CA1222438A

CA1222438A - Unified test means for ion determination

Info

Publication number: CA1222438A
Application number: CA000452893A
Authority: CA
Inventors: Steven C. Charlton; Roger L. Fleming; Arthur L.Y. Lau; Paul Hemmes
Original assignee: Miles Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1983-05-12
Filing date: 1984-04-26
Publication date: 1987-06-02
Also published as: DK161262B; ATE48918T1; ZA843496B; NO841748L; US4645744A; NO162132C; EP0125555A3; AU2794184A; ES8507691A1; JPS59211864A; ES532280A0; FI77937C; AU552420B2; NO162132B; FI841884A0; EP0125555B1; JPH0423753B2; DK161262C; FI77937B; EP0125555A2

Abstract

ABSTRACT

A test means, test device and method for use for determining the presence of an ion in a test sample are disclosed. The test means comprises a substantially nonpolar, nonporous carrier matrix incorporated with an ionophore capable of forming a complex with the specific ion, and a reporter substance capable of interacting with the complex of ionophore and ion to produce a detectable response.
The test device comprises an elongated support member having a upper, substantially flat face to which is affixed the test means. The test means and test device are used by contacting either with the test sample, and observing a detectable response.

Description

_3_ 1. INTRODUCTION

The present invention relates to -the measurement of ions, in particular ions in aqueous solution, and to a test means or device for performing such measure-ments. The invention provides a quick facile way of assaying such ions whereby results are available to the assayist momentarily after merely contacting a test sample solution with the test means or device.
There is no need for cumbersome, expensive electronic equipment such as ion-specific electrodes, flame photometers, atomic absorption spectrophotometers or the like. Nor is it necessary to resort to time-consuming wet chemistry techniques such as titration and other laboratory procedures. The present in-vention enables the analyst to merely contact the test sample with a strip device or similar test means configuration, and observe any color change.
The determination of aqueous ion concentration has application in numerous technologies. In the water purificati.on art, calcium concentration must be carefully monitored to assess the degree of sa-t-uration of an ion exchange resin deionizer. Measure-ment of sodium and other ions in seawater is impor-tant i.n the preparation of drinking water aboard a ship at sea. Measurement of the potassium level in blood aids the physician in diagnosis of conditions leading to muscle irritability and excitatory changes in myo-cardial function. Such conditions include oliguria, anuria, urinary obstruction and renal failure due to shock.
Needless to say, a quick facile method for determining ion concentration would greatly enhance the state of these technologies, as well as any ~2~2~

others where such rapid, accurate determinations would be beneficial. Thus, for example, if a medical laboratory technician could accurately measure the potassium or calcium level of a serum or whole blood sample in a matter of seconds or minutes, not only would such rapid results aid the physician in diag-nosis, but also laboratory efficiency would increase manifold.

2. BACKGROUND OF THE INVENTION

Prior to the present invention, methods for determining ions in solution included flame photo-metry, atomic absorption photometry and ion-specific electrodes. The use of certain compounds and com-positions which selectively isolate certain ions from the sample solution has become popular in ion-specific electrodes. These substances, known as ionophores, have the capability of selectively iso-lating ions from their counterions thereby causing a charge separation and a eorresponding change in eleetrieal conductivity in -the phase containing the ionophore. Illustrative of the ion/ionophore pheno-menon inelude ion assays utilizing membrane eleetrodes, liquid/liquid partitioning and fluoreseenee.

2.1 Ion-Speeifie Electrodes When two solutions having different eoncentra-tions of ions are separated by an elec-trieally eon-duetive membrane, an elee-trieal potential (EMF~ is generated. The EMF developed by sueh system is a funetion of eoneentration or ionic activity. This phenomenon is expressed mathematically by the well-known Nernst Equation -5~ 43~

= RT
ln YlCl Y2C2 (1) in which ~ is the EMF of the particular system, F is the Faraday Constan-t [23,062.4 ~ 0.003 calories (volt equiv.) ], R is the gas constant, T is the temperature in C and y and c are, respectively, the activity coefficients and molal concentrations of the ion under study, the subscript 1 designates the solution on one side of the membrane, the subscript 2 denoting the solution on the other side, and n is the number of electrons transEerred in the reaction.
In such membrane separation cells, the membrane can be a simple fritted glass barrier, allowing a sma].l but measurable degree of ion diffusion from one solution to the other. Alternatively, a nonporous, electrically nonconductive film, such as polyvinyl chloride, impregnated with an ionophore can be em-ployed. In the absence of the ionophore the film is an insulator and no EMF can be measured; when blended wi-th an ionophore, charged ions are bound to the film and a small, measurable current can be induced to flow. Because the ionophore is selective in lts affinity, and thus will bind only certain specific ions, such cells are ion selective. Any measureable EMF is due solely to the presence oE those ions.
Thus, a cell for determining potassium ions (K ) can be produced through use of an ionophore specific for K , e.g. valinomycin. In the presence of potas-sium, valinomycin produces a conductive path across a membrane by binding and transporting K , thus -6~ 38 allowing a small current to flow. A reference con-centration o~ K is placed on one side of the mem-brane and the test sample on the other. The EMF
developed is measured and used to calculate the unknown concentration from e~uation (1). ~ecause K
binds to the valinomycin membrane, the conductive path only appears for IC . Therefore, -the only EMF
developed is attributable solely to the K concen-tration gradient across the membrane.
The curren-t flowing across the membrane is so small that no significant quantity of K or counter-ion is transported through it. Electrical neutrality of the membrane is maintained either by a reverse flow of hydrogen ions, or by a parallel flow of OH .
This anion effect can reduce the specificity of the electrode -towards the intended ion and is an inter~
ference to be minimized.
A major difficulty in the use of such ion-selective electrodes has been the marked reduction of accuracy and speed of response over time. Further, small changes in ion concentration produce such small changes in EMF that sophisticated voltmeter equipment is required.
It has been known that certain antibiotics, such as valinomycin, have an effect on the electrical properties of phospholipid bilayer membranes (bio-logical membranes), such that these antibiotics solubilize cations wi-thin the membrane, in the form of mobile charged couples, thereby providing a "car~
rier" mechanism by which cations can cross the in-su:Lating hydrocarbon interior of the membrane. Such complexes have the sole purpose of carrying the charge of the complex through the membrane such that a voltage differential can be determined between solutions on either side of the membrane.

