key: cord-1046759-7wm6ubow
authors: Wei, Feng; Cui, Xinyu; Wang, Zhao; Dong, Changchang; Li, Jiadong; Han, Xiaojun
title: Recoverable peroxidase-like Fe(3)O(4)@MoS(2)-Ag nanozyme with enhanced antibacterial ability
date: 2020-10-06
journal: Chem Eng J
DOI: 10.1016/j.cej.2020.127240
sha: 4e81dec75eddbbece29d399e4d42a4535a672ae8
doc_id: 1046759
cord_uid: 7wm6ubow

Antibacterial agents with enzyme-like properties and bacteria-binding ability have provided an alternative method to efficiently disinfect drug-resistance microorganism. Herein, a Fe(3)O(4)@MoS(2)-Ag nanozyme with defect-rich rough surface was constructed by a simple hydrothermal method and in-situ photodeposition of Ag nanoparticles. The nanozyme exhibited good antibacterial performance against E. coli (∼ 69.4%) by the ROS destruction and release of Ag(+), while the nanozyme could further achieve an excellent synergistic disinfection (∼ 100 %) by combining with the near-infrared photothermal property of Fe(3)O(4)@MoS(2)-Ag. The antibacterial mechanism study showed that the antibacterial process was determined by the collaborative work of peroxidase-like activity, photothermal effect and leakage of Ag(+). The defect-rich rough surface of MoS(2) layers facilitated the capture of bacteria, which enhanced the accurate and rapid attack of ·OH and Ag(+) to the membrane of E. coli with the assistance of local hyperthermia. This method showed broad-spectrum antibacterial performance against Gram-negative bacteria, Gram-positive bacteria, drug-resistant bacteria and fungal bacteria. Meanwhile, the magnetism of Fe(3)O(4) was used to recycle the nanozyme. This work provided the motivation to engineer the nanozyme for efficient disinfection treatment.

The increase of drug-resistant microorganism, caused by over usage of antibiotics, has become a serious public threat to human [1, 2] . Therefore, enormous attention has been paid to urgent developing various antibacterial agents with broad-spectrum antimicrobial property and minor side effect [3] . As with the development of nanotechnology, noble metals, especially Ag nanoparticles, have been widely applied due to their potent antibacterial properties dominated by local releasing metal ions [4, 5] . Nevertheless, the effective application has been limited by the easy aggregation of small nanoparticles, which requires suitable matrix for accurate design of the nanoparticle loading. Moreover, the excessive leakage of metal ions can inevitably result in the toxicity to the organism [6] . To avoid the above issues, considerable efforts have been dedicated in alternative strategies without detrimental effects [7] . Recently, the property of natural enzymes to produce reactive oxygen species (ROS) has been used in defecting microorganism [8] . Unfortunately, the natural enzyme always suffers from high cost and environment-dependence [9, 10] . The further attraction has been focused on the construction of artificial nanozymes which endow stable materials with promising ROS production features [11] . Some inorganic materials, such as ferroferric oxide [12] , graphene quantum dots [13] and cerium dioxide [14] , have shown strong intrinsic peroxidase-like properties, which can mimic enzymes and effectively catalyze concentration of H 2 O 2 into highly active ·OH [15] . These nanozymes exhibited excellent antibacterial activity in disinfection treatment using ·OH to destroy the membranes of bacteria via oxidization [16] . However, the effective bacteriotoxic application of nanozymes still needs extensive improvement in many aspects [17] . For instance, the low bacteria-binding ability of most artificial enzymes as well as the short lifetime and poor diffusivity of ROS greatly hindered their interaction with bacteria and limited the disinfection performance. Hence, it is still a great challenge to develop creative strategies to address the aforementioned issues and limitations.

