Phosphotungstic acid hydrate

Polymyxin B sulphate (Pol-B) was purchased from Alfa Aesar®. Sodium poly(styrene sulphonate) 70 KDa average molecular weight (PSS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), phosphotungstic acid hydrate and Luria Bertani (LB) broth (Miller) were bought from Sigma-Aldrich Co. Nylon 0.45 μm syringe filters were purchased from Camlab Ltd. Carbon-coated copper TEM grids (200 mesh) were purchased from Agar Scientific Ltd. Float-a-Lyzer® G2 (20 KDa MWCO) dialysis membranes were purchased from Spectrum Laboratories Inc. Dulbecco’s phosphate buffered saline (DPBS), 9.5 mM in phosphate and without Ca²⁺ and Mg²⁺, was bought from Lonza. Agar was purchased from Sigma-Aldrich Co.

Sodium dodecylbenzenesulfonate (NaDBS, 99%), ethylene glycol (EG, 99.8%), tetrabutylammonium hexafluorophosphate (TBAPF₆, >99%), phosphotungstic acid hydrate (PTA, PW₁₂O₄₀³⁻, reagent grade), and propylene carbonate (PC, 99%) were supplied from Sigma-Aldrich (Taufkirchen, Germany). Commercial Durapore (Millipore, 0.22 μm, Sigma-Aldrich) was used as supplied. Pyrrole (Py, 98%) from Sigma-Aldrich was distilled prior to use and stored at a low temperature (−20 °C) in the dark under a nitrogen atmosphere. Milli-Q+ (Tallinn, Estonia) deionized water was used as supplied.

Titanium (IV) oxide (P25, TiO₂, 99.5%), phosphotungstic acid hydrate (H₃O₄₀PW₁₂·xH₂O, M_w = 2880.05), zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O, ≥99.0%), iron(III) nitrate nonahydrate (Fe(NO₃)₃·9H₂O, ≥98%), ammonium metavanadate(V) (NH₄VO₃, 99%), cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O, 98%), cerium(III) nitrate hexahydrate (Ce(NO₃)₃·6H₂O, 99%), gallium(III) nitrate hydrate (Ga(NO₃)₃·xH₂O, 99.9%), copper(II) nitrate trihydrate (Cu(NO₃)₂·3H₂O, 99–104%), silver nitrate (AgNO₃, ≥99.0%), palladium(II) nitrate hydrate (Pd(NO₃)₂·xH₂O, 99.9%) and dimethyl carbonate (DMC, ≥99.0%) were purchased from Sigma-Aldrich and used without further purification. ¹³C labeled carbon dioxide (¹³CO₂, 99 atom% ¹³C - <3 atom% ¹⁸O) was purchased from Cortecnet.

The following chemicals were obtained from commercial suppliers and used as received: the polymer poly(d,l-lactic-co-glycolic acid) 50 : 50 (PLGA; M_W 38 000–54 000 Da, T_g 46–50 °C), under the commercial name of Resomer® RG 504 was purchased from Boehringer Ingelheim (Ingelheim, Germany) and Evonik Industries (Evonik Röhm GmbH, Germany). Sodium cholate, chloroauric acid (HAuCl₄·3H₂O), sodium citrate tribasic dehydrate ≥99.0% (Na₃C₆H₅O₇·2H₂O), PVA (polyvinyl alcohol) M_W 85 000–124 000 Da, 97–99% hydrolyzed, taurocholic acid sodium salt hydrate ≥95% (TLC), ethyl acetate ACS reagent, dodecanethiol, phosphotungstic acid hydrate as a contrast agent for microscopy and fluorescent molecule pyrene ≥99.0% were purchased from Sigma Aldrich (St Louis, MO, USA).

Polymyxin B sulphate (Pol-B) was purchased from Alfa Aesar®. Sodium poly(styrene sulphonate) (PSS) of 32, 17 and 10 kDa average Mw (GPC standards), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), phosphotungstic acid hydrate, agar and Luria Bertani (LB) broth (Miller) were bought from Sigma-Aldrich Ltd. Nylon 0.45 µm syringe filters were purchased from Camlab Ltd. Carbon-coated nickel TEM grids (200 mesh) were purchased from agar Scientific Ltd. Dulbecco’s phosphate buffered saline (DPBS), 9.5 mM in phosphate and without Ca²⁺ and Mg²⁺, was bought from Lonza.

For virus stability tests, bare and salt-coated filters were exposed to CA/09 H1N1 aerosols and, after 5 and 60 min incubation, virus was recovered by suspension of the filters, as described above. To study the effects of the coating formulation during aerosol drying independently from crystal growth, bare and salt-coated filters were immersed in DI water and removed after 60 min. Subsequently, virus was incubated in the obtained suspension for 60 min. Additionally, the virus suspension was centrifuged at 19,800 g and 4 °C for 10 min to collect the samples and suspend them in DI water. For TEM analysis (200 kV, JEOL JEM 2100; JEOL, Peabody, MA), samples were deposited on copper grid (Electron Microscopy Sciences, Hatfield, PA) and negatively stained with solution comprised of phosphotungstic acid hydrate (1.5 w/v%, pH = 7.0; Sigma-Aldrich, Oakville, Canada).
To identify the morphology of salt-coated filters and recrystallized salts, SEM/EDX analysis was performed for bare and salt-coated filters after coating with 10 nm thick gold layer. Scanning electron microscopy analysis (Hitachi S-3000N; Hitachi, Toronto, Canada) was operated in secondary electron mode at 20 kV and EDX analysis was obtained with EDX detector (Oxford Instruments, Concord, MA).

Swarming motility was determined by transferring 5 µL of an overnight culture of each strain onto the surface of trypticase soy broth (Difco Laboratories) solidified with 0.25% agar [44 (link)]. The plates were incubated at 3 and 37 °C for 6 weeks and 24 h, respectively.
The presence of flagella was examined using a Tecnai 12 transmission electron microscope (Philips Electron Optics, Eindhoven, The Netherlands) on BHI cultures grown to mid-logarithmic growth phase at 3 and 37 °C. Cells from 1 ml of each culture were fixed with 5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA), incubated at room temperature for 2 h and washed with 1 ml autoclaved water. The cells were then prepared on carbon-coated grids and negatively stained with 1% phosphotungstic acid hydrate (Sigma-Aldrich).