Superior visible light antimicrobial performance of facet engineered cobalt doped TiO2 mesocrystals in pathogenic bacterium and fungi

Pristine and Co-doped TiO2 mesocrystals have been synthesized via a simple sol–gel method and their antimicrobial activity has been investigated. The antimicrobial performance was evaluated in terms of zone of inhibition, minimum inhibitory concentration (MIC), antibiofilm activity, and effect of UV illumination in liquid media. The Co-doped TiO2 mesocrystals showed very promising MIC of 0.390 μg/mL and 0.781 μg/mL for P. mirabilis and P. mirabilis, respectively. Additionally, the material showed an MIC of 12.5 μg/mL against C. albicans, suggesting its use as antifungal agent. Upon the addition of 10.0 µg/mL of Co-doped TiO2 mesocrystals, the biofilm inhibition% reaches 84.43% for P. aeruginosa, 78.58% for P. mirabilis, and 77.81% for S. typhi, which can be ascribed to the created active oxygen species that decompose the tested microbial cells upon illumination. Thus the fabricated Co-doped TiO2 mesocrystals exhibit sufficient antimicrobial features under visible light, qualifying them for use as antimicrobial agents against pathogenic bacteria and fungi and subsequently inhibit their hazardous effects.

In the last decades, a lot of efforts have been devoted towards the synthesis of nanomaterials with unique physical, chemical, and biological characteristics compared to their bulk counterparts 1,2 . Cölfen et al. first introduced a new class of materials known as mesocrystal 3 . Mesocrystals were proposed to form upon the addition of highly oriented small particles, thus the resulting larger crystals would have single-crystal orientation 4 . Their positive effects in improving charge carriers separation made them good candidates for many applications, such as photocatalysis 5 , sensing, and energy storage and conversion 6 . For instance, TiO 2 mesocrystals become a research hotspot for biomedical and food applications due to their antimicrobial characteristics 7 . Besides, their chemical stability, abundance, low cost, eco-friendly made them good candidates for photovoltaics 8 , hydrogen production 9 and wastewater treatment 10 . Upon irradiated by UV light, the anatase phase of TiO 2 can oxidize and reduce oxygen and water to produce reactive oxygen species (ROS), such as superoxide radicals and hydroxyl radicals 11 . These ROS play a key role in destroying pathogenic bacteria and fungi by damaging their critical molecular components 12,13 . However, the practical application of TiO 2 photocatalysts is limited by their wide bandgap energy (3.0-3.2 eV), limited to the UV region of the light spectrum with low efficiency of solar light energy utilization [14][15][16][17][18] . To enhance the photocatalytic response of TiO 2 , many strategies have been implemented, such as metal and nonmetal doping, annealing in reducing atmosphere, creating defects in the crystal lattice, and coupling with various light harvesters 16,19,20 . Doping TiO 2 with transition metals, such as Fe, Ni, Cr, and Co, was shown to enhance its photoactivity by creating shallow states that suppress the e-h pairs recombination 21,22 . Moreover, multi-doping with two or three metal or non-metal elements, such as N, C, and Ce or Co, Cu, Ir, C, and Ti 3+ , was shown to improve the conductivity and balance the deficiencies of individual dopants 23,24 . Consequently, multi-doped TiO 2 has gained much attention compared to the singly and doubly-doped counterparts. The origin of the super reactivity of multi-doped TiO 2 seems to be the synergistic effect of the dopants in narrowing the Scientific Reports | (2021) 11:5609 | https://doi.org/10.1038/s41598-021-84989-x www.nature.com/scientificreports/ bandgap, enhancing the concentration of reactive radical species 25 , and enhancing visible light absorption 24 . In this regard, identifying a one-step synthesis method of multi-doped TiO 2 is extremely desirable, which remains a challenge to be realized. Herein, we report on the successful fabrication of cobalt, Ti 3+ , and carbon multi-doped TiO 2 mesocrystals via an in-situ sol-gel process. The antimicrobial behavior of the fabricated mesocrystals was investigated at ambient conditions and under light illumination. Finally, the reaction mechanism of TiO 2 and multi-doped TiO 2 mesocrystals-treated microbial cells was suggested and discussed in details. Thus, the innovative points of this research include the one-pot synthesis with controlled amount of dopants, the defective structures and how defects played a role in the antibacterial properties as well as the superior dual bacterial and fungi inhibition functions.

