Vcx 750

The effect of different concentrations of PS microplastics on LDH release was measured using the CyQUANT LDH Cytotoxicity assay kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. After incubation for 24 h, 50 μl of the supernatant was added to a 96-well plate. For maximum LDH release, 10⁸ CFU/ml cells were lysed in an ice bath using a tip sonicator (VCX 750, Sonics & Materials, Inc., USA) at a duty cycle of 16.7% (10 s sonication and 50 s rest) for 20 min. The sonicated sample was added to a 96 well-plate, followed by addition of 50 μl of the substrate mixture and incubation without agitation at ambient temperature in the dark for 30 min. Then, 50 μl of the stop solution was added, absorbance was acquired with a spectrophotometer at 490 nm and 680 nm (background), and the difference in absorbance at these two wavelengths was determined. The LDH release was calculated using the LDH activity of the PS-treated sample (LDH_T), spontaneous LDH activity (LDH_S), and maximum LDH activity (LDH_M):
$$LDH(\%)=\frac{LD{H}_T-LD{H}_S}{LD{H}_M-LD{H}_S}\times 100$$
The LDH release of each sample (LDH_Sample) was then expressed relative to the negative control (LDH_NC) that had no microplastics:
$$\text{Relative }LDH(\%)=\frac{LD{H}_{Sample}}{LD{H}_{NC}}\times 100$$

SbSI nanowires were prepared via a sonochemical method [42 (link)] from the constituents, i.e., the elements—antimony, sulfur, and iodine weighed in the stoichiometric ratio. The component mixture was immersed at room temperature and ambient pressure in ethanol. The reagents were put into a polyethylene/polypropylene cylinder. The vessel was closed during the experiment to prevent volatilization of the precipitant during the longer test times. The cylinder was partly submerged in water in a cup-horn ultrasonic reactor (750 Watt ultrasonic processor VCX-750 with a sealed VC-334 converter (Sonics & Materials, Inc., Newtown, CT, USA)). The reagents were irradiated by ultrasounds with 20 kHz frequency and 565 W/cm² power density for 2 h. The temperature of sonolysis was 323 K. Further experimental details of the applied procedure are described in previous works [42 (link),43 (link)]. When the sonochemical process was completed, the ethanol was evaporated off giving a SbSI xerogel.
The structure of individual SbSI nanowire was analyzed using a high-resolution transmission electron microscopy (HRTEM). These investigations were completed at 300 kV accelerating voltage on a JEOL-JEM 3010 microscope (Peabody, MA, USA) with point-to-point resolution of 0.17 nm. The procedure of sample preparation was the same as described in [42 (link),43 (link)]. The morphology of aligned SbSI nanowires was studied at acceleration voltage of 10 kV on Phenom Pro X (Thermo Fisher Scientific, Waltham, MA, USA) scanning electron microscope (SEM).
The optical diffuse reflection spectroscopy (DRS) was carried out using the apparatus described in [42 (link)]. DRS spectra were recorded at 296 K in the wavelength range from 350 to 1000 nm. The diffuse reflectance values R_d were converted to the Kubelka–Munk function (F_K–M) [44 ], known to be proportional to the absorption coefficient α.
The procedure of the FE-PV device preparation can be summarized as follows. Alumina chip #103 (Electronics Design Center, Case Western Reserve University, Cleveland, OH, USA) was used as substrate. It was equipped with platinum interdigitated electrodes separated by a gap of 250 μm. A substrate with platinum electrodes was chosen due to the fact that the energy level of the Pt electrode is close to Fermi energy in the p-type SbSI semiconductor [45 (link),46 (link)], which should influence a relatively high zero-bias photocurrent [38 (link)]. Symmetric electrodes were used to eliminate the effects of different work functions and asymmetric Schottky-Ohmic contacts on the photovoltaic properties of SbSI. In the first step of device preparation, SbSI xerogel was dispersed uniformly in toluene using the ultrasonic reactor IS-UZP-2 (InterSonic, Olsztyn, Poland). Then a droplet of dispersed solution was placed onto the #103 chip using an insulin syringe equipped with a 31 G needle. The direct current (DC) electric field of 5∙× 10⁵ V/m was applied to the electrodes during the deposition of SbSI sol in order to align the nanowires perpendicularly to the electrodes. Each single coating process was followed by sample drying for toluene evaporation. It was realized at room temperature for 5 min in 830-ABC/EXP glove box (Plas-Labs Products). The steps from sol deposition to sample drying were repeated 15 times. The SbSI FE-PV device, prepared according to the procedure mentioned above, had lateral architecture, in which a photocurrent could be measured along the polarization direction.
Dark current and the photocurrent were detected by a Keithley 6430 Sub-Femtoamp Remote SourceMeter equipped with a low noise probe station (Tektronix, Inc., Beaverton, OR, USA). The acquisition of the data was realized using a PC computer with a GPIB (General Purpose Interface Bus) and an appropriate program in LabView (National Instruments, Austin, TX, USA). All electrical measurements were performed in a test chamber in a vacuum (p = 2·× 10⁻³ Pa) produced by TW70H turbomolecular vacuum pump (Prevac, Rogow, Poland) in order to eliminate the influence of humidity [47 (link)] and gas adsorption [2 (link),7 (link)] on electrical properties of SbSI nanowires. A constant operating temperature T = 268 K of the SbSI FE-PV device was maintained using a HAAKE DC30 thermostat with a Kessel HAAKE K20 circulator (Thermo Scientific, Waltham, MA, USA), and Pt-100 sensor with 211 temperature controller (Lake Shore, Columbus, OH, USA). Measurements of photocurrent were carried out under illumination with monochromatic light (λ = 488 nm) from Reliant 50 s argon laser (Laser Physics, Milton Green, UK), which covered the whole sample area between the electrodes. The neutral filters UV–NIR-FILTER-250–2000 nm (Quartzglas-Substrate, Oriel) were applied to change the optical power density (P_opt). The values of P_opt were determined using S-2387 silicon photodiode (Hamamatsu, Hamamatsu City, Japan) in a short-circuit regime with a Keithley 6517A electrometer. Prior to the photocurrent measurements, the SbSI FE-PV device was poled by cooling from 320 to 268 K under an external electric field of ±10⁶ V/m to align the polarization in one direction, hereafter indicated as positive (+P) or negative poling (−P).

