Enzyme sensors, membrane

Koncki R., Mohr G., Wolfbeis O.S., Enzyme sensor for urea based on novel pH bulk optode membrane, Biosens. Bioelectr. 1995 10 653-659. 

The first enzyme biosensor was a glucose sensor reported by Clark in 1962 [194], This biosensor measured the product of glucose oxidation by GOD using an electrode which was a remarkable achievement even though the enzyme was not immobilized on the electrode. Updark and Hicks have developed an improved enzyme sensor using enzyme immobilization [194], The sensor combined the membrane-immobilized GOD with an oxygen electrode, and oxygen measurements were carried out before and after the enzyme reaction. Their report showed the importance of biomaterial immobilization to enhance the stability of a biosensor. 

Electron Transfer Type of Dehydrogenase Sensors To fabricate an enzyme sensor for fructose, we found that a molecular interface of polypyrrole was not sufficient to realize high sensitivity and stability. We thus incorporated mediators (ferricyanide and ferrocene) in the enzyme-interface for the effective and the most sensitive detection of fructose in two different ways (l) two step method first, a monolayer FDH was electrochemically adsorbed on the electrode surface by electrostatic interaction, then entrapment of mediator and electro-polymerization of pyrrole in thin membrane was simultaneously performed in a separate solution containing mediator and pyrrole, (2) one-step method co-immobilization of mediator and enzyme and polymerization of pyrrole was simultaneously done in a solution containing enzyme enzyme, mediator and pyrrole as illustrated in Fig.22. 

It is difficult to incorporate dehydrogenases that are coupled with NAD(P) into amperometric enzyme sensors owing to the irreversible electrochemical reaction of NAD. We have developed an amperometric dehydrogenase sensor for ethanol in which NAD is electrochemically regenerated within a membrane matrix. 

An electron transfer type of enzyme sensor was thus fabricated by a electrochemical process. Although no appreciable leakage of ADH and MB from the membrane matrix was detected, NAD leaked slightly. To prevent this leakage, the ADH-MB-NAD/polypyrrole electrode was coated with Nation. A calibration curve is presented in Fig.25 for ethanol determination in an aquous solution with the enzyme sensor. Ethanol is selectively and sensitively determined in the concentration range from 0.1 nM to 10 mM. 

In a further development, an ADH-MB-NAD/polypyrrole electrode, a platinum counter electrode and an Ag/AgCl reference electrode were assembled and covered with a gas-permeable polymer membrane to form an gaseous ethanol sensor. This appears to be the first time that a complete enzyme sensor for gaseous ethanol has been fabricated in such a manner with NAD incorporated in immobilized form. 

Many different types of techniques for protein immobilization have been developed using, in most cases, enzyme sensors. Early studies of enzyme biosensors often employed thick polymer membranes (thickness 0.01-1 mm) in which enzymes are physically entrapped or chemically anchored. The electrode surface was covered with the enzyme-immobilized polymer membranes to prepare electrochemical enzyme sensors. Although these biosensors functioned appropriately to... 

The glucose oxidation reaction in the enzyme immobilized membrane of the glucose sensors is performed according to the following reaction. 

Sterilization of tylon Enzyme Electrodes. Sterilization of in vivo electrodes is essential for clinical use and advisable for applica-tions in the food industry. It was thus of interest to study the behaviour of various carbohydrate sensor membranes before and after irradiation with 60Co-y radiation (Figure 1). Thus, after each membrane calibration in a batch mode, the membrane was detached, placed in a sealed glass tube with phosphate buffer (pH 7) and irradiated to set doses. The membrane was then reattached to the... 

Haga et al. developed another type of immunosensor by combining an enzyme membrane immunoassay and an enzyme sensor using oxygen electrodes (HI). In this assay antigen molecules (theophylline) are attached on the surface of the liposomes and an enzyme (horseradish peroxidase) is encapsulated in the sensitized liposome. When antibody (antitheophylline antibody) and complement are added, the enzyme is released by the liposome lysis. The enzyme activity with the NADH-NAD reaction can be determined by the oxygen electrode. When antigen is added, it competitively binds to antibodies, then liposome lysis and enzyme activity are decreased. The sensitivity of this method for theophylline determination was reported as 0.7 ng/ml. 

