Performance of a Flow‐Through Enzyme Reactor Prepared from a Silica Monolith and an α‐Poly(D‐Lysine)‐Enzyme Conjugate

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α‐poly(D‐lysine) chains of ≈1000 repeating units length, PDL, via a bis‐aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL‐BAH‐HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4 °C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro‐ and mesoporous silica monolith with pore sizes of 20–30 µm as well as below 30 nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP‐containing monolith can be used as flow‐through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer‐enzyme conjugates with approximately the same average polymer chain length, the PDL‐BAH‐HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces. Horseradish peroxidase (HRP) is covalently attached to some of the amino groups of commercial poly(D‐lysine) via a bis‐aryl hydrazone bond. The obtained conjugate is immobilized by simple adsorption inside a silica monolith and used as stable flow‐through HRP reactor at pH ≈7. Complete desorption of the conjugate at pH 5 and monolith reuse at pH >5 are demonstrated.