Electronic origin of antimicrobial activity owing to surface effect


Preparation of npAu and npAu-Pt

A 100-nm-thick pure gold film (>99.9 mass%) was sputtered on a 50 × 50 × 1.2 mm glass substrate. 150-nm-thick Au0.3Ag0.7, (Au0.5Pt0.5)25Ag75, and (Au0.9Pt0.1)25Ag75 films were then sputtered on the pure gold film. Nanoporous Au and Au-Pt specimens were fabricated by dealloying (free corrosion) of these films at 253 K for 24 h in 69 mass% HNO3. Also, a nanoporous Au specimen with a larger pore of 50 nm was fabricated by dealloying at 298 K. Nanoporous Au and Au-Pt made of Au0.3Ag0.7, (Au0.5Pt0.5)25Ag75 and (Au0.9Pt0.1)25Ag75 were denominated “npAu”, “npAu-Pt0.5” and “npAu-Pt0.1”, respectively. Immediately after dealloying, the specimens were thoroughly rinsed more than 10 times with pure water. A flat Au (fAu) specimen, which was fabricated by sputtering of pure gold, was used as a reference inert substrate. The microstructures of npAu-Pt and npAu specimens were observed by scanning electron microscopy (SEM; SU-6600 by Hitachi High-Technologies Corporation). The average ligament sizes were calculated by measuring the diameter of >50 ligament, while the average pore sizes were calculated by averaging >50 spacing between ligaments, except for npAu-Pt0.5 sample whose ligaments and pores were too small to observe clearly by SEM. X-ray diffraction (XRD; X’Pert Pro by PANalytical) measurements were performed on the npAu-Pt, npAu and fAu specimens. Their chemical compositions were investigated by energy-dispersive X-ray (EDX; XFlash 5010, Bruker AXS, Germany) spectroscopy.

Bacterial strain

Type strains of E. coli (K-12, NBRC 3301) were supplied by the National Institute of Technology and Evaluation (Tokyo, Japan). We incubated the bacteria in Luria-Bertani (LB) medium at 308 K for 44 h before treating them in antimicrobial tests. Casein-peptone glucose yeast extract LB (Wako Pure Chemical Industries Ltd., Osaka, Japan) was used for the incubation.

Tests of antimicrobial activity (AA)

The antimicrobial properties of npAu-Pt, npAu and fAu were investigated mainly according to the Japanese Industrial Standard (JIS) “Antibacterial products-Test for antibacterial activity and efficacy”31. First, one quantity of platinum loop of bacteria incubated in the medium was removed from the colony and placed in 5 mL of 1/500 nutrient broth, followed by vortex mixing. Second, 400 μL of the bacterial suspension was dropped onto the samples and then a 40 × 40 mm PE film covered the bacterial suspension. In this way, bacterial suspensions were incubated on the specimen for 24 h in humidity-controlled incubators at 308 K and at a relative humidity (RH) of 50%. The RH is 90% in JIS; however, the AAs for fAu and npAu were almost zero at an RH of 90%11. Therefore, the AA tests were carried out at the intermediate RH of 50%. Third, the incubated bacteria were recovered using 10 mL of Soybean Casein Digest Broth with Lectithin & Polysorbate 80 (SCDLP) medium and diluted 10-fold in phosphate-buffered saline (PBS). The diluted PBS was mixed in LB medium to make a 10-fold dilution series of LB pour plates. These were then incubated at 308 K for 48 h. The number of colonies in the LB pour plates was then counted. Viable bacteria counts (VBCs) were statistically analyzed by the one-way analysis of variance followed by a post-hoc test. The AA was given by



in which N0 is the viable bacteria count for fAu (as a control sample) and N is the viable bacteria count for npAu-Pt or npAu. The mean value of AA was obtained from 5 repeated tests. All results are expressed as mean ± standard deviation.

Inductively coupled plasma (ICP) atomic emission spectrophotometry measurements

The culturing solution was suspended on the npAu-Pt substrate for 24 h, and the sample was then analyzed using ICP atomic emission spectroscopy. The concentrations of silver, gold, and platinum ions in the culturing solutions were found to be < 0.05 ppm of the apparatus detection limit. At least approximately 1 ppm is necessary for realizing the antimicrobial properties of Ag ions4,32,33,34. Therefore, the effect of Ag ion dissolution on AA could be ignored.

Ultraviolet photoelectron spectrometery (UPS) measurements

WFs of npAu-Pt, npAu and fAu were measured with a PHI 5000 VersaProbe II Scanning ECSA Microprobe system (ULVAC-PHI, Chigasaki, Japan). A windowless helium discharge light source that provided He1 emission at 21.22 eV was used. The diameter of a vacuum-ultraviolet (VUV) light beam was 5 mm and the incident angle was 45°. The samples were biased at −5 V dc to drive low-energy secondary electrons into the detector to prevent signal cut-off owing to the detector. The work function (WF) ({rm{Phi }}), can be given by

$${E}_{{rm{fermi}}}-{E}_{{rm{cutoff}}}={rm{Phi }}-hnu ,,$$


in which Efermi is the binding energy of the electron at fermi level, Ecutoff is the energy of the low-energy secondary electron, and hv is the photon energy (21.22 eV). Before the measurements, the surfaces were cleaned by removing organic molecules using gas cluster ion beam (GCIB) of Ar emission for 5 minutes.

