Controlled protein orientation and protein immobilization on biosensor surfaces
When aiming to enhance efficiency and robustness of biosensors, the orientation of the surface immobilized biorecognition element is a crucial factor to consider as well as the nature of its bond to the surface. A robust surface in terms of stability and regeneration possibilities can be obtained by covalently immobilizing the biorecognition molecule. The requirement of high biorecognition activity can be met partly by controlling the orientation of the recognition molecule (ligand) so that the availability for the analyte interaction is optimized. Ideally the covalent immobilization needed for robustness must not affect the protein conformation or in any other way reduce the activity.
Controlled orientation and photoimmobilization by “CAP” surface chemistry
In the project “Chelation Assisted Photoimmobilization” (“CAP”) a self assembled monolayer (SAM) of three different components (OEG, NTA and BP, explained below) is used to covalently immobilize preoriented proteins to biosensor surfaces in a two step method. The oligo(ethylene glycol) (OEG) reduces nonspecific binding, while the other two components are used for CAP.
The first step involves orientation of biomolecules modified with a hexahistidine sequence (His-tag) to surface bound nitrilotriacetic acid (NTA). The NTA chelation of nickel ions (Ni2+) enables the non-covalent binding of histidines to the Ni-NTA complex - a property often used in protein chromatography, but here used for preorientation.
The second step involves photoactivation of a surface bound photolabile functional group, benzophenone (BP), which upon UV light activation forms a covalent bond to the preoriented protein by insertion in C-H bonds, preferably in the His-tag so that the conformation of the protein is unaffected.
The surface chemistry will be used for creation of protein microarrays. At the moment we are developing an experimental set-up for the real time detection of the photoimmobilization process by combining the UV source with our imaging SPR instrument.
Bui, L. Synthesis of Substituted Alkanethiols Intended for Protein Immobilization: Chelate Associated Photochemistry (CAP). Linköping University Electronic Press; 2009. Linköping Studies in Science and Technology. Licentiate thesis no. 1411.
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Last updated: 06/01/10