IONOPHORES AND ION-SELECTIVE MEMBRANES CONTAINING THE SAME

Abstract: Ionophores for use in sensors for the potentiometric determination of the ion content or activity of a sample and the concentration of other components of a sample. Membranes having the ionophores are also disclosed.

Description: IONOPHORES AND ION-SELECTIVE MEMBRANES CONTAINING THE SAME Description Background Ion-Selective Electrodes Ion-selective electrodes respond preferentially or selectively to a particular ionic species in a liquid. They are often used in potentiometric measurement of the activity of an ion in a liquid sample. Potentiometric measurement determines the difference in electrical potential between two electrodes which, in contact with a liquid, form an electrochemical cell. The half cell potential of one electrode - the reference electrode - is essen- tially constant and that of the other electrode - the indicator electrode - varies with the ionic activity of the liquid being analyzed. The elec¬ trical potential across the electrodes is propor¬ tional to the logarithm of the activity of ions in solution to which the ion-selective electrode responds. The Nernst equation describes the loga¬ rithmic relationship. The difference in electrical potential can be determined using a potentiometric measuring device, such as an electrometer. Several types of ion-selective electrodes are available and include, for example, conventional glass electrodes for pH determinations which are widely used in laboratories. Glass electrodes are based on alkali ion silicate compositions. Elec¬ trodes for the determination of pH can be made of lithium silicates or borosilicate glass which is permeable to hydrogen ions (H ) but not to anions or to other cations. If a thin layer of a glass selectively permeable to H is positioned between two solutions of different H concentrations, H ions will move across the glass from the solution- of high concentration to that of low H concentration. Such movement results in the addition of a positive -t- ion to the solution of low H concentration, leaves a negative ion on the opposite side of the glass and produces an electric potential across the glass. Immersion of a pH independent reference electrode into the solution completes the circuit and the potential difference can be measured. Another type of ion-selective electrode uses liquid ion exchangers and is supported by inert polymers, such as " cellulose acetate, or in polyvinyl chloride films. An important example of this type 2+ of electrode is a Ca -responsive electrode which is based on calcium salts of diesters of oil-soluble phosphoric acids. U.S. Patents 3,429,785; 3,438,886; and 3,445,365 describe ion-sensitive electrodes which have membranes made of a porous inert substance filled with an ion-exchange organic liquid, which are selectively responsive to divalent cations /(e.g., rC-a2+, M ..g2- ) . Neutral carrier-based sensors for both mono- valent and divalent cations are similar to ion- exchanger-based electrodes. Both can involve ion-exchange sites, especially negative mobile sites resulting from mediators or negative fixed sites arising from hydrolysis of support materials. Neutral carriers, which can be cyclic or open-chain, are generally hydrophobic complex formers with cations. Such compounds result in selective extrac¬ tion, and therefore selective permeability, for K , 4- 2-t- Na and Ca , which would not otherwise dissolve in the membrane phase as simple inorganic salts. An example of such a carrier is valinomycin, which can be used in electrodes selectively responsive to . Electrodes for the determination of the H content of a liquid sample have been described by others. They commonly contain a plastic membrane which has an ion-selective component (an ionophore) and a solvent/plasticizer compound in which the ion-selective component can be dissolved. In addition to glasses selectively permeable to H ions, hydrogen ionophores such as lipophilic deri- vatives of uncouplers of oxidative phosphorylation and lipophilic tertiary amines have been used. Simon et al, for example, have described a micro pH electrode containing tridodecylamine (C. -H- j .) 3 N or trilauryl amine) as the hydrogen ionophore. Analytical Chemistry 53: 2267-2270 (1981) . The authors report that this electrode gives excellent response to changes in pH between 5.5 and 12. However, at least two important dis¬ advantages result from the fact that the membrane phase must contain dissolved carbon dioxide (C0_) in order to function properly. First, electrode performance (i.e., CO- content) may be seriously affected by changes in temperature and pressure. Second, mass production is also very difficult because manufacture must be carried out under carefully controlled conditions; in addition, the dissolved C0„ diffuses out during storage of the electrodes. Other types of ionophores are based on un¬ couplers of oxidative phosphorylation in mito- chondria. An example is described by Finkelstein. Biochimica Biophysica Acta, 205: 1-6 (1970) . Weak-acid uncouplers of phosphorylation, such as 2,4-dinitrophenol and m-chlorophenylhydrazone mesoxalonitrile, act as H ion carriers. They are, however, unsuitable as components of membranes incorporated into ion-selective electrodes because of their finite water solubilities (i.e., they would not remain membrane bound and would leach out of the membrane) . Brown et al. describe a pH sensor claimed to be suitable for chronic intravascular implantation. The pH-sensitive element is a thin film of an elastomeric polymer which is made ion permselective through the addition of a hydrophobic, lipophilic specific H -ion carrier. The carrier used is p-octadecyloxy-m-chlorophenylhydrazone mesoxalonitrile (OCPH, which is a higher molecular weight homolog of the weak-acid uncoupler m-chlorophenylhydrazone mesoxalonitrile) . Brown and co-workers state that although the OCPH molecule acted as a mobile H carrier in a variety of elas¬ tomers, pH response characteristics of sufficiently good quality for practical use were only obtained using polymer matrices especially designed for the purpose. U.S. Patent No. 3,743,588 (1973) ; O.H. LeBlanc, J.F. Brown et al. Journal of Applied Physiology, 40; 644-647 (1976) . The preparation of polymers which can be used with the OCPH molecule is described by Vaughn. U.S. Patent No. 3,189,662 (1965) ; H.A. Vaughn and Ξ.P. Goldberg, Polymer Letters, ]_ι 569-572 (1969) . In U.S. Patent 3,691,047 (1972) Ross and Martin describe an ion-sensitive membrane for potenti¬ ometric electrodes. The membrane is described as a gelled mixture in which the solid phase is described as being polymer (e.g., cellulose triacetate) and the liquid phase as an organic ion-exchange material dissolved in an organic solvent (e.g., calcium (bis-dibromoleylphosphate) - dissolved in dioctyl- phenylphosphonate) . The ion-sensitive electrode is said to have a membrane whose major constituent is. . ' an organic ion-exchanger which is dissolved in a non-volatile organic solvent. In U.S. Patent 4,214,968 (1980) , Battaglia et al. describe a dry-operative ion-selective electrode for use in determining ion content of liquids. The electrode is said to be comprised of a dry internal reference electrode in contact with a hydrophobic ion-selective membrane. The internal reference electrode is a dried metal/metal-salt reference half cell or a dried redox couple reference electrode and is wetted upon application of a liquid sample. The ion-selective membrane includes an ion carrier (e.g., valinomycin) which is dissolved in a carrier solvent dispersed in a hydrophobic binder. In U.S. Patent 4,184,936 (1980), Paul and Babaoglu describe a device for determining ion activity of a liquid sample. The device is de¬ scribed as having an ion-selective membrane which is coated over an internal reference element (made of an electrolyte-containing layer, a metal salt and a metal layer) and a support. The two electrodes of the device are said to be solid and, preferably, dried. In U.S. Patent 4,053,381 (1977), Hamblen et al. describe another device for measuring ion activity in liquids. The electrodes which are a component of the device preferably include at least one ion- selective electrode in which the internal reference electrode has several layers. The layers include a metal layer, a layer of an insoluble salt of the metai and an electrolyte-containing layer which is preferably dried. The claimed device includes solid electrodes. At the present time, there are many types of ion-selective electrodes available for the measure- ment of the ion content of a liquid. These ion- selective electrodes, however, have limitations. These limitations include the requirement for membranes comprised of specially designed polymer matrices; utilization of ionophores which require pre-neutralization with base to improve membrane sensitivity and reduce response time; the need for storage under well-controlled conditions; and loss of sensitivity and reliability during storage. In addition, ion-selective electrodes now available for pH determination require relatively large samples (i.e., 1.0 ml. or more) for accurate operation and are made of glass, which is costly and cannot be incorporated into an electrode suitable for automa¬ tic processing of samples of very small size. There is a need for an ion-selective electrode which can be used to provide accurate, rapid measurement of the ion content of smaller samples (e.g., of microliter size) than those presently available. There is also a need for an ion- - selective electrode which can provide an accurate and quicker measurement of other constituents of smaller samples than is possible with presently available electrodes. Differential Measurement Techniques The technique of differential potentiometric measurement depends on potential differences arising between two identical electrochemical half-cells immersed in solutions of different activity sepa¬ rated by a salt bridge. The two half-cells together comprise a concentration cell. In the present case the activity of one half-cell (a 1 ) is fixed (reference) while that of the other (a ) (sample) is variable such that the emf of the concentration cell may be defined as: 3 - Ecel , l , = E-2-E-1 = nF lna 2 - τnF lna ι1 where and , E. = emf of reference half-cell Em = emf of sample half-cell E° = standard reference electrode potential R = molar gas constant; 8.314 volt-coulombs/degree(K) T = absolute temperature, °K n = charge on the ion F = Faraday constant; 96,493 coulmbs a, and a- = ionic activity of reference sample In the case of a cation such as- potassium (K ) , if the half-cell a., is defined as a reference half-cell and assigned a zero potential then the emf of the concentration cell will be positive if a„^ a. and negative if a 2 <^a.. Since a. is fixed, the equation for the cell potential contains only one unknown (a„) ; upon measuring E .. the equation may be solved for a_ . Differential measurement techniques have been used to measure indirectly the concentration or activity of constituents of biological fluids other than H +, including other ions such as sodium (Na+) , + ++ — potassium (K ) , calcium (Ca ) and chloride (Cl ) . In addition, such techniques often make use of bio¬ sensors or enzyme electrodes, which include a biological catalyst (e.g., immobilized enzymes, cells, layers of tissue) coupled to an electrode sensitive to a product or co-substrate of the biologically catalyzed reaction. The concentration of enzymes or of substrates can be determined using differential measurement techniques. For example, many enzyme reactions result in the production of an acid or a base. Iσnization of the acid or base in turn results in liberation or uptake of H and a change -in the pH of the solution. The measured change in H concentration or pH can be the basis for a stoichiometric determination of the concen¬ tration of substances (e.g., glucose, urea, etc.) which liberate or take up hydrogen ions. In the case of differential pH measurement, each half-cell would contain a pH electrode. The hydrogen ion activity of the a., half-cell would be fixed and that of a_ (sample) would vary depending on the pH of the sample. The emf measured across the cell by means of an electrometer could then be used to calculate the hydrogen ion activity of a_ . In the case of a differential measurement requiring an enzyme, the enzyme would be placed in either one or both half-cells and the sample would be added to both half-cells or the