3.3 Photodynamic antimicrobial activity of the CDs and effect on
ROS levels
It has been reported that CDs with superior optical property can induce
phototoxic reactions under light irradiation, which we think may be
capable of photodynamic killing of bacteria.[34]To this end, we further investigated if the antibacterial effect of the
CDs could be enhanced under 365
nm-UV light (Figure 4a ). The result show that compared with
that with the non-luminescent CDs (Figure 4b ), the UV light
irradiation (Figure 4c ) made a negligible light effect on the
survival rate of E. coli ;
while the bacterial survival rate was greatly reduced under light
irradiation. It has been indeed reported that the CDs can initiate the
efficient charge separation under
photoirradiation.[35] Therefore, it is deducible
that the photoinduced redox potential facilitates the transfer of charge
or energy to a substance or to molecular oxygen to produce more ROS, and
causes aggravated damage to the bacteria.[34, 36]
In addition, the survival rate of E. coli strongly depends on CD
concentration and incubation time. The higher the concentration of the
CDs, the lower the survival rate. A similar trend was confirmed with
increasing incubation time. The reduced survival rate of E. coliwith increasing the CD amount and incubation time can be rationalized by
the sufficient production and accumulation of ROS under the conditions.
Similar results were attained for
the S. aureus group under dark (Figure 4d ) or UV light
irradiation (Figure 4e ).
Namely, the survival rate ofS. aureus is also dependent on the concentration and incubation
time of the CDs. Therefore, photoirradiation can effectively boost the
antibacterial ability of the CD, regardless of the type of bacteria.
As the luminescence of the CDs under light excitation associate with
recombination of photoinduced electrons and
holes,[37] a high quantum yield of the CDs may
lead to further enhanced antibacterial effect. We next produced CDs in
parallel using PEI of different molecular weights, that is 2 kDa, 10 kDa
and 25 kDa, respectively, and their quantum yields were calculated to be
5.7, 18.6, and 21.3% (Figure S5 ), respectively. Interestingly,
the CDs with a higher quantum
yield exhibited a stronger antibacterial effect under the identical
treatment conditions, which has been validated for both E. coli(Gram-negative) and S. aureus (Gram-positive) (Figure
S5 ). This can be ascribed to the fact that, upon absorption of photons,
the CDs with a higher quantum yield can produce more electrons and
holes. This behavior could promote energy transfer to produce active
substances such as ROS, exerting greater killing
effects.[38]
We next became interested in the molecular mechanism of the photodynamic
killing by the CDs.
To
test whether the CDs generate ROS under light irradiation, which is the
mostly considered cause of bacterial
death,[39]DCFH-DA was used to serve as the probe to detect intracellular ROS,
which can be oxidized into highly fluorescent 2’,7’-dichlorofluorescein
by ROS.[40] Our results reveal that compared with
the E. coli group treated in the dark, the CDs under
light irradiation produced more
ROS, and the generation of ROS was boosted with increasing doses of the
CDs (Figure 4f ). Similar results were attained for the S.
aureus group (Figure 4g ). To test whether1O2 is involved in the produced
ROS,[41] DBPF as the indicator was employed. The
probing mechanism for 1O2 lies in its
decomposition by DBPF into 1,2-dibenzoylbenzene, leading to a decrease
in UV absorbance intensity.[42] As shown inFigure S6 , the absorption peak of the mixture of the CDs and
DPBF at 410 nm decreases significantly as the irradiation time
increases; whereas no change in the absorbance of the mixture was
attained in the dark. We further found that neither the pure CDs nor
DPBF exhibit a decay in absorbance at 410 nm, regardless of whether
there was a light (Figure 4h ). The results point to the
generation of 1O2 by the CDs under
light irradiation. Indeed, the CDs undergo charge separation upon
photoexcitation, and generate electrons and holes on the CDs to drive
redox reactions listed below, a phenomenon similar to those observed in
semiconductor nanomaterials.[43]
\(CDs+hv\rightarrow CDs+h^{+}+e^{-}\) (ⅰ)
\(H_{2}O+h^{+}\rightarrow H^{+}+\ \bullet OH\) (ⅱ)
\(O_{2}+e^{-}\rightarrow\ O_{2}^{\ -}\)(ⅲ)
\(O_{2}^{\ -}+2H^{+}+\ e^{-}\rightarrow\ H_{2}O_{2}\) (ⅳ)
\(H_{2}O_{2}+O_{2}\rightarrow 2OH+O_{2}\ \) (ⅴ)