Materials and methods
Titanium n-propoxide (Ti(O-n-Pr) 4 , 98%), cobalt nitrate hexahydrate (Co (NO 3 ) 2 ·6H 2 O), Formamide (FA: H 2 N-CHO), and hydrofluoric acid (HF 40%) were purchased from Sigma-Aldrich. Microbiological media ingredients were purchased from Oxford, and reagents used in the biological tests were obtained from Sigma-Aldrich. All the other chemicals were of pure grade and used as received without any further purification. All the solutions were prepared using distilled water (DW).

Physicochemical characterizations and antimicrobial activities of TiO 2 and Co-doped TiO 2 mesocrystals.
The crystal properties of the as-synthesized samples were investigated by X-ray diffraction (XRD) patterns recorded on PANalytical X'Pert PRO X-ray diffractometer with Cu Kα radiation (λ = 0.15418 nm, 2θ range = 5°:80°, step size = 0.04°, and scan-step time = 0.5 s). Raman measurements were performed on a Raman microscope (Pro Raman-L Analyzer) with an excitation laser beam wavelength of 532 nm. Fourier transform infrared (FTIR) spectra were recorded on Nicolet 380 Thermo-Scientific in the range of 400-4000 cm −1 . The elemental composition was assessed using Thermo-Scientific ESCALAB 250Xi X-ray photoelectron spectroscopy (XPS). The morphological analysis of the as-synthesized nanoparticles was performed using a Zeiss SEM Ultra 60 field-emission scanning electron microscope (FESEM) operating at an accelerating voltage of 5 kV. The nanostructure of the samples was investigated using JOEL JEM-2100 high-resolution transmission electron microscope (HR-TEM) operating at an accelerating voltage of 200 kV; the sample was prepared by dispersing the TiO 2 powder in ethanol followed by dropping a small amount on a standard copper TEM grid containing lacy carbon. The UV-Vis absorption spectra of samples were collected using a Shimadzu UV-2600 UV-Vis-NIR spectrophotometer. The photoluminescence spectra (PL) were recorded using Thermo-Scientific LUMINA fluorescence spectrometer. The antimicrobial potential of as-synthesized TiO 2 mesocrystals, Co-doped TiO 2 mesocrystals, and Co 2+ ions against different pathogenic microbes (yeast and bacteria) are examined via employing the agar-disc diffusion method 12 . Firstly, the as-synthesized TiO 2 mesocrystals, and Co 2+ ions are dissolved into distilled water with concentrations 0.01 mg/mL; 10 ppm. The activity of the as-synthesized compounds are examined against different types of bacteria, namely Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA), Proteus vulgaris, Salmonella typhi, and Proteus mirabilis. The examined multi-drug resistance bacteria were tested by Vitek two systems (bioMarieux and Marcy-LEtoile, France). Most of them were resistant to antibiotics like Cefapirin, Ciprofloxacin, Amikacin, Norfloxacin, Amoxicillin, Cefoxitin, Gentamicin, Ampicillin, and Cefotaxime. In the microbiological experiments, we performed the biosafety Level-2 (BSL-2). It should be noted that all the inoculums are established and fixed from 2-5 × 10 8 CFU/mL (0.5 McFarland; at 600 nm). The inhibition of the bacterial growth was defined by the zone of inhibition (ZOI) after 24 h of incubation. Additionally, the antifungal potential of the as-synthesized TiO 2 mesocrystals, and Co 2+ ions is examined against pathogenic unicellular fungi (Candida albicans and Candida tropicalic). After that, the inoculums of the tested yeast cells are set from 1-4 × 10 7 CFU/mL. Finally, Nystatin (NS) and Amoxicillin (AX) are conducted as standard antibiotics. AX is similar to penicillin in its bactericidal action against susceptible bacteria during the stage of active multiplication. It acts via the inhibition of cell wall biosynthesis that leads to the death of the bacteria. While, NS is an antifungal that is both fungi-static and fungicidal in vitro against a wide-variety of yeasts and yeast-like fungi. It exerts its antifungal effects via disruption of the fungal cell membrane.
The minimum inhibitory concentrations (MIC) investigation is completed in Luria-Bertani (LB) broth within a serial dilution. Briefly, a positive control (the microorganism and the nutrient), a negative control (the nutrient solely), and the examined TiO 2 mesocrystals (beginning with 0.1 mg/mL concentration; 100 ppm) are applied; MIC is defined following 24 h at 37 °C. The inoculums of the tested bacteria are at 3-5 × 10 8 CFU/mL and 2-3 × 10 7 CFU/mL to Candida species. MIC is defined by operating ELISA plate (at 600 nm). Finally, the results are statistically analyzed by applying ONE WAY ANOVA, the least significant difference (LSD), and Duncan's multiple ranges, which are calculated by special software (SPSS version 15).