sample would be added to one-half cell and a calibrator would be added to the other half-cell. As a result, when the enzyme reacts with the substrate in the sample, a decrease or increase in pH occurs; the magnitude of this change is directly proportional to the amount of substrate in the sample. Similarly, a substrate could be substituted for the enzyme and the cell used to measure the enzyme activity of a given sample. For example, Nilsson and co-workers describe the development of enzyme electrodes in which hydrogen ion glass electrodes are used to make enzyme-pH electrodes for the determination of glucose, urea and penicillin in solutions. The enzymes used for the determination are glucose oxidase, urease and pehici linase, respectively. Nilsson, H. et al. , Biochimica et Biophysica Acta, 3_20_:529-534 (1973). Mosca and co-workers also describe the determ¬ ination of glucose by means of differential pH measurements. The technique is based on the mea¬ surement of the change in pH produced by the hexo- kinase catalyzed reaction between glucose and ATP. They describe two systems said to be useful for determining the difference in pH between two 1-ml. aqueous samples. The concentration of glucose is calculated from the measured change in pH by means of an equation derived by the authors. Mosca, A. et al. , Analytical Biochemistry, 112:287-294 (1981). Differential measurement of pH to determine glucose in whole blood and plasma and development of an automated system for doing so is subsequently described by this group. Luzzana, M. et al. , Clinical Chemistry, 29:80-85 (1983). The same"apparatus and a differential pH technique are described for use in measuring lipase activity of biological fluids such as serum, plasma and duodenal juice. Ceriotta, F. et al. , Clinical Chemistry, 3_1:257-260 (1985). A refinement in the differential pH measurement technique is said to serve as the basis for the determination of urea, creatinine and glucose in plasma and whole blood. The enzymes urease, creatinine iminohydrolase and hexokinase, respectively, are used for the determin¬ ations. The concentrations of the three substrates are calculated based on the observed changes in pH of the solutions after reactions have occurred. In U.S. Patent 4,353,867 (1982), Luzzana describes a method and apparatus for the determin¬ ation of substances, such as glucose, urea and enzymes in biological solutions (e.g., blood, serum, urine) . The method uses differential pH measurement in which two glass pH electrodes are placed in separate solutions; the change of pH in the solu¬ tions after reagents are added is determined; and the concentration of the substance of interest is calculated from the observed pH change. The appa¬ ratus is comprised of a sample cuvette; a cell having two glass capillary electrodes; a means for measuring pH at the two electrodes; and an elec¬ tronic means for calculating the concentration of the substance from the pH measurements. Immunosensors Electrochemical immunosensors may be described as either potentiometric or amperometric. Potenti¬ ometric immunosensors may be used to measure either antibodies or antigens. They may be described as either direct or indirect and can be either mem¬ branes or solid electrodes. An example of a direct potentiometric immunosensor for the determination of an antigen is described by Yamamoto, et al. An antibody, anti-hCG, is immobilized on a titanium wire. The anti-hCG electrode and a reference elec¬ trode are placed in a buffer solution. The antigen, hCG, is added and as the antigen binds to the immobilized antibody, the potential difference between the two electrodes changes until equilibrium is attained. The equilibrium potential difference is directly proportional to the concentration of the antigen. Yamamoto, et al. , Clinical Chemistry, 26: 1569-1572 (1980) . The exact nature of the potenti- ometric response is not fully understood, but it is generally acknowledged to involve a surface charge neutralization or redistribution. Another example of a direct potentiometric immunosensor for the determination of an antibody is described by Keating and Rechnitz. This type of immunosensor responds to specific antibodies through modulation of a background potential, fixed by a marker ion, in such a manner that the potential change is proportional to the concentration of antibody. An immunosensor for the determination of digoxin antibody is described wherein digoxin is coupled to the marker ion carrier molecule benzo- 15-crown-5. The resulting conjugate is incorporated into a polyvinylchloride membrane. Keating, M.Y. and Rechnitz, G., Analytical Chemistry, 56; 801-806 (1984) . An antibody-selective potentiometric elec- trode for antibody determination is also described by Rechnitz and Solsky in U.S. Patent 4,402,819. A serious limitation to the routine clinical use of this method is the necessity of maintaining a constant background level of the marker ion such that biological samples would have to by dialyzed before analysis. Indirect potentiometric immunosens.ors are enzyme linked and are analogous to other enzyme immunoassay methods except that the electrochemical sensor is used to measure the product of the enzyme- substrate reaction. Both homogeneous and hetero¬ geneous potentiometric enzyme immunoassays have been described. For example, Boitieux and coworkers describe a heterogeneous potentiometric enzyme- linked immunoassay for the determination of estradiol. Boitieux et al. , Clinical Chemica Acta, 113: 175-182 (1981). The antibody to estradiol is immobilized onto a porous gelatin membrane. The membrane is incubated with peroxidase-labelled estradiol and free estradiol. After washing, the membrane is fixed onto an iodide sensitive elec¬ trode. The peroxidase activity is determined in the presence of hydrogen peroxide and iodide ion. The iodide selective electrode potential is a function of the estradiol concentration. A homogenous potentiometric enzyme immuno¬ assay for human IgG has been described by Fonong and Rechnitz. The method is based on the inhibition, by IgG, of CO- production by beta-ketoadipic acid catalyzed by chloroperoxidase enzyme conjugated to IgG antibody. Fonong and Rechnitz, G., Analytical Chemistry, 56: 2586-2590 (1984) . If the enzyme-IgG conjugate is incubated with a sample containing the antigen (IgG) before reaction with the enzyme substrate, the observed rate of CO- liberation, measured with a potentiometric CO- gas-sensing electrode, will be decreased. The decrease in activity is proportional to the concentration of IgG in the sample. Amperometric enzyme immunosensors are analogous to the potentiometric immunosensors except that an amperometric sensor, usually an oxygen electrode, is used to measure enzyme activity. For example, Aizawa, Morioko and Suzuki have described an amper- ometric immunosensor for the determination of the tumor antigen alpha-fetoprotein (AFP) . Aizawa et al. , Analytica Chimica Acta, 15: 61-67 (1980). Anti-AFP is covalently immobilized on a porous membrane. The membrane is incubated with catalase- labelled AFP and free AFP. After competitive binding the membrane is examined for catalase activity by amperometric measurement of oxygen after addition of hydrogen peroxide. In a similar manner, Boitieux and co-workers describe an amperometric enzyme immunoassay for the determination of hepatitis B surface antigen. Boitieux et al. , Clinical Chemistry, 25: 318-321 (1979) . A limitation of enzyme-linked amperometric sensors is the necessity of synthesizing the enzyme conjugates and the high cost of currently available amperometric sensors such as oxygen electrodes. Disclosure of the Invention The present invention is a sensor for the potentiometric determination of the ion content or activity of a sample and the concentration of other components of a sample through the use of ion- selective electrodes. Ionophores for use in ion- selective membranes of such sensors are also the subject of the invention. The sensor is especially suited for rapid determination of the hydrogen ion content or pH of biological fluids; the concentra¬ tion of other ions in biological fluids; and, through differential pH measurements or immunoassay techniques, the concentration of other components (e.g., glucose, urea, triglycerides, uric acid, enzymes such as aspartate amino transferase (AST) , alanine amino transferase (ALT) , amylase, creatine kinase (CK) , alkaline phosphatase and drugs) of bio¬ logical fluids. It is particularly useful for automated handling or processing. The sensor is comprised of ion-selective electrodes which are held in a frame and have porous material (e.g., microporous polyethylene such as that manufactured by Porex Technologies Corp.) between them. The porous material provides a means for ionic flow between the electrodes once a sample is applied at the electrodes. The ion-selective electrodes are comprised of a membrane which is selectively permeable to the ion or other substance whose concentration is to be determined and a reference electrode. The membrane does not require preconditioning before use. The selectively permeable membrane is comprised of an ion-selective compound (or ionophore) and a thermoplastic resin or a plastic material, which can all be dissolved in an organic solvent. In addi¬ tion, it can also include a plasticizer. The ion-selective component for a sensor which can be used, for example, to measure hydrogen ion activity of a sample is a compound having the following general formula: wherein: R.. , R_ and R-. are independently selected and each can be: 1) a halogen; 2) an alkyl group having from 4-18 carbon atoms; 3) a halogen-substi-. tuted alkyl group; 4) an alkoxy group; 5) a halogen- substituted alkoxy group; 6) an acid group repre¬ sented by -CO-R. wherein R. is alkyl having 1-18 carbons; or 7) a keto group represented by -COR., wherein R- is selected from the groups defined for R ; or hydrogen. In an embodiment of the present invention, the membrane comprises an organic plastic matrix, polyvinyl chloride (PVC) , which contains the ion- selective compound 2-octadecyloxy-5-carbethoxy- phenylhydrazone mesoxalonitrile. The ion-selective compound, the PVC and a plasticizer, which in this embodiment is 2-nitrophenyloctylether, are all soluble in the solvent tetrahydrofuran (THF) . These components comprise the membrane formulation. In a preferred embodiment, the membrane com¬ prises about 10-40% PVC by weight, and its thickness is greater than 1 mil. In a particularly preferred embodiment, the membrane is made of about 20-35% PVC by weight and is about 3-15 mils thick. The mem- brane which is described can be used as a component of the ion-selective electrode of the present invention. It can also be incorporated into the sensor claimed in United States Serial No. 750,525, filed on June 27, 1985 and the sensor claimed in continuation-in-part application of United States Serial No. 750,525, Attorney's Docket No. IC84-2A, filed concurrently herewith. In addition, it can be incorporated into commercially available electrode bodies. The sensor of this invention also has an internal reference element or material which in¬ cludes a known concentration of the sample component whose concentration is to be determined. Brief Description of the Drawings Figure 1 is a perspective view of the top of a sensor having ion-selective electrodes and a handle which has a magnetic stripe in which data is recorded. Figure 2 is a perspective view of the bottom of a sensor having ion-selective electrodes and a handle which has a magnetic stripe in which data is recorded. Figure 3 is a perspective view showing the individual components of a sensor which can be used to determine the hydrogen ion activity (pH) of a sample or the concentration of other ions in a sample. Figure 4 presents schematic representations of ion selective electrode configurations. Figure 5 shows an ion-selective membrane of this invention inserted into a commercially avail¬ able barrel-type electrode. Figure 6 is a perspective view showing the individual components of a sensor which can be used to determine the activity or concentration of a component of a sample by a differential measurement technique. Best Mode for Carrying Out the Invention The sensor which is the subject of this inven¬ tion is used for the potentiometric determination of the ion content of a sample or the concentration of other components of a sample. It is particularly useful in the rapid determination of the hydrogen ion