Rapid green assembly of antimicrobial nanobunches

Antimicrobial nanobunches with different amounts of chitosan-capped Ag were prepared by continuous gas-liquid green route under ultrasound irradiation. Spark-produced aerosol Cu nanoparticles were directly injected into an ultrasound Ag(I)-chitosan reaction cell for efficient hydrosolization of the Cu particles and the subsequent incorporation of Ag and chitosan on Cu. Subsequently, electrospraying was used to form of chitosan-capped Cu-Ag nanobunch coatings. The time required for reducing the bacterial proliferation to 50% dropped to ~1 h at a nanobunch concentration of 10 μg mL−1 from the 2.0 min Ag(I) reaction time, and was further decreased to ~0.5 h by increasing the concentration of the nanobunches to 90 μg mL−1. The nanobunches were directly coated onto the substrate using an electrospray device to fabricate transparent films and composite fibers. The antimicrobial activity of the composite carbon fibers was then evaluated via the disc diffusion method.

1 and 2 were injected with the aid of a peristaltic pump (323Du/MC4, Watson-Marlow Bredel Pump, US) at constant rates of 0.42 and 2.08 mL min -1 , respectively. 85 milligrams of AgNO 3 (205052, Sigma-Aldrich, US), used as a precursor of Ag, was dissolved in 5 mL of deionized water (Solution 1). 0.25 grams of chitosan (41796, Sigma-Aldrich, US), used both as a reducing agent and a stabilizing agent, was dissolved in 25 mL of 1% acetic acid solution (03888, Sigma-Aldrich, US) (Solution 2). An ultrasound probe (VCX 750, 20 kHz, Sonics & Materials Inc., US) was immersed into the solution. The probe acted as an ultrasound irradiator (10 W mL -1 input power density) and the active part of the probe was the planar circular surface, of area 1.3 cm 2 , at the bottom of the probe. Upon the start of reactions, the pale yellow solution changed to light brown indicating the reduction of Ag(I). The Cu particles from gas-phase acted as seeds and became kinetically capable of incoming Ag ions onto the seed particles to create Cu-Ag nanobunches. Because the reduction rate of the Ag ions is faster (Ag + /Ag 0 =0.78 V) than that of Cu 2+ , the reduction of the Ag ion is preferred to that of the Cu particles. Ag layers which are formed on the surface of the Cu particles in the reaction act as the active sites for the further deposition of Ag species.
Chitosan-capped Cu-Ag nanobunches were atomized via electrospray, by means of the equipment assembled in-house. In brief, the equipment consisted of another peristaltic pump (07522-30, Masterflex, US), a stainless-steel nozzle (inner diameter 0.3 mm), a high-voltage power supply (10/40A, Trek, US), and a stainless steel plate placed directly below the nozzle as the grounded count electrode at a distance of 6 mm between the capillary and the substrate.
To find the resultant electrical field required for a stable meniscus, the following equation was employed: where E 0 is the onset electric field, γ is the surface tension of the solution, θ is the angle of the Taylor cone, ε 0 is the permittivity of the vacuum, and r is the radius of the capillary. As shown in Fig. S1, the prepared solution forms a stable jet, which is much smaller than the nozzle diameter at a position below the nozzle (monitored with a high-speed camera, Motion Pro, US). The chitosan-capped nanobunch coating after solvent evaporation was formed on a substrate (glass plate or carbon fiber) contacted with a plate heater. The prepared solution was electrosprayed (16 μL min -1 ) using the pump to perform a coating with chitosan-capped Cu-Ag nanobunches on a glass plate (7059, Corning, US). The electrosprayed nanobunches were also directly deposited on carbon fibers to evaluate their antimicrobial activity via the disc diffusion method.
The bacteria were grown overnight in Luria-Broth at 37 o C for the antimicrobial tests. The bacterial count was standardized to ~10 6 cfu mL -1 by measuring their optical density and maintained at 37 o C for 2 h. The initial concentration of bacteria was determined by the agar plate method. An aliquot of 50 mL of the bacteria broth was poured onto the nanobunch surface and covered with a glass for a specific time ranging from 10 to 300 min at 37 o C. After the time elapsed, the samples were washed with a solution of 0.9 wt % of NaCl and 1.0 wt% of Tween solution, an aliquot of 40 μL was extracted, transferred onto a nutrient agar plate and incubated for 16 h at 37 o C before the colonies were counted. The minimum inhibitory concentration was also obtained using E. coli and S. aureus in nutrient agar medium after 24 h of incubation.