Antibiofilm activities of Co-doped TiO 2 mesocrystals. Moreover, a qualitative measurement regarding the biofilm inhibition was defined as stated by G. Christensen et al. 27  www.nature.com/scientificreports/ biofilm which was performed at the tube wall in the absence and presence of the synthesized TiO 2 mesocrystals was established. The antibiofilm of the as-synthesized TiO 2 mesocrystals (at 10.0 µg/mL) was examined toward the selected bacteria and Candida spp., and was determined and compared with the control (non-treated one). Briefly, 5 mL of the nutrient broth medium was added inside all tubes, and the examined bacteria and yeast were inoculated after adjusted 0.5 McFarland to be 1-2.5 × 10 8 CFU/mL. After that, they were incubated at 37.0 ± 0.5 °C for 24 h. The contents presented in control and treated tubes were discarded, mixed with Phosphate Buffer Saline (PBS; pH 7.0), and finally desiccated. Then, the bacterial and yeast cells which adhered to the tube walls were fixed with 5 mL sodium acetate (3.0%) for about 15 min, and finally, they were rinsed with de-ionized water. Biofilms which introduced inside tubes were stained with 15 mL Crystal Violet (CV; 0.1%) and washed with de-ionized water to remove the rest of the CV. It must be noted that, for the semi-quantitative antibiofilm estimation, 5 mL of the absolute ethanol was inserted to dissolve the stained bacterial and yeast biofilms [28][29][30] .
The O.D. of the stained bacterial and yeast biofilms with CV was examined by UV-Vis. spectrophotometer at 570.0 nm. The bacterial and yeast biofilms inhibition percentage was estimated by applying the following relation (Eq. 1) 31 : Effect of UV-irradiation on the antimicrobial abilities of the prepared TiO 2 mesocrystals, and Co-doped TiO 2 mesocrystals. Furthermore, the antibacterial activity of the as-synthesized TiO 2 nanoparticles with and without UV illumination was assessed against the tested pathogenic microbes Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans strains using the optical density method 32 . The tested microorganisms were stimulated in nutrient broth (NB) overnight at 37 °C. Firstly, 0.5 mL of the overnight culture were inoculated to 5 mL NB tubes that adjusted after 2 h of incubation to standard 0.5 McFarland concentration that standardly equals 1.5 × 10 8 CFU of bacteria and 0.400 equal (1 × 10 4 cells/mL) of C. albicans. 100 µL of Co-doped TiO 2 mesocrystals were added into the tubes and then incubated at 37 °C for 60 min. While tubes without Co-doped TiO 2 mesocrystals were inoculated with bacteria and used as the positive control (subject to UV), tubes without UV illumination were used as the negative control. Typically, 10-W low-pressure mercury lamp was horizontally-placed on the laminar flow and employed as the UV-irradiation source, where 90% of the emitted irradiation was at the specific wavelength (600 nm for bacteria and 630 nm for the fungi). Finally, test tubes were subject to UV-irradiation for 1 h at a distance of about 61 cm. After the incubation, the turbidity of the medium was measured at λ of 600 nm for bacteria and 630 nm for the fungi.

Reaction mechanism using SEM/EDX analysis of TiO 2 mesocrystals, and Co-doped TiO 2 mesocrystals-treated microbial cells. The sensitive bacterial cells (from the antibiofilm results) were cleaned
with Physiological Buffer Saline (PBS) three-times and finally, fixed by 3.5% glutaraldehyde solution. The maintained microbial units were repeatedly-rinsed by PBS and regularly-dried with different concentrations of ethyl alcohol like 30, 50, 70, 90, and 100% for 15 min at 27 ± 2 °C. Following that, the prepared samples were fixed on an aluminum piece regarding SEM/EDX analysis. The morphological features of the control (non-treated P. aeruginosa), TiO 2 mesocrystals, and Co-doped TiO 2 mesocrystals-treated P. aeruginosa were examined by SEM/ EDX investigation.

