Determination of antibacterial and photothermal properties of novel composites based on graphene oxide/reduced graphene oxide, gold nanoparticles, and graphene quantum dots

Description

Methods (English)

The UV-Vis absorption spectra were recorded on LLG-uniSPEC 2 spectrophotometer. Samples were recorded in quartz cuvettes at room temperature from water dispersion. Photoluminescence measurements were performed on FluoroMax^® Plus spectrofluorometer (HORIBA Scientific). Emission spectra were measured using a CW 150 W xenon arc lamp as an excitation source and an R928P photomultiplier tube as a detector. The excitation and emission slit widths were both 3 nm. Raman spectra were recorded on a DXR Raman microscope (Thermo Scientific). Each spectrum was obtained at room temperature by using a 532 nm excitation line with a power of 2 mW. The spectral resolution was 1 cm^-1 and the acquisition time was 10 × 10 s. The confocal pinhole diameter was 50 µm. Each sample was recorded at four different places and an average spectra was used. Fourier transform infrared (FTIR) spectra were recorded in the ATR mode using a Nicolet 6700 FTIR Spectrometer (Thermo Scientific) at 4 cm^-1 resolution, in the wavenumber range 500-4000 cm^-1. The XRD analyses were performed on a Philips PW 1050 powder diffractometer with Ni-filtered Cu-Kα radiation (λ = 0.15418 nm). The diffraction intensity was measured by the scanning technique in the 5-90° 2θ range (step size of 0.05° and a counting time of 60 s per step). A high-resolution transmission electron microscope (HR-TEM) JEOL JEM-2100F operated at an accelerating voltage of 200 kV was used to analyze the structure and morphology of the composites. All samples for TEM were dispersed in ethanol and a drop of dispersion was deposited on lacey carbon copper grids (200 mesh). Contact angle measurements were carried out by using the sessile drop method on the Theta Lite contact angle meter (Biolin Scientific). Thin films were made by drop-casting the composite water dispersions on freshly cleaned microscope glass slides. Each thin film was made by dropping a total volume of 250 mL of 1 mg mL^-1 dispersion in increments of 50 µL. After each increment deposition, the glass slide was left to dry on a hot plate at 60 °C. For the data acquisition, ∼6 μL of deionized water (MilliQ 18.2 mΩ cm^-1) was carefully dropped on the samples using a micro syringe. All measurements were performed at ambient conditions (25 °C) and immediately after droplet stability. The data were analyzed using OneAttension software (version 4.0.3).

Photothermal conversion efficiency measurements were performed in 1 × 1 × 4.5 cm spectrometer quartz cuvettes at room temperature (19.0 °C). Stable homogeneous dispersions of the samples in water with a concentration of 1 mg mL^-1 were exposed to 808 nm continuous wave (CW) laser radiation with TEM00 mode radial distribution, slightly elliptical, with major and minor axes of 2.69 mm and 2.44 mm, respectively. The laser power was 770±10 mW and the laser power density was 14.95 W cm^-2. The center of the laser spot was placed at a fixed position in the center of the cuvette. The temperature evolution was recorded by a thermocouple (accuracy 0.1 °C) every 30 s. The samples were irradiated for 10 min (heating cycle) until they reached thermal equilibrium. Then, the laser was switched off to allow the sample to cool down to room temperature and the temperature was monitored for the following 15 min (cooling cycle).

The ability of GO composites to generate singlet oxygen under blue light illumination was estimated by measuring the photobleaching of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA). ABDA is considered a selective singlet-oxygen probe. Upon reaction with this radical, degradation of ABDA molecules is observed as a reduction of the absorption band at λ=378 nm. For illumination, a blue light (470 nm) was used and positioned 12 cm from the upper surface of the sample dispersions. To overcome the issue of ABDA insolubility in water, the probe (2.7 mg) was dispersed in DMSO (10.7 mL). Then, demineralized water was added to achieve the final concentration of 104 μM. The composites were added to the ABDA solution: 150 μL of water dispersion of the composites in the concentration of 1 mg mL^-1 was mixed with 2.850 mL of ABDA stock solution. Each sample was illuminated with a blue-illumination source (λ=470 nm), and UV-Vis absorption spectra were recorded every 20 min. Blank control was ABDA stock solution mixed with 150 μL of demineralized water. All spectra were recorded using a LLG-uniSPEC 2 Spectrophotometer (Lab Logistic group, Mekenheim, Germany) at 20 °C, in air. The wavelength range was 300-550 nm.

The minimum inhibitory concentration (MIC) values were determined according to the standard broth micro-dilution assays, recommended by the National Committee for Clinical Laboratory Standards for Bacteria (M07). Antimicrobial activity was tested against 8 bacterial strains: Staphylococcus aureus NCTC 6571, S. aureus MRSA ATCC 43300, Enterococcus faecium ATCC 6057, Pseudomonas aeruginosa ATCC 10332, Klebsiella pneumonie ATCC BAA 2146, Listeria monocytogenes NCTC 11994, Escherichia coli NCTC 9001, and Acinetobacter baumanii ATCC 19606. For the cultivation of bacterial test organisms, Luria-Bertani broth was used, and cultures were diluted to the concentration of 5 × 10⁵ colony-forming units (CFU) mL^-1 for bacteria. Colloids were serially diluted in media to the concentration range of 500 - 3.91 µg mL^-1. Plates were exposed to blue light for 60 min and further incubated in the dark. After incubation at 37 °C for 24 h, the minimum inhibitory concentrations were determined by measuring absorbance at 625 nm using a plate reader (Epoch Microplate Spectrophotometer, BioTek Instruments, Inc., USA).