Simultaneous Evaluation of Electrochemical Impedance and Cell Membrane Damage to Understand Antibacterial Mechanism on Nanostructured Surface
Authors: Masuda Kyosuke , Oda Kosuke , Fujino Yusuke , Kojima Hiroaki , Yamashita Ichiro , Shimizu Tomohiro , Shingubara Shoso , Ito Takeshi 
Published: December 2020

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.

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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