-7- ~ 3~

U.S. Patent No. 3,562,129, describes the use of porous membranes impregnated with macrocyclic deri-vatives of amino and oxy-acids in ion-sensitive electrodes. Materials used to form the membrane are glass frits and other porous membranes. Such electrodes are said to be effective in measuriny ion activities.
United States Patent No. 4,053,381, issued to Hamblen, et al., discloses similar technology, and utitlizes an ion specific membrane having ion mobility across it.

2.2 Liquid/Liquid Partitioning Another known application of ionophores in ion determination is through liquid/liquid partitioning.
In this procedure, a hydrophobic ionophore is dis-solved in an organic solvent immiscible with water.
Eisenman, et al., J. Membrane Biol. 1:294-345 (1969) disclose the selective extraction of cations from aqueous solutions into organic solvents via macro-tetralide actin an-tibiotics. This technique involves merely shaking an organic solvent phase containing the antlbiotics with aqueous solutions containing cationic sal-ts of lipid-soluble colored anions, such as picrates and dinitrophenolates. The intensity of color of the organic phase is then measured spectro-25 ~ photometrically to indicate how much salt has been extracted. Phase transfer has also been studied by Dix, et al., Angew, Chem. Int. Ed. Engl. 17:857 (1978) and in reviews including Bergermeister, et al., Top.
Cur.. Chem. 69:91 (1977); Yu, et al., "Membrane Active Complexones," Elsevier, Amsterdam (1974); and Duncan, "Calcium in Biological Systems," Cambridge University Press (1976).

-8 ~ 3~

Sumiyoshi, et al., Talanta, 24, 763-765 (1977) describes another method useful for determining K in serum. In this technique serum is deproteinated by trichloracetic acid, an indicator dye is added, and shaken with a solvent such as chloroform containing valinomycin.
Par-titioning of a compound is rapid and effec-tive between liquids, as shown by Eisenman, because of the mobility of the ionophore carrier and ions, which allows the transported species to diffuse rapidly away from the interface. Such a mechanism is normally impossible in the solid phase, because of the rigidity, immobility and essentially zero dif-fusion of materials in a solid phase.

2.3 Fluorescent Anions Yet another approach to the measurement of ion activity in aqueous solutions utilizes fluorescent anions. Feinstein, et al., Proc. Nat. Acad. Sci.
U.S.A., 68, 2037-2041 (1971). It is stated that the presence of cation/ionophore complexes in organic solvents are known, but that complex formation in purely aqueous media had theretofore not been detected.
Feinstein, et al., demonstrated the existence of such complexes in water through the use of the fluorescent 25 ~ sa:Lts 1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl sulfonate.
It was found that interaction of the ionophore/--cation complexes with the fluorescent dyes produced enhanced fluorescence emission, increased lifetime and polarization, and significant blue-shift at the emission maxima of the fluorescence spectrum. At constant concentrations of ionophore and fluorophore, the intensity of fluorescence emission was found to be a function of cation concentration.

--9- ~L2~j~L~3~3 2.4 Chromophore-labeled Ionophore An ion assay is disclosed in U.S. Patent No.
4,367,072 which makes use of a conjugate of an ionO-phore covalently bound to a chromophore material. In use, the conjugate is added to a liquid sample and the appearance of color in the solution is monitored spectrophotometrically.
The disclosure is limited to a solution assay, and it appears that insufficient color develops to enable direct visual observation. Moreover, the stoichiometric ra-tio of chromophore to ionophore is fixed in such a system due to -the direct bonding between these molecules. Because of this direct bonding it is impossible to regualte color intensity;
the ratio of ionophore to chromophore is fixed.

2.5 Summary To summarize the background of technological developments leading up to the present invention, many methods are known for assaying ions in solution.
Instrumental methods include such sophisticated techniques as ion-specific potentiometry, flame photometry and atomic absorption photometry. The use of ionophores which selectively complex with specific ions has led to four basic approaches: ion selective 25 . electrodes, liquid/liquid partitioning, fluorescence enhancement, and chromophore-labeled ionophore con-jugates.
None of these approaches, however, affords the assayist simple, fast analysis results through contacting a test sample solution with a test means or device. The present invention, on the other hand, permits the assayist to merely contact the sample with a dip-and-read test strip or device of similar ~Z~ 38 configuration, and observe a change in color or other detectable response. Moreover, the degree of such response can be regulated by varying the stoichiometry of the reacta.nts which produce it.

3. BRIEF DESCRIPTION OF THE DRAWINGS

Figures I-VII are graphical representations of the data obtained in Examples 9.2-9.8, respectively.
In Figure I the detection of potassium using naph-tho-15-crown-5 as the ionophore in Example 9.2 is shown.
Figure II is a graphical representation of the data obtained :in Example 9.3 in which both naphtho-15-crown-5 and valinomycin are present in the test means as ionophores Figure III depicts the results of Example 9.4 in which equal amounts of naphtho-15-crown-5 and valinomyc.in are utilized in the test means.
Figure IV shows the results of using only ~alinomycin as the ionophore in a potassium test means, as described in Example 9.5.
Figure V shows the results of using dipentyl phthalate as a plasticizer in the test means of Example 9.6.
Data from Example 9.7, in which a test means responsive to sodium ion concentration is described, is por-trayed graphically in Figure VI.
Figure VII is a plot of the data obtained in Example 9.8, in which -the reporter substance is tetrabromophenolph-thalein ethyl ester.

~2~%~

~. SUMMARY OF THE INVENTION

The present invention resides in the discovery of a new test means for detecting the presence of a specific ion in an aqueous test sample and to determining its concentration. The test means com-prises a substantially nonpolar, nonporous carrier matrix which is incorporated with an ionophore capable of selectively forming a complex with the ion under analysis. In addition, the carrier matrix is incorporated with a reporter substance which is capable of producing a detectable response, such as a change in or appearance of color or fluorescence.
A test device which utilizes the test means comprises an elongated support member, such as a plastic film, to one flat side of which is affixed the test means.
In use the sample is contacted with the test means or device, and the presence and/or concentration of the ion is then determined by observing any detectable response produced.
The test means and device of the present invention provide rapid results, sufficient detectable res-ponse forming in most instances in at least a few minutes. No cumbersome, expensive testing equipment 25 ~ is required in addition to the present invention.
Moreover, it has been found that the color or other response produced in the test means is stable, in some instances for a period of days, such that a number of used test means can be set aside for reading at some future time.

3~3 5. DEFINITIONS

Certain terms used in the present discussion should at this point be mentioned to assure that the reader is of the same mind as the author as to -their respective meanings. Thus the following definitions are provided to clarify the scope of the present invention, and to enable its formulation and use.

