Enzyme, membrane immobilized, deposition method

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 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. 

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. 

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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