Biocatalytic Mechano-Responsive Polyelectrolyte Multilayers Based on Covalently Cross-linked Enzymes

The polyelectrolytes poly(L-lysine) (PLL, Mw = 2.60 × 10 Da) and hyaluronic acid (HA, Mw = 1.32 × 10 Da) were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France) and Lifecore Biomedical (Chaska, USA) respectively. Coupling agents as N,N′Dicyclohexylcarbodiimide 99% (DCC), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride ≥ 98% (EDC), N-Hydroxysuccinimide 98% (NHS), and NHydroxysulfosuccinimide sodium salt (sulfo-NHS) ≥ 98% were all purchased from Sigma Aldrich. The enzyme β-galactosidase (β-Gal) from Escherichia coli Grade VI, lyophilized powder, 250-600 units/mg protein and the enzyme substrate fluorescein di(β-Dgalactopyranoside) (FDG) were purchased from Sigma Aldrich. 2,4,6Trinitrobenzenesulfonic acid (TNBS), Tris(2-carboxyethyl)phosphine hydrochloride powder, ≥ 98% (TCEP), tris(hydroxymethyl)aminomethane (TRIS) and 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), sodium chloride (NaCl), sodium bicarbonate (NaHCO3) anhydrous solvents for synthesis dichloromethane (CH2Cl2), N,NDimethylformamide (DMF) were all purchased from Sigma Aldrich. Dialyzed cellulose ester membrane (MWCO 3500 Da) was purchased from Carl Roth (Lauterbourg, France) and bicinchoninic assay protein quantification kit was purchased from Uptima (Montluçon, France). Thiopyridone mercaptopropionic acid (TP-MPA) was synthesized by the procedure describe by Xie et al. Maleimide succinimide ester was synthesized by the procedure describe by Thibaudeau et al. Poly(dimethylsiloxane) (PDMS) sheets of 250 μm thickness (Specialty Manufacturing Inc., Saginaw, USA) and circular glass slides with 12-mm diameter and 150-μm thickness (Menzel-Gläser, Braunschweig, Germany) were chosen as substrates for the construction of polyelectrolyte multilayer (PEM) films.