activity (or pH) of or concentration of other ions in a biological fluid (e.g., blood) and in measuring concentrations of other components (e.g., glucose, urea, triglycerides, uric acid, enzymes, such as aspartate amino transferase (AST) , alanine amino transferase (ALT) , amylase, creatinine kinase (CK) , alkaline phosphatase and drugs) in biological fluids. Ionophores selective for sample components and incorporated into ion-selective membranes of such sensors are also the subject of this invention. The sensor can now be further described with reference to the figures. Figure ' s 1 through 3 show a sensor having ion-selective electrodes and a handle having a magnetic stripe in which data are recorded. The sensor represented in these figures can be used to determine the hydrogen ion content or pH of a sample or the concentration of other ions in a sample. These figures will now be referred to in describing a sensor to be used for the determination of hydro- gen ion content of a sample. It is to be under¬ stood, however, that the sensor can be used for determination of other ions or other components as well. The sensor 30 is comprised of ion-select.ive electrodes which are held in a frame or body 10 which has upper section 12 and lower section 14. In addition, the sensor 30 has a handle 5 bearing a magnetic stripe 8 on its lower surface. Upper section 12 of frame 10 has four openings 4 and three grooves 6, which are positioned between the openings. Lower section 14 of frame 10 has four openings 7 and three grooves (not shown) positioned between the openings. Upper section 12 and lower section 14 are in such a relationship that openings 4 and openings 7 are aligned and grooves 6 in the upper section and the grooves in the lower section are aligned. As a result, openings 4 and 7 form four openings 11 and grooves 6 of the upper section and the grooves in the lower section define spaces between three of the openings 11. Upper section 12 has small holes 15 located between openings 4. Holes 15 allow the passage of air. The ion-selec¬ tive electrodes are located within the openings 11 and are connected by a porous material 17 which is cylindrical or rod shaped and provides a means for ionic flow between the electrodes upon the applica¬ tion of a sample at the electrodes. The cylindrical or rod-shaped porous material 17 is located in the spaces between openings 11. Upper section 12 and lower section 14 are sealed, for example by being ultrasonically welded under pressure, such ' that there is no leakage of sample or solutions onto porous material 17 or between or into the two sections of frame or body 10. As shown in Figure 3, sensor 30 has four positions at which ion-selective electrodes can be located. Although there will generally be an ion-selective electrode at each of these positions, these can be used in various combinations depending on the analytical information desired. Two, three or all four electrodes can be used. Accordingly, the position of grooves 6 in upper section 12 and the grooves in lower section 14 will vary as needed for the analysis being carried out. For example, grooves 6 in the upper section 12 can be positioned as shown in Figure 3; the grooves in lower section 14 will be positioned so that spaces will be defined between the three electrodes, as described above, when upper section 12 and lower section 14 are joined. Alternatively, two grooves 6 can be posi¬ tioned in parallel to one another in upper section 12 and the grooves in lower section 14 positioned correspondingly. Porous rods 17 can be hydrophilic and conductive or hydrophobic and nonconductive. The use of conductive or nonconductive material between a pair of electrodes is determined by the analyses to be carried out. The various combina¬ tions of membranes are best described by example. In the first example, as represented in Figure 4a, three membranes are active and all three contain the same membrane. Positions a and b have known concentrations of the ion to be determined and position c has the sample, which has an unknown concentration of the ion of interest. The emf developed between the solutions in a and b can be used to calibrate the sensor; the slope value is then used to determine the concentration of the sample from the emf developed between the solutions in b and c. In addition, because the concentrations of ions in a and b are known, it is possible, using predetermined slope values, to calculate what the potential difference between a and b should be and to compare this value with the measured potential difference or to calculate a concentration for a and determine how this varies from the known value. In a clinical chemistry sense, the solution in a would then be used as a control. The a, b, c configuration can also be used in such a manner that replicate samples can be analyzed. This is represented in Figure 4b. In this case the same sample is placed in positions a and c and reference solution in b. Using a pre- determined slope value, it is possible to determine simultaneously two values for the same sample. It is also possible to determine simultaneously two different analytes. This is represented in Figure 4c. For instance, a and d can be membranes selective for one analyte and b and c can be mem¬ branes selective for another analyte. Useful combinations might be sodium/potassium, pH/calcium, glucose/urea and urea/creatinine. In these in¬ stances predetermined calibration data are required. An enzyme/substrate sensor can be constructed as represented in Figure 4d. In this case, a,b,c and d can all be the same membrane; for example, they can all be for determination of pH. However, a and d would contain, in addition to the pH membrane, an immobilized enzyme. An unknown analyte sample concentration is added to both c and d; a known concentration of the same analyte is added to a and b. The enzymes in a and d act on the substrate and produce a pH change proportional to the concentra- tion of the analyte in the sample. If it is assumed that all the membranes are identical, the emf developed between a and b (the reference solution) can be used to calibrate the electrodes. The calibration data obtained for a and b may then be used to calculate the sample concentration in c and d. An enzyme/substrate sensor can be constructed as represented in Figure 4k. In this case, a, b and σ can all be the same membrane; for example, they can all be for determination of pH. However, a, b and c would contain, in addition to the pH membrane, an immobilized substrate. The substrate concentra¬ tions in a and b are different; the concentration of substrate in c can be the same as in a or b or can be different. An unknown analyte enzyme sample concentration of an enzyme to be measured is added to both c and d; a known concentration of the enzyme to be measured is added to a and b. The enzymes in a, b and c act on the substrate in a, b and c and produce a pH change proportional to the concentra- tion of the enzyme in the sample. If it is assumed that all the membranes are identical, the emf developed between a and b (the reference solution) can be used to calibrate the electrodes. The calibration data obtained for a and b may then be used to calculate the enzyme sample concentration in c. Examples of substrates which can be immobilized in an enzyme/substrate sensor such as that repre¬ sented in Figure 4k are amino acids, such as L-alanine and L-aspartate, creatine and starch. These can be used, respectively, for the determination in a sample of alanine aminotransferase, aspartate aminotransferase, creatine kinase and amylase. As described in Example 8, an enzyme/ substrate such as that represented in Figure 4k can also be used to determine the creatinine concentration of a sample. If one were using a kinetic rate method, this methodology, in effect, allows one to "calibrate out" any temperature effects on the enzymatic reaction rates. In the case of an endpoint or equilibrium measurement, effects of temperature in the Nernst equation could be normalized. The porous rods 17 between the electrodes can be conductive or nonconductive; the use of conduc- tive or nonconductive material between a pair of electrodes is determined by the analysis to be carried out. The porous material can be of a microporous plastic such as nylon, polypropylene, polyethylene, polyvinylidene fluoride, ethylene-vinyl acetate, styrene-acrylonitrile or polytetrafluoroethylene. The porous material can also be cellulosic in nature such as rolled filter paper or longitudinal fiber bundles. In one embodiment the porous material consists of crylindrically-shaped pieces of ultra- high molecular weight microporous polyethylene (POREX Technologies, Fairburn, GA) . This material is by nature hydrophobic such that it will not wet with water. The porous material can be rendered hydrophilic by soaking it in a solution (about 0.1% to 0.6% by volu e) of a surfactant. Surfactants which have been found to be useful are the nonionic surfactants such as the octylphenoxypolyethoxyethanol family of surfactants which includes Triton X-100 (Rohm & Haas Co.), polyoxyethylene ethers such as Brij 35, polyoxyethylene sorbitan derivatives such as Tween 20, fluoraliphatic polymeric esters such as 3M's Fluorad FC-171 and surfactants such as Sherex Chemical Co.'s Arosurf 66 PE-12. Anionic and σationic surfactants can also be used. The porous material without wetting agent or surfactant is nonconductive. The absence of porous material between two electrodes results, of course, in nonconductivity. The ion-selective electrodes are comprised of an ion-selective membrane 18 and a reference elec¬ trode 22 which is a silver/silver chloride material. The ion-selective membrane 18 is made of an ion- selective compound (an ionophore) , a thermoplastic resin and a plasticizer, all of which are soluble in an organic solvent. It is held in place in the ion-selective electrode by a retainer means, such as retainer ring 24. There is, thus, a mechanical seal between the retainer means and the lower section of the sensor body such that there can be no leakage from the membrane. An important characteristic of the sensor is that the ion-selective membranes incorporated in it do not require preconditioning (e.g., soaking in a solution of the ion to be measured) before the sensor can be used. In the case of the hydrogen ion-selective membrane, the ion-selective compound which is incor¬ porated into the membrane has the general formula: In this compound, R,, R- and R- represent components of the compound which can be the same or different. R-, R- and R- can be: 1) a halogen; 2) an alkyl group having 4-18 carbon atoms; 3) a halogen-substi¬ tuted alkyl group; 4) an alkoxy group; 5) a halogen- substituted alkoxy group; 6) an acid group repre¬ sented by -CO-R. wherein R. is an alkyl having 1-18 carbon atoms; or 7) a keto group represented by -COR-. wherein R, is selected from the groups defined for R.; or g) hydrogen. Derivatives of this general formula which are particularly preferred as components of the hydrogen-ion-sensing membrane include: a. 2-trifluoromethyl-4-octadecyloxyphenyl- hydrazone mesoxalonitrile b. 2-octadecyloxy-5-trifluoromethylphenylhydrazone mesoxalonitrile - 2 m ~- m N » . fJ , ^ ° C ' a ?"37 c. 2-octadecyloxy-5-carbethoxyphenylhydrazorie mesoxalonitrile; and d. 2-octadecyloxy-4-fluorophenylhydrazone mesoxalonitrile. Membranes in which the ion-selective compound is carbethoxyphenylhydrazone mesoxalonitrile are particularly useful in that they exhibit response times of one minute or less; yield stable potentials and exhibit slopes on the order of 57-59 mv per decade change in hydrogen ion concentration and do not require any preconditioning. It is not clear why such membranes have excellent performance characteristics. However, it might be attributable to the presence of an electron-withdrawing ester group meta to the anilino group and its proton, which is apparently more acidic and, therefore, more easily exchanged. In addition to the ion-selective compound described above, the ion-selective membrane 18 of this invention also comprises a plastic or a thermo¬ plastic resin and, optionally, a plasticizer. The plastic used can be any plastic which can be used to form a film by solvent casting. For example, the plastic can be polyvinyl chloride, polyvinyl ace¬ tate, silicone rubber or cellulose acetate. This membrane component serves the purpose of providing support and form to the membrane, and acts as a matrix into which the