Chemiluminescence flow biosensor for hydrogen peroxide using DNAzyme immobilized on eggshell membrane as a thermally stable biocatalyst

A DNAzyme-based chemiluminescence (CL) biosensor for sensitive detection of hydrogen peroxide (H2O2) has been developed. The complexation of hemin with a guanine-rich single-stranded nucleic acid yields the DNAzyme with peroxidase catalytic activity. The DNAzyme catalyzes the oxidation of luminol by H2O2 and the generation of CL. The DNAzyme is immobilized on eggshell membrane, and then packed into mini-column as CL flow cell. In the presence of luminol, H2O2 passed through the CL flow cell to produce CL emission, and then was sensed. The response to H2O2 concentration was linear in the range of 1 × 10−5–1 × 10−7 M with a detection limit of 5 × 10−8 M (3σ). Compared to the HRP-based biosensor for H2O2, the DNAzyme possessed high catalytic activity in strong alkaline medium, which was well compatible with the luminol CL system. The immobilized DNAzyme could be stored at room temperature (∼20 °C) and exhibited good stability with a shelf life of at least 3 months. A complete analysis, including sample and washing, could be performed in 1 min with a relative standard deviation of less than 2%. This biosensor for H2O2 has the potential to serve as a general platform for design sensors for other molecules (such as glucose and uric acid).