Urea

Indomethacin (Ind) and urea were purchased from Fagron (Cracow, Poland), Compritol 888 ATO (glyceryl behenate) from Gattefossé (Saint-Priest, France), and Tween 80 (polysorbate 80) and citric acid from Sigma-Aldrich (St. Louis, MO, USA). Lysozyme (LS) from chicken egg white was obtained from Pol-Aura (Olsztyn, Poland). Sodium chloride, potassium chloride, ammonium chloride, magnesium chloride, and anhydrous calcium chloride were sourced from POCH (Gliwice, Poland), sodium bicarbonate from Chempur (Piekary Śląskie, Poland), and lactic acid 80% from B&K (Bytom, Poland). All other chemicals used were of analytical reagent grade. A Milli-Q system (Millipore, Milford, MA, USA) was employed for obtaining high-quality water.

All chemicals were of analytical grade and water was ultrapure Milli-Q laboratory grade. Potassium hexacyano trihydrate (K₄Fe(CN)₆·3H₂O), potassium hexacyano dihydrate (K₃Fe(CN)₆), sodium phosphate dibasic dihydrate (Na₂HPO₄·2H₂O) and sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O) were purchased from Riedel de Haën; fetal bovine serum (FBS) was purchased from Alfa Aesar; sulphuric acid (H₂SO₄) was obtained from BDH; urea was from Fagron; proteinase K and (3-acrylamidopropyl)trimethylammonium chloride (solution 75 wt% in H₂O) (AMPTMA) was acquired from Sigma-Aldrich; carcinoembryonic antigen (CEA) was obtained from EastCostBio; Cancer Antigen 125 (CA-125) was from Hytest; CA 15-3 from host human (reference MBS536585) was purchased from MyBioSource.
For the calibration curves, CA 15-3 standard solutions ranging from 0.001 U mL⁻¹ and 100 U mL⁻¹ were used, prepared in PB buffer (pH 5.8). Each solution was incubated for 20 min at the electrode surface. Selectivity studies were conducted by competitive assay in which CA 15-3 (30 U mL⁻¹) were mixed with CEA (2.5 ng mL⁻¹), CA 125 (35 U mL⁻¹), and urea (0.2 mg mL⁻¹). All these solutions were prepared in PB buffer, at pH 5.8, in triplicate.