Analyzers using enzyme electrodes

Fig. 137. Scheme of an on-line bedside analyzer for parallel determination of glucose, lactate, and pyruvate using enzyme electrodes. (Redrawn from Mascini, 1987). 

First commercial enzyme electrode (for glucose) and glucose analyzer using the electrode (Yellow Springs Instruments) ... 

In the earlv 1960s. a promising approach to glucose monitoring was rlcreloped in the form of an enzyme electrode lhal used oxidation of glucose by the enzyme GOD, This approach has been incorporated into a leu clinical analyzers lor blood glucose determination... 

The gas-sensing electrodes also are used for the potentiometric measurement of biologically important species. An enzyme is immobilized at or near the gas probe. The gas sensor measures the amount of characteristic gas produced by the reaction of the analyzed substance with the enzyme. For example, an enzyme electrode for urea [NH2C(0)NH2] determination is constructed by the immobilization of urease onto the surface of an ammonia-selective electrode. When the electrode is inserted into a solution that contains urea, the enzyme catalyzes its conversion to ammonia ... 

A great number of enzymes have already been combined with electrodes. In some cases, several possible enzymatic routes may be selected, depending on the substrate to be analyzed. One such example is that used to determine aspartame (Fig. 3) (18-20). Enzyme electrodes use, ideally, only one enzyme and monitor the main substrate-enzyme reaction, for example, glucose oxidase-glucose. If the main substrate-enzyme reaction is not electrochemically detectable or if signal amplification is required, bi- or trienzymatic sequences may be applied. 

Recently, Du et al. reported an AChE electrochemical sensor based on enzyme-induced growth of gold nanoparticles (AuNPs) without the addition of any gold nano-seeds [17], They have successfully used [Fe (CN)6]3 74 as a probe to indicate the process of electron transfer across the interface and also analyze the enzyme inhibitor quantitatively. Initially, cleaned gold electrode was coated with 0.5 % (w/v) chitosan solution and AChE was later immobilized onto this chitosan modified gold electrode. CV results show that, AuNPs presence increases the peak current and decreases the peak separation. This confirms the presence of AuNPs on the chitosan modified electrode surface. However, the peak current of... 

The best-known enzyme electrode is that used to analyze for urea in blood. The enzyme urease is immobilized in a polyacrylamide hydrophilic gel and fixed at the bottom of a glass electrode whose characteristics make it an NH4 ISE. Alternatively, the ISE can be a composite system designed to detect NH3. In the presence of the enzyme, urea is hydrolyzed according to the reaction... 

In most amperometric cytochrome b2 electrodes the reaction is followed by anodic oxidation of ferrocyanide at a potential of +0.25 V or above. The first of such sensors was assembled by Williams et al. (1970), who immobilized the enzyme (from baker s yeast) physically at the tip of a platinum electrode within a nylon net of 0.15 mm thickness. The large layer thickness resulted in a response time of 3-10 min. Owing to the low specific enzyme activity used, the sensor was kinetically controlled. Therefore the linear measuring range extended only up to 0.1 Km-A similar sensor has been applied by Durliat et al. (1979) to continuous lactate analysis. The enzyme was contained in a reaction chamber of 1 pi volume in front of the electrode. This principle has also been employed in the first commercial lactate analyzer using an enzyme electrode (Roche LA 640, see Section 5.2.3.3X With a sensor stability of 30 days and a C V below 5%, 20-30 samples/h can be processed with this device. 

Similar to other analyzers the Glukometer GKM (Fig. 128) is particularly well suited to the analysis of single samples and small sample series. The measuring and sample solutions are injected by using pipettes. The preparation of the enzyme electrode requires about 3 min the... 

The first enzyme electrode-based lactate analyzer was developed in 1976 by La Roche (Switzerland) (see Table 23). It uses cytochrome b2 in a tiny reaction chamber on top of a platinum electrode polarized at +0.25-0.40V. The solution for blood sample pretreatment recommended by the manufacturer has been improved by Soutter et al. (1978) by addition of cetyltrimethylammonium bromide. This compound hemo-lyzes the sample, stabilizes the lactate content, and leads to a good correlation with the spectrophotometric reference method using deproteinized blood ... 

Geppert and Asperger (1987) employed an enzyme electrode to control the concentration of glucose in various bioprocesses. The samples were continuously withdrawn, deaerated with nitrogen, and diluted, without separation of biomass. Glucose was analyzed by discrete measurement with the use of the artificial electron acceptor benzoquinone. The method required correction of the measurement by means of an enzyme-free sensor. 

Example 3.5 looked at comparing two means for the glucose levels in soft drinks being analyzed by a spectroscopic enzyme assay and an alternative enzyme electrode method. The conclusion of the analyses, using a t-test, was that the two means were different. The analytical laboratory therefore decided to check each method relative to an AOAC (Association of Official Analytical Chemists) method that employed HPLC. The analytical results for six replicate measurements (units mM) using each method are ... 

Other biosensor-based diagnostic instruments, such as the enzyme-electrode-based analyzers of YSI for glucose and lactate, are utilized routinely in many clinical laboratories. i-Stat recently introduced a portable analyzer for bedside use which utilizes enzyme-electrode-based assays for glucose and urea, as well as chemical sensor tests for nitrogen, sodium, potassium, and chloride. 

In order to utilize enzymes for analytical purposes, one must either have a concentration of enzyme far in excess of that of the substrate, or a known quantity of enzyme. The excess quantity of enzyme is useful and practical where small samples are to be analyzed. Such is the case for enzyme electrodes, where an excess of enzyme is still a very small quantity of material, i.e., a few milligrams. However, maintaining this excess at a fixed location, i.e., at the electrode-test solution interface, requires some method of immobilizing the enzyme. 

Immobilized enzyme electrode for the determination of D-glucose Active immobilized enzyme for clinical analysis of D-glucose used in microcolumn form as part of an Autoanalyzer continuous flow system Immobilized enzyme reactor for determination of D-glucose in a continuous flow analyzer... 

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