rod-shaped bacterium which is cylindrical in size, 1.0-2.0 µm in length, about 0.5 µm micrometers in radius. E-Coli bacteria easily attach to the pristine polycarbonate [4] which is a hydrophobic surface with water contact angle of about 91 degrees.
Figure 2 shows the SEM images of the non-irradiated and irradiated samples at the mentioned irradiation conditions. Laser irradiation leads to morphological changes and formation of nanostructures on the surface. The formation of these nanostructures affects the adhesion of the bacteria to the surface and as it is seen from the figures, the bacterial adhesion to the surface decreases with increasing the number of pulses from 5000 to 1000 and no bacterium is seen on the surface irradiated with 15000 pulses.
3.3 Topography investigation
Fig. 3. AFM images of the a) non-irradiated polycarbonate sample and polycarbonate samples irradiated at the fluence of 15mJ/cm2 with b)5000, c)10000 and d)15000 laser pulses at the repetition rate of 10 Hz.
As it was shown in Fig. 2, nanostructures/roughness are formed on the surface following laser irradiation and as it was mentioned, surface roughness is an important parameter that influence the bacterial adhesion rate on a surface. AFM analysis of the irradiated samples was done to investigated the changes in the surface roughness following laser irradiation. Figure 3 shows images of the topographical features of the irradiated surfaces.
The average roughness of the samples irradiated with 5000, 10000 and 15000 pulses at a repetition rate of 10 Hz is about 56.02, 56.81 and 26.43 nm respectively. These results show that the roughness on the surface for the samples irradiated with 5000 and 10000 pulses is almost the same; while it decreases for the irradiated sample with 15000 pulses. On the other hand, according to the SEM results, the lowest bacterial adhesion is observed for the irradiated surface with 15000 pulses which has the minimum roughness among the irradiated samples.
3.4 Infrared spectroscopy
Chemical property of the surface is another important parameter that affects the wettability of the surface and thus the bacteria-surface interactions. As it was seen from the Fig. 1, laser irradiation led to an increase in the hydrophilicity of the surface.
Attenuated total reflectance (ATR) spectroscopy was done to investigate the formation of polar functional group on the surface. Figure 4 shows IR spectra of the samples irradiated with various number of pulses at the fluence of 15 mJ/cm2 and the pulse repetition rate of 10 Hz.
Fig. 4. Transmission FTIR-ATR spectrum of the polycarbonate samples irradiated at the fluence of 15mJ/cm2 with various number of laser pulses at the repetition rate of 10 Hz.
The absorption bands around 2900 cm−1 and 700 cm-1 correspond to stretching vibration and bending vibration of C-H bond, respectively. The observed bond at around 1490 cm−1 is related to C- H bending vibrations of methylene groups. C-C aromatic bending vibration and C-O stretching vibrations band of ethers occur on 1500-1700cm−1 and 1770cm−1, respectively. Comparison of the IR spectra of the irradiated and non-irradiated samples does not show any significant difference in the IR spectra of the samples. However, the peak intensities change following laser irradiation.
4. Discussion
Bacterial adhesion is a complex physical and chemical process which is affected by properties of the surface and the bacteria. On the other word, the bacteria species and the environment are important factors affect transport of bacteria towards a solid surface. Again, the bacterial mobility determines different forces that lead to its migration to the surface. Gravitational force, Brownian motion and hydrodynamic forces, are responsible for the migration of the motile bacteria to the surface [26–28].
Based on the classical DLVO (Derjaguin, Landau, Verwey, Overbeek) theory, when bacteria reach a surface, the attractive Lifshitz van der Waals (LW) and the electrostatic double layer (EL) interactions, which can be either attractive or repulsive depending on the surface charge, may happen. The strength of these interactions varies with the separation distance between bacteria and substrate surface. There is a secondary energy separation distance of where reversible adhesion occurs [29,30], after that, if the bacterium can move closer to the substrate (overcoming the barrier energy), an irreversible adhesion is formed. Finally, the transition from reversible to irreversible adhesion requires a stronger interaction between the bacteria and the surface. In addition to the bacterial surface appendages, surface properties have great effect on bacterial adhesion during bacterial adhesion process. On the other word, the relationship between bacterial adhesion and surface properties is the basis of anti-bacterial surface design.