During the disinfection process, the interaction between bacteria and antibacterial agents is well recognized to be one of the necessary steps to determine the disinfection performance [18, 19] . In this regard, the adhesive ability of the antibacterial agents 4 plays a significant role in effective capture and exact attack process. Inspired by the natural trapping system, the rough material surface with protuberance or pili, such as pollen [20] , spike on the coronaviruses and flagella of the bacteria, exhibits better adhesion towards the substrate compared to the flat surface. Studies also have shown that the promoted adhesive ability of nanomaterials mainly originates from regulating the topological structures on the surface to increase the roughness [21, 22] . Lately, remarkable works have been done by fabricating defect-rich 2D-layers MoS 2 on the surface of Cu nanowires to develop a multifunctional artificial nanozyme with good capture ability to integrate the advantages of single component [23] . As a typical material of transition metal dichalcogenides (TMDs), MoS 2 possesses 2D-layer structure with huge specific surface area, facile surface modification and good biocompatibility, making it a proper candidate as well as a supportive matrix for other material attachment [24] in the fields of catalysis [25] , energy storage [26] and hydrogen evolution [27] . The excellent absorption in near-infrared range and the friendly elements of Mo and S to human body enable it accessible in photothermal therapy (PTT) [28] , which can also be applied in disinfection treatment as a noninvasive method [29] [30] [31] . Meanwhile, the peroxidase-like property of MoS 2 was explored in the colorimetric detection of H 2 O 2 and glucose [32] , which is rarely reported in antibacterial treatment. Therefore, the smart engineered nanozymes by combination of PTT and peroxidase-like properties can provide an intriguing alternative for disinfection treatment.

The recent reported literatures has combined different bactericidal modalities (such as metal ions, ROS, hyperthermia) to achieve the synergistic effect with decreased dose of antibacterial agents and increased efficiency. However, the long distance between antibacterial agents and bacteria still limited their interaction and negatively influenced the disinfection efficiency. To solve the aforementioned issues, herein, a Fe 3 O 4 @MoS 2 -Ag with rough surface was constructed by a simple hydrothermal method and in-situ photodeposition of Ag nanoparticles as shown in Scheme 1. It was found that the surface topologies of Fe 3 O 4 nanoparticles were modified by defect-rich MoS 2 layers vertically growing on the surface, which showed excellent bacteria-binding ability. Notably, the combination of Fe 3 O 4 and MoS 2 further enhanced the intrinsic peroxidase-like properties. Moreover, the hyperthermia by the photothermal property of Fe 3 O 4 @MoS 2 -Ag assisted to prohibit the bacterial growth.

The magnetism of Fe 3 O 4 enable the nanozyme to be easily recycled. The advantages were: i) the intrinsic POD-like property of Fe 3 O 4 @MoS 2 -Ag could catalytic low concentration of H 2 O 2 into toxic ·OH, which showed a great potential in inflammation treatment; ii) the released Ag + played an auxiliary role to attack the bacterial membranes; iii) the photothermal effect of Fe 3 O 4 @MoS 2 -Ag not only generated hyperthermia but also improved the POD-like property; iv) the topological structure and S-vacancy of MoS 2 nanosheets endowed Fe 3 O 4 @MoS 2 -Ag with potent adhesion ability to bacteria by forming chemical bonds, which shorten the diffusion distance of short-life ·OH radicals and further enhance the antibacterial effect. Therefore, this work provided a promising strategy for rapid and effective disinfection treatment and show great potentials in practical inflammation treatment. The in-situ loading of Ag was prepared by photo-deposition following our previous method [35] . Specifically, 100 mg Fe 3 O 4 @MoS 2 powder was fully dispersed in 100 mL methanol by sonication for 20 min. The designed volume (46.7, 93.5, 280.4 and 467.3 μL corresponding to 0.5, 1, 3 and 5% compared to Fe 3 O 4 @MoS 2 powder mass) of AgNO 3 aqueous solution (0.1 mol/L) was slowly dropped into the suspension with the mechanical stirring for 15 min to electrostatically attract Ag + onto MoS 2 sheets, followed by bubbling N 2 (99.999%) into the mixture for 30 min to remove O 2 . The photo-deposition process was conducted using a 300 W mercury lamp for 3 h. After washing with distilled water for 3 times and collecting by magnetism, the final product was dried in the vacuum oven at 60 ℃ overnight. The samples with different mass percentage of loading Ag compared to Fe 3 O 4 @MoS 2 powder were termed as solution was added to above mixture to start the catalysis process. The kinetics were investigated using below Michaelis-Menton equation.

where V o represents the initial velocity, V max refers to the maximum reaction velocity, and [S] is the concentration of substrate.

The production of ·OH was assessed by the reaction between TPA and ·OH to produce 2-hydroxyl terephthalic acid, which was monitored by PL with the excitement wavelength at 315 nm and emission wavelength at 435 nm. All mixtures were incubated in PBS (pH of 7.4) at room temperature for 6 h with TPA (0.5 mmol/L), H 2 O 2 (1 mmol/L) and as-prepared samples (100 μg/mL). The mixtures without as-prepared samples were taken as control.