ion-selective compound is - incorporated. In addition, it serves as a barrier to water because it is a hydrophobic material. In one embodiment, the thermoplastic resin used in the membrane is polyvinyl chloride. Others which can be used include: cellulose acetate, polyvinyl acetate and silicone r-ubber. The plasticizer, which is an optional component of the membrane, can be any nonvolatile material suitable for the general purpose of facilitating the compounding or production of the membrane formula¬ tion and improving the membrane's flexibility. It also contributes to the dissolution of the iono- phore. It can be, for example, one or more of the following, used alone or in combination: phthal- ates; adipates; sebacates; aliphatic and aromatic ethers; aliphatic and aromatic phosphates; aliphatic and aromatic esters; and nitrated aliphatic and aromatic ethers. In one embodiment of this inven¬ tion, the plasticizer is 2-nitrophenyloctylether. The components of the ion-selective membrane 18 can be present in varying amounts. The plastic used can comprise about 10-30% by weight of the membrane and in one embodiment is about 28% by weight. The ion-selective compound can be from about 1% to about 10% by weight of the membrane and in general will be about 3% to about 6% by weight. The plasticizer can be from about 50% to about 80% by weight of the membrane and generally comprises about 70% by weight. The ion-specific membrane 18 incorporated into the ion-selective electrodes of this invention will generally be of a thickness greater than 1 mil and preferably will be from about 3 to about 15 mils in thickness. The ion-specific membrane of this invention can be made by at least two different methods: solvent casting and dip coating. Solvent casting is illus¬ trated in Example 2 and in general includes the following steps: 1. preparation of the hydrogen ion-specific compound by: a) alkylation of a nitrated phenolic material to produce an ether; b) reduction of the nitro group to an amino group; c) precipitation of the hydrochloride salt of the amine; and d) con¬ version of the hydrochloride salt to the mesoxalonitrile derivative; 2. dissolution of the ion-specific compound; a plasticizer and a plastic material in a volatile (organic) solvent; and 3. removal of the volatile organic solvent (e.g., by evaporation). After the membrane has been formed, it is shaped (e.g., by cutting, use of a punch-type instrument, etc.) into an appropriate configuration and incorporated into an electrode body, such as the electrode body of the present invention or an Orion electrode body. (Orion Research, Inc., Cambridge, MA. catalogue #950015) . Figure 5 shows the Orion- electrode body 61 with the ion-selective membrane 62 of this invention, held in the electrode by means of an O-ring 63. A second 0-ring (not shown) prevents sample leakage around the membrane. The ion-selective membrane can also be formed by means of a dip coating or a continuous coating process method. In this method, a base material, such as a nylon mesh or a polyester mesh (e.g., Pecap 355, Tetko, Inc., Elmsford, NY)) is placed in a solvent bath (e.g., ketone, such as 4 methyl-2 pentanone) and the air trapped within the mesh removed. This can be done, for example, ultrasoni¬ cally using a Branson ultrasonic cleaner; treatment takes about 10-15 seconds. While still wet with solvent, the mesh is placed in the membrane formula¬ tion; after it is removed, it is scraped to remove excess formulation. The mesh containing the formu¬ lation is then dried; this can be done, for example, at room temperature or in warm air (e.g., 35-50 °C) . The internal reference material consists of a solution of the salt of the ion to be determined. The counter ion consists of chloride if the refer¬ ence electrode is silver/silver chloride. A gelling agent such as agar or agarose can be used to fix the reference material in place. In the case of the pH sensor, the reference material is a buffer solution. In one embodiment, the pH reference material con¬ sists of 50 ml glycerol, 50 ml of an aqueous solution consisting of pH 7.4 phosphate-buffered saline (SIGMA Chemical Co., St. Louis, MO) and 2.0g of agar. The mixture is heated to dissolve the agar and then placed in the well behind the membrane. After a period of time, normally 5-10 minutes, the mixture gels. For a potassium sensor the aqueous solution can be 50 ml of 0.2 M KC1; for the sodium and chloride sensors, the aqueous solution can be 0.2 M NaCl. ' To modify the sensor of Figures 1-3 for the determination of other ions, it is necessary to change at least one of the membrane components and to modify the internal reference material to include the ion to be determined. For example, for potas- sium determinations, the membrane would include an ionophore specific for potassium, such as valinomy- cin, a crown ether derivative of benzo-15-crown-5 or some other neutral carrier. The internal reference material would consist of a potassium chloride solution with or without a gelling agent. To modify the sensor of Figures 1-3 for the determination of sodium, the membrane would include an ionophore for sodium, such as monensin, a crown ether derivative of benzo-12-crown-4 or some other sodium neutral carrier (e.g., as described by Simon et al., Analytical Chem. , 51: 351-353 (1979)) . The internal reference material would include sodium chloride. To modify the sensor of Figures 1-3 for the determination of chloride, the membrane would include an ionophore for chloride such as the quaternary ammonium salt Aliquat 336. The internal reference material would include chloride ion. As mentioned previously, sensor 30 has a handle 5 which has a magnetic stripe 8. This stripe is very similar to the stripes commonly found on credit cards and bears data recorded onto the stripe at the time of manufacture. The data includes, for ex¬ ample, information on test type, expiration date of the test card and "housekeeping" data. 0 ' • An independent function of the magnetic stripe is to prevent reuse of the disposable test cards which are designed for single use only. This is accomplished by destroying the coded information as the test card is removed from the instrument. The " 5 error checking functions will reject the card if an attempt is made to reuse it. Figure 6 shows a sensor of the present inven¬ tion which can be used to determine the activity or concentration of components of samples by means of a 0 differential measurement technique. Components of biological fluids (e.g., blood, serum, plasma, urine, saliva, cerebrospinal fluid) which can be measured include, for example, glucose, urea, triglycerides, creatinine, lipase, uric acid and 5 other enzymes (e.g., alanine aminotransferase (SGPT or ALT) ; alkaline phosphatase (ALP) ; creatinine kinase; aspartate aminotransferase (SGOT or AST)). The sensor 70 is comprised of ion-selective electrodes, which are held in a frame or body 40 0 which has an upper section 42 and a lower section 44. Upper section 42 of frame 40 has four openings 34 and three grooves 36, which are positioned between the openings. Lower section 44 of frame 40 has four openings 35 and three grooves positioned between the openings. Upper section 42 and lower section 44 are in such a relationship that openings 34 and openings 35 are aligned and grooves 36 of the upper section 42 and the grooves in the lower section 44 are aligned. As a result, openings 34 and openings 35 form openings 39 and grooves 36 of the upper section 42 and the grooves in the lower section 44 define spaces between three of the openings 39.. Upper section 42 has small holes 43 . located between openings 34. Holes 43 allow air to enter space 41. The ion-selective electrodes are located within the openings 39 and are connected by a porous material 46 which is cylindrical or rod shaped. Porous material 46 provides a means for ionic flow between the electrodes upon the application of a sample at the electrodes. The cylindrical or rod-shaped porous material 46 is located in the spaces 41 between openings 39. As described for the sensor of Figures 1-3, fewer than or more than the four ion-selective electrodes depicted in Figure 6 can be used. Also as previously described, the porous material between the electrodes can be hydrophilic or hydrophobic; the type of analysis determines this characteristic. For example, if an enzyme substrate is to be mea- sured using the sensor represented in Figure 6 and a reference sample is to be used, then the configura¬ tion could be as shown in Figure 4d. The ion-selective electrodes are comprised of a layer 48 having enzyme or substrate immobilized on it; an ion-selective membrane 50; a membrane 52 placed between the two; an internal reference material 54 and a reference electrode 56. There can be an additional membrane 58 in proximity with layer 48. The ion-selective membrane can be selective for H or ammonium ions (NH. ) , as in the case of a urea " electrode. The H selective membrane is made as described above for the pH' sensor. In the case of the ammonium-selective membrane, the ionophore is an ammonium-selective material such as nonactin. The ion-selective membrane is held in place in the ion-selective electrode by a retainer means, such as retainer ring 60. Layer 48 has at least one enzyme or substrate immobilized on it. Layer 48 can be, for example, a nitrocellulose membrane (e.g., type AE100, Schleicher and Schuell, Inc. Keene, NH) having a thickness of about 180 microns. It can also be made of nylon, cellulose, cellulose acetate, glass fiber or any porous material which can serve as a solid support for the immobilization of the enzyme or substrate. The enzyme or substrate can be immobil¬ ized by physical restraint (e.g., adsorption onto the solid phase) or by chemical restraint (e.g. " , covalent attachment or crosslinking) . Layer 48 is separated from the ion-selective membrane 50 by means of a membrane 52, which is generally thin (e.g., less than 20 microns thick) and can be, for example, a dialysis membrane (e.g., Spectra Por 2, Spectrum Medical Industries, Inc., Los Angeles) . Membrane 52 serves the purposes of, for example, preserving the activity of the immobil¬ ized enzyme of layer 48 by preventing the migration of components of the ion-selective membrane 50 into layer 48; migration might cause de ' naturation or inactiva ion of the enzyme. Membrane 58 is optional and its presence will be determined by what component of a sample is being measured and/or how that component is being mea- sured. The function of membrane 58 is to aid in the physical restraint (immobilization) of the enzyme or substrate of layer 48 and/or to serve as a diffu- sional barrier to the substrate. For example, to measure the concentration of a substrate of the enzyme immobilized in layer 48, it may be desirable to make a kinetic measurement, rather than an end point measurement. To extend the range of linearity for a kinetic measurement, it is necessary that the enzyme reaction be diffusion limited. In this instance membrane 58 will serve both purposes. If, however, an end point measurement is desired a diffusional barrier would not be required and membrane 58 is not needed. Membrane 58 can be made of regenerated cellulose (dialysis membrane) or other ultrafiltration or reverse osmosis membranous material which can serve as a diffusional barrier to the substrate. The internal reference material 54 is comprised of a buffer solution. In one embodiment, it is comprised of a filling solution of pH 7.4 phosphate buffered saline. In a preferred embodiment, it is a gel comprised of about 50 ml. pH 7.4 phosphate buffered saline, about 50 ml. glycerol and about 2.0 g agarose. Reference electrode 56 is an electrode with a fixed potential which exhibits no variation in liquid junction potential when the sample solution is varied or replaced by a' calibrating solution. The main requirements for satisfactory reference electrode performance are reversibility (in the electrochemical sense) , reproducibility and stabil¬ ity. Three types of reference electrode systems are in current use: metal amalgams, such as the saturated calomel electrode (SEC); redox couples, such as a quinhydrone or ferri-ferrocyanide elec¬ trode; and metal/metal halide electrodes, such as the silver-silver chloride reference electrode. In the present invention, the reference electrode will generally be a silver/silver chloride button. The subject of this invention is illustrated by the following examples, which are not to be consi¬ dered limiting in any way. Example 1 - Preparation of the Hydrogen Ion Specific Compound 2-octadecyloxy-5-carbethoxyphenylhydrazone mesoxalonitrile The hydrogen ion specific compound 2-octadecyl- oxy-5-carbethoxyphenylhydrazone mesoxalonitrile is prepared in the following manner. First, ethyl-4- octadecyloxy-3-nitrobenzoate is prepared according to the general procedure described in Organic Synthesis, Coll. vol. 3, pp 140-141. The following mixture is refluxed for 72 hours in a l-liter-3-neck flask fitted with a mechanical stirrer: 25g (0.12 moles) of ethγl-4-hydroxy-3- nitrobenzoate Lancaster Synthesis, Windham, NH catalogue #6451 41g (0.12 moles) of 96% 1-bromooctadecane Aldrich Chemical Co., Milwaukee, WI. catalogue #19,949-4 16.4g of anhydrous potassium carbonate 500 ml. of dry acetone At the end of the reflux period, the reaction mixture is cooled to room temperature (i.e., about 20-27°C) and filtered to remove salts. The filtrate is evaporated using Buchi-Brinkman rotary evapora¬ tor. The residue is dissolved in 500 ml of toluene and washed in turn four (4) times with 400 ml of 5% aqueous sodium hydrogen carbonate and then one (1) time with 400 ml of saturated aqueous sodium chlor- ide. The toluene layer is dried over anhydrous sodium sulfate and evaporated to dryness; this produces an oil which crystallizes on standing. The solid is recrystallized from hexane with charcoal clarification; the yield is 36g (66% yield) of ethyl-4-octadecyloxy-3-nitrobenzoate ( .p. 48-56°) . The nitrocompound is reduced to the amino derivative by catalytic hydrogenation. To 200 ml of hexane is added lOg of the above nitro compound along with 0.5g 10% palladium on carbon. The mixture is placed on a Parr hydrogenator, heated to 40°C under 60 psig of hydrogen and shaken in the machine for 18 hours. The reaction mixture is ' filtered through Celite filter aid medium (usually diatomaceous earth (Manville) ) while hot (e.g., about 50°C) , and the solution is treated with HC1 gas to precipitate the hydrochloride salt of the amine in nearly quantitative yield (m.p. 141-145°) . The hydrochloride salt of ethyl-4-octadecyloxy- 3--aminobenzoate is converted to the mesoxalonitrile derivative according to a modification of the procedure of Brown et al. (U.S. Patent No. 3,743,588). 8.5g (0.018 moles) of the above hydro- chloride salt is dissolved in 1 liter of dimethyl formamide (DMF) with warming and brought to a temperature of about 0°. 3 ml of concentrated HC1 is added to the cold solution, followed by 1.3g of sodium nitrite dissolved in 500 ml of DMF. The reaction mixture is stirred magnetically for 1 hour. 1.15 ml of prewarmed malononitrile is then added and the mixture is stirred magnetically for about 10 minutes. Triethylamine is added in sufficient quantity to produce a strongly basic solution (e.g., pH greater than 9) . The mixture is allowed to warm to room temperature and stirred for 18 hrs. The reaction mixture is acidified (to a pH of about 2.3) with concentrated HCl, and a precipitate results. The precipitate is filtered, washed with water and recrystallized from methanol. The result is about 7.0g, (76%) of 2-octadecyloxy-5-carbethoxyphenyl- hydrazone mesoxalonitrile (m.p. 76-77°) . The other derivatives shown in Table 1 were prepared in a similar manner. TABLE l Slope IonoDhore % Plasticizer % PVC % pH 4-9 mv Membrane Rl R2 R3 (by weight) -(by weight) (by weight) 27.3 51 1 0C lg H 37 COOEt H 4.5 68.2 2 0C 18 H 37 H F 2.3 69.8 27.9 50 Example 2 - Solvent Casting of a PVC Membrane and Incorporation Into an Ion-Selective Electrode 1.5 ml of 2-nitrophenyloctylether (Fluka Chemical Corp., Hauppauge, NY, cat #73732) and 0.10g of the hydrogen ionophore (such as that prepared according to the method illustrated in Example 1) are dissolved in 10 ml of tetrahydrofuran. To this solution is added 0.6g of powdered polyvinyl chlor¬ ide of very high molecular weight and of density of ' 1.385 g/σc (Aldrich Chemical Co., Milwaukee, WI , cat #18,261-3). The mixture is shaken (e.g., by vortex- ing) until the PVC is dissolved. The PVC solution is poured onto a 1/4" thick sheet of stress-relieved polypropylene and allowed to remain under conditions which result in the evaporation of the tetrahydro¬ furan. For example, evaporation can occur at room temperature under a fume hood so that the tetra¬ hydrofuran is removed as it evaporates. The product formed is a plastic membrane in which the ion- specific compound is incorporated. Disks are cut from the membrane using a #7 cork borer (id 0.5in) and mounted in an Orion electrode body (Orion Research Inc., Cambridge, MA, cat #950015). The internal reference electrode is a silver/silver chloride reference electrode and the internal filling solution is pH 7.4 phosphate buffered saline (SIGMA Chemical Co., St. Louis, MO, cat #1000-3). Assessment of the pH-sensing capability of the electrodes was carried out. All measurements were made versus a double junction Ag/AgCl reference electrode (Corning, cat #476067) in 250 ml beakers with magnetic stirring. For comparison a combina¬ tion pH/reference electrode was dipped into the test solution to record pH changes, such that mv changes in the membranes can be related to pH changes measured with the glass electrode. The membrane of the present invention exhibited good mechanical strength, as indicated by the fact that it did not tear or break when stretched. It also exhibited good analytical performance, yielding slope values of 55-58 mv per decade over the pH range of 6 to 8 with performance decreasing somewhat on either side of this range. For a monovalent ion, the theoretir- cal slope is ±59.1 mv at 25°C. In addition, the response time or the time to reach equilibrium was less than one minute and the electrodes required no preconditioning. The properties of the above membrane were compared to those of a membrane prepared in an identical manner except that the hydrogen ion selective component was the previously described 3-chloro-4-octadecyloxyphenylhydrazone mesoxaloni¬ trile. In contrast to the previous membrane, the membrane prepared from the chlorinated mesoxaloni¬ trile gave diminished performance; slope values were 40 mv or less over the same pH range, indicating a loss in sensitivity. The performance of the various hydrogen iono¬ phores is summarized in Table 1. The results show that the three derivatives with the 18 carbon lipophilic chain ortho to the ionophoric site outperformed the derivative in which the lipophilic chain was para. This suggests that perhaps the lipophilic chain adjacent to the site of hydrogen ion exchange enhances the response towards hydrogen ions. The lipophilic chain could form a lipophilic pocket around the active site, thereby increasing the selectivity towards hydrogen ions. Example 3 - Preparation of an Enzyme Electrode for the Measurement of Urea A sensor as shown in Figure 6 is used for the determination of urea. A urea-selective sensor is made by incorporating a second membrane containing the enzyme urease in the ion-selective electrodes of the sensor, which can be either a pH or an ammonium ion-(NH .) membrane. Urease containing membranes are present in electrodes a, b and c shown in Figure 4f. The two membranes are separated by a third membrane (e.g., membrane 52 of Figure 6). Samples to be tested (e.g., blood, plasma) and calibrant samples are added at the electrodes as shown in Figure 4e. A and B are wicks made of porous material and have been made conductive by the addition of a wetting agent, a, b, and c are ammonium ion- selective electrodes. Two different calibrant samples are added at a and b, which have urease. Patient sample is added at c, which also has urease. Reactions catalyzed by urease occur at a, b and c. This allows the comparison of the patient sample with two calibrant samples, thus allowing for the calibration of the test card. The following reactions occur where urease is present: H 2 As a result, an ammonium selective membrane can be used to detect the change in ammonium ion (NH. ) concentration or a pH selective membrane can be used to detect the increase in pH which occurs because of - the production of OH . A urease containing membrane is prepared as follows: A 5 mg/ml urease.(300 units/mg New England Enzyme Center, Boston, MA) solution is prepared in pH 7.4 phosphate buffered saline (Sigma Chemical Co., Cat. # 1000-3): other buffers found to be useful are 10 mmol/L sodium Hepes in 0.1 M NaCl (pH 7.4) and 10 mmol/L NaH j PO, 10 mmol/L tris base in 0.1 M NaCl (Ph 7.4) . A 47 mm diameter disk of 12 micron pore size nitrocellulose membrane (Schleicher & Sσhuell, Inc., Keene, NH, 03421 Grade AE 100) is soaked in 700 ul of the enzyme solution for 5 minutes at room temperature. The membrane is removed from the solution and the excess fluid scrapped off with a stirring rod. The membrane is placed on a hydrophobic surface (e.g., a sheet of polypropylene) and air dried at room temperature for 30 minutes. The membrane is stored in a closed container at 4°C. Disks are cut from the membrane and placed in the electrodes at a, b and c; see Figure 4f. Sample solution (25 micro liters) is placed in c and calibrating solutions can be placed in a and b. The urease in a, b and c acts on the urea in the calibrants and sample liberating both ammonia and CO^. The increase in pH or ammonia is measured. For example, a pH 7.5 buffer was prepared containing 40 mmol/L NaH 2 PO., 40 mmol/L tris base and 100 mmol/L NaCl. Buffer solutions containing 5 mmol/L urea and 10 mmol/L urea were prepared. After approximately one minute a 25 mv difference was observed when the 5 mmol/L urea solution was placed in a and c and buffer containing no urea was placed in b. A -50 mv difference was observed with the 10 mmol/L solution of urea. No difference in emf was observed in the absence of urea. Similar behavior was observed when the pH membrane was replaced with an ammonium selective nonactin membrane. Example 4 Preparation of an Enzyme Electrode for the Measurement of Glucose Referring to Figure 4e, cells a, b and c each contain a membrane with immobilized glucose oxidase and catalase. The following reactions occur where glucose oxidase (GOD) and catalase are present: Glucose + H 2 0+0 2 GOD -^Gluconic acid (H + ) + H 2 0 2 H 2 0 2 catalase -^ 1/2 0 2 + H 2 0 The purpose of the catalase is to recycle the oxygen consumed in the oxidation of glucose, thereby extending the linear range within which glucose may be measured. The glucose oxidase/σatalase membrane was prepared as follows: The previously described nitro cellulose membrane was soaked in 700 micro liters of a solution consisting of 5 mg/ml glucose oxidase (300 units/mg, Boehringer Mannhein) and 1 mg/ml. catalase (40,000 units/mg Sigma Cat. # C-100) in pH 7.4 phosphate buffered saline. The membrane was dried as in example 3. Enzyme disks were placed in a, b and c. The concentration of glucose was found to be proportional to the potential difference developed between b and c. ' Another method for the determination of glucose uses hexokinase/ATP as the enzyme system. Referring to Figure 4e, cells a, b and c each contain a membrane with immobilized hexokinase, ATP and a magnesium salt such as the chloride, acetate or sulfate. The following reactions occur where hexokinase, ATP and magnesium,ion are present: G lucose + ATP ^Glucose-6-phosphate + A DP + H + The hexokinase/ATP membrane was prepared as follows: The previously described nitrocelluloase membrane was soaked in 1.0ml of a solution consisting of lOg/L magnesium acetate, 50mg/ml ATP and 10 mg/ml hexokinase in a buffer consisting of 10% glycerol, 0.25% Triton X-100 and 0.005M triethanolamine pH 7.8. The concentration of glucose in the sample was found to be proportional to the potential difference developed between b and c. Example 5 Preparation of an Enzyme Electrode for the Measurement of Triglycerides Referring to Figure 4j, membranes contain¬ ing enzyme, in this case lipase, are placed in cells a, b and c . The following reaction occurs where lipase is present: Triglycerides + H^O lipase ^ glycerol + fatty acids (H ) As in the previous examples the membranes were prepared by soaking a nitrocellulose membrane in a 5 mg/ml. buffered solution of lipase (Sigma Chemical Co., Cat. # L4384) . The decrease in pH which occurs upon addition of a sample containing triglycerides is detected with the pH electrode. Example 6 Preparation of an Enzyme Electrode for the Measurement of Uric Acid As in the previous examples and referring to Figure 4e, an electrode