Recombinant bovine odorant binding protein (bOBP) is a very promising platform for building protein-based biosensors. The protein possesses a broad binding specificity for hydrophobic molecules with affinities in the sub-micromolar range. Previous work has shown non-covalent binding of 1-aminoanthracene (1-AMA) in the internal cavities of bOBP that results in a large enhancement of fluorescence intensity. We have shown fluorescence titrations of recombinant bOBP with 1-AMA yielded a single type of binding site with a Kd of 0.16 ± 0.023 μM. Competitive displacement assays between 1-AMA and other ligands such as thymol were established and the results indicated their binding to bOBP. The strategy of competitive binding with 1-AMA was thus employed to quantify thymol concentration for the bOBP biosensors.
Ten different solid supports for the bOBP biosensors were examined for their biocompatibility with bOBP function using 1-AMA as a probe. The result was that nitrocellulose was chosen as the best membrane for immobilization, probably due to its 3-D micro-porous matrix (sponge structure) that provides a much larger surface area for protein binding compared with 2-D surfaces. It was found that the optimum operating concentrations of bOBP and 1-AMA and the method for the immobilization was incubation of nitrocellulose with the complex of 100μM bOBP and 100μM 1-AMA solution. The amounts of the total and functional protein binding to nitrocellulose were 7 ± 0.1 and 7 ± 0.4 nmol bOBP per cm2 of membrane, respectively.
A fibre-optic biosensor based on bOBP has therefore been constructed. It has been an extrinsic sensor with bOBP immobilized on a nitrocellulose membrane placed at the tip of a probe of a bifurcated fibre-optic bundle that was in turn connected to the LLS-385 LED light source and the HR2000 spectrometer. The light emitted by fluorescent 1-AMA bound bOBP was detected by 2048-element CCD array of the spectrometer. The LODs for thymol in the liquid phase were found to be 14 ± 6 μM (calculated as S/N = 3), which is less than the guideline values considered to be toxic to humans. Moreover, this fibre-optic bOBP biosensor was also capable of sensing thymol vapour, and some potential uses of this sensor will be described.