Bactericidal effect derived from nanostructured surface was evaluated in the point of view of the motility of E. coli. The results suggest that the properties strongly depend on bacterial motility.
Fine particle bombarding (FPB) is typically utilized to modify metal surfaces by bombarding them with fine particles at high-speed. The diameters of the particles range from several to tens of micrometers. FPB forms fine microscale concavities and convexities on a surface. As FPB-treated surfaces are widely used in the food industry, the influence of bacteria on their surface must be considered. In this study, we examined the antibacterial activity of microscale rough surfaces formed by FPB. We applied FPB to a stainless-steel surface and evaluated the antibacterial effect of FPB-treated surfaces based on JIS Z 2801 (a modified test method from ISO 22196:2007). Our results indicated that the FPB-treated surfaces (FPB-1 (avg. pitch: 0.72 µm) and FPB-2 (avg. pitch: 3.56 µm)) exhibited antibacterial activity both against Escherichia coli and Staphylococcus aureus. Graphical Abstract
In recent years, biomimetics or biomimicry have been paid attention which focus on biotic micro and nano structures. These structures show excellent and multiple functions. It is reported that the wings of cicada and dragonfly have countless nanostructures which exhibit antibacterial and bactericidal activities. These structures kill bacteria physically1-3). One of acceptable mechanism of bactericidal effect is proposed as below: the cell membrane is stretched by the interaction between the structure and bacteria, which induce the cell membrane to be deformed. Then the intracellular fluid leaks which make the cell death4). However, the detailed mechanism has not been cleared. In this study, electrochemical impedance spectroscopy (EIS) measurement and fluorescence microscopy were performed simultaneously to analyze the mechanism of cell adhesion and deformation on the nanostructure surface. The experimental method is described as below. A working electrode (WE) with the diameter of 100 µm was fabricated on a Si substrate. The WE had gold nanostructure arrays which were fabricated by using electron beam lithography and pulse deposition. In order to observe the surface of the WE by a fluorescent microscope, a counter electrode (CE) composed of Au thin film and a reference electrode (RE) were formed on a glass substrate by using photolithography and sputtering. Here, Ag/AgCl paste was solidified and used for the RE. These substrates were opposed to each other as shown in Fig. 1-a, and silicon rubber with a sample chamber with a thickness of 500 µm was sandwiched between these substrates and fixed with screws. An electrolyte solution (PBS (pH = 7.4) containing NaCl (0.15 M) and Fe(CN)6 3- / 4- (1 mM)) was injected through a hole provided on the glass substrate. Escherichia coli (E. coli) was used as a model bacteria and concentration of E. coli was adjusted to OD600 = 0.2. We used DNA staining reagent of SYTO 9 and PI. SYTO 9 diffuses into the cell cytoplasm through the cell membrane and stains DNA green. In contrast, PI enters the cell cytoplasm and stains DNA red when the cell membrane is damaged. Therefore, adhered cells without membrane damage were colored green. After mixing the bacterial broth and the electrolyte solution, the mixture was injected through a hole Then, AC impedance measurement and time-lapse observation using the fluorescence microscope were simultaneously performed every 5 minutes to evaluate changes over time. The obtained fluorescence images are summarized in Table 1, and Fig. 1-b shows the change rate of Rct over time. From Table 1, the number of E. coli stained red increased with the lapse of time on the pillared electrode. E. coli stained green was hardly observed. On the other hand, microscopic observation showed adsorption and desorption of E. coli which stained green on the flat electrode, Here, E. coli stained red could not be observed. According to Fig. 1-b, it was found that the change rate of Rct on the pillared electrode increased gradually and was much larger comparing with that on the flat electrode. In the case of the flat electrode, it is considered that the increase and decrease of impedance was due to adsorption and desorption of E. coli, respectively. In the case of the pillared electrode, the cell membrane was damaged and died after E. coli attached on the WE. The impedance continued to rise because E. coli died and remained on the electrode. At this time, only the attachment process of E. coli could be analyzed. In the future, we will aim for single-cell level analysis by reducing the electrode diameter. We plan to make measurements closer to real time and report more detailed analysis results. 【 References】 1) E. P. Ivanova, et al.:Small 8, p. 2489-2494 (2012) 2) E. P. Ivanova, et al.:Nature Communications 4, p. 1-7 (2013) 3) A. Tripathy, et al.:Advances in Colloid and Interface Science, 248, p. 85-104 (2017) 4) K. Nakade, et al.:ACS Applied Nano Materials, p. 5736-5741 (2018) Figure 1
