Facile fabrication of Tl4HgI6 nanostructures as a novel antibacterial and antibiolm agents

In the present study, Tl4HgI6 nanostructures have been successfully fabricated through a simple precipitation route. The impact of TlI stoichiometric ratio to HgI2, and kind of surfactants was explored on purity, structure, and shape of samples. The as-fabricated Tl4HgI6 was characterized via XRD, EDX, FESEM, TEM, HRTEM, and Raman spectroscopy. Raman spectrometry corroborated the XRD outcomes, and revealed that the Tl4HgI6 nanoparticles were successfully fabricated. The structure, shape, and scale of the products were studied through FESEM images. It was observed that different factors have a notable aspect on the morphology and size of the products. The maximum antibacterial activity of Tl4HgI6 was perceived against S. aureus, E.coli and M. catarils. These outcomes demonstrate that Tl4HgI6 displays ecient bactericidal activity against Gram-positive and Gram-negative microorganisms. The anti-biolm activity revealed that the best reduction of biolm was recognized in higher Tl4HgI6 concentrations (2×MIC). Tl4HgI6 at 2×MIC concentration inhibits biolm production by S. aureus and E. feacalis with an inhibition percentage of 95% and 90%, respectively.


Abstract
In the present study, Tl4HgI6 nanostructures have been successfully fabricated through a simple precipitation route. The impact of TlI stoichiometric ratio to HgI2, and kind of surfactants was explored on purity, structure, and shape of samples. The as-fabricated Tl4HgI6 was characterized via XRD, EDX, FESEM, TEM, HRTEM, and Raman spectroscopy. Raman spectrometry corroborated the XRD outcomes, and revealed that the Tl4HgI6 nanoparticles were successfully fabricated. The structure, shape, and scale of the products were studied through FESEM images. It was observed that different factors have a notable aspect on the morphology and size of the products. The maximum antibacterial activity of Tl4HgI6 was perceived against S. aureus, E.coli and M. catarils. These outcomes demonstrate that Tl4HgI6 displays e cient bactericidal activity against Gram-positive and Gram-negative microorganisms. The anti-bio lm activity revealed that the best reduction of bio lm was recognized in higher Tl4HgI6 concentrations (2×MIC). Tl4HgI6 at 2×MIC concentration inhibits bio lm production by S. aureus and E.

1-Introduction
Antibacterial resistance has appeared as a worldwide menace to community health and has turned to the principal reason for fatality. Staphylococcus aureus is the principal cause of nosocomial diseases correlated with enhanced incidence [1]. Methicillin-resistant staphylococcus aureus is entirely accountable for multiple serious nosocomial diseases in humans which enhances the therapy continuation and remedial expenses [2]. They are identi ed to induce diverse moderate to persistent diseases in animals. Persistence against the nal usage of medicines including, linezolid and vancomycin have appeared in the clinical isolates of staphylococcus aureus which possess more aggravated the situation [3]. Moreover, the resistance issue is prevented via the capability of Staphylococcus aureus to produce bio lms on abiotic and biotic super cies mainly on various therapeutic implanted devices [4]. Staphylococcus aureus bio lm formation is an even more important menace owing to enhanced sensitivity against the host defense system and antibacterial [5]. Bio lms are complicated to use and examined as a microbial repository for distribution to different body sections [6].
These contagions may be caused by replacing the implanted medical device, thereby making extended suffering at the patient's additional medical expenses [7]. By growing fatality rate because of AMR, there is a necessity to seek new approaches to improve antimicrobials to resist bio lms and multidrugresistance associated infections [8]. Scientists are utilizing different strategies to recognize novel compounds or novel antibiotics with antibacterial activity. Several preventions of diverse passage, antibacterial peptides obtained from organic and inorganic manufactured combination, and various ecosystems by antibacterial activity have been recognized and investigated [9,10]. In search of discovering novel materials having antibacterial activity has attracted much attention.
Fabrication of an advanced group of nano-materials amidst distinctive formation and optoelectronic/electronic features is one example of the extensive demands inside the factual community. For a while, scientists dealt with the unique crystal semi-conductive substances with an extended forbidden band. The extended forbidden band semi-conductors possess great denseness that can apply within many areas, including the non-expansion of nuclear materials, medical imaging, homeland security, etc [11,12]. One of the interesting and inspiring features is a classi cation of B 4 AC 6 crystalline solid, with B = In, Tl, A = Pb, Hg, Cd, Ge, Zn, Mg, and C = Cl, Br, I. Lately, semi-conductor ternary iodides including, Tl 4 HgI 6 , Tl 4 CdI 6 , Tl 4 PbI 6 , and In 4 CdI 6 , have been drawn consideration to the scientists [13]. A large number of ternary halides, for instance, Tl 4 HgI 6 discloses a noteworthy conductivity potential and exposes the solidity ionic conduction including heavy metals (like Tl) [12][13][14][15][16][17]. Additionally, Tl 4 HgI 6 shows potential applications in temperature sensors, ionizing radiation detectors, and ionic conductors. Tl 4 HgI 6 is a direct forbidden band semi-conductor with an energy gap of about 2.150 eV and reveals cathode luminescence and photoluminescence [18]. To date, Tl 4 HgI 6 has been fabricated via the Bridgman-Stock burger method and solid-state route below hard conditions and high temperature [19,20]. Precipitation is an e cient procedure for fabrication nanomaterials since this method furnishes a facile, clean and suitable approach, and possesses numerous bene ts, including low cost, simple component adjustment, eco-friendly, and high homogeneity of the acquired nanomaterials [21].
In the current report, Tl 4 HgI 6 nanostructures were fabricated via the co-precipitation route as a new method that furnishes the small size and high homogeneity product. The effects of multiple factors including, distinct surfactants and molar concentration ratio of TlI to HgI 2 were examined on purity and structure of samples. Finally, the samples were examined to reach the best position via EDX, XRD, SEM, Raman spectrometry and TEM for the fabrication of the desired product. The antibacterial and antibio lm activities of Tl 4 HgI 6 were studied. The outcomes demonstrate that Tl 4 HgI 6 displays e cient bactericidal activity against Gram-positive and Gram-negative microorganisms.

