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)