Entrapment polyacrylamide

The mode of immobilization, as well as the source and extent of purification of the enzyme, are important factors in determining the lifetime of the bio-catalyst. Generally, the lifetime of a soluble enzyme electrode is about one week or 25-50 assays, and the physically entrapped polyacrylamide electrodes are satisfactory for about 50-100 assays, depending primarily on the degree of care exercised in the preparation of the polymer. The chemically attached enzyme can be kept for years, if used infrequendy. In frequent use, the GOD electrode has a lifetime of over one year and can be used for over 1000 assays. For 1-amino acid oxidase or uricase (100) biosensors, about 200-1000 assays per electrode can be obtained, depending on the immobilization technique. 

To develop a continuous process, the immobilisation of aminoacylase of Aspergillus oryzae by a variety of methods was studied, for example ionic binding to DEAE-Sephadex, covalent binding to iodo-acetyl cellulose and entrapment in polyacrylamide gel. Ionic binding to DEAE-Sephadex was chosen because the method of preparation was easy, activity was high and stable, and regeneration was possible. 

The entrapment method is based on confining the enzyme within the lattice of a polymeric matrix. Polyacrylamide gels have successfully yielded stable enzyme films with a high retention of activity... 

Except the physical entrapment of horseradish peroxidase in a polyacrylamide gel used by Freeman and Seitz23, immobilization of the... 

This process involves the suspension of the biocatalyst in a monomer solution which is polymerized, and the enzymes are entrapped within the polymer lattice during the crosslinking process. This method differs from the covalent binding that the enzyme itself does not bind to the gel matrix. Due to the size of the biomolecule it will not diffuse out of the polymer network but small substrate or product molecules can transfer across or within it to ensure the continuous transformation. For sensing purposes, the polymer matrix can be formed directly on the surface of the fiber, or polymerized onto a transparent support (for instance, glass) that is then coupled to the fiber. The most popular matrices include polyacrylamide (Figure 5), silicone rubber, poly(vinyl alcohol), starch and polyurethane. 

Mullen et al. (1989) reported that Bacillus cereus, B. subtilis, E. coli and P. aeruginosa were able to sorb an average of 89% of the total Ag+ and 12-27% of the total Cd2+, Cu2+ and La3+ from a ImM solution. Using polyacrylamide-entrapped cells of Brevibacterium sp strain PBZ, Simine et al. (1998) measured a sorption capacity of 40 mg g-1 and 13 mg g-1 dry biomass for Pb and Cd, respectively. Hall et al. (2001) isolated two bacterial strains of P. syringae that were tolerant to 1000 mg L-1 Cu. Similarly, Amoroso et al. (2001) were able to obtain Streptomyces spp. strains R22 and R25 with a high tolerance to Cr from sediments of the Sail River, Argentina. The cells of R22 and R25 could accumulate 10.0 and 5.6 mg Cr g-1 dry weight, respectively, from a concentration of 50 mg Cr mL 1. Cell fractionation studies with strain R22 showed that most of the chromium... 

Enzymes can be immobilized by matrix entrapment, by microencapsulation, by physical or ionic adsorption, by covalent binding to organic or inorganic polymer-carriers, or by whole cell immobilization (5 ). Particularly impressive is the great number of chemical reactions developed for the covalent binding of enzymes to inorganic carriers such as glass, to natural polymers such as cellulose or Sepharose, and to synthetic polymers such as nylon, polyacrylamide, and other vinyl polymers and... 

Many commercially important polymers are produced via emulsion polymerization. This is also one of the most common methods to produce dye-doped beads. A dye is added to the mixture of monomers prior to initiating the polymerization and is either noncovalently entrapped or is copolymerized. The second method ensures that no leaching will occur from the particle but requires modification of the dye (typically by providing it with a double bond). This method is most common for preparation of pH-sensitive beads where a pH indicator is entrapped inside cross-linked polyacrylamide particles. The size of the beads can be tuned over a wide range so that preparation of both nano- and microbeads is possible. Despite thorough washing the surfactants are rather difficult to remove completely and their traces can influence the performance of some biological systems. 

Similarly to dyes, some fluorescent proteins can be incorporated into polymeric beads to be used as an alternative for ion sensing. For example, a reporter protein (composed of a phosphate-binding protein, a FRET donor (cyan fluorescent protein) and a FRET acceptor (yellow fluorescent protein)) was incorporated into polyacrylamide nanobeads by Sun et al. [46]. FRET was inhibited upon binding of phosphate. Kopelman and co-workers [47] used a similar approach to design a nanosensor for copper ions. They have found that fluorescence of red fluorescent protein DsRed (commonly used as a label) is reversibly quenched by Cu2+ and Cu+. Both DsRed and Alexa Fluor 488 (used as a reference) were entrapped into polyacrylamide nanobeads. Typically, up to 2 ppb of copper ions can be reliably measured. It should be mentioned, that in contrast to much more robust dyes, mild conditions upon polymerization and purification are very important for immobilization of the biomolecule to avoid degradation. 

A variety of techniques has been used varying from simple adsorption to entrapment within a polymer matrix but the commonest method is the covalent linking of the ligand to a suitable polymer, such as dextran, agarose or polyacrylamide. 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Entrapment of enzyme molecule ( y) cross-linked polyacrylamide I prepared by mixing enzyme h acrylamide, methylene bisacrylamide, and catalyst... 

Membranes can be used as well as a supporting material for immobilisation (e.g. polycarbonate membranes with created amino groups on the surface that allow covalent binding with glutaraldehyde). Entrapment of enzymes on electrode surfaces can be carried out with polymeric membranes such as polyacrylamide and gelatine, or by electropolymerisation of small monomers (o-phenylenediamine). Enzyme encapsulation within a sol-gel matrix has also been reported. 

Crosslinking or entrapment Entrapment in dextran matrix, polyacrylamide or DNA crosslinked by using some agents such as glutar aldehyde [43] ... 

---