Antibacterial effects of carbon quantum dots@hematite nanostructures deposited on titanium against Gram-positive and Gram-negative bacteria

Thin films of nanostructured hematite (α-Fe 2 O 3 ) and carbon quantum dots-incorporated hematite (CQDs@α-Fe 2 O 3 ) were uniformly grown on titanium substrate and their antibacterial properties against Gram-positive ( S. aureus ) and Gram-negative ( E. coli ) bacteria were studied under dark and illumination conditions. The surface morphology of the samples was investigated with FE-SEM and HRTEM. The antimicrobial investigations demonstrate that α-Fe 2 O 3 and CQDs@α-Fe 2 O 3 are toxic to the selected microorganisms and the samples exhibit sustainable antibacterial activity against Gram-positive bacterial strains compared to Gram-negative bacteria mainly due to the presence of extra outer membrane layer in Gram negative bacteria. Based on the results of the antibacterial activity of α-Fe 2 O 3 and CQDs@α-Fe 2 O 3 under dark and the light irradiation conditions, it was concluded that the preferred mechanism of bactericidal activity of hematite-based thin films is both the penetration of iron cations into the bacteria cell via their membrane and the generation of reactive oxygen species. These results indicate that CQDs@α-Fe 2 O 3 nanoparticulates provide insight into the development of visible-light antimicrobial materials for their potential bactericidal applications.


Introduction
Increasing bacterial resistance through evolutionary processes towards conventional antibiotics such as penicillin and amoxicillin is a major global health concern in current era [1,2]. These antibiotics are chemically modified natural compounds including β-lactams and are the backbone to combat contagious diseases. Although these antibiotics have transformed modern medicine and saved millions of lives [2], however, the broad use and abuse have resulted in emergence of bacterial resistance and adverse side effects [3]. So, the treatment of bacterial infections utilizing these classical antibiotics is certainly becoming more serious global problem [1,4].
In this regard, among all the new developed strategies, the use of nanostructured metal oxides can be emerged as a promising alternative with very distinct pathways of bactericidal action [5][6][7][8]. In fact, owing to their multiple mechanisms of bactericidal action, metal oxides would serve as a new class of effective disinfectants with different mechanism compared to the conventional antibiotics. In addition, besides to their use as antimicrobial compounds, they may have diverse applications in food packaging as well as the coating agents on medical devices and implants to make them resistant against microbial infections [3]. Titanium-based alloys are biocompatible and are finding ever-increasing applications as biomaterials in orthopedic implant materials and devices due to their excellent mechanical and biological performance [9].
The mechanism of the bacteria killing under the irradiation of semiconductor-based materials is mainly the photogeneration of electron-hole (e --h + ) pairs at the surface of illuminated sample.
The electrons in the conduction band of the semiconductor can react with molecular oxygen and produce superoxide anion (O2 •-) through a reductive process, and holes in the valence band can oxidize water to generate reactive hydroxyl radicals ( • OH) [23]. However, the disadvantage of using TiO2 and ZnO is their wide bandgap that UV light is required to activate the photocatalyst and initiate the killing of the bacteria. So, from this point of view, the investigation of the antibacterial activity of the visible light active materials such as hematite is important.  [3,32,33]. So, they can locally kill bacteria or slow down their growth, without being in general toxic to surrounding tissue. Although the antibacterial activity of maghemite and magnetite have been widely studied, the bactericidal action of hematite is still under investigation. Interestingly, it was reported that hematite is nontoxic to beneficial soil microbes and does not cause lysis of red blood cells [36]. Finally, hematite have already been approved by the Food and Drug Administration (FDA) for food and medical applications [3,27,28]. So, it seems that there is still a sustained interest in the development of antibacterial activity of hematite-based materials under dark and the illumination conditions.
In this work, regarding the above-mentioned issues on α-Fe2O3 and CQDs and based on our

Fabrication of Ti/α-Fe2O3 and Ti/CQD@α-Fe2O3
For the synthesis of carbon quantum dots (CQDs), an electrochemical method was used as reported elsewhere [46] by using two graphite rods as anode and cathode. Briefly, the graphite electrodes were immersed in a 100 ml solution of ethanol:H2O (99:1) containing 0.30 g of sodium hydroxide (NaOH), and a constant current intensity of 180 mA cm -2 was applied for 24 h. The obtained soluble CQDs were then separated by silica-gel column chromatography after the treatment with MgSO4 [46].
Ti/α-Fe2O3 and Ti/CQD@α-Fe2O3 samples were fabricated by the hydrothermal method as reported in our previous work [35].

