IUVSTA 15th International Vacuum Congress (IVC-15), AVS 48th International Symposium (AVS-48), 11th International Conference on Solid Surfaces (ICSS-11)
    Biomaterials Friday Sessions
       Session BI-FrM

Paper BI-FrM4
A "Label-Free" Microchip Array Based Protein-Binding Assay using Surface Plasmon Resonance Imaging

Friday, November 2, 2001, 9:20 am, Room 102

Session: Biosensors
Presenter: C.E.J. Dentinger, Zyomyx, Inc.
Authors: C.E.J. Dentinger, Zyomyx, Inc.
D. Martin, Zyomyx, Inc.
P. Wagner, Zyomyx, Inc.
Correspondent: Click to Email
We demonstrate a microchip array based antibody binding assay that relies on imaging surface plasmon resonance (SPR) for detection of protein binding. Since SPR is sensitive to the index of refraction near a gold surface this assay does not require the binding proteins to be labeled (e.g. fluorescently or with a radio label), making it a "label free" detection technique. For our imaging SPR assay we have developed a method of specifically immobilizing proteins on surfaces that resist non-specific protein adsorption. These surfaces consist of a gold substrate coated with an omega functionalized alkanethiol self-assembled monolayer (asymNHS). The succinimidic ester head group of this monolayer is then reacted with biotin-LC-PEO-amine to make a biotin-coated surface. This surface will bind streptavidin in a manner that leaves some of streptavidin's binding pockets open to immobilize a wide variety of biotinylated proteins. The asymNHS, biotin-LC-PEO-amine, and streptavidin layers are characterized with ellipsometry, and Fourier transform infrared reflection adsorption spectroscopy (FTIRRAS). The "label-free" assay is then performed by immobilizing biotinylated proteins in an array format and monitoring the different antibody-antigen interactions on this array by imaging SPR. We have shown that this surface will bind a second protein only to the areas where the biotinylated proteins are immobilized. We have also demonstrated that with our imaging SPR we have the ability to watch the binding assays in real time.