5.1 The -term "ionophore" includes molecules capable of selectively forming a complex with a particular ion to th~ substantial exclusion of others. For example the cyclic polypeptide valino-mycin, binds selectively to potassium ions in solu-tion to form a cationic complex. Also included in the term are crown ethers, cryptands and podands.

5.2 ~s used herein, "substantially nonpolar" is intended as meaning that quality of a substance not to exhibit a substanital dipole moment or electrical polarity. In particular, it includes non-ionic substances, and substances which are dielectric.

5.3 The term "nonporous" is intended to mean substantially impervious to the flow of water. Thus a nonporous carrier matrix is one which precludes the passage of wa-ter through it, one side to the other. For example, a polyvinyl chloride film would be considered for the purposes herein as being non-porous.

~2~2~3~3 5.4 A "reporter substance" is one which is capable of interacting with an ionophore/ion complex to produce a color change or other detectable response.
Thus, the reporter can be an ionic dye such that when the dye is in its ionized state i-t is a counter ion, i.e., opposite in charge, to the ion to be analyzed. Some examples of these are Erythrosin B, 7-amino-4-trifluoromethyl coumarin and 2,6-dich-loroindophenol sodium salt. The reporter also in-cludes phenolic compounds such as p-nitrophenol, which are relatively colorless in the non-ionized state, but which color upon ionization. The reporter substance can also be one which can txigger a detect-able response together with other components. For example, the iodide ion can produce a detectable response by interacting wi-th the ionophore/ion com-plex in the presence oE starch and an oxidizing agent.

5.5 By "interacting" is meant any coaction between a repor-ter substance and an ionophore/ion complex which leads to a detectable response. An example of the reporter substance interacting with the complex is in the case where the reporter is changed by the complex from a colorless to a colored state, such as in the case of p-nitrophenol.

5.6 The term "detectable response" is meant herein as a change in or occurrence of a parametex in a test means system which is capable of being perceived, ei-ther by direct observation or instrumen-tally, and which is a function of the presence of a specific ion in an aqueous test sample. Some detect-able responses are the change in or appearance of color, fluorescence, reflectance, pH, chemilumines-cence and infrared spectra.

3~

5.7 sy the term "intermediate alkyl" as used herein is meant an a]kyl group having from about 4 to about 12 carbon atoms. It includes normal and branched isomers. It may be unsubstituted or it may be sub-stituted, provided any such substitution not inter-fere with -the operation of the presently claimed test means or device in its capability to detect ions.

5.8 The term "lower alkyl", as used in the present disclosure, is meant an alkyl moiety con-taining about 1-4 carbon atoms. Included in the meaning of lower alkyl are methyl, ethyl, n-propyl, lsopropyl, n-butyl, sec-butyl and tert-butyl. These may be unsubstituted, or they may be substituted provided any such substituents not interfere with the operation or functioning of the presently claimed test means or device in its capability to detect ions.

5.9 By "pseudohalogen" is meant atoms or ~roups of atoms which, when attached to an unsaturated or aromatic ring system, affect the electrophilicity or nucleophilicity of the ring system, and/or have an abillty to distribute an electrical change through delocalization or resonance, in a fashion similar to the halogens. Thus, whereas halogen signifies Group VII a-toms such as F, Cl, and I pseudohalogens embrace such moieties as -CN, -SCN, -OCN, -N3, -COR, -COOR, 3, CC13, -NO2, -SO2CF3, -SO CH a d -SO2C6H4CH3, in which R is alkyl or aryl.

-15- 1~2438 6. TEIE TEST MEANS

The present test means comprises three basic elements: a substantially nonpolar, nonporous car-rier matrix; an ionophore; and a reporter substance.
When an aqueous test sample contalns an ion capable of specifically complexing with the ionophore, the ion can then enter the matrix and interact with the reporter substance, thereby producing a detectable response.

6.1 The Carrier Matrix In order for the test means to provide a detect-able response solely as a result of the presence of a specific ion, it is necessary that other ions be substantially excluded from entering the carrier matrix. This is because lt is -the ionophore/ion complex which triggers the detectable response in conjuction with the reporter substance. Accordingly, the carrier matrix must be fabricated from a material which is both nonpolar and nonporous. Exemplary of such materials are films of such polymers as poly-vinyl fluoride, polyvinyl chloride, vinyl chloride/-vinyl acetate copolymer, vinyl chloride/vinylidene-chloride copolymer, vinyl chloride/vinyl acetate/-vinyl alcohol terpolymer, vinylidene chloride/acry-lonitrile copolymer, and polyurethane. Of course, many other polymeric materials would be suitable for use in the present invention, and the identification oE such materials would be well within the skill of the art, given the present disclosure.
Other, nonpolymeric, materials would include ceramic substances, a painted substance (in which the paint layer would be the carrier matrix), glass-like substances, and other nonpolar materials.

~2~3a It is xequired that the carrier matrix be non-porous and nonpolar, because the ion must not be able -to substantially penetrate the matrix unless it is that particular ion or ions for which the ionophore has complexing affinity. The concept of a nonporous matrix, of course does not exclude microscopic porosity. It is clear from the foregoing remarks as well as the very nature of the invention, that some porosity could be possible provided the analyte ion be precluded from permeation of the carrier ma-trix to a sufficient degree to cause the detectable response to occur, absent the presence of the ionophore.
The composition of the carrier matrix in the present invention is to be carefully distinguished over prior ar-t test means whereby porous materials such as paper were used. In that type of device, it is required that any test sample to which the device is exposed be capable of permeating the entire reagent area. Such test devices function on entirely dif-ferent principles from the present one, and a paper carrier matrix is not considered as within the scope of the present invention unless such paper matrix be rendered substantially nonpolar and nonporous, i.e., such as by polymer or wax impregnation.
Thus, the carrier matrix is one which is not wetted by water, i.e., one which precludes substan-tial penetration by the aqueous test sample. More-over, it is intended that both the ionophore and reporter substance become virtually insoluble in the aqueous test sample due to their being entrapped with the carrier matrix. The requirement of nonporosity of the carrier matrix is to preclude dissolution or leaching of ionophore or the reporter substance, as well as to prevent permeation by test sample com-ponents other than the ionic analyte.