Antibacterial test
The antibacterial activities of the Ti/α-Fe2O3 and Ti/CQD@α-Fe2O3 samples against Grampositive Staphylococcus aureus (S. aureus, ATCC 6538, PTCC 1112) and Gram-negative Escherichia coli (E. coli, ATCC 25922, PTCC 1399) bacteria were studied under dark and illumination conditions by using an antibacterial drop test. 100 µL of saline solution containing bacteria was spread on the surface of each sample in a sterilized Petri dish in incubator. After 24 h, the bacteria were transferred to a nutrient agar plate and incubated at 37 ºC for 24 h for counting the surviving bacterial colonies using an optical microscope. The tests were also examined under the illumination conditions. After the spread of saline solution containing bacteria on the surface of sample, the sample was irradiated by a 30 mW cm -2 tungsten lamp at room temperature in incubator. Finally, after 24 h light irradiation, the bacteria were transferred to a phosphate buffer saline solution and the bacterial suspension was spread on a nutrient agar plate and incubated at 37 ºC for 24 h for counting the surviving bacterial colonies. The reported data were the mean values of three distinct experiments. The uncoated Ti sheet was used as a control sample in all the antibacterial tests.

Bactericidal effects of samples against S. aureus and E. coli under dark conditions
The bactericidal activity of the prepared Ti/α-Fe2O3 and Ti/CQD@α-Fe2O3 samples was assessed against two pathogenic bacterial strains including S. aureus and E. coli under dark conditions. As Table 1 shows, the bactericidal activity of Ti/α-Fe2O3 against E. coli was low (~10%).

Fig. 2 and
In contrast, about 65% of the S. aureus bacteria was inactivated by Ti/α-Fe2O3 sample. By using Ti/CQD@α-Fe2O3 sample, about 20% of E. coli and 70% of S. aureus bacteria were inactivated under dark conditions. The higher resistance of Gram negative strain against hematite-based compounds and more antibacterial activity of the samples against Gram positive bacterial strains is mainly due to the presence of extra outer membrane layer in Gram negative bacteria [27]. So, hematite has more prominent toxicity against Gram positive bacterial strains and the Gram negative strain has higher resistance against hematite.  under the light illumination, it is expected that the higher amounts of ROS would be generated and consequently, lower percent of the bacteria would be survived. The results ( Fig. 4 and Table   1) showed that the inactivation of E. coli with Ti/α-Fe2O3 under the light illumination is about 50% higher than over dark conditions. Also, the photoinactivation of E. coli with Ti/CQD@α-Fe2O3 sample under the light illumination is higher compared to dark conditions (Table 1)    As we reported recently in our previous work [35], under the light illumination, CQDs as conductive nano-scaffold have the pivotal role in CQD@α-Fe2O3 to overcome the short holediffusion length and poor hole mobility limitations of hematite. So, higher amounts of holes can diffuse toward the surface of Ti/CQD@α-Fe2O3 sample and consequently, higher amounts of reactive • OH radicals will be produced at the surface of Ti/CQD@α-Fe2O3 sample. In fact, based on these studies, the probable mechanism is the h + transfer (or h + hopping) from valence band (VB) of α-Fe2O3 to the HOMO of CQDs. Fig. 5 shows the bacteria inactivation kinetics by Ti/CQD@α-Fe2O3 sample under visible light irradiation.

Fig. 5
The FE-SEM images of native and treated S. aureus and E. coli bacteria strains with Ti/CQD@α-Fe2O3 sample are shown in Fig. 6. The iron cations can penetrate into the bacteria cell via their membrane. So, it can be concluded that the prevailing mechanism is the diffusion of Fe 3+ ions inside the bacteria and producing ROS radicals.

Fig. 6
To investigate the stability of Ti/CQD@α-Fe2O3 samples in antibacterial tests, the experiments were performed for three cycles. Based on the obtained results, E. coli and S. aureus were inactivated 31±6% and 78±5%, respectively, indicating that the sample had almost its bactericidal activity and the bacterial inactivation kinetics are not slow down. These obtained results indicate that the fabricated samples are promising candidates for antibacterial applications in food packaging and orthopedic implants.