Evolution 260 bio spectrophotometer

The compounds were dissolved in PBS buffer with 2% DMSO, and their UV-visible spectra were taken with a Thermofisher Scientific Evolution 260 Bio Spectrophotometer (Rodano, MI, Italy), in a 700 μL quartz cuvette 1 cm long. The compounds were incubated in a standardized chamber with a temperature fixed at 37 °C at atmospheric pressure.

Sodium alginate (20 mg/ml) was mixed with kanamycin disulfate (50 mg/ml) in a volume ratio of 1:1 ratio (10 ml each). Resultant solution with alginate polymer was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and the pellet was washed three times with 5 ml of sterile 0.1 M (pH 7.4) phosphate-buffered saline (PBS) buffer. Six pellets were made and incubated with 2 ml of 1 M NaOH at 10°, 20°, 40°, 60°, 80° and 100°C for 30 min. After incubation, tubes were centrifuged at 10000 rpm for 10 min and absorption spectra of supernatants (Fig 2) were measured using a spectrophotometer (Thermo Scientific Evolution 260 Bio Spectrophotometer).

The prepared cryogel samples (P1-K ÷ P9-K) were tested for their capacity to retain PG as the model antibiotic. In this respect, 0.02 mol/L PG solution was prepared by the dissolution of 0.356 g of PG into 50 mL of H₂O under magnetic stirring (200 rpm), while ensuring light protection. Each cryogel sample (approximately 0.02 g) was contacted with a volume of 10 mL of PG solution. At different time intervals (5, 15, 30, 60, 120, 180, 1440 min), the liquid phase (supernatant) was tested by UV–Vis spectroscopy at λ = 322 nm (specific wavelength of PG) [62 (link)] in order to evaluate the retention capacity of cryogels for PG (q, mmol PG/g cryogel) as given in Equation (2), where Ci (mmol/L) and Cf (mmol/L) are the initial and final concentrations of PG in the supernatant, Vs (L) and Mcryogel (g) represent the volume of the PG solution and the weight of the dried cryogel taken into account. The UV–Vis spectra were recorded using a UV–Vis ThermoScientific EVOLUTION 260 BioSpectrophotometer.
$$q=\left(Ci-Cf\right)\ast Vs/Mcryogel$$
The adsorption mechanism was analyzed using a pseudo-second-order kinetic model, described by Ho and McKay [66 (link)], as presented in Equation (3).
$$\frac{1}{q_t}=\frac{t}{q_e}+\frac{1}{k_2{q}_e^2}$$
where q_e and q_t are the adsorption capacity (mg/g) at equilibrium and at time t (min), respectively, and k₂ (g mg⁻¹ min⁻¹) is the pseudo-second-order adsorption rate constant.

UV-melting experiments were carried out on a Thermo Scientific Evolution 260 Bio spectrophotometer. Quartz cuvettes (Hellma) were used with a path length of 1 cm in 10 mM sodium phosphate buffer with 100 mM sodium chloride. Oligonucleotides of 1 μM concentration were scanned at a rate of 1°C/min (for both heating and cooling), at a wavelength of 260 nm. Melting temperatures were determined by the first derivative maxima of the hexic equation fit. The DNA melting curves are included as Supplementary Figures S2 and S13.

These experiments were carried out in a PBS buffer with 5% of DMSO. Ligands concentration was 40 μM for Ga1–Ga4, In1, and In2, whereas it was 20 μM for In3 and In4. Titrant solution containing the metal nitrates in the same solvent was 400 μM concentrated for Ga1–Ga4, In1, and In2, whereas they were 200 μM for In3 and In4. Additions were executed using Eppendorf™ micropipettes sensible to 10 μL in a progressive manner: we started with 20 μL for the first 10 times, up to 40 μL and 100 μL additions at the end of each titration. UV-visible spectra were taken in a 4 mL quartz cuvette 1 cm long with a a Thermofisher Scientific Evolution 260 Bio Spectrophotometer (Rodano, MI, Italy).

Protein concentration was determined by the Pierce™ Micro BCA Protein Assay Kit (mBCA kit, Thermo Fisher Scientific) according to the manufacturer’s instructions and using the provided Pierce™ Bovine Serum Albumin (BSA, Thermo Fisher Scientific) as standard. Each sample was diluted in duplicate, preparing a 5-point calibration curve using a BSA starting solution of 20 µg/mL. Then, the three reagents of the mBCA kit are properly mixed following the manufacturer’s instructions and added to the sample, which is heated at 60 °C in a bath of water for 1 h. The samples are finally transferred to a plastic disposable cuvette and read at the spectrophotometer at 562 nm (Evolution 260 Bio Spectrophotometer, Thermo Scientific, Thermo INSIGHT software 2.1.175). The final protein concentration (µg/mL) is quantified based on the BSA calibration curve.