Fig. 1. Schematic view of an enzyme sensor a, transducer b, biocatalyst layer c, permselective membrane d, soludon.

Fig. 5. Structure of FET sensor with two discrete FET chips 1, connectingwire 2, epoxy resin 3, platinum wire 4, FET chip 5, enzyme-immobilized membrane 6, epoxy laminate. (Reproduced from Nakako et al. (26), with permission.)...

Rnzyme-aontaining Membranes for Molecular Recognition. Updike et al. applied an enzyme-containing synthetic membrane to form an enzyme electrode, that Is, an "enzyme sensor," which was the first configuration that realized the concept of a biosensor (79). The... 

In the early stage of Investigation, GOD was entrapped In a thin membrane of polyacrylamide gel. The gel membrane provided poor permeability to substrates, which resulted In a slow sensor response. Great effort has been made to Improve the membrane matrix (80-82). The response time of the sensor has been reduced to less than 10 seconds (82). Some of these enzyme sensors for glucose have been commercialized. 

In a similar manner, enzyme-containing membranes have been coupled with electrochemical devices such as an oxygen electrode to form enzyme sensors for a variety of biochemical and clinical applications (Table II). Considerable effort has been concentrated on the development of these enzyme sensors (83-86). 

Microbial Cell-containing Membranes for Molecular Recognition. Suzuki et al. (87) have proposed a miocrobial sensor which consists of membrane-bound microbial cells and an electrochemical device. The assemblies of microbial sensors are similar to enzyme sensors. Two types of microbial sensors have been developed as presented In Figure 9. The first monitors the respiration activity of membrane-bound microbial cells with a Clark-type oxygen electrode. The... 

Okahata, Y, Tsuruta, T., Ijiro, K., and Ariga, K., Langmuir-Blodgett films of an enzyme-lipid complex for sensor membranes, Langmuir, 4, 1373-1375, 1998. 

Owing to the excess of enzyme in the membrane a diffusion limited enzyme sensor has a higher functional stability than a kinetically controlled one. With the former, 2000-10 000 measurements per enzyme membrane can be performed, while kinetically controlled sensors typically permit only 200-500 measurements. 

Besides the differential equations the complete formulation of the model requires a set of initial and boundary conditions. These must reflect the situation at the interface between measuring solution and enzyme electrode membrane and between membrane and sensor. For the models considered, it is assumed that the measuring solution is perfectly mixed and contains a large amount of substrate as compared to the substrate converted in the enzyme membrane. It has been shown experimentally (Carr and Bowers, 1980) that in measuring solutions diffusion is much more rapid than in membranes. A boundary layer effect is not considered. On the sensor side all electrode-inactive substances fulfill zero flux conditions. If the model contains more than one layer the transfer between the layers may be modeled by using relations of mass conservation. The respective equations will be given in the following sections. 

Arnold et al. (1987) described an optoelectronic ethanol sensor based on fluorimetric detection of NADH formed in the reaction catalyzed by ADH. The enzyme was fixed to the inner surface of a membrane permeable to volatile substances, which separated the sample from the internal sensor solution. This solution contained NADH and semicarbazide, so that no reagent had to be added to the sample. The arrangement was named an internal optical enzyme sensor . 

For application in enzyme electrodes COD has been mainly immobilized by surface fixation (Table 7). As early as 1977, Clark had patented the polarographic analysis of free and esterified cholesterol by means of free as well as immobilized COD and CEH with anodic H2O2 indication. Cholesterol in food and serum samples has been determined by using COD bound to a collagen membrane via glutaraldehyde and coupled to a Pt electrode (Clark, 1978). A similar probe has been devised by Bertrand et al. (1979). In this sensor the enzyme membrane was not protected by a semipermeable membrane. Interferences were compensated for by difference measurements between an enzyme sensor and an enzyme-free membrane electrode. The lower detection limit was 0.05... 

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