Results and discussion
Physicochemical characterization. Figure 1a depicts the XRD patterns of both bare and Co-doped TiO 2 mesocrystals calcinated at 400 °C. Both samples have tetragonal anatase phase with a space group I41/amd (Ref card No.: 04-014-5762). No diffraction peaks for cobalt or cobalt oxide were detected in the Co-doped TiO 2 sample, which may be related to the low cobalt content 33 . However, the main (1 0 1) diffraction peak is shifted to lower 2 θ (inset in Fig. 1a), indicating the incorporation of foreign species into the TiO 2 lattice, thus changing the Ti 4+ local structure 34 . Besides, it was found that there is a low intensity peak at 26.7° in the diffraction pattern of the bare TiO 2 sample, which can be attributed to the main plane (110) of the rutile phase. The intensity of this peak was enhanced after the insertion of cobalt ions as observed in the diffraction pattern of the Co-doped TiO 2 sample. The percentage of both anatase and rutile phases was calculated using Eqs. (2) and (3) 20 : where I A and I R are the intensities of XRD peaks of anatase and rutile at 25.3° and 26.7° peaks, respectively. It was found that bare TiO 2 sample is composed of 99% anatase and only 1% rutile, while the Co-doped TiO 2 sample is composed of 93.3% anatase and 6.7% rutile. The increase of rutile% reveals that Co can act as a rutile stabilizer 35 .
Although it is generally accepted for pure phases that anatase exhibits a higher photocatalytic activity compared to rutile TiO 2 , the existence of very small percent of the rutile phase causes an enhancement in the photocatalytic activity of the samples, even in the case of bare TiO 2 mesocrystals, due to the synergistic effects between the two phases compared to pure phases 19 .
The mean crystallite sizes of bare TiO 2 and Co-doped TiO 2 were estimated to be 69 and 40 nm, respectively as determined using Debey-Scherrer formula (Eq. 4), with L being the mean crystallite size (nm), k the Scherrer constant related to the crystallite shape (k = 0.9), λ the X-ray wavelength in nanometer (nm), β the full width at half-maximum of the peak in radians, and θ the diffraction angle.
(1) www.nature.com/scientificreports/ Note the decrease in crystallinity upon Co doping, which can be ascribed to the difference between the ionic charge of Ti (+4) and Co (+2). Moreover, Co doping is expected to cause a slight change in the lattice constants. The lattice parameters (a and c) and cell volume (V) were estimated based on Bragg's law and a formula for a tetragonal system (Eq. 5) for both bare TiO 2 and Co-doped TiO 2 samples. www.nature.com/scientificreports/ It was observed that the unit cell volume has been enlarged from 135.66 to 136.612, and the lattice constant "a" from 3.78 to 3.794 for bare TiO 2 and Co-doped TiO 2 , respectively. These results may indicate the successful incorporation of Co 2+ into the anatase TiO 2 lattice.
Raman spectroscopy is an effective tool to elucidate the structural changes in materials upon doping. Based on group theory, anatase TiO 2 exhibits six Raman-active vibrational modes (3 E g + 2B 1g + A 1g ), as displayed in Fig. 1b. The six allowed modes of anatase single crystal were reported by Oshaka 36 , where the bands E g1 (at 144 cm −1 ), E g2 (at 196 cm −1 ), and E g3 (at 632 cm −1 ), for Eg modes are related to the O-Ti-O symmetric stretching vibration. The two B 1g modes: B 1g1 at 392 and B1 g2 at 511 cm −1 are related to the O-Ti-O symmetric bending vibration, and the one A1g mode at 512 cm −1 is related to the O-Ti-O anti-symmetric bending vibration 37,38 . In the case of Co-doped TiO 2 sample, it is found that the main peaks E g1 and E g2 , located at 144 cm −1 and 196 cm −1 , are shifted to higher wavenumbers (147.2 and 199.2 cm −1 ). However, the E g3 peak (632 cm −1 ) is shifted to a lower wavenumber (629.8 cm −1 ) compared to that of bare TiO 2 . Besides, the B 1g and A 1g peaks are shifted to 389.6 and 509.7 cm −1 , respectively. Furthermore, no bands appeared related to any cobalt oxide phase, probably due to the low Co content in TiO 2 lattice 39 . As the ionic radius of Co 2+ (0.70 Å) is larger than that of Ti 4+ (0.64 Å), the insertion of Co as a dopant should lead to a structural distortion and induce oxygen vacancies, which can be the main reason of the observed peak shift. Moreover, electron-phonon coupling is one of the physical parameters used to understand the electron transport and the existence of oxygen vacancies in the lattice of metal oxides 20 . The electron-phonon coupling is related to the phonon linewidth (FWHM) and can be estimated from the energy-time uncertainty relation (Eq. 6) 40 : where τ is the phonon lifetime, c is the speed of light (3 × 10 8 m/s), and r is the FWHM of the Raman peak in units of cm −1 . The estimated phonon lifetime is found to decrease from 2.49 to 2.11 ps upon cobalt doping.