Liposome is well known as mimicking a cell, which is composed of lipid bilayer membrane. Liposome is spherical shape and formed by amphiphilic molecules, meaning that outer layer is composed of hydrophilic molecules and inner layer is composed of hydrophobic molecules. Evaluation of adsorption and rupture of liposome on a surface is very important to drug delivery system (DDS) or understand the capability of the cell. Many studies reported the adsorption and interaction of lipid bilayer using highly sensitive sensing methods without labeling such as quartz crystal microbalance (QCM), plasmonic devices such as SPR, LSPR and SERS, and electrochemical impedance spectroscopy (EIS). We focus on EIS as an evaluation tool for adsorption and rupture of liposome, since EIS is highly sensitive to the surface condition of the electrode and electrochemical measurement is simple. EIS is very sensitive to the surface condition of the electrode. We report here that surface charge is very important to evaluate the adsorption and interaction of liposome on the electrode surface, which are controlled by using self–assembled monolayer (SAM). So far as we know, there are little reports focusing on surface charge evaluate the adsorption and interaction of liposome on the electrode surface. Here, we report the effect of surface charge on electrochemical reactions due to SAMs which were coated on the Au electrode. Liposome preparation was described below. Thin organic film was formed with 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). Then we obtained solution including DOPC which diameter was lower than 220 nm because the solution was filtered 20 times through the syringe filter (pore diameter was 220 nm). We used 1-Dodecanethiol (DDT), 11-Mercapto-1-undecanol (MUD or 11–HUT: 11–Hydroxy–1–undecanthiol) and 11-Amino-1-undecanthiol (11–AUT) for the SAM. These chemicals have same straight–chain alkane with thiol group to bind with Au surface, but head group is changed as CH3 for DDT meaning that the surface is neutral and hydrophobic, CH2OH for MUD meaning that the surface is charged negative and hydrophilic and CH2NH2 for 11–AUT meaning that the surface is charged positive and hydrophilic. Thus, we can discuss about the characteristics of Rct dependent with head group of SAM on the detection of liposome adsorption and rupture. As the results, most sensitive surface was obtained on use of 11–AUT in this study. In addition, we estimated the critical micelle concentration (CMC) of a surfactant, Triton–X 100, on the rupture process of liposome. As an example of results, we show two type of hydrophilic surface, type A and B. Type A was coated with 11-Mercapto-1-undecanol, which surface had hydroxy group and was negatively charged. Type B was coated with 11-Amino-1-undecanol, which surface had amino group and was positively charged. At first, we discussed about the results on type A. Fig. 1(Type A) shows Nyquist plots according to adsorption and rupture of liposome. After the adsorption of liposome, Rct decreased drastically. This characteristic was caused by surface charge after the liposome adsorption. As written above, the hydroxyl surface was charged negatively which repel the redox probe. After the liposome adsorption, surface charge changed to positive because the surface of DOPC was charged positively, which attracted the redox probes by charge polarity. After adding the surfactant, Rct increased and was almost same as that before the liposome adsorption. Next, we discussed about the results on type B. Fig. 1(Type B) shows Nyquist plots according to adsorption and rupture of liposome. After the adsorption of liposome, Rct increased drastically due to steric barrier of liposome. In this case, liposome adhered on the surface by hydrophilic interaction. Surface charge was not changed via liposome adsorption. After adding the surfactant, Rct decreased and was almost twice comparing with that before the liposome adsorption. This data show that ruptured liposome and/or surfactant adhered on the electrode surface. As discussed above, the amino surface was superior to detect adhesion and rupture of liposome comparing with other electrodes which coated with/without methyl and hydroxy SAM, when [Fe(CN)6]3-/4- was used as redox probes because of mixed surface conditions such as hydrophilicity and electric charge. Figure 1
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.