The Adsorption Mechanism of Hexahistidine on Solid Surfaces

LAURA E. VALENTI; CARLA E. GIACOMELLI; CARLOS P. DE PAULI

Iguazú, Brasil

Conferencia; 11st International Conference of Surface and Colloid Science; 2003

The reproducibility and specificity of solid-phase immunodiagnostic tests have been highly improved by adsorbing recombinant antigens that specifically recognize a particular antibody. These antigens are commonly engineered to terminate with a strech of six histidines, incorporated in the primary sequence to facilitate purification by immobilized metal ion-affinity chromatography. The histidine residues not only bind with high affinity to metal ions immobilize on a sorbent surface but also adsorbs on negative solid surfaces at acid pH values, when the residues are positively charged. Inducing the adsorption of the recombinant antigens through the histidine residues provides a stable and strong interaction with the sorbent surface and controls the orientation of the adsorbed molecules. This site-oriented methodology has been recently incorporated to improve the performance of immunoassays. However, the fundamental aspects of the adsorption of recombinant antigens on solid sorbent surfaces have received a little (if any) attention. The aim of this study is to determine the adsorption mechanism of the hexahistidine peptide (His₆) on silica surfaces. Such a simple system, is a model to better understand the more complex recombinant antigen-surface interactions and will be applied as the starting point to optimize the adsorption conditions in the development of an immunoassay. The kinetics of the His₆ adsorption process was followed by reflectometry, that is very suitable for measurements on the time scale of seconds to minutes. Therefore, the transport from the solution to the interface and the first peptide-surface attachment were determined at different experimental conditions. The limiting step rate was evaluated at different hexahistidine concentrations and pH values. The electrostatic contribution to the overall mechanism was analised as a function of the pH. The first steps of the adsorption process are mainly driven by this contribution, i.e. the adsorption rate is higher at lower pH values