Page 114 - IJB-8-1

Antibacterial Performance of Graphene Oxide-loaded Nickel
2.5. Antibacterial activity assessment The bacterial suspension was diluted to 10 CFU/mL.
6
After the incubation, the bacteria on the samples were
The antibacterial activity was assessed by agar plate collected by PBS (2000 μL). A fluorescence microplate
assessment, and cell live/dead staining was used to reader (Tecan Infinite, 200Pro, Switzerland) was used to
directly observe whether the bacteria were alive or quantitatively determine the intracellular ROS level of
dead. Escherichia coli (E. coli, ATCC25922) and bacteria in PBS. The excitation light wavelength was set
Staphylococcus aureus (S. aureus, ATCC25923) were to 488 nm.
used in this experiment. Luria-Broth was used for
bacterial culture. Prior the experiment, a single colony 2.7. Measurement of nickel ion release
of bacteria was added into the LB broth and cultivated
overnight using a constant temperature shaker (220 rpm, The nickel ion releasing ability of the samples was
37℃) to obtain a bacterial solution for testing. Glass detected, and PBS was used to simulate the living
without antibacterial activity was used as a blank control environment of the bacteria. Samples were immersed
sample, and the samples were disinfected with 75% in PBS (10 mL) for 2 h at 37℃. The concentration of
ethanol for 30 min. nickel ions in the resultant solution was determined by
In agar plate assessment, the bacterial culture inductively coupled plasma mass spectroscopy (Agilent,
solution was diluted to ~1 × 10 CFU/mL with phosphate Agilent 7700/7800, USA).
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buffer solution (PBS). Bacterial suspension (100 μL) 3. Results and discussion
was inoculated on the samples and incubated at 37℃
for 2 h. After incubation, the samples were soaked in 3.1. Surface characterizations of the samples
PBS (30 mL), and shaken using a shaker (220 rpm) for
10 min to rinse and collect the bacteria on the surfaces Figure 3A shows the photos of the samples. The
of the samples. Subsequently, the samples flushing liquid uncoated substrate and the WNC possess a shiny surface.
(100 μL) was introduced to the LB agar culture plate and The introduction of Nip and a magnetic field caused the
cultured for 24 h. GNC and PNC to be darkened. Under the effect of GO in
For cell live/dead staining, bacterial suspension the plating bath, the surface of GNC is darker than PNC.
(100 μL) with a cell density of 2 – 3 × 10 CFU/mL Raman spectroscopy is commonly used to characterize
9
was inoculated on the samples and incubated for 2 h at GO. In the Raman spectrum of the GO, the peaks at
−1
37℃. Such a high concentration of bacteria is helpful 1356 cm corresponding to D band due to the edges and
−1
for observation under a fluorescence microscope. After defects and 1611 cm corresponds to the G band, depicting
[44]
2
the incubation, the bacteria on the samples were stained the in-plane stretching vibrations of C-sp atoms . The
with cell live/dead kit containing 4’, 6-diamidino-2- D and G peaks of GO can be clearly observed on the
phenylindole (DAPI) and propidium iodide (PI), and then Raman spectrum of GNC, but no characteristic peaks of
incubated for 15 min in the dark at 37℃. DAPI can pass GO can be found on WNC and PNC (Figure 3B). The
through intact cell membranes and bind to bacterial DNA. presence of the D and G peaks in the Raman spectrum
confirms the existence of GO on the surface of GNC .
[45]
All of the bacteria were stained with DAPI and fluoresced Figure 3C shows a typical morphology of nickel
blue, while only cells with damaged membranes can be coating with large grains and cellular bulges. Observed
stained as red by PI. Subsequently, PBS (200 μL) was under high magnification, the surface of the WNC is flat
used to rinse excess stain. Finally, a laser microscope
(Olympus, IX83, Japan) was used to observe the and dense. WNC is prepared by the traditional Watts nickel
bath, so WNC possess a low surface roughness. However,
experimental result.
due to the introduction of Nip and a magnetic field, a large
2.6. Intracellular reactive oxygen species (ROS) number of coral-like clusters on the surface of the PNC
assay were observed (Figure 3D). This unique morphology
appear due to the external magnetic field trapping the
2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA) ferromagnetic Nip in the bath and the mutual repulsive
was used to investigate the intracellular ROS levels magnetic force existing between the adjacent clusters .
[40]
in the bacteria collected from the samples. DCFH- According to the principle of minimum energy, the
DA can be transformed into non-fluorescent DCFH by distribution of particles along the lines of magnetic force
intracellular esterase and DCFH can be oxidized by ROS has a tendency of peaking [46,47] . Affected by the tip effect,
into fluorescent DCF. Because the incubation time of the the electric field at the protrusion is more concentrated, the
bacteria on the surface of samples is within 2 h, to record current density is higher, and the deposition speed is faster.
the accumulation of intracellular ROS, DCFH-DA 10 μM Finally, coral-like clusters are formed on the coating. The
is dispersed in the bacterial suspension in advance when gap between the protrusions is approximately 20 μm, and
the bacteria are inoculated on the surface of the samples. the surface roughness of the coating is greatly improved.

100 International Journal of Bioprinting (2022)–Volume 8, Issue 1