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Antibacterial Performance of Graphene Oxide-loaded Nickel
were not stained as red by PI. However, almost all E. coli does not exhibit any sharp edges (Figure 3F). GO was
observed on the surface of GNC was stained as red by PI, anchored on the coating and does not appear to be a free
implying that almost all E. coli died. Fluorescence images state, so that the wrapping mechanism or physical cutting
show that GNC can indeed effectively inactivate E. coli has a limited influence on the antibacterial activity. Since
on its surface. The order of the antibacterial activity of the these effects are excluded as the origin of the bacterial
samples against E. coli is consistent with the agar plate activity of GO, the ROS dependent oxidative stress effect
assessment, and GNC has better antibacterial activity and electron transfer may be the antibacterial mechanism
against E. coli than the other samples. of GO on the surface of GNC [19,56-58] .
Similar results obtained for S. aureus are shown in To detect the level of ROS during the incubation of
Figure 6B. The ability of GNC to inactivate S. aureus is bacteria on the coated samples, DCFH-DA was used as
more significant compared with other samples. Most of an indicator of intracellular ROS, as shown in Figure 7.
S. aureus died on the surface of GNC. However, after the In the experiment of two types of bacteria, a low level of
same incubation time, a small part of S. aureus was not ROS was detected in the bacterial liquid recovered from
stained red on the surface of GNC. This shows that the the TI at the two detection points. Such a low level of ROS
S. aureus has not been completely killed S. aureus has a did not show an evident inhibitory effect on the bacteria,
higher cell viability on GNC, which may be due to that which is consistent with the results of antibacterial
Gram-positive bacteria (S. aureus) have thicker cell walls experiment. However, high levels of intracellular ROS
compared to Gram-negative bacteria (E. coli). Combined were detected in WNC, PNC and GNC, indicating that
with the results of the agar plate assessment, although the antibacterial ability of coated samples is related to the
the S. aureus on the surface of GNC was not completely oxidative stress effect of intracellular ROS. The nickel
stained as red, the cell vitality of S. aureus had been lost, ions released by the metallic nickel on the coating may
and the remaining bacteria could not form dense colonies induce the production of ROS. Nickel ions can cause
on the agar plate. It was concluded that GNC can be used cells to produce high levels of intracellular ROS, causing
as an effective antibacterial platform that causes severe damage to cell lipids and DNA, decreasing intracellular
membrane damage to bacteria and causes bacteria death. enzyme activity and leading to cell apoptosis [59-61] .
To further explore the relationship between the
3.4. Effect of ROS and nickel ions release of nickel ions from the samples and the level of
As shown by the above experiments, WNC, PNC, and ROS, an inductively coupled plasma mass spectrometer
GNC all exhibit better antibacterial performance than was used to detect the nickel ion release rate of the
TI. GO was not detected on the surface of WNC and samples. To avoid interference from the culture medium
PNC, so the antibacterial activity of WNC and PNC and the metabolites of bacteria, the same amount of
mainly comes from the metallic nickel of the coating. PBS (10 mL) was used to soak the coated samples to
Due to the incomplete coverage of GO, the bare metallic simulate the incubational environment of bacteria. The
nickel on the surface of GNC may also participate in the results for the concentration of nickel ions are shown
antibacterial process of GNC. Metal materials are widely in Figure 8. After immersing for 2 h, the concentration
used for antibacterial [50,51] . As a traditional inorganic of nickel ions released by WNC was only 4.49 μg/mL,
bactericide method, metal ions have been reported to while the corresponding values for PNC and GNC were
inactivate the oxidase involved in bacterial respiration 10.43 μg/mL and 8.6 μg/mL, respectively. The highest
and induce the production of ROS [52-55] . For GNC, few concentration of nickel ions released from PNC was
layers of GO were used in this experiment, and the GO mainly due to the larger actual contact area between the
on the surface of GNC shows a soft fibrous shape and rough surface of the PNC and the liquid that promoted the

A B

Figure 7. Quantitative analysis of the reactive oxygen species intensity. (A) Escherichia coli. (B) Staphylococcus aureus.

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