An antibacterial platform based on capacitive carbon-doped TiO2 nanotubes after direct or alternating current charging

Electrical interactions between bacteria and the environment are delicate and essential. In this study, an external electrical current is applied to capacitive titania nanotubes doped with carbon (TNT-C) to evaluate the effects on bacteria killing and the underlying mechanism is investigated. When TNT-C is charged, post-charging antibacterial effects proportional to the capacitance are observed. This capacitance-based antibacterial system works well with both direct and alternating current (DC, AC) and the higher discharging capacity in the positive DC (DC+) group leads to better antibacterial performance. Extracellular electron transfer observed during early contact contributes to the surface-dependent post-charging antibacterial process. Physiologically, the electrical interaction deforms the bacteria morphology and elevates the intracellular reactive oxygen species level without impairing the growth of osteoblasts. Our finding spurs the design of light-independent antibacterial materials and provides insights into the use of electricity to modify biomaterials to complement other bacteria killing measures such as light irradiation.

space between the two TNT-C pieces were filled with the LB medium and the two samples were connected to the respective power supplier. In the DC charging system (IT6123, ITECH, Nanjing, China), the sample connected to the anode was named DC+ and the sample connected to the cathode was named DC-. After charging with DC at 2 V for 15 min, the samples were removed and put on a petri dish before 400 µL of the bacteria solution were spread on the surface for the following antibacterial process. For the AC treatment, the TNT-C pieces were connected to the two ends of the AC (FY3200S, FeelTech, Shenzhen, China) power system with no discrimination. In the AC charging system, both charged samples were named AC and used for the subsequent antibacterial tests as mentioned above.
With reference to the electrochemical results, the CV curves were acquired between the range of -1~1 V (Fig. 2a) and 2 V was selected as the charging voltage to assess the performance. The post-charging antibacterial rates of TNT-C-15 triggered by AC at different frequencies showed no significant differences (Supplementary Figure 9). 50 Hz was selected as the model AC power supply because it is commonly used.

Antibacterial effects of TNT-C-15 on Staphylococcus epidermidis and
Pseudomonas aeruginosa. Two additional strains of bacteria (S. epidermidis, pAO1 and P. aeruginosa, clinical isolate) were included in our study to confirm the capacitance-dependent antibacterial effects of TNT-C-15. In brief, the pure bacteria in LB were cultivated overnight in a rotating shaker at 37 ºC, twice diluted, and cultivated to a concentration of 2-3×10 9 CFU mL -1 (OD 600 =0.3 for S. epidermidis and OD 600 = 1.0 for P. aeruginosa). The bacteria solution with a concentration of 2-3×10 5 mL -1 was prepared for the subsequent antibacterial test.

Anti-biofilm tests. E. coli were cultivated on various samples up to 48 h.
During the process of bacteria cultivation, the samples except TNT control were charged every 8 h. In the quantitative analysis, the specimens were gently rinsed in PBS, stained by 0.1% crystal violet for 20 min, rinsed in a deionized water bath, and the bound crystal violet was eluted by 1 mL of 100% alcohol. Afterwards, the optical density of eluates was determined on a multimode reader (BioTek, US) at 590 nm 1 . In the qualitative analysis, the samples with adhered bacteria were fixed and dehydrated prior to SEM observation. Besides, the biofilms were stained with the LIVE/DEAD® BacLight™ Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR) before the 3D morphology was examined by confocal scanning laser microscopy.

Cyclic antibacterial tests.
After each cycle of post-charging treatment, the bacteria were collected from the samples and the samples were recharged for another 15 min. Afterwards, the pre-collected bacteria were again spread on the charged samples to evaluate the cyclic antibacterial effect. The CFU counting method was used to quantitatively calculate the antibacterial rates and the detailed antibacterial procedures are described in the Methods section. 5. Recyclable platform as capacitive materials. After the first antibacterial process, the samples were cleaned ultrasonically in acetone, alcohol, and deionized water, and dried in nitrogen to remove the remained bacteria. CV was carried out from -1 V to 1 V at a scanning rate of 0.1 V s -1 and GCD tests were performed at a constant charging current of 2.5 mA cm -2 .
6. Membrane potential test. The membrane potential of the bacteria was measured with a membrane potential kit (B34950, Invitrogen, USA). The bacteria treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) served as the positive group for membrane destruction. After the post-charging treatment, 4 µL of DiOC 2 (3) (3,3′-diethyloxacarbocyanine Iodide, 3 mM) were added to the sample 13 and after 15 min, the bacteria were collected and assayed by flow cytometry. An excitation wavelength of 488 nm was used to excite DiOC 2 (3) and the green and red fluorescence was monitored simultaneously using the 530 nm and 610 nm bandpass filters, respectively.
The degree of membrane depolarization was characterized by the red/green fluorescence ratio 2 .