2~3~
6.2 Ionophores The ionophore element of the present invention is indeed a concept which is broad in scope, as characterized by the definition of the term in para-graph 6.1, supra. It includes multidentate cyclic compounds which contain donor atoms in their cyclic chains. Such multidentate cyclic compounds can be monocyclic or polycyclic. Alternatively, the ionophore can be an open chain containing donor atoms. Thus, included in the term are monocylcic systems which are ion-specific, termed coronands; polycyclic ion-specific compounds known as cryptands; and open chain structures, known as podands, which are capable of selectively complexing with ionsO

6.2.1 Coronands The coronands are monocyclic compounds which contain donor atoms which are electron rich or de-ficient and which are capable of complexing with particular cations and anions because of their unique structures. Included in this term are the crown ethers in which the ~nocyclic chain contains oxygen as the donor atoms. Other coronands are compounds which contain as assortment of electron rich atoms such as oxygen, sulfur and nitrogen. Because of the 25 , unique sizes and geometries of particular coronands, they are adaptable to complexing with various ions, In so complexing, the electron rich atoms, such as the oxygens in a crown ether, orient towards the electron deficient cation. The carbon atom segments of the ch~in are simultaneously projected in a direction out-wards from the ion. Thus, the resultant complex is changed in the center, but is hydrophobic at its perimeter .

-18- ~ 38 6.2.2 Cryptands The cryptands are the polycyclic analogues of the coronands. Accordingly, they include bicyclic and tricyclic multidentate compounds. In the cryp-tands, the cyclic arrangement of donor atoms is three dimensional in space, as opposed to the substantially planar configuration of the coronand. A cryptand is capable of virtually enveloping the ion in three dimensional fashion and, hence, is capabel of strong bonds to the ion in forming the complex. Like in the coronands, the donor atoms can include such atoms as oxygen, nitrogen and sulfur.

6.2.3 Podands Ions can also be selectively complexed with noncyclic compounds. For example, a linear chain which contains a regular sequence of electron rich atoms such as oxygen has the capability of associat-ing with positively charged ions to form complexes, not entirely unlike the coronands and cryptands. The main structural difference between podands and the other -two ionophores is the openness of the struc-ture. Thus, podands can be subcategorized into mono-podands, dipodands, tripodands,... . A monopodand, therefore, is a single organic chain containing donor atoms, a dipodand is two such chains attached to a central atom or group of atoms, and is capable of variable spacial orientation, and a tripodand is three such chains.

' ~, -19- ~22Z~

6.2.4 Specific Ionophores Some of the ionophores whieh have been found to be especially useful with the instant invention are tabulated herein along with the eations with whlch they are capable of selectively eomplexi.ng.

Ionophore Cation Valinomycin K+

4,7,13,16,2].-Pentaoxa-l,10-diaza-bicyclo~8,8,5]trieosane (Krypto-fix 221) Na 4,7,13,16,21,24-Hexaoxa-l,10-diaza-bicyclo[8,8,81hexacosane (Krypto-fix 222) K
4,7,13,18-Tetraoxa-l,10-diaza-bicyclo[8,5,5]eicosane (Krypto-fix 211) Li 12-Crown-4 Li 15-Crown-5 Na ,K
18-Crown-6 K
Dibenzo-18-crown-6 K
Dicyclohexano-18-crown-6 K

Kryptofix is a registered trademark of E. Merck, Darmstast, Germany 6.3 The Reporter Substance Given the presence of the ion of interest in the test solution, it is the reporter substance which provides the detectable response as a result of its interacting with the ionophore/ion complex. The reporter substance can range in eomposition from a single compound, such as ehromogenie eounterion, to a mixture of reactive speeies whieh produee a deteetable produet when their reaetion ehain is -20- ~22438 triggered by the complex. Thus, it can be seen that when no analyte ion is present the reporter substance remains dormant; no detectable response is observed.
Alternatively, when the particular ion under sur-veillance is present, lt is enabled by the ionophore to enter the carrier ma-trix to form a complex, which complex interacts with the reporter substance and induces it to undergo a detectable change.
In the case where the reporter is a single compound, it can include a salt or other dissociable compound, such that upon dissociation a colored ionic species is formed. Depending on the charge of the analyte ion, an ionic reporter is chosen such that the colored ion is opposite in charge to the analyte.
Also useful is a dissociable compound in which the counterion to the analyte is fluorescent. Examples of such chromophoric and fluorophoric reporter sub-stances are dichlorophenolindophenol, fluorescein and its derivatives, 8-anilino-1-naphthalenesulfonic acid, 7-amino-4-trifluoromethyl coumarinr Erythrosin B, Orange IV, Phloxine B, and Eosin Y. Structures of Erythrosin B, Phloxine B and Eosin Y are given in "Aldrich Handbook of Fine Chemicals", Aldrich Chemical Company, Milwaukee (1983). The structure of Orange IV can be found in "The Merck Index", 9th ed., Merck & Co., Inc. Rahway (1976).
Where the reporter substance comprises a mixture of reactive species, great latitude is possible in selecting an appropriate combination of ingredients.
For example, one system could be iodide ion, starch and an oxidizing agent. Such a system could be utilized in a test means in which the carrier matrix -21~ 2~3~

contains (in addition to ionophore) starch and the oxidi~er. Iodide could then be added to the test sample. In the presence of an analyte ion, formation of the ion/ionophoric complex would induce iodide to associate with the matrix, whereupon it would be conver-ted -to free iodine, thus indicating a positive test.
Yet another example of a reaction sequence use-ful as the reporter substance is one which involves the dissociation of a proton from a phenol, thus initiating a coupling reaction to form a colored product. The so~called Gibbs Reaction is typical of such a reaction sequence, in which 2,5-cyclohexa-diene-l-one-2,6-dihalo-4-haloimine couples with a phenol to form a colored reaction product X ~ X ~ ~+ X ~ X t HCl NX OH N

(I) (II) R ~ (III) In this reaction sequence R can be any 2, 3, 5, and/or 6 position substituent which will not hinder the overall reaction sequence. Thus R is H, lower or intermediate alkyl or aryl, or R can form a fused ring system at either the 2,3- or 5,6-positions. X
is halogen such as F, Cl, Br and I, or X can be a -22- ~222~3~

pseudohalogen. This kind of reporter substance can be utilized by incorporating compounds having the structures (I) and (II) directly wi-th the carrier matrix.
Still another utilization of the Gibbs chemistry involves compounds having a structure such as (III) in its nonionized form. The formation of the ion/-ionophore complex results in an interaction such that reporter substance (III) yields observable color in and of itself. This phenomenon can be thou~ht of as proceeding in accordance with -2:3- ~22~,3~

OH
Rn - ~1 Rn ~
Il P
X~ X~X

O~ O

R -- $ n ~3 X~X ' X~X

-24- ~2~2438 in which R is lower alkyl or aryl, or any two of R
together form a fused ring system, n is 0-4 and X is as defined above. Especially preferred is a compound having the structure OH
~_ R
N

Cl~l Yet another preferred reporter substance is a compound having the structure OH
R* ~ R

1~
C~ ~ Cl in which R* is an intermediate alkyl groupr i.e., having 4-12 carbon atoms, and in which R is H or lower alkyl. Compounds such as these have been found to be especially resistant to possible interference due to the presence of serum albumin in the test sample. Preferred among these types of reporter ~2~438 substances is that in which R* is n-decyl and R is methyl. Tetrabromophenolphthalein alkyl esters have also been found to be preferred reporter substances.

