Ammonium ISE

Although rum ammonia levels are not routinely measured, it is a useful indicator of Reye s syndrome and should be monitored in newborns at risk of developing hyperammonemia Ammonia is produced in many analytically useful enzyme reactions and the ammonium ISE has been used as the base sensor in several enzyme electrodes (see next section). In addition to valinomycin, other antibiotics such as the nonactin homalogs and gramicidins also behave as ionophores. The nonactin homolo were originally studied for their ability to selectively bind potassiiun ions It was then discovered that ammonium ions were preferred over potassium ions, and the selectivity coefficient Knh+ = 0.12 was reported. Since ammonia is present at fairly low levels in serum, this selectivity is not sufficient to to accurately measure NH4 in the presence of K. An extra measure of selectivity can be gained by using a gas permeable membrane to separate the ammonia gas from the sample matrix... 

Ammonium ISEs have been developed using a mixture of nonactin and monactin XXIII they can be used for the determination of NHj in serum [186] (see fig. 7.6), in urine [80], in water [40, 136], as well as for kinetic study of the decomposition of arginine [159]. Unfortunately the detection limit is 50 times greater than that of the ammonium gas probe [40] (see section 4.4). 

Fig. 45.1. Calibration curve obtained for the ammonium ISE in respect of its primary ion.

Figure 45.3 shows the response surface obtained for ammonium ISE in presence of potassium ion as interference. This surface has been generated from the experimental data of the seven previous calibration sequences, fitting them to the Nicolsky-Eisenman equation with SigmaPlot 2000 software and plotted with 3D visualization options. The experimental data also appear in the figure as black symbols. In the same way, the fitted response surfaces for potassium and generic ISEs can be easily generated. 

The sensor was improved by using the non-actin-based ammonium ISE. This ionophore has selectivity constants Rnh4,k of 0.15 and RNH4,Na of 1.3 X 10, thus partially eliminating the response of these ions by the sensor. Several articles have been published on this principle, taking into account the residual effect of sodium and potassium. 

The transducer can be an ammonium ISE ionophore (nonactin-based), a gas sensor for ammonia or carbon dioxide, and an iodide-selective sensor. The latter can be coupled with the enzymes L-aminooxidase (l-AOx) and peroxidase (e.g., coimmobilized in a polyacrylamide gel), which catalyses reactions [V] and [VI] ... 

Fig. 8 Design of potentiometric urea sensor based on the immobilized enzyme urease on the surface of a polymer membrane type ammonium ISE.

All ammonium ISEs are based on the macrotetrolide antibiotics and their major applications relate to enzyme studies. PVC membranes with nonactin (18) (containing up to 28% monactin 19) have been fabricated with a wide range of solvents, namely diethyl-, dibutyl- and dioctyl phthalates dioctyl adipate dibutyl-, dioctyl- and diethylhexyl sebacates diethylhexyl- and tripentyl phosphates di-octylphenyl phosphonate and 2-nitrophenyl phenyl ether (49,50). In terms of selectivity, dioctyl adipate (DOA) was the best solvent, and the nonactin sensor could be conserved by fixing the mass ratios of nonactin DOA PVC at 1 300 160. The Nerntian slope was 57.5mV decade and kRH4,K = 0 H (50). 

In a second system reported by the same laboratory, a sensor array incorporating a urea biosensor was proposed for the determination of urea in blood serum [19]. Similarly to the previous case, the urease enzyme was immobilized employing BS A and glutaraldehyde onto an ammonium ISE, taking profit of the appearance of ammonium product after the enzyme hydrolysis of urea. As before, the array was complemented by ISEs for K", Na and with the idea of counterbalancing any interference by alkaline ions. Authors achieved correct prediction of urea and potassium, not sodium or ammonium initially present. Correlation and prediction... 

Fig. 10. Response of an ammonium ion-selective electrode which was made from nonactin dissolved in a PVC membrane mounted in a Phillips electrode body (ISE-561, Glasblaserie Mitller, Zurich) referenced against an SCE to a spHt-stream EIA system, (a) EIA response to injected standards containing both glutamine and ammonium chloride lines A—E contain both glutamine and ammonium chloride E—G contain only glutamine, (b) Strip-chart...

Low sulfate selectivity of the ion-selective electrodes (ISE) based on lipophilic quaternary ammonium salts (QAS) is usually explained by unfavorable ratio of sulfate hydration and solvation energies. We have been shown that another reason does exist as well namely, low efficiency of sulfate-QAS cation interaction caused by steric hindrance for simultaneous approach of two QAS cations, containing four long-chain hydrocarbon substituents, to sulfate ion. 

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. 

The electrolytes Na", and Cl are second only to glucose in being the most frequently run hospital tests. Many clinical chemistry analyzers now contain an ISE module for electrolyte analysis. Most commonly the module will consist of a Na -glass electrode, a valinomycin/PVC electrode, a Ag/AgCl pellet or a quaternary ammonium ion/PVC electrode and a reference electrode. A selective electrode for the bicarbonate ion continues to elude workers in the field. An indirect measurement of HCOf must be made. The sample is usually reacted with acid to evolve carbon dioxide gas which is measured with a traditional Severinghaus type CO2 electrode. Alternatively, the sample is treated with base to convert HCO to CO3 and a carbonate ion-selective electrode is used In this manner, the complete primary electrolyte profile is obtained electrochemically. 