The FT-IR spectra of the bare and Co-doped TiO 2 mesocrystals are shown in Fig. 1c. The broad peaks located at 3847, 3837, and 3800 cm −1 are ascribed to Ti 3+41 . Besides, the broad peaks at 3435 and 3750 cm −1 are likely due to stretching vibrations of adsorbed O-H groups, while the peak at 1748 cm −1 arises from Ti-O-C vibration, confirming the effective interaction between Ti and C. The peak at 1643 cm −1 is from the H-O-H bending mode 42 . The peak located at 1427 cm −1 is assigned to the Ti-O vibrations on the {001} facets 43 , in good agreement with the XRD results. Also, the peak at 1024 cm −1 is due to Ti-F vibrations in both samples. Moreover, the Ti-O stretching and Ti-O-Ti bridging have appeared between 521 and 460 cm −1 . Thus, the FT-IR spectra reveals the presence of Ti 3+ in the prepared TiO 2 mesocrystals.
In order to investigate the bonding states of the elements on the surface of bare and Co-doped TiO 2 , XPS analysis was carried and the data are presented in Fig. 2. The full-scan XPS survey of both samples (Fig. 2a) revealed peaks that are exclusively related to Ti, O, and C, elements in addition to an extra peak of Co in the Codoped TiO 2 sample. Figure 2b shows the high-resolution XPS spectra of Ti 2p, where Ti 2p 3/2 and Ti 2p 3/2 peaks are observed at 459.1 eV and 464.7 eV, respectively for the bare TiO 2 sample. The O1s HR-XPS spectrum (Fig. 2c) is fitted into two-sub peaks centered at 530.26 eV and 532.17 eV for bare TiO 2 , and 529.78 and 531.4 eV for Codoped TiO 2 . The peaks at 530.26 eV and 529.78 eV are related to Ti-O and surface OHgroups 44 . Additionally, the other oxygen peak (530.3 eV) in Co-doped TiO 2 is originated from the presence of Co-O bond 45 . Moreover, the C 1 s peak (Fig. 2d) can be de-convoluted into three peaks in both samples, one located at 284.69 eV and 284.54 eV and others at 285.82 eV, 286.39 eV, 288.67 eV and 288.42 eV for bare and Co-doped TiO 2 , respectively. The main peak corresponds to C-C bond that exists in carbon species and the others at higher energy could arise from C-O and C=O bond in TiO 2 , revealing interstitial and/or substitutional C 46 . Finally, the Co 2p peaks appeared at 781.8 and 796.42 eV (Fig. 2e) correspond to Co 2+ 2p 3/2 and Co 2+ 2p 1/2 , respectively. Notably, all elements binding energies (Ti, O, and C) in Co-doped TiO 2 sample exhibited a slight negative shift when compared to the bare TiO 2 sample, which can be related to the doping of cobalt ions in TiO 2 lattice where the Co has a higher electronegativity than Ti 47 . Figure 3a,b shows the representative FESEM images of the prepared undoped and cobalt-doped TiO 2 mesocrystals, respectively, revealing agglomerates of highly connected of small particles with homogenous size distribution. Upon Co-doping, a notable decrease in the size of the particles was observed, revealing the effect of cobalt insertion on retarding the TiO 2 growth 48 . The EDX analysis (insets in Fig. 3e,f) reveals the presence of Ti, O, C, and Co without any impurities. Figure 3c-f depicts typical HR-TEM images of the TiO 2 mesocrystals before and after doping with cobalt, viewed along a square surface of {001} crystallographic direction. Note also the presence of a carbon shell as indicated by an arrow in Fig. 3e,f. The transparent carbon layer is uniform and continuously surrounding the TiO 2 mesocrystals 49,50 . Raman spectroscopy was used to confirm the presence of the residual carbon (insets in Fig. 3e,f), where the peaks at 1400 and 1290 cm −1 are mainly originating from sp 3 hybridization (D-band) and the planar configuration of the sp 2 -bonded carbon structure (G-band), respectively 51 .
