Potentiometric enzyme electrodes

The potentiometric enzyme electrodes are made from a basic sensing electrode which is modified with a selective biocatalytic layer coating their measuring surface. The layer of their surface catalyzes a reaction of the analyte. The local concentration change resulted by the reaction is detected by the potentiometric sensor. The first potentiometric biosensor has been reported by Guilbault and coworkers [17, 30]. From that time very high numbers of enzyme electrodes or electrometric biosensors have been reported. Potentiometric detection, however, is less frequently employed in their case comparing to the amperomet-ric one. 

In the early times of biosensor research, enzyme sensors were prepared for application in clinical diagnosis. The urea enzyme electrode of Guilbault [17] was built on an ammonium 

The original electrode was developed for detecting urea in blood and urine samples. An enzyme sensor for measuring urea in milk has been reported by an Indian group [31]. The working principle is identical to that of Guilbault s sensor. The sensor prepared for milk analysis, however, is a flat cell fabricated with screen-printed technology. 

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Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

P. Mulchandani, A. Mulchandani, I. Kaneva, and W. Chen, Biosensor for direct determination of orga-nophosphate nerve agents. 1. Potentiometric enzyme electrode. Biosens. Bioelectron. 14, 77-85 (1999). 

The classic potentiometric enzyme electrode is a combination of an ion-selective electrode-based sensor and an immobilized (insolubilized) enzyme. Few of the many enzyme electrodes based on potentiometric ion- and gas-selective membrane electrode transducers have been included in commercially available instruments for routine measurements of biomolecules in complex samples such as blood, urine or bioreactor media. The main practical limitation of potentiometric enzyme electrodes for this purpose is their poor selectivity, which does not arise from the biocatalytic reaction, but from the response of the base ion or gas transducer to endogenous ionic and gaseous species in the sample. 

Fig. 6.27 Schematic of potentiometric enzyme electrode for urea, based on ammonium ion-selective electrode...

Because each enzyme sensor has its own unique response, it is necessary to construct the calibration curve for each sensor separately. In other words, there is no general theoretical response relationship, in the same sense as the Nernst equation is. As always, the best way to reduce interferences is to use two sensors and measure them differentially. Thus, it is possible to prepare two identical enzyme sensors and either omit or deactivate the enzyme in one of them. This sensor then acts as a reference. If the calibration curve is constructed by plotting the difference of the two outputs as the function of concentration of the substrate, the effects of variations in the composition of the sample as well as temperature and light variations can be substantially reduced. Examples of potentiometric enzyme electrodes are listed in Table 6.5. 

Fig. 6. Typical response curve of a potentiometric enzyme electrode.

Figure 4 14 Potentiometric enzyme electrode for determination of biood urea, based on urease enzyme immobilized on the surface of an ammonium ion-seiective polymeric membrane electrode.

The determination of oxalate in urine is required for diagnosis of renal calculus and hyperoxaluria. For enzymatic oxalate determination, oxalate decarboxylase (EC 4.1.1.2) has been employed in enzyme thermistors (Danielsson etal., 1981), enzyme reactors (Lindberg, 1983), and potentiometric enzyme electrodes (Kobos and Ramsey, 1980). 

The pH increase caused by urea hydrolysis can also be indicated by using pH sensitive glass or metal oxide electrodes. Owing to the dissociation equilibria of NH4 and HCO3 in the neutral range approximately 1 mole of OH- ions is formed per mole of urea. Blaedel et al. (1972) showed that in diffusion controlled potentiometric enzyme electrodes, i.e. when the substrate is completely consumed within the enzyme membrane, the product concentration at the electrode surface depends... 

In potentiometric enzyme electrodes lyases producing carbon dioxide or ammonia are used as terminal enzymes of sequences. In fact, the term enzyme sequence electrode was introduced on the occasion of the design of a potentiometric D-gluconate sensor containing gluconate kinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) (Jensen and Rechnitz, 1979). The authors found that for such a sensor to function the optimal pH values of the enzymes and the transducer should be close to each other. Furthermore, cofactors, if necessary, must not react with one another nor with constituents of the sample. It was concluded that the rate of substance conversion in multiple steps cannot exceed that of the terminal enzyme reaction. A linear concentration dependence is obtained when an excess of all enzymes of the sequence is provided, i.e. complete conversion occurs of all substrates within the enzyme membrane. Different permeabilities of the different substrates results in different sensitivities. This is particularly important with combinations of disaccharidases and oxidases, where the substrate is cleaved to two monosaccharides of approximately the same molecular size. The above... 