7. THE TEST DEVICE

The test means described above can be used by itself or it can be mounted at one end of an elong-ated support member, the other end serving as a handle. Such a test device can be held at the handle end, while the other end beariny the test means is contacted with the test sample.
Useful materials for the support member include films of a myriad of plastics or polymers. Examples include such polymeric materials as cellulose ace-tate, polyethylene terephthalate, polycarbonates and polystyrene. The support can be opaque or it can transmit light or other energy. Preferred supports include transparent materials capable of transmitting electromagnetic radiation of a wavelength in the range of about 200 nanometers (nm) to 900 nm. The support need not, of course, transmit over the entire 200-900 nm region, although for fluorometric detec-tion of analytical results it is desirable that the support be transparent over a band wider than, or at ' least equal to the absorption and emission spectra of the fluorescent materials used for detection. It may also be desirable to have a support that transmits one or more narrow wavelength bands and is opaque to adjacent wavelength bands. This could be accom-plished, for example, by impregnating or coating the support with one or more colorants having suitable absorption characteristics.

-26- ~2~ 8 To prepare a test device of the present inven-tion, a small rectangle of the test means, l.e., a carrier matrix incorporated with an ionophore, a reporter substance and possible other ingredients, is affixed to an elongated suppor-t member having an upper substantially flat face, such as an oblong piece of polystyrene film. The test means piece is affixed to the flat face at one end, leaving the other end of the polystyrene to serve as a convenient handle.
The test means can be affixed byany means compatible with the intended use. A preferred method is by using a double faced adhesive tape between the test means square and the support member. One such tape is known as Double Stick~, available from 3M
Company.

8. USE OF THE INVENTION

The test means and device of the present inven-tion can be adapted for use in carrying out a wide variety of chemical analyses, not only in the field of clinical chemistry, but in chemical research and chemical process control laboratories. They are well suited for use in clinical testing of body fluids . such as blood, blood serum and urine, since in this work a large number of repetitive tests are fre-quently conducted, and test results are often needed a very short kime after the sample is taken. In the field of blood analysis, for example, the invention can be adapted for use in carrying out quantitative analysis for many of the ionic blood components of clinical interest.
The test means (and test device) is used by contacting it with the test sample, and observing a -27- ~2~43~

detectable response. If the ion under analysis is present in the test sample, the complex of ionophore and ion will interact with the reporter substance and a detectable response will appear. Where the reporter substance, for example, is a dissociable sal-t pro-ducing a colored counterion of the analyte, the observable response will be the appearance of or change in color in the test means. Where the re-porter substance is a fluorophore such as fluoro-scein, a fluorescence spectrophotometer can be uti-lized to measure the de-tectable response formed in the test means (here, the appearance of or change in fluorescence). Other techniques useful in observing a detectable response include reflectance spectro-photometry, absorption spectrophotometry and light transmission measurements.
When the test sample is blood serum, trans-mission techniques can be used to detect and quantify the presence of any reaction products, the formation of which serves as -the detectable response. In this case radiant energy such as ultraviolet, visible or infrared radiation, is directed onto one surface of the test means and the output of that energy from the opposite surface is measured. Generally, electro-magnetic radiation in the range of from about 200 to about 900 nm has been found useful for such measure-ments, although any radiation permeating the test means and which is capable of signifying the occur-rence or extent of the response can be used.
Various calibration techniques are applicable as a control for the analysis. For example, a sample of analyte standard solution can be applied to a separate test means as a comparison or to permit the use of differential measurements in the analysis.

-28~ 2~38 9. EXAMPLES

The ~ollowing Examples are provided to further assist the reader in making and using the present invention. Thus, preferred embodiments are described in experimental detail and analyzed as to the re-sults. The Examples are meant to be illustrative only, and are in no way intended as limiting the scope of the invention described and claimed herein.

9.1 Preparation of 7-(n-Decyl)-2-methyl-4-(3',5'-dichlorophen-4-one)-indonaphthol The captioned compound (hereafter 7-decyl-MEDPIN) was prepared for use as a reporter substance in the present test means and test device. The reaction pathway is depicted in the following se-quence, in which R* is n-decyl.

~Z2Z~38 ~ >
o=~ ~ ~
cr~ ~
"~ o /
., o ~- n ~
o \ / ~a o \ ~

~ ~ ~o ~

~to \o O ¦ ~ ~
o~b P
~ ~ \ . J

-30- ~ZX2~38 ~o o~

o~ w ,_ ., -31~ 438 Preparation of ~-(p-n-Decylbenzoyl)-propionic Acid A mixture of 26.2 grams (g~ phenyl-n-decane (1.2 mole), 120 g succinic anhydride (1.2 mole) and 360 mililiters (mL) nitroethane in a 5 liter (L) three-necked flask equipped with HCl outlet and mechanical stirrer was cooled to 0C in an ice-bath. To this mixture 360 g AlC13 (2.7 moles) was added slowly over 1/2 hour with stirring. Evolution of HC1 was ob-served when about half of the AlC1 was added. After the addition, the ice bath was removed, the reaction mixture was allowed to stand at room temperature (RT) for 5 minutes. The mixture was then heated over a steam bath. The heatiny and stirring was continued until the vigorous evolution of HCl ceased (about 30 minutes). The reaction was cooled in an ice bath while 2 L of ice water was added followed by 600 ml of concentrated EiCl. This was stirred at RT for 2 hours until all the dark brown solid was hydrolyzed.
The insoluble product was recovered by filtration.
The solid was then recrystallized twice with acetic acid (250 ml. each time) to give about 320 g (85%
yield) of product (dried in vacuum over KOH). TLC:
Rf 0.43 in 1:1 (v/v) ethylacetate:toluene (silica gel plate).
Analysis: Calculated for C20H30O3: C, 75.42;
H, 9.50.
Found: C, 76.02;
H, 9.89.

