Enzyme, membrane immobilized, deposition

Different approaches have been reported for enzyme immobilization and membrane deposition including drop-on techniques [42, 51], ink-jet printing [52] and photolithographically patterned enzyme membranes [53,54]. 

The three enzyme membrane deposition methods can be adapted for the preparation of different enzyme-immobilized membranes on a single FET chip simply by repeating the cycle of coating, irradiation, and development procedures in the cases of the first and third methods, and by the injection of a different enzyme-immobilizing solution into a different micropool in the case of the second method. 

The micropool injection method was developed by Miyahara et al. (20), and independendy by Kimura and his collaborators (15,16, 43). It was successfully used for a monolithic enzymatically coupled FET, sensitive to urea and glucose, and for the urea-, glucose-, and potassium-sensitive trifunctional FET biosensor (see Section 2.3). The thickness of an enzyme membrane is about 10 pm, and the thickness of an enzyme-immobilized membrane prepared by the ink jet-micropool injection method is 0.1 -1 pm. The lift-off method was developed by Kimura and Kuriyama s group and used for the deposition of a urease- and a glucose oxidase-immobilized membrane (16, 17) (see Fig. 8(3)). The enzyme membrane thickness is similar to the thickness of the film resist layer, about 1 pm thick. 

Enzyme membrane patterning procedures were utilized similar to the monofunctional FET biosensors, except that the membranes were developed in water instead of a glutaraldehyde solution. The center FET element was used as a reference FET employing a bovine serum albumini-immobilized membrane. After the three membranes were deposited on the FET surface, they were cross-linked by immersing them in a glutaraldehyde solution to make them mechanically strong. 

The design and implementation of a portable fiber-optic cholinesterase biosensor for the detection and determination of pesticides carbaryl and dichlorvos was presented by Andreou81. The sensing bioactive material was a three-layer sandwich. The enzyme cholinesterase was immobilized on the outer layer, consisting of hydrophilic modified polyvinylidenefluoride membrane. The membrane was in contact with an intermediate sol-gel layer that incorporated bromocresol purple, deposited on an inner disk. The sensor operated in a static mode at room temperature and the rate of the inhibited reaction served as an analytical signal. This method was successfully applied to the direct analysis of natural water samples (detection and determination of these pesticides), without sample pretreatment, and since the biosensor setup is fully portable (in a small case), it is suitable for in-field use. 

Polymerization of the D-glucan chains occurs by way of a multi-subunit, enzyme complex embedded in the plasma membrane an almost simultaneous association, by means of hydrogen bonds, of the newly formed chains results in formation of partially crystalline microfibrils. This mechanism of polymerization and crystallization results in the creation of microfibrils whose chains are oriented parallel (cellulose I). In A. xylinum, the complex is apparently immobile, but, in cells in which cellulose is deposited as a cell-wall constituent, it seems probable that the force generated by polymerization of the relatively rigid microfibrils propels the complex through the fluid-mosaic membrane. The direction of motion may be guided through the influence of microtubules. 

Semiconductor fabrication techniques have also been successfully applied to the construction of conventional transducers sensitive to hydrogen peroxide, oxygen, and carbon dioxide, A hydrogen peroxide-sensitive silicon chip was made by using metal deposition techniques (28,29). The combination of the hydrogen peroxide-sensitive transducer and enzyme-immobilized membranes gave a miniaturized and multifunctional biosensor. Similarly, an oxygen- and a carbon dioxide-sensitive device was made cmd applied to the construction of biosensors (25, 30, 31). 

The enzymatically coupled FET is reviewed in this chapter (32). First, a brief review is given. Determining how to deposit an enzyme-immobilized membrane on the surface of a FET was one of the most difficult problems to solve before tin enzymatically coupled FET could be developed. Therefore, some enzyme-immobilized membrane deposition methods and the photolithographic enzyme-immobilized membrcme patterning method developed by the authors are described in detail. Concomitantly, the performances of some FET biosensors with an enzyme-immobilized membrane made by this method are described. Finally, recent applications of an enzymatically coupled FET are surveyed. 

The advanced type of enzymatically coupled FET utilizes an integrated FET transducer chip with several FET elements closely spaced to each other (see Section 2.3 and Fig. 7). The monolithic enzymatically coupled FET requires that small and well-defined enzyme-immobilized membranes be patterned on the specific areas of such a FET chip. In addition, the method for depositing the enzyme on the membrtme should be compatible with mass-production processes. Therefore, a more sophisticated procedure is needed to deposit enzyme on the membranes used in the monolithic enzymatically coupled FET. 

The selective UV-inactivation method is effective for the deposition of a single enzyme-immobilized membrame on a FET, but it is not possible to deposit several different enzyme-immobilized membranes on one FET chip. Other photolithographic methods, outlined in Fig. 8, do not have this disadvantage. The methods are applicable to the simultaneous deposition of different membranes on a FET chip. In other words, these methods are usable for the preparation of a multifunctional enzymatically coupled FET. 

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