Modification of the surface morphology and roughness can reduce the contact area between bacteria and the surface and weaken the initial adhesion force. On the other hand, changing surface wettability can decrease/increase the bacterial adhesion.
As it is clear from the Fig. 1, the wettability of the samples irradiated by laser, decreases drastically. The reason for this can be explained by two important parameters of the surface chemistry and the roughness. The results of the surface characterization with scanning electron and atomic force microscopy, shows the formation of nano-structures on the surface which their roughness vary from ∼ 26 nm to ∼56 nm. On the other hand, the IR spectra of the irradiated samples show no difference with the pure sample in terms of the formation of polar groups. However, on the other hand, according to the Cassie-Baxter or Wensel theory, changing the surface wettability with the surface roughness can be explained by the air trapping or entering the liquid in the grooves on the micro/nanostructures surface. Therefore, here, increasing the surface hydrophilicity despite no changes in the surface chemistry, can be explained by the water penetration in to the formed nano-structures on the surface following irradiation. Generally, superhydrophobic surfaces could hinder the bacterial adhesion [31,32]. But the surface patterning is also a physical surface modification technique, which determines the contact area and the adhesion force between bacterial cell and surface. The interaction between bacteria and patterned surfaces depends on the bacteria properties as well as the geometry and the size of the structures on the surface [33]. The result of this experiment shows that the adhesion of E-coli bacteria to the surface decreases after laser irradiation. However, the results are greatly influenced by number of laser pulses. The results of the AFM analysis show that, although the average size of the nanostructures is almost the same for the samples irradiated with 5000 and 10000 pulses, it decreases for the sample irradiated with 15000 pulses. In addition, the distribution of the nano-structures is different for samples irradiated with 15000 pulses, which may affect the surface contact area and energy and then the bacteria- surface interaction forces.
It is important to note that, killing the bacteria is may be occurred due to the surface bacterium interactions. As an example, in bacteria interaction with the nanostructured surface, the bacteria tend to increase the contact area with multiple points of anchoring and settle on the nanostructures. In this stretching process, bacterial wall rupturing can take place when the wall of the bacteria reaches the threshold limit of the pressure induced by the nanostructures [34]. It means that, sharp nano-scale structures can pierce the bacterial wall upon contact or rupture it.
However, the anti-bacterial behavior of the irradiated surface with the lowest size of nano-structures, compared with non-irradiated surface with minimum roughness and irradiated surfaces with bigger size of nano-structures shows that there is a relation between the size of the roughness and the antibacterial properties of the surface, which is not related to the size of bacterium but may be related to the bacterial surface appendages.
Table 1 show the results for laser irradiation of PC samples with different irradiation conditions. At it can be seen from the table, there are two different regimes for antibacterial properties of the surface. The adhesion of the bacteria to the surface is hindered for super-hydrophobic and super-hydrophilic surfaces regardless of the surface morphologies. However, no changes in the chemistry of the surface (regarding to the formation of the polar groups) according to the IR spectra of the samples shows that the main factor for changing the surface wettability could be the formation of micro/nano structures. Again, according to the various surface energies obtained for the antibacterial surfaces, the reason for antibacterial property of the micro-structured surfaces with super-hydrophobic properties can be explained with entrapping the air pockets between the structures and the hindering the surface- bacteria interaction [35]. On the other hand, rupturing the bacterium following interaction with nanostructures formed on the super-hydrophilic PC may be the reason for antibacterial properties of the surface.
Table 1. Results of laser irradiation of polycarbonate samples with different irradiation conditions
5. Conclusion
Due to the many problems of bacterial attachment to the surfaces followed by biofilm formation and increasing the human diseases, antibacterial surfaces fabrication in an efficient and essential work. The surface wettability, morphology and roughness are supposed to be the most effective parameters affect the bacterial adhesion on the surface. The laser treatment as a standard method can change the surface properties directly and has very good potential for reducing the bacterial adhesion to the surface.
In this paper, antibacterial polycarbonate surface was fabricated by nanosecond laser treatment. Both chemical and morphological changes of the irradiated samples were investigated. The results show that the main factors affect the bacterial adhesion reduction of the irradiated sample are morphological changes and nano-structure formation which may lead to long-last antibacterial properties of the surface time.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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