2-1-Precursors
Each alchemic agent utilized in the present paper was provided within the analytic standard. Mercury The thallium(I) iodide was fabricated through a co-precipitation route TlNO 3 and LiI.2H 2 O as commencement substances. In the rst place, the stoichiometric quantity of TlNO 3 was dissolved in distilled water and the determined quantity of surfactant was a xed to the above mixture. In the end, the solution of LiI.2H 2 O was added to the above solution to acquire yellow precipitation.

2-2-2-Fabrication from Mercury(II) iodide
The mercury(II) iodide was acquired by combining the LiI.2H 2 O among a stoichiometry quantity of Hg(COOH) 2 (through examining the coveted molar percentage ratio of TlI to HgI 2 under several tests). The orange precipitate was obtained by adding LiI to Hg(COOH) 2 solution.

2-2-3-Fabrication from Tl 4 HgI 6
The mixture including of TlI was added into the HgI 2 beaker and stirred at 25 ℃ for 2 h. The orange precipitate was separated, puri ed utilizing ethanol and distilled water and eventually evaporated into an oven at 75 °C. Different conditions was used in sequence to reach the best state. Table 1 clari es the various states for the fabrication of Tl 4 HgI 6 .

2-3-1-Determination of the Minimum Inhibitory Concentration (MIC)
The broth microdilution technique was utilized to survey the minimum inhibitory concentration (MIC) as claimed to the Clinical and Laboratory Standards Institute (CLSI) guidelines [22]. In synopsis, Mueller Hinton broth such as twofold concentration increases of sample 6 was a xed to every well. Mixtures for every bacterium equal to a 0.50 McFarland standard were further thinned and a xed to the plates to reach a speci c inoculum of 105×5 CFU/ml. Then, all the plates were hatched aerobically at 37°C for 18-20 hours. The minimum concentration of the samples that limited the extension of bacteria was recognized as MIC. Besides, MIC measurements were accomplished by Nystatin, Rifampin, and Gentamicin employed as positive inhibitor controls for fungus, Gram-negative, and Gram-positive bacteria.

2-3-2-Determination of the Minimum Bactericidal Concentration (MBC)
The MBC was estimated by the CLSI guidelines. In synopsis, after the completion of the MIC experiment, 10 L from each clean well-including Tl 4 HgI 6 was subcultured to Mueller Hinton agar (Becton Dickinson, USA). The plates were incubated at 37°C in air for one day. The colony-forming units (CFU) were determined visually. The MBC was employed as the concentration at which a three-log decrease in bacterial extension (>99.9%) was identi ed related to the initial inoculum. Experiments were performed ve times.