First-principles calculations of Au surfaces

We performed first-principles calculations for geometry optimization calculation of Au surface models by using the Cambridge Serial Total Energy Package (CASTEP)35, in which a plane-wave basis set was used to calculate the electronic properties based on density functional theory (DFT)36,37. The Perdew-Burke-Ernzerhof functional (PBE) version of the generalized gradient approximation38 was used to represent exchange and correlation interactions within the DFT. Ultrasoft pseudopotentials39 were used for all elements in the calculations. The cutoff energy was set to 320 eV and the Brillouin zone was sampled using 5 × 5 × 1 Monkhorst-Pack k-point meshes in all calculations40. Periodic boundary conditions were applied in the x, y, and z directions for all of the calculations.

A slab geometry with 4 atomic layers of 4 × 4 and a vacuum layer of 30 Å was used to model npAu-Pt, npAu and fAu surfaces (Supplemental Fig. 4). In the models, Ag atoms were not considered because effects of Ag atoms on the AA were ignorable. The atoms at the top three layers were relaxed to their equilibrium positions and the atoms at the bottom layer were frozen at their bulk positions in the models. Nanoporous metals have large lattice strains of up to 10% at the surfaces26,28. A previous study12 showed that a cell wall was hyperpolarized when the cell wall was adsorbed on the (111) surface of npAu with 5% compressive lattice strain. Thus, the Au (111) surface with 5% compressive lattice strain was used as the npAu model in the present study. To create a npAu-Pt model, three Au atoms of the first layer in the npAu model were substituted by Pt atoms. The Pt concentration in the Au-Pt model almost corresponded to the experimental one, which was detected by XPS for npAu-Pt0.5. The WF was calculated with these surface models, in which the WF was defined as the energy difference between the electrostatic potential at the middle of the vacuum region and the Fermi energy14.

Molecular dynamics simulation and first-principles calculations of hyperpolarization of peptidoglycan

The hyperpolarization of peptidoglycan interacting with npAu-Pt, npAu or fAu was calculated by first-principles calculations and molecular dynamics (MD) simulations with the same methods used in a previous study12. A scaffold model of peptidoglycan was constructed. The peptidoglycan was immersed in a spherical water solvation, where the center of water solvent was positioned at the mass center of peptidoglycan and the diameter of the spherical solvent water was 50.0 nm. Counter ions of 43 Na+ and 43 Cl were added to neutralize the system. The system was energy-minimized using the steepest decent algorism (200,000 steps) and the conjugate gradient algorism (100,000 steps). MD simulations were performed with a time step of 2.0 fs. The system was gradually heated from 5 to 300 K for 4 ps to activate thermal motion in the system. The system was equilibrated for 1 ns to obtain a stable structure of peptidoglycan with a constant number of particles, volume and temperature (NVT). Finally, the 10 ns NVT simulations were performed.

An interaction between MurNAc, which is a part of peptidoglycan, and the Au surface was calculated by first-principles calculations. A (4sqrt{3}times 3sqrt{3}) unit cell, which consisted of four Au layers, with a lattice strain of −5% was used as a npAu surface model (Supplemental Fig. 5). A vacuum gap of 15 Å was added to create the surface. For the npAu-Pt model, 12 Au atoms of the surface layers were substituted by Pt atoms, in which the replaced positions were the same as those in Supplemental Fig. 4 (Supplemental Fig. 5). The geometry optimization calculations were performed on the Au surface models by first-principles calculations using the Dmol3 code41,42. In the DMol3 method, the physical wave functions were expanded in terms of the accurate numerical basis sets. The exchange-correlation energies were treated according to the generalized gradient approximation (GGA) with the Perdew-Wang 1991 (PW91) approximation43 to deal with the core (DNP). The ultrasoft pseudopotentials39 represented in reciprocal space were used for all elements in the calculations. Optical Bloch equation (OBE) calculations were used to set the van der Waals interactions into calculations. A Fermi smearing of 0.005 hartree (1 hartree = 27.2114 eV) was adopted. A Brillouin zone of 2 × 2 × 1 using a Monkhorst-Pack k-point mesh40 was used. The bottom layer of the cell was frozen during geometry optimization calculations. MurNAc was positioned to be the atop site12.

After the geometry optimizations of a MurNAc molecule located on the atop site of the Au surface model, a MurNAc molecule was put back at the same position in the original peptidoglycan model, and 1 ns MD simulations were performed again, in which the atomic positions of the MurNAc molecule were fixed during the calculations. Then, electrostatic potentials of the obtained peptidoglycan were calculated by solving the Poisson Boltzmann equation using the finite difference method implemented in the Delphi program44,45. The values of the atomic radii and partial atomic charges were taken from the CHARMM parameter set. The peptidoglycan was divided into a three-dimensional cubical grid and the electrostatic potential at each grid point was computed.

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