~, :

-32- ~2~3~

Preparation of 4-(p-n-decyl-phenyl)-butyric acid Twenty grams of Pd/C (palladium-saturated carbon obtained from Aldrich Chemical Co., catalogue No.
20,569-9) and ~-(p-n decyl-benzoyl)-propionic acid (150 g 0.47 moles) were mixed with acetic acid (350 mL) in a 1 L Paar bomb. The reaction was started at 50 psi (pounds per inch) H2 pressure and 50C. A
sudden increase in temperature accompanied by a drop in H volume was observed. Thin layer chromatography reaction mixture indicated complete reaction. The catalyst was removed by glass fiber filtration while ho-t~ The filtrate was allowed to crystallize at RT.
The crystalline product was recovered by filtration.
A second crop of product which formed after the filtrate was chilled was also recovered. The total yield was 100 g (68%) after drying under a vacuum over KOH. The melting point was 67-69C.
TLC: Rf 0.68 in 1:1 (v/v) e~hylacetate:toluene (silica gel plate) Analysis Calculated for: C20H32O2: C, 78.90;
H, 10.50.
Found: C, 78.39;
H, 10.70.

Preparation of 7-n-Decyl-1-tetralone A mixture of p-deeyl-phenyl butyric acid (30 g, 98~7 mmoles) and polyphosphoric acid (150 g) was heated in an oil bath until all solid was melted.
The heating was elevated to 150C (internal temp.) and the mixture was stirred vigorously for 30 minutes.
The reaction was -then cooled to RT and treated with 300 mL ice water and 150 mL ethyl ether. After the mixture was stirred for 30 minutes at RT, the aqueous layer was separated and washed twice with 150 mL
ethyl ether. The combined organic phases were washed -33- ~ 3~

with 150 mL saturated aqueous NaCl. Ether was re-moved by evaporation and the residue was distilled on a Kugelrohr distillation apparatus (Aldrich Chemical Co.). The product had a boiling point of 190-200C/
0.1 mm Hg. The yield was 11 g (39%) of pale yellow oil.
TLC- Rf - 0.34 in toluene (silica gel plates) Analysis Calculated for: C20H30O: C, 83.90;
H, 10.70.
Found: C, 85.63;
H, 10.83.

Preparation of 2-Hydroxymethylene-7-n-decyl-1-tetralone A mixture of sodium methoxide t5.4 g 40.5 moles), ethyl formate (7.4 g, 100 mmoles) and 100 mL
dry toluene was cooled in an ice bath under inert atmosphere (N2). A solution of the 7-decyl-tetralone (11.5 g, 40 mmoles) in 100 mL dry toluene was added with rapid stirring. The ice bath was removed and the reaction was stirred at RT for 4 hours. The reaction mixture was treated with 100 mL water and 100 mL 6N HCl. The organic layer was separated and washed twice with 50 mL saturated NaCl, dried over anhydrous Na2SO4, filtered and evaporated to remove 25 , all the toluene. The oily residue was used for the next reaction without further purification.
Tl.C: Rf = 0.56 in toluene (silica gel plates), the spot turned dark-brown after spray with 5% FeC13 solution.

~34~~ ~2~8 Preparatlon of 2-Benzoyloxyme-thylene-7-n-decyl-l-tetralone The oily residue from the previous reaction step was mixed with dry pyridine (120 mL). The solution was stirred under nitrogen at 0C (ice bath). The solution was treated with 30 mL of benzoy] chloride.
Af-ter the addition of the benzoyl chloride, insoluble pyridinium chloride was observed in the mixture. The reaction was stirred at RT for two hours. The pro-duct was poured into ice water (400 mL) with vigorous stirring. The light cream color solid was recovered by filtration, and washed well with cold water. The slightly wet solid was recrystallized from hot ab-solute ethanol (120 mL). White solid (14 g, 87% yield based on the 7-decyl-1-tetralone) was recovered. The melting point was 64-66C.
TLC: Rf = 0.40 in toluene (silica gel plates) Analysis Calculated for: C28H34O3: C, 80.34;
H, 8.19.
Found: C, 80.05;
El, 8.27.

Preparation of 7-n-Decyl-2-methyl-1-naphthol To a mixture of 2-benzoyloxymethylene-7-(n-decyl)-l-tetralone (14 g, 33.5 mmoles) and Pd/C (3.5 25 , g) under inert atmosphere was added cyclohexene (175 mL). The mixture was heated to reflux while main-taining the inert atmosphere. The conversion of starting material to product was determined by TLC
after 3 hours. After all the starting material reacted, the mixture was cooled down to RT. The catalyst was removed by filtration and washed twice with 50 mL hot toluene. The combined filtrate was ~2~:~3~3 evaporated to a small volume. The product was puri-fied wi-th a Prep-500 silica gel column (a high pre-ssure sllica gel preparative column, obtained from Waters Association, Milford, MA). Toluene was used as the mobile phase. The product fractions were pooled and evaporated to dryness under vacuum over-night. Cream white solid (9.0 g 90% yield) was recovered: The melting point was 65-67C.
TLC: Rf = 0.65 in toluene (silica gel plates).
Pink color developed when the product spo-t was irradiated with short UV light.
Analysis Calculated for: C21H30O: C, 84.51;
H, 10.13.
Found: C, 84.49;
H, 10.72.

Preparation of 7-(n-Decyl)-2-methyl-4-(3',5'-dich-lorophen-4'-one)-indonaphthol 7-Decylmethyl-l-naphthol (4.5 g, 15.1 mmoles) and 2,6-dichloroquinone-4-chloroimide (3.0 g, 14.3 mmoles) were dissolved in acetone (150 mL). The solution was treated with 700 mL potassium carbonate solution (0.1 M, pH = 10.0). The solution was stirred vigorously at RT for 10 min. The pH of the reaction mixture was adjusted to 2.8 with HCl (1.0 N). The mixture was stirred for 15 minutes. The red solid was recovered by filtration and washed well with water. The solid was dissolved in toluene and filtered with glass fiber paper to remove any in-soluble materials. The filtrate was concentrated and purified with Prep-500 silica gel column, using toluene as -the mobile phase. Product fractions were pooled and evaporated to dryness. The residue was crystallized with n-hexane (100 mL) to give product (3.9 g, 58% yield).

