## 3 Discussion  
The global burden of antibiotic resistance continues to grow, and the development of new antibacterial agents has been inadequate in tackling this growing threat. In particular, _P. aeruginosa_ is not only a leading opportunistic human pathogen but also a prevalent food spoilage bacterium in various food products, especially in those with higher water content [38]. Herein, we utilize aPDI via the excitation of CHL with red light (\(\uplambda\) 623 nm, 295.55 J/m\({}^{2}\)/s) individually and together with PEI against _P. aeruginosa_ planktonic cells, biofilm and persister cells. This study demonstrates that PEI enhanced the efficiency of CHL in the eradication of the planktonic free cells and biofilm cells, and more than fourfold of the antimicrobial concentrations (CHL/PEI > 500/100 ug mL\({}^{-1}\)) are required to eradicate the bacteria in biofilm (>99.99%, >4 log\({}_{10}\) reduction) compared to planktonic cells (CHL/PEI 125/100 ug mL\({}^{-1}\)). In addition, the CHL/PEI 125/100 ug mL\({}^{-1}\) combination is capable  
Figure 9: Influence of different concentrations of chlorophyllin (CHL) and 100 ug mL\({}^{-1}\) polyethyleneimine (PEI) on growing and persister cells after exposure to red LEDs for 15 min (**a**) and 30 min (**b**). The dashed line corresponds to a 4 log\({}_{10}\) reduction (99.99% killing efficacy). Data represent the mean value of three biological replicates. Error bars represent the standard deviations. \(\star\)_t_-test: _p_-value < 0.05.  
of detaching the established biofilm by approximately 76%. Surprisingly, the CHL/PEI (31.25/100 \(\mu\)g mL\({}^{-1}\)) combination was more effective against persister cells compared to the growing cells by 1.7 \(\pm\) 0.12 and 0.4 \(\pm\) 0.1 log\({}_{10}\) reduction, respectively, after 15 min of exposure to red LEDs, suggesting that the combinations can overcome the tolerance caused by the dormant cells. CHL is a negatively charged PS and has electronic absorption at longer wavelengths (660 nm), as shown in Figure S1. Thus, the long absorption wavelength allows CHL to be a potential candidate for deep penetration [28]. Under the light irradiation of PS with an appropriate wavelength, the PS can undergo an electron transfer reaction (Type I mechanism) with triplet (ground-state) oxygen (\({}^{3}\)O\({}_{2}\)) to produce reactive oxygen species (ROS) such as superoxide radical anion O\({}_{2}\)\({}^{-}\), hydrogen peroxide H\({}_{2}\)O\({}_{2}\), hydroxyl radical HO\({}^{\bullet}\) or energy transfer reactions (Type II mechanism) to activate (ground-state) oxygen to form singlet (excited-state) oxygen (\({}^{1}\)O\({}_{2}\)) [39]. Singlet (excited-state) oxygen has a short half-time of about 200 ns in cells [40], ranging from 100 ns in the lipid areas of membranes to 250 ns in the cytoplasm [41]. The possible diffusion distance of \({}^{1}\)O\({}_{2}\) in a biological system could be up to 10 nm [40]. Therefore, it reacts with the immediate neighborhood target molecules [40]. The chlorophyll triplet state has an even longer lifetime (a few \(\mu\)s) and under O\({}_{2}\)-saturated conditions, can react with \({}^{3}\)O\({}_{2}\) to produce the very reactive \({}^{1}\)O\({}_{2}\) if no efficient quenchers are around [40]. [PERSON] and colleagues [42] found that the irradiation of CHL with visible light for 30 min generated ROS including \({}^{1}\)O\({}_{2}\) and HO\({}^{\bullet}\). Furthermore, [PERSON] and colleagues [24] found that a tiny part of CHL bound to _Salmonella enterica_ serovar Typhimurium and the presence of an \({}^{1}\)O\({}_{2}\) scavenger (sodium azide NaN\({}_{3}\)) protected the cells from the phototoxic effect of CHL. It is well documented that the outer membrane of the Gram-negative bacteria acts as a physical barrier against PSs reaching the cell membrane.