Canavalia ensiformis

The reaction mixture consisting of 25 µL enzyme solution (urease from Canavalia ensiformis, Sigma, 1U final concentration) and 5 µL of test compounds dissolved in water (10–300.0 µM final concentration) was preincubated at 37 °C for 60 min in 96-well plates. Then 55 µL of phosphate buffer solution with 100 µM urea was added to each well and incubated at 37 °C for 10 min. The urease inhibitory activity was estimated by determining of ammonia production using indophenol method [26 (link)]. Briefly, 45 µL of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The absorbance was measured after 50 min at 630 nm using a microplate reader Multiskan FC (Thermo Scientific, Canada). All the reactions were performed in triplicate in a final volume of 200 µL. The pH was maintained 7.3–7.5 in all assays. DMSO 5% was used as a positive control.

Metaphase and interphase chromosome preparations were made from short-term peripheral blood lymphocyte or primary fibroblast cell cultures following standard protocols [23 (link),25 (link),35 (link)]. Alpaca blood lymphocytes were stimulated into proliferation with concanavalin A, a mitogen from Canavalia ensiformis (20 μg/mL; Sigma Aldrich, St. Louis, MO, USA). Cells were harvested with demecolcine solution (0.1 μg/mL; Sigma Aldrich), treated with optimal hypotonic solution (Rainbow Scientific, Maple Avenue, Windsor, CT, USA), and fixed in 3:1 methanol/acetic acid. Approximately 10 µL of fixed cell suspension was dropped on precleaned wet glass slides at room temperature and air dried. The quality and quantity of metaphase spreads was evaluated under phase contrast microscope.

A reaction mixture consisting of 25 µL enzyme solution (urease from Canavalia ensiformis, Sigma, 1U final concentration) and 5 µL of test compounds dissolved in water (10–300.0 µM final concentration) was preincubated at 37 °C for 60 min in 96-well plates. Then, 55 µL of phosphate buffer solution with 100 µM urea was added to each well and incubated at 37 °C for 10 min. The urease-inhibitory activity was estimated by determining ammonia production using the indophenol method. Briefly, 45 µL of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaClO) were added to each well. The absorbance was measured after 50 min at 630 nm using a MultiskanFS microplate reader (Thermo Scientific Inc., Beverly, MA, USA). All reactions were performed in triplicate in a final volume of 200 µL. The pH was maintained at 7.3–7.5 in all assays. DMSO 5% was used as a positive control.

The test to determine the lowest urease concentration detectable (producing a color change) in the RUT employed the urease in Canavalia ensiformis (Jack bean) powder (Sigma-Aldrich). The urease was diluted in distilled water at concentrations of 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 mg/mL; approximately 60 μL of each concentration was used in the developed RUT assays. These assays were observed for a color change at 1, 3, 5, 10, 20, and 30 min and 1, 2, 3, 4, 5, 6, and 24 h. The negative control was 60 μL of Deionized (DI) water, which substituted for the urease.

Penicillinase from Bacillus cereus (1500–3000 Units/mg protein, Sigma Aldrich, Darmstadt, Germany) and urease from Canavalia ensiformis (75,265 Units/g solid, from Jack bean, Sigma Aldrich, Germany) were applied as the model enzymes. In order to immobilize them on the biotinylated TMV particles, they were conjugated with streptavidin (SA), allowing strong attachment via SA-biotin high affinity binding. The streptavidin conjugation was performed by utilizing a commercial conjugation kit (LYNX Streptavidin rapid conjugation kit, Bio-Rad, Great Britain). The conjugation of penicillinase was performed as described in [65 (link)], using a molar ratio between penicillinase and SA of 1:25 in the reaction mixture. For the urease conjugation, the same protocol was applied with a molar ratio of 1:10. The streptavidin-conjugated enzymes (SA-enzymes) were stored in stock solutions (10 mM phosphate buffered saline (PBS)) with a concentration of 600 Units/mL (SA-penicillinase) and of 2000 Units/mL (SA-urease) at 4 °C. For the fabrication of the bi-enzyme biosensors, both enzyme solutions were mixed in a ratio of 1:1, resulting in an enzyme cocktail containing 300 Units/mL SA-penicillinase and 1000 Units/mL SA-urease.