4
The generation of intracelluar reactive oxygen species (ROS) generation in nanobunch treated bacterial cells was determined using a cell-permeant fluorescent dye 2',7'dichlorodihydrofluorescein diacetate (H 2 DCFDA), an intracellular ROS indicator. S1 All experiments were performed in triplicate, and the results were reported as average values and standard deviations.

FIG. S1
Schematic diagram of continuous gas-liquid hybrid chemical route to fabricate chitosancapped Cu-Ag nanobunches through a serial system consisting of a spark plasma, an ultrasound Ag(I) cell, and an electrospray device with a heated ground plate A size distribution of the spark-produced Cu nanoparticles. Representative TEM images of the Cu particles are also shown as insets.
In the case of pure Cu colloidal solution, 0.1 mL of the purified solution was dropped on the TEM grid, and then dried to remove solvent from the drop on the grid for scanning SEM and TEM observations. According to the inset in Fig. S2, the most frequently observed lattice spacing was approximately 0.21 nm, which can be matched to the (111)   The stretching vibrations of N-H and O-H were assigned to the 3,200 cm -1 and 3,400 cm -1 broad peaks, respectively, and the bands at 2,940 cm -1 and 2,960 cm -1 were attributed to the CH 2 -and CH 3 -bends, respectively. The peaks around 1,580 cm -1 corresponding to the N-H bending vibration S3 moved to 1,570 cm -1 and their intensity decreased. This suggests that the Ag atoms obtained from Ag ions in the presence of chitosan could be conjugated with nitrogen atoms during ultrasonification. Thus, the vibration intensity of the N-H bond could be reduced by increasing the molecular weight owing to the Ag conjugation. S4 In addition, the peak at around 1,660 cm -1 corresponds to the N-H scissoring from the primary amine groups that are being dissociated, and the intensity decreases at around 3,420 cm -1 of the O-H stretching from the hydroxyl were identified when the Ag atoms were capped by chitosan molecules, suggesting that Ag atoms were conjugated with the amino and hydroxyl groups of chitosan, as shown in Fig. S5b. Photographs of glass discs as untreated, chitosan-coated, and chitosan-capped Cu-Ag nanobunch-coated on a mobile display device, and on white and printed papers.
The figure shows that the nanobunch-coated glass plate is transparent, although there are differences in the transparency among the bare, chitosan-coated, and the nanobunch-coated glass plates.

FIG. S7
Representative batch growth profiles in the presence of chitosan-capped Cu-Ag nanobunches with increasing reaction time ranging from 0.5-5.0 min. SEM images of E. coli before and after treatment with the Cu-Ag nanobunches (2.0 min reaction time) are also shown as insets.

Clean
Chitosan Chitosan-conjugated Cu-Ag The high antimicrobial activity of nanobunches corresponds to the efficient diffusion of the metallic ions, which introduces more significant inactivation of DNA and relevant cellular components that subsequently generates pits in the bacterial cell walls, resulting in enhanced diffusion into the cells (inset of Fig. S7). S5 The wrinkled membranes on the nanobunchtreated cells verify the ability of the nanobunches to disrupt the membrane. A larger number of metallic ion diffusion points of the nanobunches (Fig. S9) owing to the smaller primary metallic particle sizes and anisotropic overall shapes might induce higher antimicrobial activity compared to the spherical Ag particles.

FIG. S9
Different antimicrobial activities with spherical Ag nanoparticles and Cu-Ag nanobunches toward E. coli. A larger number of bumps (favorable for diffusing Ag ions onto cell membranes) on the nanobunches than those on the Ag nanoparticles might be more suitable for efficient inactivation of cell proliferation. Generation of intracellular ROS in the bacterial cell suspensions treated with chitosan-capped nanobunches (2 min) including pure chitosan, Ag, and Cu-Ag samples (at 50 μg mL -1 for 4 h incubation).
Chitosan provided a higher affinity than the nanobunches themselves with the negatively charged biological membranes. This suggests that the chitosan-capped nanobunches are not cytotoxic and are likely suitable as antimicrobial coatings of biomedical devices.