TLC: RF = 0.26 in toluene (silica gel plates).
Brown color spot, turn purple-blue after treated with 0.1 N NaOH on the plates.
Analysis Calculated for: C27H31NO2C12: C, 68.64;
H, 6.57; N, 2.97.
Found: C, 68.88, H, 6.85; N, 2.97.

This product, 7-decyl-MEDPIN, was used in the following experiments as the reporter substance.

9.2 Naphtho-15-crown-5 as Ionophore An acetone mixture was prepared containing 10.8 mg (milligrams) 7-decyl-MEDPIN and 24 mg naphtho-15-crown-5 (2,3,5,6,8,9,11,12-octahydronaphtho[2,3-b]-1,~,7,10,13-pen-taoxacyclopentadecane). Solvent was removed under a stream of nitrogen gas. Then dried solids were combined with 4 g of a film solution com-prising 70% by weight cyclohexanone, 12% by weight polyvinyl chloride/polyvinylidene copolymer, 18~ by weight diethyl phthalate, and 60 (uL) Triton X-100 (a 1% by weight solution of nonionic detergent in acetone; available from Rohm and Haas, Co.). The mixture was homogenized on a vortex mixer, and then spread into a thin film on a piece of KODAR~ cyclo-hexanone/dimethylene terephthalate copolymer (Lustro Co.) channeled plastic, using a doc-tor blade having a 10 mil (0.01 inch) gap. The dried film has a thick-ness of about 3 mils.
The test means was evaluated with aqueous test samples containing various KCl concentrations. Each sample was 15.56 ~ NaCl and 88.89 CAPS buffer [3-(cyclo-hexylamino)-propanesulfonic acid] and was adjusted to pH 10 with LiOH. The respective KCl concentrations were 0, 0.33, 0.67 and 1.0 m~.

.~ , :

-37~ 38 The evaluations were conducted by innoculating a section of the test means film with ~0 microliters (~L) of test sample and the change in refIectance monitored for one minute in a SERALYZER reflectance Photometer (Ames Division of Miles Laboratories, Inc.).
The reflectance values (R) were conver-ted to (K/S) in accordance with (K/S) = (1 R) _ in which R is the fraction of reflectance from the test device, K is a constant, and S is the light scattering coefficient of the particular reflecting medium. The above equation is a simplified form of the well-known Kubelka-Munk equation (See Gustav Kortùm, I'Reflectance Spectroscopy", pp. 106-111, Springer Verlaz, New York (1969). The data is tabu-lated below as (K/S) with respect to time.

[K ]mM (K/S) second 0 0.001151 0.33 0.007679 0 ~7 0.01295 1.O O.01909 As can be seen from the table t rate of change in (K/S) with time varies in accordance with potassium concentration. The data is shown graphically in Figure I, and demonstrates easy differentiation of various K concentration levels.

-38- ~2243~

9.3 Potassium Test Means Using Naphtho-15-crown-5 and Valinomycin as Ionophores A test means film was prepared and evaluated as in example 9.2 except that 6 mg of naphtho-15-crown-5 was replaced by 6 mg valinomycin. The data is re-ported in the following table:
.~ --1 [K ]mM (K/S)second 0.001182 0.67 0.01331 1.0 0.01857 The data shows a direct correlation between potassium concentration and rate of change of (K/S~, as is clearly depicted by the graphical depiction of the da-ta in Figure II.

9.4 Potassium Test Means Using Equal Amounts of Naphtho-15-crown-5 and Vali.nomycin as Ionophores A test means film was prepared and evaluated as in Example 9.2 except that 12 mg of naphtho-15-crown-

5 was replaced by 12 mg valinomycin. The data is reported in the following table:

[K ]mM (K/S) second 0 0.0007449 0.33 0.008314 0.67 0.01251 1.0 0.01898 -39- ~2243~

The data shows a direct correlation between potassium concentration and rate of change of (K/S).
Thls is clearly shown by the graphical plot of the data in Figure III.

9.5 Potassium Test Means Using Valinomycin as Ionophore A test means film was prepared and evaluated as in Example 9.2 except that the amount of 7-decyl-MEDPIN was 5.4 mg, the ratio of polyvinyl chloride/-polyvinylidene chloride copolymer to diethylphthalate was adjusted to 8.55:21.45 by weight, and the 12 mg naphtho-15-crown-5 was replaced by 12 mg valinomycin.
The aqueous test samples contained KCl at con-centra-tions of 0, 0.33, 0.67, 1.0, 2.0 and 3.0 mM~
In addition, each solution contained 46.67 mM NaCl, 66.67 mM CAPS and was titrated to pH 10 with LiOH.
The reflectance data is reported in the fol-lowing table:

[K ]mM (K/S) second 0 0.001008 0-33 0.01090 0.67 0.01787 1~0 0.02872 2.0 0.04321 3.0 0.05330 As can be seen from the data, the test means exhibited a direct correlation between potassium concentration and the rate of change of (K/S) with time. As the plot of the data in Figure IV shows, easy differentiation between K concentration levels was obtained.

-40~ 243~

9.6 Potassium Test Means Using Dipentyl Phthalate as Plasticizer A test means film was prepared and evaluated as in Example 9.5 except that the diethyl phthalate was replaced by an equal weight of dipentyl phthalate.
Aqueous test samples were as in Example 9.5 and contained KCl as indicated in the table of data below:

[K ]mM (K/S) second 0 0.0005o7o 0.33 0.004041 0.67 0.007020 1.5 0.01391 3.0 0.02195 The data shows a direct correlation between potassium concentration and the rate of change of (K/S) with time. The data is plotted in Figure V, which portrays the ease of differentiation of various potassium levels using the test means film of the present example.