2-3-3-Agar disk diffusion procedure
Sterilized supplying empty plates soaked with sample 6, and saturated plates (20 μl) were placed on Mueller-Hinton agar plates and incubated at 37°C for 18-20 hours. Bactericidal activities were determined by tracing the cleared area of inhibition to the most near millimeter (mm). Nystatin, Rifampin, and Gentamicin were employed as positive inhibitor controls for fungal, Gram-negative, and Gram-positive bacteria.

2-4-Bio lm formation inhibition procedure
Bio lm production was estimated as speci ed via Kolter and O'Toole with some adjustments. 100 μl of each Tl 4 HgI 6 concentration was a xed to the sterilized 96-well polystyrene microplate. The bacterial culture strains for bio lm assays were provided from the cells were cultured overnight at 37 °C and resuspended at an optical density at 600 nm (OD600) of 0.2 in 1 ml of tryptic soy broth (TSB). A 100μl of bacterial suspension and 100 μl of each Tl 4 HgI 6 concentration are dispersed into every microplate well.
Bio lm formation was measured via estimating the bio lm absorption rate of crystal violet (CV) stained bio lm at 570 nm and with entire feasible counts. After one day of the subjection, planktonic cells in microplate wells are removed via rinsing by phosphate-buffered saline (PBS) (pH= 7.2) and dried Adherent microorganisms were set via combining 200 μl of 96% ethanol for 15 minutes. Next, microplate wells stained by CV (Gram staining set for microscopy-Merck, Germany) stained microplates wells are rinsed twice with PBS to remove CV stain and the microplate were dried at room temperature. To estimate the adherent cell absorbance, the CV was re-solubilized with 200 μl of 33% acetic acid glacial (Merck, Germany) and the absorbance was determined by a microplate reader (ELX-800, Biotec, India) at 570 nm.
The bio lm inhibition rates in the attendance of various Tl 4 HgI 6 concentrations were measured by using the ratio among the amounts of OD570nm without and with Tl 4 HgI 6 [23].

3-1-Structural determination
The X-ray diffraction patterns of specimens 3, 5, and 6 are demonstrated in Figure 1 (a-c). As shown in Figure 1a, a product was achieved with high purity and good crystallinity when the mole ratio of TlI : 446. The crystallite size was estimated by Scherrer equation, D g = Kλ/βcosθ, θ is the Bragg angle, λ is the wavelength of X-ray, K is a non-dimensional shape factor, which usually is nearly 0.9, and β is the region of the detected diffraction line on its half intensity maximum [24]. The grain sizes of the specimens are in the extent of 20-37 nm.