The PBS solution of different samples (100 μg/mL) were irradiated under an 808 nm NIR laser (MLL-III from Changchun, China) at a density of 1.0 W/cm 2 for 15 min with the temperature recording every 30 sec by a thermal probe (FLIR E6).

Gram-negative E.coli was taken as the target bacteria to investigate the bacteriabinding ability. The bacteria were cultured on the LB solid plate, and single colony was extracted and slowly dropped into the fluid nutrient medium. The fresh E.coli suspension can be obtained after incubation at 37 ℃ for 12 h in the water shaking bath.

The E.coli was collected by centrifuging (5000 rpm, 3 min) and the E.coli suspension (~10 7 cfu/mL) was formed by diluting with PBS (pH of 7.4). All the glassware in the experiments were kept at 121 ℃ for 20 min to guarantee sterility.

The different as-prepared samples (final concentration of 100 μg/mL) were mixed with 1 mL bacterial suspension containing ~10 7 cfu/mL of E.coli. The mixture was incubated at room temperature in shaking bath for 30 min. The bacteria stuck onto the as-prepared samples were removed by magnetism. The bacteria mixed with MoS 2 was collected by centrifuging at 1000 rpm for 1 min. The bacteria left in the suspension were withdrawn and diluted to proper concentrations, followed by being spread onto the sterilized LB agar plates. The viable colonies were enumerated and calculated.

The bacterial suspension (~10 7 cfu/mL) was incubated with different concentrations ( covering Fe 3 O 4 (diameter of ~ 428.9 nm) by SEM and TEM images ( Fig. 1c and d) .

The Fe 3 O 4 nanoparticle was employed as the support with irregular and curvy MoS 2 nanosheets vertically and densely growing on the surface to increase the exposed edges of MoS 2 layers and form the rough surface, followed by loading Ag nanoparticles on MoS 2 sheet surface (Fig. 1e) . The morphology of MoS 2 was shown in Fig. S1a and b with sheet structure. The high resolution TEM image (Fig. 1f) The kinetic study of the catalytic process in our work was analyzed according to

Michaelis-Menton equation. The Michaelis Menten constant (K m ) was calculated through the Lineweaver Burk plot: (Table S1 ). Normally, the lower K m represents the stronger attraction between the catalyst and the substrate, while the higher V max refers to the better catalytic ability. The whole catalytic process should be assessed by combining K m and V max .

Compared to the data of HRP in previous reports [45] , both Fe 3 O 4 @MoS 2 and sheets engineered on Fe 3 O 4 nanoparticles, which would be an essential part for the highly efficient attacking during disinfection process. Besides, the interaction between different samples and bacteria was further investigated. The cell wall of E. coli was mainly composed of peptidoglycan with amino acid residues with negative charge (Fig.   S12 ), which acted as the functional sites to tightly combine with S-vacancy of S-defect MoS 2 to form chemical bonds [47, 48] . It is also confirmed by phase transfer of samples from water to oil phase with oleylamine (-NH 2 ) and oleic acid (-COOH) as the hydrophobic ligands. Fig. S13 showed great affinity of Fe 3 O 4 @MoS 2 to -NH 2 and Fig. 4a 1%Ag to bacteria facilitated the precise and rapid attacking of ·OH caused by peroxidase-like property and Ag + leaking from the catalyst surface assisted by the local hyperthermia under 808 nm NIR irradiation, thus leading to the deformation and disruption of bacterial membrane with leakage of cytoplasm. Fe 3 O 4 @MoS 2 -1%Ag was recycled by external magnetic field and reuse to disinfect E.coli, which showed excellent inactivation rate (~95%) and stability after 5 times reuse (Fig. S18) .

The broad-spectrum antibacterial performance of Fe 3 O 4 @MoS 2 -1%Ag was confirmed by their disinfection effect on S. aureus, B. subtilis, MRSA and C. albicans. 

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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work

A nanozyme of Fe 3 O 4 @MoS 2 -Ag with topological surface is successfully fabricated

The defect-rich rough surface enhances the adhesive ability against E.coli

The nanozyme possesses high peroxidase-like properties to convert H 2 O 2 into ·OH

The nanozyme has combined antibacterial ability of ·OH, PTT, and Ag +