Novel surface plasmon resonance sensor for the detection of heme at biological levels via highly selective recognition by apo-hemoglobin

We have developed a novel surface plasmon resonance (SPR) biosensor for heme detection that utilizes the reconstitution of the heme cofactor with apohemoglobin (apoHb), hemoglobin from which the heme has been removed, as the sensing mechanism. The binding is highly specific, efficient and generated very strong SPR signals. This is the first report that uses immobilization of the apoprotein in a hydrophilic polymer matrix and senses the corresponding cofactor by SPR. This is also the first report of high sensitivity heme detection in real time by SPR and the sensing surface is re-generated many times without loss of sensitivity or selectivity. The sensing surface was fabricated by covalent immobilization of hemoglobin in a polyacrylic acid matrix in situ, which allowed for a high concentration of protein to be located in the plasmon detection range on the Au chip. Removal of the heme from the hemoglobin–polymer conjugate (Hb–PAA) resulted in a surface anchored apoHb–polymer conjugate. The limit of detection was approximately 2 μM or 1.30 μg/mL, which is relevant for biological heme levels (1–50 μM for hemolytic pathological conditions). This apoHb–polyacrylic acid system demonstrates a new concept in SPR detection with the use of protein cofactor binding pockets for analyte detection. The methodology that we developed here may be extended for the detection of a number of other cofactor molecules with high sensitivity, selectivity and low detection limits. In future, such sensors could be useful for the development of point-of-care devices to detect biologically important small molecules.