Electrodes enzyme loading

In ion-selective FETs (ISFETs), an ion-selective membrane replaces the gate electrode. When an enzyme-loaded gel is combined with the membrane, the device can be used to measure substrates which enzymically generate charged species. 

An alternate method to produce templated electrodes is the use of chemical reduction of the monomer in the presence of a track-etched or alumina membrane. Parthasarathy et al. [46] have produced enzyme-loaded nanotubules by a combination of both electrochemical and chemical deposition. Initially, the alumina membrane was sealed at one end with a thick Au film (Figure 1.9a), after which the membrane was placed into a mixture of pyrrole and Et4NBF4. The pyrrole was then electropolymerized to form a small plug of polypyrrole at the closed end of the alumina membrane (Figure 1.9b). Subsequently, the membrane was placed into a... 

The increase of the measuring value of the amperometric glucose electrode with increasing substrate concentration reflects the course of a Michaelis-Menten curve and reaches a concentration-independent saturation corresponding to the maximum rate, vmax. The sensitivity of the GOD electrode depends on the enzyme loading (Fig. 30) (Scheller et al., 1988). The substrate concentration giving rise to the half-maximum current in air saturated solution is between 1.4 and 1.8 mmol/1 glucose. 

Fig. 30. Dependence of the stationary current of a GOD electrode on glucose concentration at different enzyme loadings. Electrode surface 0.22 mm2 electrode potential +600 mV vs. Ag/AgCl conditions as in Fig. 28 curves 1-3 oxygen-saturated solution curves 4—6 air-saturated solution.

The highest sample frequency achieved up to now has been obtained in a computer-controlled flow injection analysis (FIA) system (Olsson et al., 1986b) using an amperometric flow-through cell (Bertermann et al., 1981). A Pt electrode of 0.5 mm diameter (VEB Metra Radebeul, GDR) has been used as hydrogen peroxide detector. GOD was immobilized in polyurethane (thickness, 0.02 mm, enzyme loading, 50 U/cm2) and... 

Laval etal. (1984) bound LDH covalently to electrochemically pretreated carbon. The enzyme was fixed by carbodiimide coupling simultaneously with anodic oxidation of the electrode surface. The total amount of immobilized LDH was determined fluorimetrically after removal from the electrode and hydrolysis. The authors found that at a maximal enzyme loading of 13 pmol/cm2 six enzyme layers are formed. The immobilization yield was about 15%. The kinetic constants, pmax and. Km, were not affected by the immobilization. The obtained enzyme loading factor of 10-3 indicates that diffusion in the enzyme layer was of minor influence on the response of the sensor. The layer behaved like a kinetically controlled enzyme membrane, i.e., the NADH oxidation current was proportional to the substrate concentration only far below Km- With increasing enzyme loading the sensitivity for NADH decreased due to masking of the electrode surface. 

ADP AFP ab as ALAT AP ASAT ATP BQ BSA CEH CK CME COD con A CV d D E E EC ECME EDTA EIA /e FAD FET FIA G GOD G6P-DH HBg HCG adenosine diphosphate a-fetoprotein antibody antigen alanine aminotranferase alkaline phosphatase aspartate aminotransferase adenosine triphosphate benzoquinone bovine serum albumin cholesterol ester hydrolase creatine kinase chemically modified electrode cholesterol oxidase concanavalin A coefficient of variation (relative standard deviation) layer thickness diffusion coefficient enzyme potential Enzyme Classification enzyme-chemically modified electrode ethylene diamine tetraacetic acid enzyme immunoassay enzyme loading factor flavin adenine dinucleotide field effect transistor flow injection analysis amplification factor glucose oxidase glucose-6-phosphate dehydrogenase hepatitis B surface antigen human chorionic gonadotropin... 

Enzyme loading test. The minimum amount of enzyme required for maximum sensitivity is determined by varying the enzyme loading (figure 17.2). This test also, reveals the enzyme limit of diffusion-controlled sensors. Owing to differences in /Ka/ values and the layer thickness, the transition from the kinetic to diffusion control of different enzyme electrodes takes place at rather... 

Figure 17.2 An enzyme loading test of a lactate oxidase membrane electrode e 2.5 mmoir lactate + 5.0 mmoir lactate (reproduced with the permission of Elsevier Science Publishers BV).

Figure 14-22. Electrochemical Lineweaver-Burk plot of GOD electrodes with different enzyme loadings, determined in air-saturated solution. Reproduced from [281] with permission from Academic Press.

Arrhenius plot of the tmperature dependence of the response of a GOD electrode with different enzyme loadings to different giucose concentrations and to hydrogen peroxide. 

The measurement of the lactate/pyruvate ratio in plasma is possible by using a lactate dehydrogenase-lactate monooxygenase sequence electrode [373]. The sensor is equally sensitive to lactate and pyruvate (Figure 14-34), because of the high enzyme loading and the... 

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