specific for uric acid is prepared by soaking a nitrocellulose membrane in a solution of uricase (50 units/ml Sigma Chemical Co., Cat. # U1878) plus catalase (1 mg/ml 40,000 units/mg Sigma Chemical Co., Cat. # C-100) . The enzyme membrane is placed in cells a, b and c. The following reactions occur where uricase and catalase are present: uric acid + 0 2 + H 2 0 uricase Allantoin + CO„ + H_0 2 H 2 0 2 catalase ^ 1/2 0 2 + H 2 0 The CO_ produced in the reaction causes a decrease in pH which is detected with the pH electrode. Example 7 Preparation of an Immunosensor for the Measurement of Theophylline In the present case, the measurement of theophylline is based on differences in immunochemical reactivity between theophylline and caffeine. Caffeine differs from theophylline by a methyl group. Theophylline and caffeine have been coupled to a crown ether moiety and each conjugate has been incorporated into a PVC membrane. The theophylline membrane forms one half-cell while the caffeine membrane forms the other half-cell. The caffeine membrane serves as a reference electrode and separately, each membrane exhibits a response to potassium ion. However, when each membrane is incorporated into a concentration cell and the same solution containing potassium ion is placed in each half-cell, the emf developed between the two half- cells is very close to zero millivolts. Although the theophylline and caffeine conjugates exhibit identical behavior in the electrochemical sense, they differ greatly in their immunochemical re¬ activity towards theophylline antibody. This difference in immunochemical reactivity and identi¬ cal ionophoric electrochemical reactivity, form the basis for a competitive binding differential potentiometric assay for theophylline. This method does not require constant ionic strength nor con¬ stant potassium ion activity (as in the Rechnitz approach) . A major advantage of the present method resides in the fact that undiluted and undialyzed serum samples may be used. The arrangement of the sensor can be as represented in Figure 41. The sensor consists of theophylline crown ether conjugate membranes in cells a and d and caffeine crown ether conjugate membranes in cells b and c, separated by porous junctions A and C. The sample containing theophylline is added to c and d while a calibrant is added to a and b. The competi¬ tive binding aspects require the presence of theo¬ phylline antibody with a low cross reactivity towards caffeine. The antibody may be added to the sample and calibrant before analysis in the sensor or the antibody may be contained within the sensor cells such that competitive binding occurs within the sensor. The competition for antibody site is set up between theophylline immobilized in the membrane and theophylline in the sample: Ab ■ + Ag(sample) + AgH^-ZZ-iAbAg(sample) + AbAg- (membrane) (membrane) As theophylline antibody binds to the theophylline immobilized in the membranes (a and c) a change in potential develops, the magnitude of which is inversely proportional to the concentration of theophylline in the sample. So as the theophylline concentration in the sample increases, less antibody is available to bind to the membrane, and therefore, less of an emf is developed. For a typical assay, potential changes due to antibody binding are normally on the order of a few millivolts. Another advantage in using the differential approach with a theophylline membrane, versus or in combination with a caffeine "reference" membrane, is that the common mode or initial starting potential is near zero millivolts and the signal to be measured is on the order of millivolts. The low common mode potential allows for greater senstivity in the measurement of the signal voltage and therefore a more accurate and precise signal measurement is possible when the background or common mode voltage is less than the signal voltage. As previously discussed, the potential difference between the theophylline and caffeine half-cells is nearly zero when treated with the same potassium ion-containing sample. However, when either half-cell is replaced with a reference electrode such as an SCE or a silver-silver chloride reference electrode, the potential difference with the same potassium ion solution increases to as much as 50 mv or more. The synthesis of the conjugates and the method of preparation of the membranes is described below: Reaction Scheme for the Preparation of Theophylline Ben-o-15-Crown-S-Conjuoate The first step in the synthesis involves blocking the 7 position in 1-methyl xanthine. The procedure of Hu, Singh and Ull an, J. Am. Chem. Soc, 5_: 1711-1713 (1980) was followed. To 700 ml of dry DMF in a 1-liter conical flask was added 3.2g (18.9 mmoles) of 98% 1-methylxanthine (Aldrich Chemical Co., cat. # 28,098-4), the mixture was stirred magnetically and warmed until dissolution occurred and then cooled to room temperature. To the stirred solution was added 2.0g (18.9 mmoles) of anhydrous sodium carbonate followed by the dropwise addition, over a period of 1 hour, of 3.0g (19.3 mmoles) of 97% chloro ethyl pivalate (Aldrich Chemical Co., cat. #14,118-6) dissolved in 50 ml of DMF. The reaction mixture was stirred overnight at room temperature. The reaction mixture was evapor¬ ated to dryness and the residue treated with 50 ml of methylene chloride. The insoluble material was collected by filtration and washed with IN HCl to give 1.24g of 1-methylxanthine. The methylene chloride solution was evaporated to dryness to yield a mixture of mono- and bis- protected xanthine. The mono-protected l-methyl-7-[ (pivaloyloxy)methyl.! xanthine was separated from the bis protected xanthine by- crystallization from ethyl acetate. By this method! there was obtained 1.5g of the mono- protected xanthine (m.p. 204-206°) . The ethyl- acetate filtrate from the isolation of the mono- protected xanthine, containing the bis-protected xanthine, was evaporated to dryness and refluxed with 20 ml of 2N sodium hydroxide for 18 hours. The reaction mixture was cooled and acidified with concentrated HCl and extracted with chloroform. Evaporation of the aqueous layer yielded 0.5g of 1-methylxanthine. The second step in the synthesis is the prepar¬ ation of 4'-amino-benzo-15-crown-5 hydrochloride. To a stirred solution of 30g (6.11 mmoles) of benzo-15-crown-5 (Parish Chemical Co., Orem, UT, cat. #1405) in 800 ml of 1:1 glacial acetic acid/ chloroform was added dropwise, over a period of 1 hour, a solution of 15 ml concentrated nitric acid dissolved in 50 ml of glacial acetic acid. The reaction mixture was stirred for one hour and then evaporated to dryness on a rotary evaporator. The residue was treated with 500 ml of 5% aqueous sodium hydrogen carbonate and the mixture partitioned with 500 ml of chloroform. The chloroform layer was washed with saturated aqueous sodium chloride and dried over anhydrous sodium sulfate. Evaporation of the chloroform yielded 30g (86%) of 4 '-nitobenzo-15- crown-5. The product was reσrystallized from ethanol and 15g (48 mmoles) dissolved in 250 ml of dioxane and catalytically reduced over Raney nickel (AJdrich Chem. Co., cat. #22,167-8) in a Paar hydrogenator at 50°C. The reaction mixture was filtered and the filtrate evaporated to dryness and the residue dissloved in 200 ml of ethyl acetate. The solution was then treated with HCl gas to precipitate the hydrochloride salt of 4' -amino-benzo-15-crown-5. Yield 9.0g (58%) . The third step in the synthesis is the prepara¬ tion of 4'-benzo-15-crown-5-(11-bromoundecanoamide) . To lOg (37.7 mmoles) of 11-bromoundecanoic acid (Aldrich Chemical Co., cat. #B8,280.4) in 100 ml of CH 2 C1 2 in a 250 conical flask was added 5.5 ml of triethylamine. The mixture was cooled to 0°C and 9.67g of N,N-Bis[2-oxo-3-oxazolidiny t phosphordi- amidic chloride (BOPCL, Chemical Dynamics Corp., cat. #12-1370-00) was added. The mixture was ' stirred for 0.5 hours then 12.06g (37.7 mmoles) of 4'-amino-benzo-15-crown hydrochloride was added along with 5.5 ml of triethylamine. To the reaction mixture was added dropwise * another 5.5 ml of triethylamine in 50 ml of CH 2 C1 2 over a period of 1 hour. The reaction mixture was allowed to warm to room temperature for 18 hours. Water (50 ml) was added and the mixture acidified with concentrated HCl. The CH 2 C1 2 layer was washed in turn 3 times with 200 ml saturated NaCl solution and 3 times with 200 ml 5% sodium hydrogen carbonate solution. The CH 2 C1 2 layer was dried over anydrous sodium sulfate and evaporated to dryness. The residue was crystallized from hexane/ethyl aσtetate to yield 8.8g (44%) of 4'-benzo-15- crown-5-(11-bromoundecanoamide) , m.p. 101-103°, analysis calaculated for C 2 -.H 4O Br 0 g , C:56.60; H:7.60, Br:15.06; N:2.62. Found: C-.56.41; H-.7.46; Br. :14.89; N:2.60. In the fourth step, the crown ether was coupled to the protected 1-methylxanthine. To 50 ml of DMF in a 125 ml conical flask was added l.Og (3.57 mmole) of l-methyl-7-[ (pivaloyloxy)methγ l xanthine. The mixture was warmed (e.g., to about 80°C with stirring) until dissolution. The solution was cooled to room temperature and 0.76g (7.1 mmole) of anhydrous sodium carbonate was added followed by the addition of 1.90g (3.57 mmole) of the crown ether amide. The reaction mixture was stirred under nitrogen for 3 days. The DMF was removed by rotary evaporation and 100 ml of water was added and the mixture extracted 3 times with 50 ml of chloroform. The combined chloroform extracts were washed 1 time with 100 ml of saturated aqueous sodium chloride and dried over anhydrous sodium sulfate. Evaporation of the chloroform yielded a pale brown oil. The fifth step is the removal of the protecting group with aqueous base. To the above oil in a 250 ml round bottom flask was added 75 ml of 0.2M NaOH and 25 ml of methyl alcohol. The mixture was refluxed for 1 hour and cooled to room temperature. The solution was acidified with concentrated HCl with cooling in an ice/acetone bath. The precipi¬ tated product was recrystallized from ethyl alcohol- water to yield 1.2g (55%) of an off-white crystal¬ line solid, m.p. 166-170°. The material was homo- genous by hplc analysis. Analysis calculated-, for C3,a1,H4,5 c N,50 o 8: C-.60.47; H-.7.37; N:11.37. Found: C:60.25; H-.7.23; N:11.09. The benzo-12-crown-4 derivative was prepared by a similar method. Preparation of Caffeine-Benzo-15-Crown-5 Conjugate The caffeine conjugate was prepared according to step 4 in the reaction scheme except that the protected 1-methyl xanthine was repl 1,7-dimethyl xanthine. To 50 ml of DMF in a 125 ml con added l.Og (5.56 mmole) of 1,7-Dime" The mixture was warmed with stirriπ solution occurred. The solution we temperature and 0.59g of anydrous ε was added followed by the addition mmole;) o t ' he previously -described 0' and the reaction mixture -stirrsέd for DMF was removed by rotϊry evagaE-ra ic water w s added and the im±x±.u.x-E ext~ with 515 ml n>± chloroform,. The ccomb;, were w-ashed 1 time with 3.00 ml cof s 5 sodium ciiloTiώe and dri-eS s ~ - ~ τ -eanhy • ΪΠI sulfate- E<sraporation of tfcfcae cdblLorc :,ed a brown oil v.'tα-i<cla was cry -+.-,. ~ -i -»*-_. f -ethyl acetate ±o yie d l.Og (299 ' %i o± pre . product was from e to l— acet, '- with D rfnajrcosl. cla_rif caticn aoa fJEJ-Hnl tr jenous by ihgple., ._ m 130-133°. CsSl.03? H;7,52r HIsll.l Cs€ffi.TSy tt5?,52y N:10.9~, Tltae Janr 5/n-4 (deιri it 5a7e ~~~~ i prepared b- a. similε 5 ~ mCm ~ -a_τ_#tϊ. ~~ ι <&£ & Drug Specific STlec z-r The Theop3-^ll-b-.e Membrane Hϋhe CttaπgsBsoeiπits for the ϋ o h*. , rembrane csras-isifcai≤l ~ _z Q. QQ '5 g theophγlL-Lne - , * jm ether © _s ~ xj- ~~ _ ~ i l , ®.a 2 g potassiiαm tetra. I(τ * ; *" .