The above authors coimmobilized choline oxidase and AChE on a nylon net which was fixed to a hydrogen peroxide probe so that the esterase was adjacent to the solution. The apparent activities were 200-400 mU/cm2 for choline oxidase and 50-100 mU/cm2 for AChE. The sensitivity of the sequence electrode for ACh was about 90% of that for choline, resulting in a detection limit of 1 pmol/l ACh. The response time was 1-2 min. The parameters of this amperometric sensor surpass those of potentiometric enzyme electrodes for ACh (see Section 3.1.25). Application to brain extract analysis has been announced. 

The first potentiometric enzyme electrode, aimed at monitoring urea, was developed by Guilbault and Montalvo [11]. In this case, urease was entrapped... 

Figure 7.11 A theoretical potentiometric enzyme electrode calibration curve based on external diffusion control of the reaction a plot of the logarithm of the product concentration in the enzyme layer versus the logarithm of the bulk substrate concentration. K = 10" the value of kaEV/PsE is given on the curve [24],...

The signal-concentration dependence for electrochemical biosensors is linear between one and three concentration decades. The lower limit of detection is at 0.2 mmol I" with potentiometric and 1 jUmoll with amperometric enzyme electrodes. The use of amplification reactions allows us to decrease the detection limit to the nanomolar to picomolar range. Whereas the response time of potentiometric enzyme electrodes averages 2-10 min, with amperometric ones an assay can be conducted within a few seconds up to 1 min. This permits up to several hundred determinations per hour to be performed. Increasing complexity of the biochemical reaction system, e.g. by coupled enzyme reactions, may bring about an increase in the overall measuring time. 

Enzymes are often employed in the chemical layer to impart the selectivity needed. We saw an example of this in Chapter 13 when discussing potentiometric enzyme electrodes. An example of an amperometric enzyme electrode is the glucose electrode, illustrated in Figure 15.4. The enzyme glucose oxidase is immobilized in a gel (e.g., acrylamide) and coated on the surface of a platinum wire cathode. The gel also contains a chloride salt and makes contact with silver-silver chloride ring to complete the electrochemical cell. Glucose oxidase enzyme catalyzes the aerobic oxidation of glucose as follows ... 

Figure 14>U. Schematical setup for measurements with potentiometric enzyme electrodes.

Potentiometric methods are less useful in metabolite assays because of the instability of the signals and the need of calibration. A special form of a potentiometric enzyme electrode, the enzymatically coupled field effect transistor is reviewed by Caras and Janata [31]. 

Amperometric electrode instruments, nonenzyme electrodes, amperometric enzyme electrodes, potentiometric enzyme electrodes Various CO2 gas sensors... 

Guilbault and Montalvo were the first, in 1969, to detail a potentiometric enzyme electrode. They described a urea biosensor based on urease immobilized at an ammonium-selective liquid membrane electrode. Since then, over hundreds of different applications have appeared in the literature, due to the significant development of ion-selective electrodes (ISEs) observed during the last 30 years. The electrodes used to assemble a potentiometric biosensor include glass electrodes for the measurement of pH or monovalent ions, ISEs sensitive to anions or cations, gas electrodes such as the CO2 or the NH3 probes, and metal electrodes able to detect redox species some of these electrodes useful in the construction of potentiometric enzyme electrodes are listed in Table 1. 

Carr PW (1977) Fourier analysis of the transient response of potentiometric enzyme electrodes. Anal Chem 49 799 02... 

Tliis work demonstrates the potential for application of potentiometric enzyme electrodes based on mediatorless enzyme electrocatalysis for fast and sensitive assay of organophosphorus pesticides. The sensing element based on screen-printed carbon material pomits mass fabrication of the electrodes at a low cost which is essential for the disposable sensor concept. The biosensor does not require any low-molecular weight mediator and can be arranged as an all-solid-state device. Such electrodes. 

Selective potentiometric sensors are used as base sensor elements in modified probes. Two types of these are mentioned here briefly the potentiometric gas sensors and the potentiometric enzyme electrodes. 

Potentiometric enzyme electrodes were the first biosensors. In such sensors, an IBS was coated by an enzyme layer acting as a biocatalyst for reaction of a specific substance. The reaction product subsequently was detected by the ISE. This feature had been transferred soon to ISFETs. A special term, ENFET, has even been informally proposed for enzyme-modified ISFETs. Attempts were also made to utilize further biological interactions for recognition of analytes and construction of potentiometric biosensors. Immunologic sensors on the basis of antigen-antibody reaction may be called IMFETs if they are built on top of a MOSFET. Immunologic reactions, however, maybe used much more efficiently in combination with other transducers besides potentiometric... 

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