Single-Step Formation of a Biorecognition Layer for Assaying Histidine-Tagged Proteins

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Abstract
The purpose of this work was to develop a simple procedure for the creation of a specific biorecognition layer for histidine-tagged (His-tagged) molecules. Such a layer was prepared by the spontaneous fusion of vesicles containing readily available plain (DOPC) and iminodiacetic acid (DOGS-NTA) phospholipids on a silica surface resulting in the formation of an NTA-containing supported lipid bilayer. The frequency surface acoustic waveguide device which supports Love waves was used to follow the real-time formation of the biorecognition layer. The mole percent of the DOGS-NTA phospholipids in the supported bilayer was optimized by following the kinetics of the fusion for the different NTA-containing lipids. Fluorescently labeled lipids were used with observations of the fluorescence recovery after photobleaching to confirm the presence of lipid bilayers. After saturating all NTA-molecules with Ni(2+), the binding of a His-tagged protein fragment within the concentration range of 0.04 and 0.4 mM to a 5 mol % DOGS-NTA/DOPC was detected; binding curves were used to calculate the apparent association constant k(on) = 2.56 x 10(4) M(-)(1) s(-)(1), dissociation constant k(off) = 1.3 x 10(-)(3) s(-)(1), and equilibrium constant k(eq) = 1.97 x 10(7) M(-)(1). The described method could find significant applications as a generic technique for preparing biorecognition layers for His-tagged proteins. In addition, the acoustic waveguide device, which provides high sensitivity together with flexibility in terms of the substrate material used, is shown to be an attractive alternative to direct optical biosensors.