QCM-D Study of Adhesion and Deformation of Bacteria Onto the Periodic Nano-Pillar Array Electrode

Abstract

Recently, periodic nanometer-order structures on cicada and dragonfly wings attract attention because of their bactericidal property. These structures kill bacteria physicochemically because cell membrane was stretched between the nanostructures after cell adhesion. However, mechanisms of the bactericidal effect have not been clarified yet. Toward the elucidation of the mechanism, we mimicked the nanostructure of the cicada wing and fabricated the periodic nano-pillar array as an electrode for quartz crystal microbalance dissipation (QCM-D). Then, we monitored frequency shifts (Δf) and resistance shifts (ΔR) according to the bacteria adhesion and bacteria death. Here, Δf changes linearly with mass change due to cell adhesion. ΔR changes linearly with viscoelastic changes on the electrode surface due to adhesion and deformation of adhered cells. We monitored these two parameters in real time on two types of measurement conditions: one was dropped only 2 µL sample solution to estimate the surface conditions until the solution was vaporized (first experiment). The other was set in a well-type jig which was filled with sample solution up to 500 µL and we monitored their shifts for 24 hours (second experiment) after the active cells were added into the jig.The periodic nano-pillar array was fabricated by using electron beam (EB) lithography and pulsed electrodeposition of Au. A quartz crystal coated with Au thin films on the double side (fundamental frequency: 9 MHz) was used as a substrate. Fabricated Au nano-pillars had the pitch of 500 nm, diameter of 280 nm and the height of 400 nm, respectively. The nanostructured surface was coated with self-assembled monolayer (SAM) to control the wettability, it was evaluated as water contact angle (WCA). WCA was changed from 80 ° to 128 ° after coating 1-Dodecanethiol, meaning hydrophobic surface, and was changed from 80 ° to 26 ° after coating Mercapto-1-undecanol, meaning hydrophilic surface. E. coli was used as a model bacterium of gram-negative bacteria. After it was cultivated until OD550=0.6, sample solution including bacteria was prepared as OD600=0.04 diluted it by PBS after a centrifugation. For the first experiment, Δf and ΔR were monitored just after the solution of 2 µL was dropped. In this case, water of 2 µL was also monitored for a reference. As results of reference experiment for three different WCA, ΔR and Δf decreased with increasing WCA. The results indicated that it might be considered as Cassie-Baxter model, meaning that water does not enter the space between the nano-pillars at the hydrophobic surface. On the other hands, it might be considered as Wenzel model at the hydrophilic surface, meaning that water enter the space between the nano-pillars and cling damply to the nanostructure surface. Vaper speed was the maximum on the hydrophilic surface because contact area between liquid and surface was largest among the experimental condition.For the second experiment, 400 µL of PBS was stored in the jig at first, then, 100 µL of sample solution including bacteria was added after the signals attributed to QCM-D became stable. We checked the time dependence resistance shift with/without nano-pillars as shown in Fig. 1(a). ΔR increased over time with nano-pillars, this characteristic means that bacteria adhered on the nano-pillars and deformed serially. However, ΔR stayed constant without nano-pillars, but some periodic changes were found. This characteristic might be due to the adhesion and desorption of bacteria from the electrode surface. Next, we discussed about the results according to the surface wettability as show in Fig. 1(b). Shape of graph for hydrophobic is very similar to that for non-coating surface. Magnitude of ΔR at 24 h on hydrophobic surface is higher than that on non-coating surface. On the contrast, shape of graph for hydrophilic is very different from others: magnitude of ΔR was saturated around 5 h after the sample injection. These results agreed with our reports describing the adhesion and bactericidal characteristics dependent with wettability which were obtained by fluorescence microscopy. In fact, the results indicated that bacteria preferred to adhere the hydrophobic surface because the flagella have hydrophobicity. Figure 1