The optical absorption of the bare and Co-doped TiO 2 mesocrystals was elucidated by recording their UV-Vis diffuse reflectance spectra (DRS) as shown in Supplementary Fig. S1a. Both samples exhibit visible light response in the wavelength range of 400-600 nm. The spectra of bare TiO 2 sample (Supplementary Fig. S1a) reveal an absorption edge at 418 nm, which is redshifted relative to that reported for P25 (TiO 2 ) 52 . The Co-doped TiO 2 sample showed an absorption edge at 424 nm, which is redshifted relative to the bare TiO 2 . The UV-visible DRS analysis illustrated that bare and Co-doped TiO 2 have bandgaps of 2.75 and 2.6 eV, respectively ( Supplementary  Fig. S1b). The observed redshift in the absorption spectra can be ascribed to Co 2+ → Ti 4+ charge-transfer 53 . Thus, photoluminescence (PL) emission spectroscopy was utilized to study the fate of the photoinduced charge carriers 54 . Oxygen vacancies and surface states play a vital role in the photocatalytic response of anatase TiO 2 55 . Supplementary Fig. S1c shows the room-temperature PL spectra of the bare and Co-doped TiO 2 samples, where eight peaks started from 396 nm and ended at 700 nm were recorded 56  Antimicrobial properties. Usually antimicrobial agents are used to hinder microbial diseases emanating from clinical poisoning, such as urinary tract infection (UTI)-causing microbes 31 . However, nanomaterials-based agents have recently received great attention as they are uniquely-applied to combat pathogenic microbes 59 . In our study, the fabricated samples were checked for their antimicrobial capabilities using the disc agar diffusion technique. The TiO 2 samples were found to deactivate a broad spectrum of the tested bacteria such as P. aeruginosa, P. mirabilis, and S. aureus. Specifically, Co-doped TiO 2 mesocrsytals showed the most powerful antibacterial effect against all examined microbes, see Fig. 4, Supplementary Fig. S2 and Supplementary Table S1. The  www.nature.com/scientificreports/ antimicrobial abilities of the samples were compared with Co 2+ ions and standard antibacterial and antifungal agents like Amoxicillin (AX; 25 μg/mL) and Nystatin (NS; 25 μg/mL). Our samples are found to be more active than the used standard antibiotics, and Co 2+ ions. Interestingly, the synthesized TiO 2 mesocrystals were found to be more active against Gram-negative bacteria than the Gram-positive counterpart because the cell wall of the Gram-negative bacteria contains a thick layer of lipopolysaccharide essentially in addition to a small layer of peptidoglycan. On contrary, Gram-positive bacteria primarily incorporate a thicker layer of peptidoglycan blocks 29 . The fabricated NPs enjoy high surface-to-volume ratio, thus can be easily combined and interact with some of the pathogenic microbes, such as yeasts, bacteria, and fungi yeast 30 .
The MIC results ranged from 0.39 to 25 μg/mL of the integrated samples against all tested microbes. The promising MIC of the Co-doped TiO 2 mesocrystals was 0.39 μg/mL (P. aeruginosa), and 0.781 μg/mL (P. mirabilis). Additionally, the synthesized Co-doped TiO 2 NPs exhibit accepted MIC of 12.5 μg/mL against C. albicans at very low NPs concentration (10.0 µg/mL), suggesting their potential use as antifungal agents. Importantly, the properties of the synthesized mesocrystals play a vital role in their antimicrobial characteristics, including their structure, purity, and size [51][52][53][54] . Various advanced mechanisms, such as reactive oxygen species (ROS) distribution (superoxide anion; O 2 − ), were proposed in the literature to explain the possible effects of a plethora of metal oxides as antibacterial agents [60][61][62][63] . However, the antimicrobial mechanism of Co-doped TiO 2 mesocrystals has not been identified yet. Thus, the interaction of Co-doped TiO 2 mesocrystals with the pathogenic microbes and the alkaline tendency have been included here to demonstrate the possible antimicrobial activity mechanism. It is suggested that Co-doped TiO 2 mesocrystals could alter the microbial morphology and their film composition, change the microbial membrane permeability, and produce the residence of oxidative stress genes via the production of H 2 O 2 60 . Note that Co 2+ ions were shown to possess antibacterial activity, where a series of Co 2+ complexes of mercapto-thiadiazole-derived furanyl, thienyl, pyrrolyl, salicylic, and pyridinyl Schiff bases exhibited in-vitro weak to moderate antibacterial potential toward Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Shigella flexneri) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureous) 64 . Another study by Gaëlle et al. 65 showed that the ligands, metal salt, and the complexes (Cobalt (II) complex [Co(phen) 3 (NO 3 ) 2 ]·2H 2 O and a novel Co (III) complex) were investigated for their antimicrobial potentials in-vitro toward pathogenic bacteria and fungi. The antimicrobial results indicated that all ligands were very effective towards the tested microbes. The antibacterial activity of pure cobalt or Co 2+ ions was attributed to the reaction with negatively-charged molecules inside the microbial cells, which in turn leads to genotoxicity and destruction of the main bacterial organelles 31 .