^rophenyl) -barrafta.,, -3D-5 ml fo±s 2-etlιylh-3xyl). s- e, 0.3 g high molecular weight PVC and 5 ml THF. The mem¬ brane can be formed by either solvent casting or dip coating as previously described. Caffeine Membrane The components for the caffeine membrane consisted of: 0.005 g caffeine-crown ether conju¬ gate, 0.02 g potassium tetra(p-chlorophenyl) borate, 0.5 ml bis-(2-ethylhexyl) sebacate, 0.3 g high molecular weight PVC and 5 ml THF. The membrane can be formed by either solvent casting or dip coating as previously described. Differential Measurement Technique One membrane each of theophylline and caffeine was mounted in an Orion 95 electrode body. The internal filling solution consisted of pH 7.4 phosphate buffered saline and the reference elec¬ trodes were silver-silver chloride wires. The two electrodes were fitted into a rubber stopper and immersed into 5.0 ml of pH 7.4 phosphate buffered saline. The solution was magnetically stirred. The emf measured between the two electrodes was 0.23 mv. To test the response to potassium ion, 100 ul of 0.2 molar KCL was pipetted into the stirred buffer solu¬ tion. A transient response was observed as evi- denced by a sharp peak (0.3 mv) on the output of a strip chart recorder. The potential difference returned to the base-line value within 30 to 60 s. The transient response to potassium is probably due to a local concentration of potassium ion near the membrane surface which disappears as the solution is mixed. To test the response to protein, ICO ul of a standard protein solution (Sigma Chemical Co., St. Louis, MO, Cat. # 540-10) consisting of 3.5 g/dl albumin and 3.0 g/dl globulin was added to the solution. The standard protein solution had no effect on the electrode potential. When, however, 100 ul of theophylline antibody (Immunotech Corp., Allston, MA, Cat. # 65i or Research Plus, Bayonne, NJ, Cat. # 01-9603-09) was added the potential difference between the two electrodes shifted by 0.85 mv over a period of about 20-30 min. A sensor of this type can. be used in a competi¬ tive binding assay. The antibody which remains unbound in the sample is measured and provides an indirect measure of antigen present in the sample. The more antibody bound by the antigen in the sample, the lower the resulting electrical signal because less antibody will be available for binding to the antigen immobilized in the sensor membrane; that is, antigen concentration will be inversely related to the magnitude of the signal. Example 8 Preparation of an Ion Selective Electrode for the Measurement of Creatinine Creatinine is an important indicator of renal function and its measurement in serum is a routinely performed blood test. Presently, in the clinical laboratory there are two ways of determining cre¬ atinine. The more widely used method is the Jaffe reaction, which is based on the production of a red colored complex between creatinine and picrate in alkaline solution. This method gives erroneous results in the presence of certain metabolites and drugs. The second method utilizes an enzyme. Although enzymic methods are highly specific, the expense and the time required to carry out a test restrict their routine use in clinical laboratories. Most recently a creatininium ion selective electrode has been descrbed. E.P. Diamondis and T.P. Hadjiiannou, Anal. Lett., 13, 1317-1332 (1980). The creatinium ion exchanger was prepared by mixing equimolar aqueous solutions of creatinine and sodium tetraphenylboron , followed by the addition of a hydrochloric acid solution which results in precipi¬ tation of the complex salt creatininium tetra- phenylboron. The salt was extracted into 2-nitro- toluene and this solution of the complex salt used as a liquid ion exchanger in an Orion 92 (Orion Re¬ search, Cambridge, MA) electrode equipped with Teflon membranes. In this type of ion selective electrode the ion exchange material is in liquid form contained in a reservoir and continuously leaches through the membrane into the sample solu¬ tion and therefore must be periodically replenished. The electrode was conditioned by soaking in 0.01 mol/L creatininium chloride for 24 hours before use. The response of the creatininium electrode at pH 3 was linear in the 10 -3 to 10-1 mol/L range with a slope of 57 mv at 20°C. The slope decreased to 37 -4 -3 mv in the 10 to 10 mol/1 range. The present invention makes use of a plastic membrane in which the ion exchange material is immobilized. When creatininium tetraphenyl boron was incorporated into a PVC membrane using 2-nitro- toluene as a plasticizer a slope of 44 mv was ob- tained between 10 -3 and 10-1 mol/L creatininium chloride and decreased to 14 mv in the 10 -4 to 10-3 mol/L range. The clinically important range for -4 -3 creatinine is 10 to 10 mol/L. The use of other nitrated plasticizers such as 2-nitro-p-cymene and 2-nitrophenyloctyl ether did not improve the sensi- tivity in the 10 -4 to 10-3 mol/L range. A significantly improved response in the 10 -4 _3 . to 10 mol/L range was obtained when a substituted ' tetraphenylboron salt was used having the general formula: where x = Cl, Br, F, CF., In one embodiment, a membrane was prepared consist¬ ing, of 0.02 g of creatininium tetra-(p-chlorphenyl) boron, 1.5 ml of 2-nitrophenyloctyl ether, 0.3 g of very high molecular weight PVC, and 5 ml of THF. The membrane was prepared either by solvent casting or by dip coating as previously described. The _3 membrane yielded a slope of 55 mv between 10 ml/L and 10 mol/L creatininium chloride and 40 mv in -4 -3 the 10 to 10 mol/L range and did not require any preconditioning. The substituted tetraphenyl boron salts exhibited much higher sensitivity in the clinical range of concentration than the unsubsti¬ tuted tetraphenyl boron salt. The performance of the various substituted tetraphenyl boron salts is summarized in Table 2. The unexpected increase in sensitivity of the substituted tetraphenyl boron salts over the un- substituted tetraphenyl boron may be attributed to an increase in lipophiliσity and differences in the nature of the ionic exchange properties of the complexes formed with the substituted tetraphenyl boron salts. In practice, the pH of the solutions containing creatinine must be decreased to a pH of 3 or less by the addition of acid. This can be accomplished by dilution of the sample with an acid solution. However, in the case of a serum sample, significant dilution may adversely effect the sensitivity of the assay due to a decrease in the concentration of creatinine. In the present case a different approach was taken. The pH of the sample containing creatinine was lowered by treating the sample with a support containing dried glycine hydrochloride. This was accomplished by dipping a membrane support such as nitrocellulose (Schleicher and Schull 12 micron, grade HE-100) into an aqueous solution of glycine hydrochloride (0.1 to 1.0 moles/L) . A sandwich was formed such that the nitrocellulose membrane covered the surface of the creatininium tetra (p-chloro- phenyl) boron PVC membrane. Referring to Figure 4k, each cell a, b and c contains a creatininium PVC membrane and a nitrocellulose membrane containing glycine hydrochloride. The serum sample is added to c and reference and calibrant solutions are added to a and b, respectively. The glycine hydrochloride converts the creatinine to creatininium hydro¬ chloride which is detected by the membrane. The emf developed between a and b may be used to calibrate the sensor. The slope from a and b and the emf developed between b and c may then be used to determine the creatinine concentration in the sample. Preparation of Creatininium Tetraphenyl Boron Salts The creatininium tetraphenyl boron salts were prepared by mixing equimolar aqueous solutions of creatininium hydrochloride (Sigma Chemical Co., St.. Louis, MO, Cat. # C-6257) and the corresponding sodium salt of the substituted tetraphenyl boron. The precipitated salts were collected by filtration, washed with water and dried. The sodium salts of the substituted tetraphenyl boron derivatives were prepared by the method of Cassoretto, McLafferty and Moore, Anal. Chem. Acta, 3_2_:376-380 (1965) and involved the following steps: ^ r l Cl ι CF 3 Table 2. Response of Creatininium Tetraphenyl Boron Salts to Creatininium Hydrochloride. X 10 "1 to 10 "3 mol/L 10 " " ' to lθ "4 mol/L Slope (mv) Slope(mv) H 50 10 F 45 26 Cl 55 36 CF-. 57 40 All mv readings shown in Table 2 are versus a double junction silver-silver chloride reference electrode. The creatininium solutions used were prepared with 0.05 M glycine-glycine hydrochloride buffer pH 3.0 containing 150 mM sodium chloride and 6 mM potassium chloride. Example 9 Preparation of an Ion Selective Electrode ' for the Measurement of Sodium A membrane selective to sodium ions was pre¬ pared using the following proportions of components: 0.2g methylated monensin, 0.04g sodium tetra- (p-chlorophenyl) borate, 2.0 ml 2-nitrophenyloctyl ether, 1.2g high molecular weight PVC, 12 ml tetrahydrofuran (THF) and 5 ml 4-methyl-2-pentanone. The membrane can be formed by either solvent casting or dip coating as previously described. The membrane was incorporated into the previously described electrode housing and tested using aqueous sodium ion containing solutions. The sensor yielded a slope of 57-60 mv per decade at 25°. Preparation of Methylated Monensin To 250 ml of 1,4-dioxane in a 500 ml round bottom flask was added 5.0 g of sodium monensin (Sigma Chemical Co., St. Louis, MO, Cat. # M2513) along with 20 ml of iodomethane. The mixture was gently refluxed for 18 hours and then evaporated to dryness on a rotary evaporator. The residue was dissolved in 200 ml of methylene chloride and washed in turn 2 times with 500 ml saturated aqueous sodium chloride, 2 times with 200 ml 5% aqueous sodium hydrogen carbonate and one time with 200 ml of saturated aqueous sodium chloride. The methylene chloride solution was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was treated with 50 ml of hexane with warming in a 50° water bath. The hexane solution was cooled in a freezer for 1-2 hours followed by filtration to remove precipitated salts. ' The hexane solution was evaporated to dryness to yield 2.5-4.0 g of a light tan oil. The oil was used to prepare the sodium selective membrane. Example 10 Preparation of an Ion Selective Electrode for the Measurement of Potassium A membrane selective to potassium ions was prepared using the following proportions of com¬ ponents: 0.2 g valinomycin, 0.04 g potassium tetra¬ phenyl borate, 2.0 ml 2-nitrophenyloctyl ether, 1.2 g high molecular weight PVC, 10 ml THF and 5 ml 4-methyl-2-pentanone. The membrane can be formed by either solvent casting or dip coating as previously described. Example 11 Preparation of an Ion Selective Electrode for the Measurement of Chloride A membrane selective to chloride ions was prepared using the following proportions of com¬ ponents: 1.0 ml Aliquat 336 (Aldrich Chemical Co., Milwaukee, WI, Cat. # 20,561-3), 2.0 ml tris (2-butoxyethyl) phosphate (Aldrich Chemical Co., Milwaukee, WI, Cat. # 13,059-1), 1.2 g very high molecular weight PVC (Aldrich Chemical Co., Mil¬ waukee, WI, Cat. # 18,261-3), 12 ml THF and 5 ml 4-methyl-2-pentanone. The membrane can be formed by either solvent casting or dip coating as previously described. Industrial Utility This invention has industrial utility in the determination of the ion content or concentration of other constituents of samples, in particular biological fluids such as blood, serum, plasma, urine, saliva and cerebrospinal fluid. It is particularly useful for the rapid determination of the ion activity of a biological sample, as well as the concentration of other sample components, such as glucose, urea, triglycerides, creatinine, uric acid, lipase, other enzymes and drugs. It is well suited for use in a clinical or research context because there is no need for preconditioning of the ion-selective membrane and because it provides results quickly. In addition, the invention can be used for the similar determination in other samples such as beverages, meats, canned and processed foods, fruit extracts, etc. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experi¬ mentation, many equivalents to the specific compo¬ nents and materials described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims: CLAIMS 1. A composition having the general formula; wherein R.. R_ and R 3 are independently selected from the group consisting of: a. a halogen; b. an alkyl group having from 4-18 carbon atoms; c. a halogen-substituted alkyl group; d. an alkoxy group; -e. a halogen-substituted alkoxy group; f. an acid group represented by the formula -C0 2 R., wherein R. is alkyl having 1-18 ♦ carbon atoms; and g. a keto group represented by the formula -COR.., wherein R- is alkyl having 1-18 carbon atoms; and h. a hydrogen atom. 2. An ionophore having the general formula: wherein R. R 2 and R- are independently selected from the group consisting of: a. a halogen; b. an alkyl group having from 4-18 carbon atoms; c. a halogen-substituted alkyl group; d. an alkoxy group; e. a halogen-substituted alkoxy group; f. an acid group represented by the formula -C0-R., wherein R. is alkyl having 1-18 carbon atoms; and g. a keto group represented by the formula -C0R_, wherein R- is alkyl having 1-18 carbon atoms; and h. a. hydrogen atom. 3. An ionophore of Claim 1 selected from the group consisting of 2-trifluoromethyl-4-octadecyloxy- phenylhydrazone mesoxalonitrile; 2-octadecyloxy-5-trifluoromethylphenylhydrazon'e mesoxalonitrile; 2- octadecyloxy-5-carbethoxyphenylhydrazone mesoxalonitrile; 2-octadecyloxy-4-fluoro- phenylhydrazone mesoxalonitrile and their derivatives. 