A chemiluminescence assay for determination of lysozyme based on the use of magnetic alginate-aptamer composition and hemin@HKUST-1

Abstract

Lysozyme aptamer-functionalized magnetic alginate hydrogel was prepared for separation and enrichment of lysozyme. Luminol-labeled aptamer was used as a signal tag, and the signal tag was adsorbed on magnetic carboxylated carbon nanotubes based on the π-interaction. When lysozyme was added, the aptamer specifically binds to the lysozyme, causing the signal tag to detach from the magnetic carboxylated carbon nanotubes. When the aptamer/lysozyme complex bound to the complementary single strand of aptamer on the hemin@HKUST-1, lysozyme was released. The released lysozyme can be recombined with the signal tag adsorbed on the magnetic carboxylated carbon nanotube, allowing more signal tag to be dispersed into the solution. Determination of lysozyme was achieved by releasing the luminol-labeled aptamer to generate a chemiluminescence signal at a wavelength of 425 nm. It was proved by experiments that the synthesized hemin@HKUST-1 had a strong catalytic effect on the luminol-NaOH-H₂O₂ system. The chemiluminescence signal was increased nearly 100 times. The complementary pairing allowed the luminol to be immobilized on the surface of hemin@HKUST-1. The generation and consumption of short-lived reactive oxygen species were concentrated on the surface of the MOFs, which improves the chemiluminescence efficiency. The introduction of hemin@HKUST-1 and DNA solved the defects of chemiluminescence analysis. The chemiluminescence assay was able to detect lysozyme with linear range of 1.05 × 10⁻⁶ U∙mg⁻¹ (6.00 × 10⁻¹³ mol∙L⁻¹)–1.25 × 10⁻² U∙mg⁻¹ (7.14 × 10⁻⁹ mol∙L⁻¹); the detection limit was 3.50 × 10⁻⁷ U∙mg⁻¹ (2.00 × 10⁻¹³ mol∙L⁻¹) (R² = 0.99). The recovery of lysozyme in spiked saliva samples was 97.4–102.8%.