Antibiofilm activity of Co-doped TiO 2 mesocrystals. The formation of biofilm in pathogenic microbes
is characterized by the exo-polysaccharide secretion 28,29 . The tube method was applied to determine the antibiofilm potential of the synthesized Co-doped TiO 2 mesocrystals against some UTI-producing microbes. Supplementary Fig. S3 shows the antibiofilm action of the Co-doped TiO 2 mesocrystals for Pseudomonas aeruginosa and Candida albicans. The complete steps are: (I) Normal microbial growth and production of the distinct ring in the lack of the synthesized Co-doped TiO 2 mesocrystals and the interference with microbial growth in the closeness of Co-doped TiO 2 mesocrystals, (II) The probability of staining of the formed biofilm with Crystal Violet (CV), which is a qualitative determination method, and (III) Eliminating and separating the adhered microbial cells after ethanol addition for semi-quantitative estimation of the biofilm hindrance % (Supplementary Table S2). Supplementary Fig. S3a shows the tube design for the determination of antibiofilm potential of Co-doped TiO 2 against P. aeruginosa, the sensitive bacteria example, which creates a thick whitish-yellow layer www.nature.com/scientificreports/ in the air-liquid interface in the lake of the mesocrystals (control). The produced matt layers were fully-adhered across the walls of the designed tubes and developed as a blue color following the staining with CV. Next, a dark blue color was created in the produced solution subsequent dissolving CV with absolute ethanol, as presented in Supplementary Fig. S3a. On the other side, in the tubes including P. aeruginosa cells and in the closeness of Codoped TiO 2 mesocrystals (10 µg/mL), a remarkable negative effect was recognized as the cells of the tested bacteria do not form biofilm layers and the ring formation was blocked. Also, the adherent cell color was quiet and the blue color was faintly-formed after ethanol addition, as displayed in Supplementary Fig. S3a. Related forms were shown for the biofilm repression of the tested yeast C. albicans as presented in Supplementary Fig. S3b. The semiquantitative determination of the inhibition percentage (%) was investigated by a UV-visible spectrophotometer. The optical density (O.D.) was estimated at 570 nm following terminating CV-stained biofilms, which were considered as a means of their creation. Supplementary Table S2 displays the inhibition% following the addition of 10.0 µg/mL Co-doped TiO 2 mesocrystals, revealing that the highest percentage for P. aeruginosa is 84.43%, for P. mirabilis is 78.58%, and for S. typhi is 77.81%. Note that Co-doped TiO 2 mesocrystals were able to control the biofilm growth at its adhesion degree, which is the first step in the antimicrobial process 66 . The change in the inhibition percentage may be ascribed to many factors such as the high potential of the antimicrobial agents to be attached to the surface due to the high surface area of the synthesized Co-doped TiO 2 mesocrystals and their particle size as well as the invasion skills and different chemical characteristics influencing the relationship and communication of Co-doped TiO 2 mesocrystals with biofilms-producing microbes 67 . Positively, the synthesized   Figure 5 shows a summarized diagram regarding the antibiofilm potential of Co-doped TiO 2 mesocrystals (as inhibition %) against different pathogenic microbes.

