Hydrogels enzyme immobilization

Kokufuta, E Jinbo, E, A Hydrogel Capable of Facilitating Polymer Diffusion through the Gel Porosity and Its Application in Enzyme Immobilization, Macromolecules 25, 3549, 1992. Kresge, CT Leonowicz, ME Roth, WJ Vartuli, JC Beck, JS, Ordered Mesoporous Molecular Sieves Synthesized by a Liquid-Crystal Template Mechanism, Nature 359, 710, 1992. 

Figure 1. Schematic of synthesis of enzyme immobilized within hydrogel exhibiting thermally reversible shrinking and swelling.

D. Ivekovic, S. Milardovic and B.S. Grabaric, Palladium hexacyanofer-rate hydrogel as a novel and simple enzyme immobilization matrix for amperometric biosensors, Biosens. Bioelectron., 20(4) (2004) 872-878. 

Another enzyme that was studied extensively in microreactors to determine kinetic parameters is the model enzyme alkaline phosphatase. Many reports have appeared that differ mainly on the types of enzyme immobilization, such as on glass [413], PDMS [393], beads [414] and in hydrogels [415]. Kerby et al. [414], for example, evaluated the difference between mass-transfer effects and reduced effidendes of the immobilized enzyme in a packed bead glass microreactor. In the absence of mass-transfer resistance, the Michaelis-Menten kinetic parameters were shown to be flow-independent and could be appropriately predicted using low substrate conversion data. 

For the repeated use of enzymes in such an analytical device, numerous techniques for enzyme immobilization including entrapment in hydrogels (membrane... 

Enzyme-substrate Hydrogel containing immobilized enzymes Substrate present, enzymatic conversion, product changes, swelling of gel, release of drug... 

Kondo, A., and Fukuda, H., Preparation of thermo-sensitive magnetic hydrogel microspheres for antibody and application to enzyme immobilization, J. Ferment. Bioeng., 41, 99, 1994. 

D Urso, E.M. Jean-Francois, J. Doillon, C.J. Fortier, G. Poly(ethylene glycol)-serum albumin hydrogel as matrix for enzyme immobilization biomedical applications. Artif. Cells Blood Substit. Immobil. Biotechnol. 1995, 23 (5), 587-595. 

Enzyme immobilization methods other than using the hydrogels (A) Since appUcations of the redox hydrogels (A) to glucose sensors was first reported by Pishko et al. in 1991, various other methods have been reported to immobilize redox polymers to construct biosensors as indicated (see Section 3.3.3.2). Other recent examples are as follows. 

To activate the hydrogel adducts for enzyme immobilization, it was necessary to reduce the nitro function to the corresponding amine. From the many techniques available to effect this modification, we selected sodium dithionite as the mildest procedure with the least potential problems with residual reagent. Since the hydrogels were quite hydrophilic it was possible to use a two phase solvent system (THF/H2O) to effect the reduction. The arylsulfonyl carbamates were reduced selectively under these conditions no reduction of p-nitrophenyl carbamate was observed. 

Enzyme immobilization is an attractive method to stabilize the enzyme against denaturating reaction conditions and to make the biocatalyst recyclable [34-37]. In combination with peroxidase-initiated polymerizations, this has been reported recently by Zhao et al. [38]. Incorporation of HRP into a hydrogel increased both storage and thermal stability of the enzyme also resistance against H202 was increased to some extent. Furthermore, the immobilized enzyme preparation could be reused at least four times, albeit with significant activity losses after each cycle. 

Other examples are enzymes immobilized on beads which are trapped in a microreactor by etched weirs [88], enzymes encapsulated in hydrogel patches or sol-gel silica [89] and enzymes attached on the surface of (porous) microstructures (for example, on porous silicon manufactured by anodization of single-crystalline silicon see Figure 1.10 [91]), of mesoporous silica or polymer monoliths or directly... 

D Urso, E.M., J. Jean-Francois and G. Fortier, New bioartificial hydrogel biomedical matrix for enzyme immobilization, Polymer Preprints, 35 (1994) 444-445. 

Carrageenan Extrusion, w/or w/o TPP Composite with nanotubes Nanoparticles microparticles hydrogel beads fibers Drug delivery Enzyme immobilization... 

Xanthan gum Extrusion cryogelation Hydrogel beads tablets cryog Drug delivery Enzyme immobilization Tissue engineering... 

Other work on polymerization in particulate form has included the preparation of polycyanoacrylate nanocapsules as potential lysomotropic carriers, the encapsulation of pancreatic islet cells in hydrogels, " suspension polymerization of glycidyl methacrylate-2-hydroxypropylene dimethacrylate copolymers as hydrophilic macroporous spheres for enzyme immobilization, and the synthesis of macro-porous hydrogels as potential haemoperfusion adsorbents. ... 

Dumitriu S, Chomet E (2000) Polyionic hydrogels as support for enzyme immobilization. Chitin Enzymol 2 527—542... 

Concerning possible applications, the proposed uses are still mainly based on the hydrogel character of ESA films, that is, on their tendency to swell efficiently in aqueous media without dissolution. As already discussed in Section IX up to the year 1999, such potential applications comprise biocompatible and nonfouling coatings [368,447,694,801,804-819], biodegradable coatings [774,814,820], enzyme immobilization or encapsulation [403,563,800,821-827], cell encapsulation... 

Many biochemical sensors depend on enzymes immobilized at an electrode surface, such that an analyte reacts to produce a compound that is readily oxidized or reduced at the electrode. Hydrogels bound to the electrode surface can be used to immobilize the enzyme without deactivating it [ 188]. In this case the gel is acting as a passive matrix but there are also systems where the responsiveness of the gel drives the sensing process. 

The fourth and probably most popular enzyme immobilization technique is the entrapment technique. In this case, monomer or low molecular weight water soluble polymers are crosslinked in the presence of the enzyme to entrapment the enzyme into the polymer matrix. This has been done with a variety of redox polymer (osmium and ferrocene-based), as well as sol-gels and other hydrogels. This technique effectively covalently links the enzyme to the electrode surface and minimizes leaching and most of these polymers are hydrogels with facile transport of substrate/product in and out of the film. However, frequently this crosslinking affects specific activity of the enzyme. 

Biocatalytic reactions performed using immobilized enzyme microreactors under continuous flow mode have been found effective for hydrolysis reactions [121,158-161], with the enzyme either trapped in the matrix [159], covalently linked to modified surface wall [160,121], enzymes entrapped in hydrogels [162], or enzymes immobilized on monolith [179]. The experimental setup consists of either chip-type microreactors with activated chaimel walls where enzymes bind, enzymes that bind to beads, enzymes entrapped in the matrix, enzymes adsorbed in nanoporous materials, and most recently, nanosprings as supports for immobilized enzymes in chip-based reactors, or enzyme immobilized monolith reactors, where support is packed inside a capillary tube (Table 10.4). 

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