The purified expression vectors were transformed into the competent BL21(DE3) E. coli strain. In addition, the pernisine^wt-containing vectors were transformed into the BL21(DE3)pLysE, BL21(DE3)pMAGIC [F^–ompT hsdS(rB^–mB^–) dcm⁺Tet^rgal λ(DE3) endA Hte] and BL21-CodonPlus(DE3)RIL [F^–ompT hsdS(rB^–mB^–) dcm⁺Tet^rgal λ(DE3) endA Hte [argU ileY leuW Cam^r]
E. coli strains and plated in the appropriate selection medium. The selected transformants were grown as a mini-scale batch (10 ml LB medium) and the plasmids were purified using GenElute plasmid miniprep kits (Sigma). The DNA was sequenced (Macrogene), and the transformants with confirmed pernisine DNA were used for large-scale expression (4.0 L LB medium).
A single colony was cultivated overnight at 37°C in 25 ml LB medium supplemented with the appropriate antibiotic, under constant agitation at 240 rpm. The next day, 475 ml fresh LB medium containing the appropriate antibiotic was added to 25 ml of the overnight culture. When the cells reached an optical density at 600 nm (OD₆₀₀) of 0.6 to 0.8, expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. The culture growth times after this induction ranged from 1 h to 4 h, as optimised initially by the detection of pernisine on dot blots. The cells were centrifuged (6,000x g, 20 min, 4°C) and resuspended in 25 ml lysis buffer (30 mM Tris-HCl, 0.3 M NaCl, 1 mg ml^-1 lysozyme, pH 7.5). The cells were then lysed by sonication (amplitude 40%; 10 s on, 10 s off; 120 s; VCX 750 by Sonics), and centrifuged (19,000x g, 20 min, 4°C). The supernatants were used for analysis and purification of pernisine. The pellets were resuspended in 4 M urea and subjected to SDS-PAGE, for determination of the insoluble pernisine fraction.
N-terminal His₆-tagged pernisine was purified using Ni²⁺-Sepharose 6 FF columns (GE Healthcare), followed by size exclusion chromatography using a HiLoad 16/60 Superdex 200 preparative grade column (GE Healthcare). The unbound samples were washed out with 20 column volumes of binding buffer (20 mM Na₂HPO₄, 0.5 M NaCl, 20 mM imidazole, pH 7.4), and the bound samples were eluted with the same buffer containing 500 mM imidazole. The eluted proteins were applied directly onto the size exclusion column, which was equilibrated with 30 mM Tris HCl, 0.3 M NaCl, pH 7.4. The pernisine fractions were collected, dialysed (SPECTRA/POR, MWCO 8–10 kDa) in 10 mM HEPES, pH 8.0, for 4 h, and lyophilised (Christ alpha 1-2LD Plus, Germany). All of the purification procedures were performed at 4°C.