Antimicrobial effect of Co-doped TiO 2 in liquid media under illumination. The comparison
between the inhibition% of P. aeruginosa, S. aureus, and C. albicans upon the use of TiO 2 and Co-doped TiO 2 mesocrystals and UV are presented in Fig. 6. Note that Co-doped TiO 2 showed higher antimicrobial activities against P. aeruginosa, S. aureus, and C. albicans colonies than pure TiO 2 , Fig. 6b-d, revealing the synergistic actions of Co doping and the TiO 2 mesocrystals. Moreover, upon UV-illumination, Co-doped TiO 2 mesocrystals exhibited even higher antimicrobial activities than that in the dark. The maximum inhibition percentage of bare TiO 2 and Co-doped TiO 2 mesocrystals under UV-illumination for P. aeruginosa at the end of the experiment was 24.24% and 50.50%, respectively (Fig. 6a,b), while it was 30.31% and 60.25% for S. aureus (Fig. 6c,d), and 39.15% and 55.55% for C. albicans (Fig. 6e,f). The observed activity under light irradiation may be related to the induced oxygen species such as OH free radicals, which caused the destruction of the microbial coenzymes and reduced their contents 19,42 . The major influences involve the creation of holes in the cell wall of the microbes, which subsequently-progressed the cell permeability and finally a cell death will occur. To confirm the induced oxygen species, electron paramagnetic resonance (EPR) spectra were collected as shown in Supplementary Fig. S4. The two samples showed signals at g = 1.95, g = 2.157, g = 2.05, and g = 2.12, confirming the presence of Ti 3+ and free oxygen radicals such as OH . , O .-, or O 2 .-68 . Notably, the Co-doped TiO 2 sample showed a higher concentration of paramagnetic centers of 4.16953 × 10 18 spin/g relative to the bare TiO 2 sample (4.09473 × 10 18 spin/g). The increased paramagnetic centers after the addition of cobalt may be related to the increase in the concentration of oxygen radicals in the TiO 6 lattice 57 . This is in agreement with the inhabitation % of Co-doped versus bare TiO 2.
SEM/EDX analysis was performed to elucidate the possible antimicrobial mechanism toward P. aeuroginosa, see Fig. 7. The SEM analysis of the control sample in the absence of any mesocrystals showed bacterial groups that are constantly-developed with typical normal bacterial surface and semi-formed biofilm, Fig. 7a. Upon TiO 2 mesocrystals treatment, noticeable morphological differences were identified in P. aeuroginosa (Fig. 7b), including the incomplete lysis of the outer surface followed by deformations of the P. aeuroginosa cells. Additionally, Co-doped www.nature.com/scientificreports/ TiO 2 mesocrystals caused the entire and complete lysis of the bacterial cell with the decrease in the whole viable number, and ultimately the biofilm growth was restrained (Fig. 7c). The EDX elemental analysis shows the presence of Ti and O elements with others from the bacteria like C, and O, along with Fe, Si, and Na form the microelement in the bacterial medium. All detected elements were located at the malformed cities and the outside surface of the P. aeuroginosa cells, validating the performance of the tested TiO 2 mesocrystals, (Fig. 7d). Finally, the EDX elemental spectra, in case of Co-doped TiO 2 , revealed Co, Ti, and O elements along with different atoms from the bacterial structure at the irregular areas and at the outside surface of the treated P. aeuroginosa cells. The schematic in Fig. 8 illustrates the possible antibacterial mechanism. We believe that Co-doped TiO 2 mesocrystals start their action by adhesion at the outer surface of the bacterial cell, causing membrane damage and altered transport activity. Then, diffusion of Co 2+ inside the bacterial cell (at pH = 3) and dividing all of the intracellular structure like mitochondria, plasmid, DNA, and other vital organelles. Afterwards, cellular toxicity occurs due to the oxidative stress generated by the production of ROS. Finally, TiO 2 mesocrystals were withstood the acidic condition inside the bacterial cells and conversion did not occur 69 but possessed the antibacterial effect by affecting the signal transduction pathways.

Conclusion
In summary, unique multi-doped TiO 2 mesocrystals have been synthesized via a facile sol-gel approach. The crystal structure, optical, and compositional properties of the materials were elucidated using XRD, Raman, FTIR, XPS UV-vis analyses. The synthesized Co-doped TiO 2 mesocrystals showed excellent antimicrobial activity compared to bare TiO 2 counterparts. The antimicrobial performance was evaluated in terms of zone of inhibition, minimum inhibitory concentration (MIC), antibiofilm activity, and photoactivity. Co-doped TiO 2 mesocrystals Figure 8. Schematic representation regarding the four prominent ways of antimicrobial potential of Co-doped TiO 2 mesocrystals, where (1) Co-doped TiO 2 mesocrystals adhere to the bacterial cell surface and results in membrane damage and altered transport activity; (2) Co-doped TiO 2 mesocrystals create and increase the ROS leading to cell damage, (3) Co-doped TiO 2 mesocrystals block the ions transport from and to the bacterial cell, and (4) Co-doped TiO 2 mesocrystals penetrate inside the bacterial cells and interact with cellular organelles and biomolecules, and thereby, affect respective cellular machinery, and modulate the cellular signal system and causing cell death. Co-doped TiO 2 mesocrystals may serve as a vehicle to effectively-deliver Co ions to the bacterial cytoplasm and membrane, where proton motive force would decrease the pH to be less than 3.0 and therefore improve the release of Co ions.