Biosensing and Protein Array Design using Lipid Assemblies

Doctoral thesis, 2005

Biomolecular sensing often includes immobilization of biomolecules, e.g. DNA, antibodies etc., to solid supports, where they act as selective recognition elements. The actual immobilization step is a non-trivial task, however, since upon immobilization, biomolecues have a high tendency to lose their native conformation and thus function. Furthermore, the high tendency of biomolecules to adsorb spontaneously on solid surfaces makes unspecific binding a severe problem, often leading to false detection signals. In this work, efforts have been made to overcome these two complications using controlled coupling strategies of biomolecules to, primarily, phospholipid bilayers supported on SiO2. After demonstrating excellent protein-repellent properties of supported phospholipid bilayers (paper I), various coupling strategies for DNA and antibodies have been evaluated with respect to sensing efficiency. In paper II biotinylated DNA was immobilized to streptavidin coupled to a biotinylated lipid bilayer. DNA-hybridization was investigated using the quartz crystal microbalance with dissipation monitoring (QCM-D) and the surface plasmon resonance (SPR) techniques. Via information gained from modeling of the QCM-D response, it was also demonstrated that the DNA-hybridization efficiency is significantly improved by reducing the surface coverage of DNA. Paper III describes coupling of single-chained antibody fragments (scFvs) via an oligohistidine tag, a supported lipid bilayer was instead modified with NTA-functionalized lipids. It was also demonstrated that scFv:s can be efficiently coupled directly from the expression supernatant, thereby avoiding time consuming protein purification steps. Independent of whether the coupling was made from the supernatant or from the purified sample, the template was proven efficient for antigen detection, in this case being cholera toxin (CT). Via a secondary amplification step utilizing GM1 containing vesicles, sub-nanomolar concentrations of CT was detectable and the introduction of a double-histidine tag, increased binding affinity was obtained, important for detection in complex solutions. In paper IV, neutravidin-based DNA-immobilization on gold spots was used for a novel DNA-array design, but with the aim to create 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 the NTA/Ni2+-based coupling described in paper III. In paper V, being an extension of paper IV, DNA-neutravidin complexes were spotted on a biotinylated PLL-g-PEG surface using a nanoplotter. Binding of DNA and scFvs to the vesicles was improved by the introduction of bivalent bonds and the protein array concept was proven to work with two different scFvs, one against CT and one against fluorescein isotiocyanate (FITC), which were detected fluorescently via Cy-5 labeled CT and Cy-3 labeled FITC-BSA.