Surface coverage dictates the surface bio-activity of d-amino acidoxidase

ELISA HERRERA; CARLA E. GIACOMELLI

COLLOIDS AND SURFACES B-BIOINTERFACES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2014 vol. 117 p. 296 - 302

0927-7765

tThis work presents a systematic study on the relationship between the adsorption mechanism and thesurface bio-activity of d-amino acid oxidase (pkDAAO). This rational approach is based on measuringthe characteristic filling and relaxation times under different experimental conditions. With such a goal,real-time adsorption?desorption experiments at different degrees of surface coverage were performedtuning the electrostatic and hydrophobic interactions by changing the pH condition for the adsorptionand the substrate properties (silica or gold). Surface bio-activity was measured in situ by amperometryusing the bio-functional surface as the working electrode and ex situ by spectrophotometry. On both solidsubstrates, pkDAAO adsorption is a transport-controlled process, even under unfavorable electrostaticinteractions (charged protein and substrate with the same sign) due to the high percentage of basicamino acids in the enzyme. On silica, the relaxation step is electrostatic in nature and occurs in thesame time-scale as filling the surface when the substrate and the enzyme are oppositely charged at lowsurface coverage. Under unfavorable electrostatic conditions, the relaxation (if any) occurs at long time.Accordingly, the bio-activity of the native pkDAAO is preserved at any surface coverage. On gold, this stepis driven by hydrophobic interactions (pH-independent) and the surface bio-activity is highly dependenton the degree of surface coverage. Under these conditions, the surface bio-activity is preserved only athigh surfaces coverage. Our results clearly indicate that pkDAAO bio-functionalized surfaces cannot becoupled to amperometry because the analyte interferes the electrochemical signal. However, this simplebio-functionalized strategy can be joined to other detection methods.