4. In a sensor for the potentiometric determina¬ tion of the ion activity of a sample, said sensor including an ionophore, the improvement comprising an ionophore of the general formula: wherein R. R 2 and R- are independently selected from the group consisting of: a. a halogen; b. an alkyl group having from 4-18 carbon atoms; c. a halogen-substituted alkyl group; d. an alkoxy group; e. a halogen-substituted alkoxy ' group; f. an acid group represented by the formula -CO-R., wherein R 4 is alkyl having 1-18 carbon atoms; g. a keto group represented by the formula -COR,., wherein R-. is alkyl having 1-18 carbon atoms; and h. a hydrogen atom. 5. In a sensor of Claim 4, the improvement comprising an ionophore selected from the group consisting of 2-trifluoromethyl-4-octadecyloxy- phenylhydrazone mesoxalonitrile; 2-octadecyloxy-5-trifluoromethylphenylhydrazone mesoxalonitrile; 2- octadecyloxy-5-carbethoxyphenylhydrazone mesoxalonitrile; 2-octadecyloxy-4-fluoro- phenylhydrazone mesoxalonitrile and their derivatives. 6. A membrane comprising: a. an ion-selective compound of the general formula: ' wherein R 1 R~ and R, are independently selected from the group consisting of: a. a halogen; b. an alkyl group having from 4-18 carbon atoms; c. a halogen-substituted alkyl group; d. an alkoxy group; e. a halogen-substituted alkoxy group; f. an acid group represented by the formula -CO_R., wherein R, is alkyl having 1-18 carbon atoms; g. a keto group represented by the formula -COR 5 , wherein R- is alkyl having 1-18 carbon atoms; and h. a hydrogen atom. 7. A membrane of Claim 6 in which ' : a. the ion-selective compound comprises from about 1 to about 10 percent by weight of the membrane and is selected from the group consisting of 2-trifluoromethyl-4- octadecyloxyphenylhydrazone mesoxaloni¬ trile; 2-octadecyloxy-5-trifluoromethylphenylhyd- razone mesoxalonitrile; 2-octa- decyloxy-5-σarbethoxyphenylhydrazone mesoxalonitrile; 2-octadecyloxy-4-fluoro- phenylhydrazone mesoxalonitrile and their derivatives; and b. the thermoplastic resin or plastic com¬ prises from about 10 to about 30 percent by weight of the membrane and is selected from the group consisting of polyvinyl chloride; cellulose acetate; polyvinyl acetate; and silicone rubber. 8. A membrane of Claim 6- which is additionally comprised of at least one plasticizer which comprises from about 50 to about 80 percent by weight of the membrane and is selected from the group consisting of phthalates; adipates; sebacates; aliphatic and aromatic ethers; aliphatic and aromatic esters; and nitrated aliphatic and aromatic ethers. 9. A membrane of Claim 8 in which the plasticizer comprises from about 50 to about 80 percent by weight of the membrane and is 2-nitrophenyl- octyl-ether. 10. In a method in which an ionophore selective for hydrogen ion is used for the potentiometric determination of the hydrogen ion activity of a sample, the improvement comprising an ionophore having the general formula: wherein R 1 R~ and R 3 are independently selected from the group consisting of: a- a halogen; b. an alkyl group having from 4-18 carbon atoms; c a halogen-substituted alkyl group; d- an alkoxy group; e. a halogen-substituted alkoxy group; f, an acid group represented by the formula -C0 2 R 4 , wherein R. is alkyl having 1-18 carbon atoms; g- a keto group represented by the formula -COR.., wherein R ς is alkyl having 1-18 carbon atoms; and h- a hydrogen atom. 11. In a sensor for the differential measurement of the activity in a sample of an enzyme or the concentration of a component of a sample other than an enzyme, the enzyme being one which catalyzes a reaction which results in a change in pH of the sample or the component being a substrate in a reaction which results in a change in pH of the sample, the improvement comprising an ionophore having the general formula: wherein R. R„ and R, are independently selected from the group consisting of: a. a halogen; b. an alkyl group having from 4-18 carbon atoms; c. a halogen-substituted alkyl group; d. an alkoxy group; e. a halogen-substituted alkoxy group; f. an acid group represented by the formula -C0 2 R,, wherein R. is alkyl having 1-18 carbon atoms; g. a keto group represented by the formula -COR.. , wherein R_ is alkyl having 1-18 carbon atoms; and h. a hydrogen atom. 12. A method of making a hydrogen ion-selective membrane comprising the steps of: aking an ionophore selective for hydrogen ion, the ionophore having the general formula: wherein R, R~ and R 3 are independently selected from the group consisting of: 1. a halogen; 2. an alkyl group having from 4-18 carbon atoms; 3. a halogen-substituted alkyl group; 4. an alkoxy group; 5. a halogen-substituted alkoxy group; 6. an acid group represented by the formula -C0 2 R., wherein R. is alkyl having 1-18 carbon atoms; 7. a keto group represented by the formula -C0R-, wherein R-. is alkyl having 1-18 carbon atoms; and 8. a hydrogen atom. b. dissolving plasticizer and the ionophore of (a) in a volatile organic solvent; and c. removing the volatile organic solvent. 13. A method of making a hydrogen ion selective membrana, comprising the steps of: a. placing a membranous material in a solvent; b. removing air trapped within the membranous material c. removing the membranous material from the solvent; d. contacting the membranous material with a mixture comprising: 1. an ionophone having the general formula: wherein R 1 R 2 and R_ are indepen¬ dently selected from the group consisting of: a. a halogen; b. an alkyl group having from 4-18 carbon atoms; c. a halogen-substituted alkyl group; d. an alkoxy group; e. a halogen-substituted alkoxy group; f. an acid group represented bv the formula -C0_ 2R4,, wherein R. is alkyl having 1-18 carbon atoms; g. a keto group represented by the formula -C0R-, wherein R 5 is alkyl having 1-18 carbon atoms; and h. a hydrogen atom; 2. a thermoplastic resin or plastic; and 3. a plasticizer; and e. drying the membranous material. 14. A compound having the general formula: wherein R is a drug; X is an oxygen atom or a nitrogen atom linked to a hydrogen atom or to an alkyl group ; and n. and n 2 are integers equal to or greater than one. 15. An ionophore having the general formulas wherein R is a drug; X is an oxygen atom or a nitrogen atom linked to a hydrogen atom or to an alkyl group ; and n. and - are integers equal to or greater than one. 16. An ionophore of Claim 15 in which R is theophylline or caffeine; X is nitrogen atom linked to a hydrogen atom; n, is equal to ten; and n 2 equals one or two. _T ~ m a a sensor for the potentiometric determina¬ tion of the concentration of a drug in a sample, said sensor comprising an ionophore, am the improvement comprising: -wherein R is a drug; X is an oxygen atom or a nitrogen atom linked to a hydrogen atom or to an alkyl group and n.. and n 2 are integers 15 equal to or greater than one. IS- compound which is a substituted tetraphenyl- Iboron salt having the general formula: wherein X can be chlorine, bromine, fluorine or a trifluoromethyl group or an electron with¬ drawing group selected from th« group consist¬ ing of amides, keto groups and esters. 19. An ionophore which is a substituted tetra- phenyl-boron salt having the general formula: wherein X can be chlorine, bromine, fluorine or a trifluoromethyl group or an electron with¬ drawing group selected from the group consist¬ ing of amides, keto groups and esters. 20. A membrane comprising: a. an ion-selective compound which is a substituted tetraphenylboron salt having the gereral formula: wherein X can be chlorine, bromine, a trifluo-ronmetyl group or an electron withdrawing group selected from the group consisting of amides, keto groups and esters; b. a thermoplastic resin or a plastic; and c. a plasticizer. 21. A membrane of Claim 20 in which the thermo¬ plastic resin or plastic is selected from the group consisting of polyvinyl chloride; cellu¬ lose acetate; polyvinyl acetate; and silicone rubber and the plasticizer is 2-nitrophenyl- octyl ether. 22. In a sensor for the potentiometric determina¬ tion of the creatinine concentration of a sample, the sensor comprising an ionophore, the improvement comprising an ionophore of the general formula: wherein R is a drug; X is an oxygen atom or a nitrogen atom linked to a hydrogen atom or to an alkyl group ; and n. and π_ are integers equal to or greater than one. 23. A method of determining the hydrogen ion activity of a sample, comprising: contacting a solution of known hydrogen ion activity with a first ion selective electrode and a second ion selective electrode, the first ion selective elec¬ trode and the second ion selective elec¬ trode being comprised of an ion selective membrane having an ionophore of the general formula: wherein R. R 2 and R_ are independently selected from the group consisting of: a. a halogen; b. an alkyl group having from 4-18 carbon atoms; c. a halogen-substituted alkyl group; d. an alkoxy group; e. a halogen-substituted alkoxy group; f. an acid group represented by the formula -C0„R. , wherein R. is alkyl having 1-18 carbon atoms; and g. a keto group represented by the formula -COR,., vherein _ is alkyl having 1-18 carbon atoms; h. a hydrcgsn atom, the first and the second ion selective electrodes having between them porous material for providing ionic flow between them; b. contacting a sample of unknown hydrogen ion activity with a third ion selective 5 electrode, the electrode being comprised of an ion selective membrane having an ionophore of the general formula of (a) and having between it and the second electrode porous material for providing 1 0 - ionic flow between the third electrode and the second electrode; c. determining potentiometrically the emf between the solution contacted with the first electrode and the solution contacted 15 with the second electrode and the emf between the solution contacted with the second electrode and the solution con¬ tacted with the third electrode; d. calibrating the sensor; and ZCD ε- determining the hydrogen ion activity of the sample from the emf developed between the solution contacted with the second electrode and the solution contacted with the third electrode. 25 24_ Α method of potentiometrically determining in a sample the activity of an enzyme which -catalyzes a reaction which produces a change in tfce pH of the sample or the concentration of a substance which is a substrate of a reaction which produces a change in the pH of the sample, comprising: a. contacting a solution having a known concentration of the enzyme or the sub- 5 strate to be determined with a first ion selective electrode and a second ion selective electrode, the first electrode being comprised of a membrane having an ionophore of Claim 2 and a membrane having „ Q- the enzyme or the substrate immobilized thereon and the second electrode being comprised of a membrane having an iono¬ phore of Claim 2' and a membrane having an inert protein immobilized thereon, the 15 first and the second electrodes having between them porous material for providing ionic flow between them; b. contacting a solution having an unknown concentration of the enzyme or the sub- 2ϋ- strate to be determined with a third ion selective electrode and a fourth ion selective eletrode, the third ion selec¬ tive eletrode being comprised of a mem¬ brane having an ionophore of Claim 2 and a 25 membrane having the enzyme or the sub¬ strate immobilized thereon and the fourth ion selective electrode being comprised of a membrane having an ionophore of Claim 2 and a membrane having immobilized thereon 30 the same inert protein as in the second electrode, the third and the fourth electrodes having between them porous material for providing ionic flow between them; c. measuring the change in potential dif- ference between the first electrode and the second electrode and between the third electrode and the fourth electrode; and d. calculating the activity of the enzyme or the concentration of the substrate in the sample.

Inventors: WILLIS JOHN P, WILLIS, JOHN, P.
Applicants: WILLIS JOHN P, WILLIS, JOHN, P.
Application year: 1986
Main classification: C07C121
IPC codes: C07C255, C07D323, C07D473, C07F5, G01N27