9.7 Sodium Test Means A solution of 10.8 mg 7-decyl-MEDPIN in acetone and a solution of 5 mg sodium Ligand I [N,N',N"-triheptyl-N,N',N"-trimethy-4,4',4"-propylidintris-3-oxa-butyramide] in tetrahydrofuran (THF) were mixed and the solvents removed under a stream of nitrogen.
To the dried solids was added 0.5 g of a film solu-tion. The latter was 70QO by weight cylohexanone, 8.55% by weight of vinylchloride/vinylidene chloride copolymer, and 21.45% by weight dipentyl phthalate.
The mixture was homogenized on a vortex -41- ~2~Z43~

mixer and the homogenate spread into a film on a piece of KODAR channeled plastic using a 10 mil doc-tor blade. The dried film had a thickness of about 3 mils.
Aqueous sodium test samples were prepared for evaluating the test means. Each contained 88.98 mM
CAPS and KOH was added to adjust the pH to 10. Sam-ples were prepared containing 11.11 mM and 22.22 mM
NaCl, respectively.
To evaluate the ability of the test means to detect sodium, 40 ~IL of a test sample was applied to a section of the test means film and reflectance at 640 nm was monitored over 2 minutes in a SERALYZER
reflectance photometer. Reflectance values were converted to (K/S) values as in Example ~.2. The rate of chanye of (K/S) with time and respective sodium concentrations are tabulated below:

[Na ]mM (K/S) second 0 0.001459 11.11 0.003148 22.22 0.004354 The data shows a direct correlation between sodium concentration and the rate of change of (K/S) ~ with time, as portrayed graphically in Figure VI.

9.8 Tetrabromophenolphthalein Ethyl Ester Used as a Reporter Substance in a Test Means for Detecting Potassium A solu-tion was prepared containing 1.8 mM
valinomycin, 5 mM te-trabromophenolphthalein ethyl ester (TBEE), 5% by weight polyvinylchloride ("high molecular weight"j Aldridge Chemical Co. Catalogue No. 18,956-1) and 13.1% by weight dipentyl phtha].ate ~2~2~3~3 in tetrahydrofuran. This solution was spread onto a polyester film using a 10 mil doctor blade, and dried.
The test means was evaluated using aqueous solutions (test samples) containing KCl at concen-trations of 0, 0.222, 0.556 and 1.111 mM, respec-tively. Each solution contained 100 mM sodium citra-te (pH = 5.3). The reflectance response oE the test means to each test sample sol.ution was monitored at 37C and 520 nm using a SERALYZER reflectance spectrophotometer. The results are tabulated below:

[K ]mM(K/S) second 1 x 10 3 0 -0.0108 0.222 0.990 0.556 2.013 1.111 4.101 The da-ta shows a direct correlation between rate of change o~ (K/S) per unit time and potassium con-centration. Figure VII presents this data graphi-cally, and demonstrates a linear relationship between actual K concentration and (K/S).

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A test means for determining the presence of an ion in a test sample, the test means comprising a substantially nonpolar, nonporous carrier matrix incorporated with an ionophore capable of forming a complex with the specific ion, and a reporter substance capable of interacting with the complex of ionophore and ion to produce a detec-table response.

2. The test means of Claim 1 in which the ion-phore is a coronand, a cryptand or a podand.

3. The test means of Claim 1 in which the ionophore is valinomycin, 4,7,13,16,21-pentaoxa-1, 10-diazabicyclo [8,8,5]tricosane, 4,7,13,16,21,24-hexaoxa-l,10-diazabicyclo [8,8,8]hexacosane, 4,7,-13,18-tetraoxa-1,10 diazabicyclo [8,5,5] eicosane, 12-crown-4, 15-crown-5, 18-crown-6, dibenzo-18-crown-6, and dicyclohexano-18-crown-6.

4. The test means o claim 1 in which the iono-phore is valinomycin.

5. The test means of Claim 1 in which the reporter substance is a compound having the structure in which X is a halogen or pseudohalogen; R is a 2-, 3-, 5-, and/or 6-position substituent selected from lower or intermediate alkyl, aryl and a fused ring at the 2,3- or 5,6- positions; and n is 0 to 4.

6. The test means of Claim 1 in which the reporter substance is a compound having the structure in which R' is H or lower alkyl, R* is H or inter-mediate alkyl and X is a halogen or pseudohalogen.

7. The test means of claim 6 in which R' is methyl and R* is n-decyl.

8. The test means of Claim 1 in which the reporter substance is one capable of producing the appearance of, or change in, fluorescence in the presence of the complex of the ionophore and ion.

9. The test means of Claim 8 in which the reporter substance is fluorescein or a derivative thereof.

10. The test means of Claim 1 in which the reporter substance comprises at least two precursor substances which are capable of participating in the formation of a detectable response in the presence of the complex of the ionophore and the ion.

11. The test means of Claim 10 in which the precur-sor substances have the respective structure in which X is halogen or a pseudohalogen, R is a 2-, 3-, 5-, and/or 6-position substituent selected from lower or inter-mediate alkyl, aryl and a fused ring at the 2,3- or 5,6-posi-tions, and n is 0-4.

12. The test means of Claim 10 in which the pre-cursor substances are starch and an oxidizing agent.

13. A test device for determining the presence of an ion in a test sample, the test device comprising an elongated support member having an upper substan-tially flat face, and the test means of any one of claims 1 to 3 affixed to the flat face of the support member.

14. A test device for determining the presence of an ion in a test sample, the test device comprising an elongated support member having an upper sub-stantially flat face, and the test means of any one of claims 4 to 6 affixed to the flat face of the support member.

15. A test device for determining the presence of an ion in a test sample, the test device comprising an elongated support member having an upper substan-tially flat face, and the test means of any one of claims 7 to 9 affixed to the flat face of the support member.

16. A test device for determining the presence of an ion in a test sample, the test device comprising an elongated support member having an upper substan-tially flat face, and the test means of any one of claims 10 to 12 affixed to the flat face of the support member.

17. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with the test means of any one of claims 1 to 3, and observing a detectable response.

18. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with the test means of any one of claims 4 to 6, and observing a detectable response.

19. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with the test means of any one of claims 7 to 9, and observing a detectable response.

20. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with the test means of any one of claims 10 to 12, and observing a detectable response.

21. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with a test device and observing a de-tectable response, said test device comprising an elongated support member having an upper sub-stantially flat face, and the test means of any one of claims 1 to 3 affixed to the flat face of the support member.

22. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with a test device and observing a de-tectable response, said test device comprising an elongated support member having an upper sub-stantially flat face, and the test means of any one of claims 4 to 6 affixed to the flat face of the support member.

23. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with a test device and observing a de-tectable response, said test device comprising an elongated support member having an upper sub-stantially flat face, and the test means of any one of claims 7 to 9 affixed to the flat face of the support member.

24. A method for determining the presence of an ion in an aqueous test sample, the method comprising contact-ing the test sample with a test device and observing a de-tectable response, said test device comprising an elongated support member having an upper sub-stantially flat face, and the test means of any one of claims 10 to 12 affixed to the flat face of the support member.