3-2-Structure and size characterization
In the current paper, diverse factors were changed to obtain the tiniest specimen amidst high uniformity and the in uence of them was investigated on structure, size and shape of specimens via the FESEM images. Figure 2 illustrates the SEM images of as-fabricated Tl 4 HgI 6 by changing the molarity of TlI and HgI 2 . As shown in Figure 2a, the micro-structures were produced when the mole ratio of TlI to HgI 2 was 1: 1 (specimen 1). Figure 2b presents the FESEM images of specimen 2 that was composed in 2: 1 mole ratio of TlI to HgI 2 . The microstructures were fabricated the same as specimen 1. The nanoparticles with highly alike and similar size particles was formed in specimen 3, when the mole ratio was 4: 1 ( Figure 2c). The uniformity in specimen 3 is fairly high and the average of particle size is about 44 nm. The microstructures were fabricated, when the mole ratio of TlI to HgI 2 was 1: 2 and the morphology is heterogeneous (Figure 2d). Therefore, the optimum mole ratio between TlI : HgI 2 was chosen 4:1. This mole ratio was utilized to explore the in uence of the different surfactants on the structure, shape, and size of the specimens. Figure 2(e-i) illustrates the FESEM images of the specimens in the existence of different surfactant. As revealed in Figure 2e, in presence of SDS, the product was made of nanostructures and the average size of nanoparticle is about 19 nm. The uniform nanoparticles with the mean size of 74 nm were composed by applying PVP as a polymeric surfactant (Figure 2f). By utilizing EDTA, the morphology of the product is heterogeneous micro-particles (Figure 2g). In the presence of NaHSal the FESEM images of the product displays bulk structures with some nanoparticles. (Figure 2h). As shown in Figure 2i, in the presence of HTAB, the product made from cubic particles with tiny nanoparticles and the average size of particles is around 22 nm. Figure 2(j-l) illustrates the EDX spectra of Tl 4 HgI 6 nanostructures (samples 3, 5, and 6, respectively). In these spectrums, the peaks of Tl, Hg, and I elements can be noticed. The results show that the purity of the products is high. Figure 3(a-d) reveals the histograms of the particle diameters of samples 3, 5, 6 and 9 estimated of its FESEM images employing Digimizer program, respectively. Figure 3a shows that 46 % of sample 3 has a size particle between 30-50 nm. 50 % of Sample 5 possesses the size distribution between 10-20 nm. The histograms of particle size of specimen 6 is demonstrated in Figure 3c. As shown in this Figure 44% of particles are between 70-90 nm. Figure 4(a-b) demonstrates TEM images of the optimum product (specimen 6). As-prepared Tl 4 HgI 6 in multiple scales 20 and 150 nm, sequentially. The average particle size from nano-Tl 4 HgI 6 was estimated at 35 nm, which is in almost correct compromise by the FESEM and XRD results. Figure 4c indicates the HRTEM of specimen 6. The crystalline planes were identi ed by the parallel lines indicate the high degree of crystallinity of the compound. The lattice fringes are clearly shown with spacing fringes of 2.95 Å, which well-matched with the crystal planes (212) of Tl 4 HgI 6 crystals. This image shows spherical particles having spherical cross-sections. Besides, it shows that the particles in the FESEM image consist of several spheres. All six bands are in accordance to Tl 4 HgI 6 [25]. Likewise the bands at 57.4 and 66.3 cm -1 are allocated to the symmetric stretching of the Tl-I [26]. Raman movement rates of nano-Tl 4 HgI 6 was correlated to the Raman movement rates of Tl 4 HgI 6 detailed in the reports. The outcomes offer a blue movement within the Raman spectra due to the stress and the captivity of phonon impact which made up through decreasing the particle size [27,28]. Figure  to Tl 4 HgI 6 [26]. Also, the bands at 51.4 cm -1 and 69.3 cm -1 were attributed to the symmetrical stretching of the Tl-I [26]. Raman movement rates of nano-Tl 4 HgI 6 were correlated to Raman movement rates of Tl 4 HgI 6 detailed in the reports [27]. The outcomes offer a blue movement within the Raman spectra due to the stress and the captivity of phonon impact which made up through decreasing the particle size [28].

3-4-1-Determination of MIC
The MICs and MBC were assessed via the broth microdilution technique and the outcomes are displayed in Table 2. The MIC of Tl 4 HgI 6 was estimated by the lowest concentration factor that ultimately inhibits visible growth. As shown in Table 2 3-4-2-Agar disk-diffusion Disk diffusion was determined by evaluating the growth inhibition zone diameters on the examined microorganisms. The inhibition zone diameter in the disk is provided in millimeters. The experiments were reiterated three times, and the outcomes are exhibited in Table 3 and Figure 6. It can be observed from the table that E. feacalis have higher inhibition growth zone than other examined bacteria. Tl 4 HgI 6 reveals remarkable antibacterial activity the inhibition zone was extremely clear, including larger diameters of 24 mm and 22 mm for bacterial media, including E. feacalis, E. coli, and S. aureus, respectively.

3-4-3-Bio lm formation inhibition
The effect of Tl 4 HgI 6 nanoparticles on bio lm production was examined with S.aureus and E.feacalis, as strong bio lm creator. As presented in Figure 7 the achieved result was depended on the concentration. The best reduction of bio lm was recognized in higher Tl 4 HgI 6 concentrations (2×MIC). Tl 4 HgI 6 at 2×MIC concentration inhibits bio lm production by S. aureus and E. feacalis with an inhibition percentage of 95% and 90%, respectively. The bio lm growth inhibition at the Tl 4 HgI 6 concentration higher than MIC con rmed that the bacterial cells in a bio lm are more persistent to antibacterial factors correlated to the planktonic cells, which is a renowned characteristic.

4-Conclusions
In summary, the co-precipitation technique was adopted to fabricate nano-Tl 4 HgI 6 for the rst time.
Several factors were developed to obtain tiny specimens and pure phase. The mole percentage ratio of TlI to HgI 2 and the variety of surfactant can in uence the particle size and purity. The function of surfactants on the particle size of the specimens was con rmed via the XRD patterns and FESEM images. The results obviously showed that the homogenous structure was produced in the presence of PVP-25000. The antibacterial and anti-bio lm activities of Tl 4 HgI 6 revealed that the maximum antibacterial activity of All authors analyzed and interpreted the results.

Figure 3
The particle size distribution of the specimens a) 3, b) 5, c) 6 and d) 9.