## 4 Discussion  
In this research, we investigated the antibacterial and antibiofouling activities of CPD/PU and C\({}_{60}\)/PU composite films. Apart from these properties, we investigated their structural, chemical, electrical and optical properties. Structural analysis of nanoparticles encapsulated into polymer films conducted by AFM showed that the CPD nanoparticles had a core-shell structure and an average diameter of 33 nm. Electrostatic force microscopy enabled the determination of the charge type, content and distribution of the CPD nanoparticles and C\({}_{60}\) clusters and showed that both filler particles were located in the interior of the polymer matrix. It was found that CPDs have much more electrons than the cluster of C\({}_{60}\) molecules located in the nearly identical polyurethane pore. Before antibacterial and antibiofouling testing, we performed the measurements of ROS production by two methods: (1) by using SOSG as a fluorescence probe and measuring its PL at 530 nm and (2) by using ABDA color and measuring decolorization vs. time. It was established that CPDs/PU and C\({}_{60}\)/PU generated singlet oxygen. Furthermore, it was found later that CPD/PU composite films also produced hydroxyl radicals. None of these composite films showed cytotoxicity against HaCaT cell lines at concentrations ranging from 12.5 to 50%. Antibacterial testing showed that both composite films had strong antibacterial activity against investigated bacteria strains under blue light irradiation for 1 h.  
The obtained results indicated that the CPD/PU composite films produced both singlet oxygen and hydroxyl radicals under blue light irradiation at 470 nm. The mechanism of ROS generation is related to negative charge transfer from photosensitizers (CPDs) to oxygen molecules. CPDs are produced from riboflavin and carbon molecules with abundant nitrogen atoms. During the carbonization process in a hydrothermal reactor, nitrogen-doped CPDs are produced. Nitrogen is a rich source of electrons that transfer charge to carbon, which is detected by EFM in the form of an electron cloud over a CPD surface. Similar to an atom, the greatest density of electrons is in the center of CPDs. Since polyurethane is a polymer with the highest oxygen diffusion rate among polymers, oxygens easily collide with CPDs, transferring to one of two detected ROS species and diffusing to the polymer surface [52]. On the other hand, during blue light irradiation of the C\({}_{60}\)/PU samples, only singlet oxygen was generated. Since the C\({}_{60}\) cluster charge was much smaller than for CPDs, the dominant way for singlet oxygen generation is energy transfer from C\({}_{60}\) molecules encapsulated in the interior of polymer films to molecular oxygen. C\({}_{60}\) is composed of pure carbon, and there is no dopant to donate extra electrons. Thus, we prepared two types of composite films with different mechanisms of ROS generation, and it would be interesting to monitor their antibacterial and antibiofouling activities on different bacterial strains. It is very interesting to emphasize that C\({}_{60}\)/PU composite films eradicated almost completely _S. flexueri_ for 1 h. _S. flexueri_ is a Gram-negative intracellular pathogen responsible for bacillary dysentery in humans [53]. Per year, more than 1 million deaths occur due to infections with _Shigella_ spp. The victims are mostly children from the developing world. The pathogenesis of _Shigella_ is based on its ability to invade the colonic epithelium, where it induces severe mucosal inflammation. We supposed that the antibacterial activity of CPD/PU composite films is predominantly based on ROS generation and further disruption of the bacterial membrane. The C\({}_{60}\)/PU composite films interacted with bacterial membranes by electrostatic forces, and the production of singlet oxygen induced higher membrane permeation compared with the CPD/PU composite films and further bacteria cell death. Antibiofouling testing showed that the CPD/PU composite films had potent activity against _P.