Evolution 220 uv visible spectrophotometer

UV-visible absorbance measurements were performed on an Evolution 220 UV-Visible Spectrophotometer (Thermo-Fisher Scientific, Dreieich, Germany). A quartz cell (Hellma Analytics, Müllheim, Germany) with 10 mm light path was used. Solutions of the cross-linkers SIA and BMPS in methanol/acetonitrile were added to a buffer solution for a final concentration of 0.03 mg/mL. Phosphate buffer (12 mM sodium phosphate, 137 mM sodium chloride) was used for the pH range 6 to 9 and citrate buffer (35 mM citric acid, 65 mM trisodium citrate) for pH 5. Different pH values were achieved by adjusting the pH with NaOH 20% and HCl 20%.

The total polyphenolic content (TPC) in S. aureus Newman cultures was determined using Folin-Ciocalteu reaction according to Łopusiewicz et al. [43 (link)] with slight modification. 20 μL of suspension (1A–C) was mixed with 1.5 mL of deionized water and 100 μL of the Folin-Ciocalteu reagent (Sigma-Aldrich, Darmstadt, Germany), stirred gently for 5 min and mixed with 300 μL of a saturated solution of sodium carbonate (Sigma-Aldrich, Darmstadt, Germany). The mixture was allowed to stand in darkness for 30 min at 40 °C, followed by an absorbance reading at 765 nm using a spectrophotometer (Evolution 220 UV-Visible spectrophotometer, Thermo Fisher Scientific Inc., Waltham, DE, USA). A calibration curve of gallic acid (Sigma-Aldrich, Germany) in water:methanol (1:1, v:v) (0, 50, 100, 200, 400 and 500 μg/mL) was prepared and TPC was calculated as milligrams of gallic acid equivalents (GAE)/gram of S. aureus cell dry mass (mg GAE/g DM). Experiments were performed in triplicate.

The DPPH radical-scavenging capacity of S. aureus samples was determined, according to Ding et al. [42 (link)]. Briefly, 1 mL of suspension (1A–C) was mixed with 2 mL of methanolic DPPH radical (0.05 mM; Sigma-Aldrich, Darmstadt, Germany) solution, stirred vigorously, and incubated in the dark at room temperature for 30 min. The absorbance of the resulting solution was measured at 517 nm using a spectrophotometer (Evolution 220 UV-Visible Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). A mixture of DPPH and water:methanol (8:2, v:v) was used as a control sample. Experiments were performed in triplicate. The scavenging activity was calculated according to Equation (1): $\mathrm{DPPH} \mathrm{free} \mathrm{radical} \mathrm{scavenging} \mathrm{activity} (\%)=\frac{{\mathrm{A}}_{\mathrm{control}}{ - \mathrm{A}}_{\mathrm{sample}}}{{\mathrm{A}}_{\mathrm{control}}}\times 100$

RP of S. aureus samples was determined according to the method reported by Ding et al. [42 (link)], with slight modification as described by Łopusiewicz et al. [43 (link)]. 0.5 mL of the suspension (1A–C) was mixed with 0.5 mL of water:methanol (8:2, v:v) solution and 0.5 mL of 1% potassium ferricyanide (Sigma-Aldrich, Darmstadt, Germany) and incubated at 50 °C for 20 min. After cooling, 1 mL of this mixture was transferred to 1 mL of 0.1% ferric chloride (Sigma-Aldrich, Darmstadt, Germany), and the absorbance was measured at 700 nm using a spectrophotometer (Evolution 220 UV-Visible spectrophotometer, Thermo Fisher Scientific Inc., Waltham, DE, USA). Experiments were performed in triplicate. The RP was calculated according to Equation (2): $\mathrm{Reducing} \mathrm{power} (\%)=\frac{{\mathrm{A}}_{\mathrm{control}}{ - \mathrm{A}}_{\mathrm{sample}}}{{\mathrm{A}}_{\mathrm{control}}}\times 100$

UV-Visible studies were carried out using the Thermo Scientific Evolution 220 UV-Visible Spectrophotometer. Data were processed with INSIGHT ^TM 2 software. Quartz cuvette cell was used for sample measurement. Absorbances were recorded over a wavelength range of 350–750 nm.
The selectivity of the synthesized calix[4]arene derivative along with its chromogenic properties were investigated using stock solutions of the ligand (1 × 10⁻⁵ mol·dm⁻³) and the anion salt (CO₃²⁻, F⁻, H₂PO₄⁻, HSO₄⁻ & Cl⁻) (1 × 10⁻⁴ mol·dm⁻³), (ammonium and potassium as counter-ions for carbonate and tetra-n-butyl ammonium as counter-ion for other anions) in dimethyl sulfoxide. Screening experiments on different cation and anion salt solutions were carried out prior to this step. A CA-AZ solution (3 cm³) was placed in quartz cell, and the absorbance spectrum was recorded against DMSO. UV-absorption measurements of the complex (ligand- ion salt) were also conducted in a solution (3 cm³) containing the same concentration of the ligand with the desired amount of salt solutions.

The optimal growth and pH were evaluated in conformity to Kavitha et al. (33 (link)). Briefly, the three bacterial isolates' fresh overnight cultures were inoculated in LB broth with varying pH levels (1–10), which was adjusted with acetic acid (99%) and 5 N NaOH. Subsequently, the inoculated broths were incubated at 37°C for 24 h, and growth was monitored with a spectrophotometer (Evolution™ 220 UV-Visible Spectrophotometer, Thermo Scientific™, USA) at 600 nm wavelength against uninoculated broth.

The enzymatic activity was quantified by determining the increase in absorbance at 348 nm (isosbestic point, ε under these conditions is 5150 M⁻¹ cm⁻¹) during 90 s produced by the p-nitrophenol released in the hydrolysis of p-NPB [85 (link)] using an Evolution 220 UV-Visible Spectrophotometer (Thermo Fisher Scientific, Miami, FL, USA). The reaction was started by adding 50 μL of the sample (free enzyme solution or, immobilized enzyme suspension) in 2.5 mL of 25 mM sodium phosphate at pH 7.0 and 25 °C containing 50 μL of p-NPB solution (at a concentration of 50 mM, dissolved in acetonitrile) under magnetic stirring and temperature control.