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Magnetically retrievable formulations of urease potentially promising for biomedical and environmental applications were constructed by immobilization of the enzyme on the surface of magnetite nanoparticles functionalized by siloxane layers with active thiol or thiol-and-alkyl moieties. The latter were deposited using a hydrolytic polycondensation reaction of tetraethoxysilane with either 3-mercaptopropyltrimethoxysilane, or with 3-mercaptopropyltrimethoxysilane and methyltriethoxysilane, or alternatively n-propyltriethoxysilane. Immobilization of urease was carried out in different ways for comparison: by adsorption, by entrapment during the hydrolytic polycondensation reaction, or by covalent bonding. For entrapment the enzyme was introduced into solution before functionalization of the magnetite. Entrapment bound high amounts of enzyme (more than 700 mg per g of carrier), but its activity was decreased compared to the native form to between 18 and 10%. In the case of covalent binding of urease using Ellman's Reagent, binding of the enzyme was almost as efficient as in the case of entrapment but its residual activity was 75%. The residual activity of urease immobilized by adsorption on the surface of thiol-functionalized particles was truly high as compared to that of the native enzyme (97%), but binding was significantly less efficient (46%). Introduction of alkyl functions permitted increase of the amounts of the adsorbed enzyme but its activity was somewhat decreased.

A sol-gel method of covalent immobilization of urease on polysiloxane matrices is developed which uses glutaraldehyde or Ellman’s reagent as binding agents. We show that the urease covalently bound to the poly(3-mercaptopropyl)siloxane matrix retains 67–84% of its activity and is stable, losing only 10% of its activity after 300 days. The urease adsorbed on the poly(3-mercaptopropyl)siloxane matrix was more active than the native urease. On the basis of the literature, we suggest that, in this case, the 3-mercaptopropyl groups of the polysiloxane matrix are brought into close proximity to the active site of urease, where they possibly act as proton donors, which results in an accelerated enzymatic reaction. Covalent immobilization of urease on the polysiloxane matrix containing 3-aminopropyl was less efficient, because the immobilized enzyme was significantly less active. At the same time, the urease adsorbed on the same matrix showed high activity (60–86%).

The possibility of “double immobilization” of urease on silica gel, involving enzyme incorporation into the polysiloxane polymer prepared by sol-gel procedure on the surface of an inorganic support was studied. The optimal composition and thickness of the polysiloxane film, providing relatively high activity of urease, were determined.

Magnetic nanoparticles modified with siloxane layers bearing amino and thiol functions have been used for immobilization of urease either by adsorption or via surface grafting. The activity of the immobilized enzyme in the hydrolysis of urea extended to the levels typical of the native enzyme, while its long-term stability in combination with magnetic retraction opened for its repeated use in both analysis and detoxification of bio-fluids. The immobilized urease revealed strongly enhanced stability and 65% activity in the presence of 0.1mmol/l of Hg(2+) or 0.3mmol/l of Cu(2+) while the native urease did not retain any activity at all. The enzyme grafting was shown to be a potentially perspective tool in alleviation of heavy metal poisoning and to be providing an opportunity for use of the developed adsorbents as both biosensors and bio-reactants for removal of urea from biofluids.

Sol-gel method was used to synthesize polysiloxane hydrogel with encapsulated urease (immobilization degree in the range of 79–88%) which preserved the enzymatic activity at the level of 56–84%. The nature of functional groups was shown to influence the pore structure parameters. Immobilization degree and preservation of adsorbed urease activity depend on the structural-adsorption characteristics of matrices. The possibility of “double immobilization” of urease on silica gel by sol-gel method and the opportunity of reuse of the synthesized formulations was investigated. Urease immobilization on the surface of magnetite (FeO·Fe2O3) was also studied by adsorption method.