Na2hpo4

All the chemicals used in the study were purchased from Sigma-Aldrich unless stated otherwise. LB medium: 10 g/l tryptone, 5 g/l yeast extract, and 10 g/l NaCl. For agar plates, 15 g/l agar was added. M9 medium: 6.8 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 0.5 g/l NaCl, 1 g/l NH₄Cl, 0.34 g/l thiamine, 0.2% casamino acids (BD Biosciences), 0.4% glucose, 2 mM MgSO₄, and 100 μM CaCl₂. The LB medium for overnight culture contained ampicillin and kanamycin at concentrations of 100 μg/ml to maintain the plasmids. The mixed medium for the microfluidic culture contained ampicillin and kanamycin at concentrations of 1 μg/ml at which the cells can grow normally and the plasmids can be maintained.

For western blot analysis, cells or kidneys were lysed in lysis buffer solution of 150 mM NaCl (Sigma-Aldrich, #s9625), 20 mM Na₂HPO₄ (BDH, #10494 L)/NaH₂PO₄ (BDH, #102455 S), 10% glycerol (Sigma-Aldrich, #G7757), 1% Triton X-100 (pH 7.2), complete protease inhibitor cocktail (Roche, #11836145001) and phosphatase inhibitors (1 mM final concentration of glycerophosphate (Sigma-Aldrich, #G9891), sodium orthovanadate (Sigma-Aldrich, #S6508) and sodium fluoride (Sigma-Aldrich, #S6521)). Total lysates were then quantified with Bio-Rad Protein Assay Dye reagent (Bio-Rad Laboratories, #500-0006), and Laemmli buffer at a final concentration of 2× was added to the samples. Proteins were next resolved in 4–12% Tris–glycine gradient gels (Life Technologies, #NP0335BOX) and then transferred onto Immobilon-P polyvinylidene fluoride membranes (Millipore, #IPVH00010). Membranes were blocked with 5% milk in Tris-buffered saline, Tween 20 (Sigma-Aldrich, #P1379) (TBS-T). All the primary antibodies for western blot analysis were diluted in 3% BSA in TBS-T. HRP-conjugated secondary antibodies were diluted 1:10,000 in 5% milk, TBS-T, and detection was performed with ECL (GE Healthcare, #RPN2106) alone or supplied with 10% SuperSignal West Femto (Thermo Fisher Scientific, #34095) when necessary.

All chemicals were used without further purification. Na₂HPO₄ (98%) and KH₂PO₄ (99%) were obtained from BDH. NaOH, EDTA, Nile blue A (98%), L-lysine, and methyl orange were purchased from International Fisher Scientific. Caffeine (99%), toluidine blue, Ni(NO₃)₂.6H₂O (98%), Cd(NO₃)₂.4H₂O (98%), and ascorbic acid were obtained from Sigma-Aldrich. Pb(NO₃)₂ (99%) was purchased from VWR Chemicals BDH and Cu(HCO₂)₂.2H₂O (99%) from Merck Chemicals GmbH. Citric acid monohydrate and HCl (36%) were purchased from J.T. Baker and Pronalys AR, respectively. The phosphate buffer solutions used in this work consisted of a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate. By varying the amount of each salt, we prepared a range of phosphate buffers with pHs between 5.0 and 9.0. Analytical solutions of NBA at various concentrations were obtained by dilution from a standard solution of a concentration of 0.01 M, using doubly distilled water.
Specimens of snail shells were collected from a local market in downtown Nkongsamba (Cameroon). The raw snail shells were washed with water, rinsed with distilled water, and dried at room temperature for two weeks. They were exploited to yield hydroxyapatite, as described in the next section.