4 2 pyridylazo resorcinol

Acetonitrile (MeCN), HCl (trace metal grade), NaClO₄ H₂O, ZnSO₄·7H₂O, 4-(2-pyridylazo)resorcinol (PAR), and Triton X-100 were from Merck Millipore. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), tryptone, yeast extract, LB Broth, agar, agarose, isopropyl-β-D-1-thiogalactopyranoside (IPTG) and sodium dodecyl sulfate (SDS) were purchased from Lab Empire. Ampicillin, chloramphenicol, 1,4-dithiothreitol (DTT) and Tris base were from Roth, pTYB21 vector and chitin resin from New England BioLabs, Chelex 100 resin from Bio-Rad, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES), 5,5ʹ-dithiobis-(2-nitrobenzoic acid) (DTNB) from TCI Europe. To eliminate trace metal ion contamination all pH buffers were treated with Chelex 100 resin (Bio-Rad) and degassed over 2 h prior to use. For the culture of E. coli, Luria–Bertani (LB) medium and agar plates were used.

The stock standard solutions of U(VI) were CPA_chem, solution of Uranium 10.00 g/L in 5% nitric acid (Stara Zagora, Bulgaria). Working standard solutions were daily prepared by appropriate dilution with deionized water (DW) (Millipore Corp., Milford, MA, USA). All reagents were of analytical-reagent grade.
Methacrylic acid (MAA), trimethylolpropane trimethacrylate (TMPTMA), 2,2′-azobisisobutyronitrile (AIBN), 4-(2-Pyridylazo)resorcinol (PAR, as Na salt), uranyl acetate (UO₂(CH₃COO)₂·6H₂O) (Merck, Darmstadt, Germany), and acetonitrile (ACN) (Labscan, Dublin, Ireland) were used to prepare the U(VI) ion-imprinted and non-imprinted polymer sorbents. Hydrochloric acid (Fisher Chemical™, Waltham, MA, USA) was used for uranium desorption. The pH value of water samples was adjusted with NH₃ or HNO₃.

H₂O₂ content was determined according to the method of Becana et al. [36 (link)]. Four roots of cucumber seedlings were homogenized in a cooled mortar with 3 mL of 5% trichloroacetic acid (TCA, Serva) and 0.1 g of active carbon. The homogenates were filtered through a membrane layer—miracloth (Biofuge fresco Heraeus, Kendro Laboratory Products, USA)—and centrifuged at 12,000 × g for 30 min at 4 °C. After centrifugation, the pellet was discarded, and the supernatant was used to determine H₂O₂ content. The reaction mixture contained 2.0 mL of a 0.1 mM potassium phosphate buffer at pH 8.4, 60 μL of the analysed extract, and 1 mL of the titanium reagent. The titanium reagent was prepared by mixing it in a 1: 1 (v/v) ratio 0.6 mM solution of 4-(2-pyridylazo) resorcinol and 0.6 mM potassium titanate oxalate (Sigma-Aldrich). The reference sample was 5% TCA. The absorbance of the complex formed was measured at a wavelength of 508 nm, and the H₂O₂ content in the tissue was read from the standard curve. The H₂O₂ content was expressed in nM·g⁻¹ dry weight of roots. The assays were performed in 3 independent experimental repetition.