Potentiometric enzyme assay

Uithoven K.A., Schmidt J.C., Ballman M.E., Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system, Biosens. Bioelectron. 2000, 14 761-770. 

Fig. 8. Layout of the Modules of an Ektachem 700 Analyzer showing the rate incubator for rate-reaction measurements, the potentiometric incubator, and colorimetric incubator. The latter two are not used for enzyme assays.

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. 

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]. 

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. 

Potentiometric devices have a relatively high sensitivity and selectivity and abroad response range. They are also simple, rehable, and inexpensive and use commonly available instrumentation. Potentiometric enzyme-based biosensor systems have been recently reviewed. The most general approach for the determination of OP compounds (OPCs) is based on their inhibition of the activity of choline esterase enzymes. The presence of low concentrations of inhibitors strongly and specifically affects the enzyme activity. Therefore, by measuring the enzyme activity, the concentration of the OPCs can be assayed. Different enzymes, such as acetylcholine (ACh), BuCh, and, most often, pH sensors, were used as transducers in potentiometric biosensors. 

The synthesis of linear 4 —> 1-a-D-glucans from D-glucopyranosyl phosphate by the action of phosphorylases has been shown by comparison of results of methylation and end-group assay and viscosity determination,209 and by potentiometric, iodine titrations82 on the product. The chain length of the enzymic product (100 to 200 D-glucose units) is less than that of the natural component. Whether this is due to impure enzymes cannot yet... 

During the past 40 years there have been numerous exciting extensions of electrochemistry to the field of analytical chemistry. A series of selective-ion potentiometric electrodes have been developed, such that most of the common ionic species can be quantitatively monitored in aqueous solution. A highly effective electrolytic moisture analyzer provides continuous online assays for water in gases. Another practical development has been the voltammetric membrane electrode for dioxygen (02), which responds linearly to the partial pressure of 02, either in the gas phase or in solution. The use of an immobilized enzyme (glucose oxidase) on an electrode sensor to assay glucose in blood is another extension of electrochemistry to practical analysis. 

Potentiometric. Not all analytes can be readily assayed via a redox enzyme and in these instances the assay schemes suggest parameters other than electron transfer which may be probed. Indeed, even where a redox enzyme is available for the analyte in question, it is not always desirable to deploy an amperometric technique. 

Potentiometry. Potentiometric methods rely on the logarithmic relationship between measured potential and analyte concentration. The most common involves an instrument called a pH-Stat , in which a glass (pH) electrode follows reactions that either consume or produce protons. Since pH changes cause changes in enzyme activity, the pH is maintained at a constant value by the addition of acid or base. The rate of titrant addition is then proportional to the rate of the enzymatic reaction. Precise measurements using the pH-Stat require low buffer concentrations in the enzymatic assay mixture. 

The sensitive part of an electrode is covered with a membrane on which the enzyme is immobilized in immunocomplexes. The enzyme-catalyzed reaction takes place near the sensor (Mattiasson and Nilsson, 1977). The method is as fast as the thermometric assay but less sensitive. Electrode-based EIA using urease conjugates have been reviewed by Meyerhoff and Rechnitz (1980). This method has reasonably low detection limits. These promising potentiometric EIA are discussed by Boiteux et al. (1981) and Gabauer and Rechnitz (1982). 

A heterogeneous EIA coupled with a potentiometric electrode permitted the assay of BSA down to 10 ng/ml and cAMP down to 10 nmol/1 (Meyerhoffand Rechnitz, 1979). Urease was used as the marker enzyme and its activity was measured by means of an ammonia gas-sensing electrode. The equilibrium of the immunological reaction at the sensor was reached rather slowly. The advantage of the rapid response of biosensors could not therefore be exploited. 

For the assay of low molecular weight metabolites usually enzymes are employed as speciHers which turn over the metabolites to form a reaction product. Its concentration can be measured potentiometrically or amperometrically [11, 12]. 

Clark et al. [32] have published in 1981 a method employing both enzymes in solution and potentiometrically measuring the concentration of the hydrogen peroxide formed. For an amperometric cholesterol assay Yao et al. [33, 34] used immobilized cholesterol esterase and cholesterol oxidase in a reactor assaying the hydrogen peroxide formed at a peroxidase electrode with the redox pair hexacyanoferrate(II)/hexacyanoferrate(III) as a mediator. 

A potentiometric electrode based on direct mediatorless bio-electrocatalysis for determination of choline and butyryl-choline has been described in our earlier work (54). Using the two enzymes peroxidase and choline oxidase in a coupled system allows determination of choline concentration as a result of the consecutive enzyme reactions. In the system choline concentration assay is based on the measurement of the rate of electrode potential increase (AE/At). 

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