AVS 50th International Symposium
    Biomaterial Interfaces Thursday Sessions
       Session BI-ThM

Paper BI-ThM8
Coupling of his-tagged scFvs to Functionalized Lipid Assemblies for Array Based Sensing

Thursday, November 6, 2003, 10:40 am, Room 318/319

Session: Biosensors
Presenter: C. Larsson, Chalmers University of Technology, Sweden
Authors: C. Larsson, Chalmers University of Technology, Sweden
F. Höök, Chalmers University of Technology, Sweden
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Lipid bilayers containing 5% NTA-lipids supported on SiO@sub 2@ have been used as a template for efficient immobilization of oligohistidine-tag containing single-chained antibody fragments (scFv) directed towards cholera toxin (CT). It was demonstrated that his-tagged scFv:s is equally efficient coupled to the NTA/Ni@super 2+@ containing lipid bilayer from a purified sample and the expression supernatant. Using the latter, time consuming protein purification steps is avoided. Independent on whether the coupling was made from the supernatant or from the purified sample, the template was proven efficient for antigen detection, in this case verified via the quartz crystal microbalance with dissipation monitoring (QCM-D) technique using the antigen CT (Mw ~85 kD). Via a secondary amplification step utilizing G@sub M1@ containing vesicles, i.e. the membrane receptor for CT, sub-nanomolar concentrations of CT was detectable with QCM-D. Furthermore, this coupling strategy was also utilized for creation of protein array templates. The template was, however, based on novel DNA-array design, using streptavidin-based DNA-immobilization on gold spots, surrounded by a pure lipid bilayer on SiO@sub 2@, but with the aim to be used as a protein array rather than a DNA array. The latter was accomplished using DNA-modified lipid vesicles, directed to predefined DNA spots via complementary hybridization, where the protein-array concept was proven utilizing scFv-modified lipid vesicles utilizing NTA/Ni@super 2+@-based coupling for highly sensitive detection of fluorescently labeled CT.