Another important bioanalytical application of voltammetric ISEs is the detection of polyions (see also above). A technique using cyclic voltammetry on micropipette electrodes filled with the organic electrolyte solutions in 1,2-dichloroethane was successfully applied for the detection of protamine [65] in saline solution and heparin in undiluted sheep plasma samples [66]. Protamine transport was facilitated with dino-nylnaphthalenesulfonic acid (DNNS). As a heparin-selective component the tetrakis-(4-chlorophenyl)borate salt of trimethyloctadecyl ammonium was used. 

Therefore, the ISE potential depends on the CO2 partial pressure with Nernstian slope. Contemporary microporous hydrophobic membranes permitted the construction of a number of gas probes, developed mainly by the Orion Research Company (for a survey see [143]. The most important among these sensors is the ammonia electrode, in which ammonia diffusing through the membrane affects the pH at a glass electrode. Other electrodes based on similar principles respond to SO2, HCN, H2S (with an internal S ISE), etc. The ammonia probe has a better detection limit than the ammonium ion ISE based on the non-actin ionophore. The response time of gas probes depends mostly on the rate of diffusion of the test gas through the microporous medium [77,143]. 

Consequently, an ISE for nitrate for example, a strongly hydrophilic ion, must have a strongly hydrophobic ion-exchanger ion. This conclusion has been demonstrated experimentally for a series of NO3 ISEs based on tetra-alkyl-ammonium salts with long alkyl chains [161] (see fig. 7.2). It was found that, in the studied series of substances, the tetradodecylammonium ion which is... 

Fig. 7.2. Calibration curves of NOj ISE containing trioctylmethyl-(l), tetraheptyH2), tetraoctyl-(3), tetradecyl-(4) and tetradodecyl-(5) ammonium ions dissolved in dibutylphthalate. All electrodes have been adjusted to the same o,ise- (According to Nielsen and Hansen [161].)...

ISEs for HCO3 and COl" [74-76, 222] are based on quaternary ammonium salts. 

Figure 4.15 — (A) Tubular flow-through electrode 1 Perspex body 2 conducting epoxy cylinder 3 mobile carrier PVC membrane 4 electric cable 5 channel (1.2 mm ID) 6 holders 7 screws 8 0-rings. (B) Schematic diagram of a system for on-line monitoring of ammonia ISE tubular flow-through ammonium ion-selective electrode R reference electrode W waste. (Reproduced from [137] with permission of the Royal Society of Chemistry).

Figure 4.16 — Schematic diagram of a split-stream FI system used for the determination of glutamine in bioreactor media C de-ionized water carrier stream R buffer diluent reagent stream S sample injection point L delay coils CPG controlled pore glass enzyme reactor ISE ammonium ion-selective membrane electrode W waste. (Reproduced from [139] with permission of the American Chemical Society).

Glasses exist that fnnction as selective electrodes for many different monovalent and some divalent cations. Alternatively, a hydrophobic membrane can be made semiper-meable if a hydrophobic molecnle called an ionophore that selectively binds an ion is dissolved in it. The selectivity of the membrane is determined by the structnre of the ionophore. Some ionophores are natnral products, such as gramicidin, which is highly specific for K+, whereas others such as crown ethers and cryptands are synthetic. Ions such as, 1, Br, and N03 can be detected using quaternary ammonium cationic surfactants as a lipid-soluble counterion. ISEs are generally sensitive in the 10 to 10 M range, but are not perfectly selective. The most typical membrane material used in ISEs is polyvinyl chloride plasticized with dialkylsebacate or other hydrophobic chemicals. 

Many of the UV-VIS spectrophotometric methods (shown in Tables 12.3 and 12.6) have been automated by using flow analyzers. Thus, nitrite and nitrate,50,82 ammonium,50,83 orthophosphate,50,84,85 silicates,50,86 chloride,50,87 cyanide,50,88 and sulfate50,89 are measured by CFA and FIA. Oxygen is measured by iodometric titration51,90 and electrochemical methods91 (Table 12.7). Other dissolved gasses (Table 12.2) are measured by ISE-based gas sensors. 

Enzyme sensors can measure analytes that are the substrates of enzymatic reactions. Thermometric sensors can measure the heat produced by the enzyme reaction [31], while optical or electrochemical transducers measure a product produced or cofactor consumed in the reaction. For example, several urea sensors are based on the hydrolysis of urea by urease producing ammonia, which can be detected by an ammonium ion-selective ISE or ISFET [48] or a conductometric device [49]. Amperometric enzyme sensors are based on the measurement of an electroactive product or cofactor [50] an example is the glucose oxidase-based sensor for glucose, the most commercially successful biosensor. Enzymes are incorporated in amperometric sensors in functionalised monolayers [51], entrapped in polymers [52], carbon pastes [53] or zeolites [54]. Other catalytic biological systems such as micro-organisms, abzymes, organelles and tissue slices have also been combined with electrochemical transducers. 

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